EP1080186A2 - Method for altering the activity of proteins of the pka signaling pathway - Google Patents

Abstract

The invention relates to methods of altering the activity of the PKA signaling pathway by altering, preferably inhibiting, the extent of phosphorylation of PKA substrates or downstream kinase substrates, particularly by the use of modified substrates, gene sequences encoding such modified substrates, vectors and host cells containing the gene sequence, inhibitors of substrate phosphorylation and use of the gene sequence, protein or inhibitors to alter the PKA signaling pathway, for example in treating immunosuppressive disorders or proliferative diseases.

Description

Method

The present invention relates to methods of altering the activity of the PKA signaling pathway by altering the extent of phosphorylation of PKA substrates or downstream kinase substrates, particularly such that the inhibition of lymphocyte activation mediated by protein kinase A is reduced or abolished. The invention also relates to modified, preferably mutated substrate gene sequences wherein the phosphorylation site of the wild type gene is mutated, the protein expressed by the mutated gene sequence, the vector containing the gene sequence, the host cell transfected with the vector, activators or inhibitors of substrate phosphorylation and use of the gene sequence, protein, activators or inhibitors to alter the PKA signaling pathway, for example in treating immunosuppressive disorders or proliferative diseases.

Cyclic AMP-dependent protein kinase (PKA) is a key negative regulator of lymphocyte function through the T- cell antigen receptor/CD3 complex (TCR/CD3) (Muraguchi et al., 1987, J. Cell . Physiol . , 131, p426-433; Blomhoff et al., 1987, J. Cell. Physiol. , 131, p426-433; Kammer, 1988, Immunol. Today, 9, p222-229; and Skalhegg et al . , 1992, J. Biol. Chem. , 267, pl5707-15714) . With few exceptions, all known actions of cAMP are mediated through cAMP-dependent protein kinase (PKA) . This broad specificity serine-threonine kinase is activated by binding of four molecules of cAMP to a regulatory (R) subunit dimer, resulting in the release of two free, active catalytic (C) subunits from an inactive, tetrameric complex (Skalhegg & Tasken, 1997, Front .Biosci. , 2: d331-d342) . PKA type I and type II are distinguished by the R subunits in the inactive tetramer. Several different R (Rlα, Rlβ, Rllα, Rllβ) and C (C , Cβ, Cy) subunits have been cloned and characterized, and constitute a number of PKA isozymes that contributes specificity to signal transduction by cAMP/PKA (Scott, 1991, Pharmacol. Ther., 50, pl23-145; and Skalhegg & Tasken, 1997, supra) .

The inventors of the immunomodulation strategies presented herein and others have previously shown that cAMP inhibits T lymphocyte proliferation induced through the T cell antigen receptor/CD3 complex (TCR/CD3) .

Agonists which directly activate adenylate cyclase or adenylate cyclase-coupled receptors, as well as cAMP analogues, interfere with T cell activation (Kammer, 1988, Immunol. Today, 9, p222-229) , and T cell stimulation with mitogens or anti-CD3 monoclonal antibodies (mAbs) leads to an increase of intracellular cAMP ( edbetter et al . , 1986, J. Immunol., 137, p3299- 3305; and Kammer et al . , 1988, Proc. Natl. Acad. Sci. USA 85, p792-796) , resulting in the rapid activation of type I isozyme of protein kinase A (Skalhegg et al . ,

1992, J. Biol. Chem., 267, pl5707-15714 ; Laxminarayana et al., 1993, J. Clin. Invest., 92, p2207-2214; and Laxminarayana & Kammer, 1996, J. Immunol., 156, p497- 506) . We have shown that T cells express both PKA type I and II. However, only the selective activation of PKA type I (RIα₂C₂) is sufficient to mediate the inhibitory effect of cAMP . In addition, we have demonstrated that PKA type I, but not PKA type II, redistribute to and colocalize with and inhibit signaling through antigen receptors on T and B cells and regulate mitogenic responses in T and B cells and acute cytotoxic responses in NK cells (Skalhegg et al . , 1992, supra; Skalhegg et al., 1994, Science, 263, p84-87) . Thus, PKA type I seems to serve as a key negative regulator of lymphocyte functions, eg. mitogenic and cytotoxic responses initiated through antigen receptors. This together with the observation that triggering of the TCR/CD3 complex leads to production of cAMP (Skalhegg et al . , 1994, Science, 263, p84-87; Ledbetter et al . , 1986,

J. Immunol., 137, p3299-3305; and Kammer et al . , 1988, Proc. atl.Acad. Sci.U.S A., 85, p792-796) , has previously led us to hypothesize that modulation of normal immune responsiveness by activation of PKA type I is a negative feedback mechanism. Dysregulation of this system may lead to immunological overshoot or impaired immune functions (Kammer et al . , 1994, J. Clin. Invest., 94, p422-430; and Aandahl et al . , 1998, FASEB J. , 12, p855- 862) .

We have previously investigated the possible role of cAMP-mediated inhibition of T cell immune function in purified T cells from HIV-infected patients prior to and during highly active antiretroviral therapy. We thus demonstrated that increased activation of PKA type I significantly inhibits T cell proliferation in cells from HIV-infected individuals independent of ongoing potent antiretroviral therapy and that this effect can be reversed by a specific antagonist of PKA type I that increases the impaired proliferation of T cells from HIV-infected patients to normal or subnormal levels (up to 300%) International Patent Application No. PCT/NO98/00134. Follow-up of patients after initiation of highly active antiretroviral treatment revealed that the majority of patients had a persistent T cell dysfunction which is normalized by incubation of T cells with Rp-8-Br-cAMPS . These observations imply that increased activation of PKA type I may contribute to the progressive T cell dysfunction in HIV-infection (Aandahl et al . , 1998, supra), and that PKA type I may be a potential target for immunomodulating therapy. Furthermore, a number of strategies, including the use of PKA type I selective antagonists, have been designed to reverse the inappropriate activation of PKA type I in immunodeficiencies and thereby restore T cell function and immune responsiveness.

Protein kinase A mediates its role as a negative regulator of lymphocyte function by phosphorylation of substrate proteins. As the effect of cAMP on inhibition of lymphocyte function is acute and mediated by protein kinase A type I that is colocalized with the antigen receptors, the important substrates are probably other signaling molecules in the proximal signaling pathway down-stream of the antigen receptor. The molecular targets for PKA-dependent phosphorylation which is associated with inhibition of T-cell receptor function remain unknown.

In Arch. Biochem. Biophys., (1997), 343:2, 194-200 (Sun et al) regulation of the enzyme C-terminal Src kinase (Csk) via PKA is described. However, in contrast to the findings described herein PKA phosphorylation was found to decrease Csk kinase activity. Furthermore, the authors did not identify or describe the actual phosphorylation sites, specific mutants of Csk which are not phosphorylated by PKA, or the role of regulation of Csk.

In WO 95/18823 (Cantley and Songyang) a method of identifying specificity of phosphorylation sites for novel or known protein kinases is described. However, substrates of PKA which are involved in PKA signalling, particularly those involved in regulation of T-cell receptor function were not identified.

Thus the object of the present invention is to provide the above mentioned substrates to identify targets for altering the activity of the PKA signaling pathway .

Surprisingly, substrates which are phosphorylated by PKA, or phosphorylated by kinases downstream in the PKA signaling pathway, have been identified, whose extent of phosphorylation may be varied to alter the activity of the PKA signaling pathway, particularly in T lymphocytes .

Thus view from a first aspect the present invention provides a method of altering the activity of the PKA signaling pathway in a cell, preferably a T lymphocyte, wherein the extent of phosphorylation of one or more PKA substrates, or kinase substrates downstream in the PKA signaling pathway is altered.

As mentioned above, PKA Type I mediates the inhibitory effect of cAMP resulting in lymphocyte dysfunction. In a preferred aspect therefore, the PKA Type I (particularly preferably the PKA Type Iα, ie. RIα₂C₂) signaling pathway is altered.

The "PKA signaling pathway" as referred to herein refers to a series of signaling events in which PKA is activated resulting in increased kinase activity of this enzyme. This signaling pathway is intended to include molecular events from activation of PKA to end effects such as reduced proliferation or IL-2 production, or intermediate effects such as inactivation of Src kinases . As referred to herein the phrase "altering the activity of the PKA signaling pathway" is intended to mean the alteration of one or more signaling elements in the pathway (e.g. to affect its enzymatic or other functional properties) is achieved which affects downstream signaling events. Alteration of the signaling elements may for example be due to the signaling element exhibiting an increase or decrease in enzymatic activity, e.g. kinase, phosphatase or GTPase activity, or alteration (negatively or positively) in the ability to form interactions with other molecules, e.g. protein-protein interactions. The ultimate effect may be to up- or down-regulate downstream events which typify PKA signaling. Preferably however, for use in particularly clinical conditions, downregulation of the PKA signaling pathway, ie . reversal of the effects of cAMP activation, e.g. to reverse lymphocyte dysfunction, is preferred.

The "extent of phosphorylation" refers to phosphorylation at the PKA phosphorylation site, or in the case of a downstream substrate, the site on that substrate phosphorylated by a kinase in the PKA signaling pathway, or at least one site if more than one site exists. Alteration of this phosphorylation refers to an increase or decrease in the amount of phosphate bound to the substrate. Clearly this would need to be determined as an average for all such substrate molecules present in the cell . Whilst this will in some cases be achieved by affecting the level of phosphorylation of endogenously present substrate, this may also be achieved by altering the level of the substrate in the cell (e.g. by altering the level of expression or adding exogenous substrates, which may be modified) . In such cases it is relevant to consider the amount of phosphorylated substrates (and whether this is increased or decreased) regardless of whether they are endogenous or exogenous in origin. PKA substrates include any proteins or peptides which are phosphorylated by PKA as part of the PKA signaling pathway. These include, for example, Csk kinase, Vav, phospholipase C-γl or 2 and Raf . Downstream kinase substrates include any substrate which is phosphorylated in the PKA signaling pathway, as an indirect consequence of activation of PKA (although PKA activation may cause an increase or decrease in phosphorylation) . Such substrates include for example substrates of Csk kinase which is activated in the PKA signaling pathway, such as Lck, or substrates of Src kinases which are inactivated in the PKA signaling pathway. In the latter case to reduce the effects of PKA signaling, increased phosphorylation of the Src kinase substrates would be desirable. Activation of T lymphocytes encompasses a pleiotropic cascade of molecular events resulting in the production of lymphokines, upregulation of receptors, and ultimately, mitosis. One of the earliest biochemical events following ligation of the antigen- specific T cell receptor (TCR) /CD3 complex is the activation of Src-family protein tyrosine kinases (PTKs) such as Lck and Fyn (Mustelin, 1994, Immunity., 1, p351- 356; and Qian & Weiss, 1997, Curr. Opin. Cell Biol., 9, p205-212) . These kinases seem to mediate the initial tyrosine phosphorylation of immunoreceptor tyrosine- based activation motifs (ITAMs) in the TCR-ζ and CD3 chains (Weiss & Littman, 1994, Cell, 76, p263-274;

Isakov et al . , 1994, J. Leukoc . Biol., 55, p265-271) . ITAM phosphorylation results in the activation of Syk- family PTKs, Zap-70 and Syk and recruitment to the phosphorylated receptors where they become auto- and/or trans-phosphorylated and thereby enzymatically activated (Weiss & Littman, 1994, supra; Isakov et al . , 1994, supra) . The coordinated activation of Src- and Syk- family PTKs eventually leads to the phosphorylation of multiple downstream substrates such as phospholipase Cγl (PLCγl) (Mustelin et al . , 1990, Science, 247, pl584- 1587; Carpenter et al . , 1993, Adv. Second Messenger Phosphoprotein Res., 28, pl79-185; and Park et al . , 1991, Proc. atl.Acad. Sci.U.S.A. , 88, p5453-5456), which in turn hydrolyzes phosphatidylinositol 4 , 5-bisphosphate to generate two second messengers, inositol 1,4,5- trisphosphate and diacylglycerol . These messengers mediate an increase in the free intracellular Ca²⁺ concentration and activation of protein kinase C (PKC) , respectively (Isakov et al . , 1994, J. Leukoc. Biol., 55, p265-271) .

The Src kinases also activatee the Ras-Raf signaling pathway (Franklin et al . , 1994, J. Clin. Invest. , 93, p2134-2140) . The Src family of tyrosine kinases are activated by the tyrosine phosphatase, CD45 (Mustelin & Altman, 1990, Oncogene, 5, p809-813), and inhibited by another tyrosine kinase, C- terminal Src kinase (Csk) (Partanen et al . , 1991, Oncogene, 6, p2013-2018; and Nada et al . , 1991, Nature, 351, p69-72) . Csk is a regulator of the Src family of tyrosine kinases. Csk phosphorylates a conserved C-terminal tyrosine residue (Y505 in Lck, Y528 in Fyn^τ) in all Src kinases. The resulting phosphotyrosine binds to the SH2 domain of the Src kinase by an intra-chain conformational change that turns off Src kinase activity and inhibits lymphocyte activation. Csk is structurally related to the family of Src kinases (Partanen et al . , 1991, supra) . These kinases are all non-receptor protein tyrosine kinases and contain SH3 , SH2 and catalytic domains. However, Csk differs from the Src kinases in that it has no N-terminal myristylation motif, no regulatory C-terminal tyrosine residue, and no tyrosine residue localized in the catalytic domain that is autophosphorylated. It has been demonstrated that knock-out of the csk gene is lethal in mouse embryos (Imamoto & Soriano, 1993, Cell, 73, plll7-1124; and Nada et al., 1993, Cell, 73, pll25-1135), but in spite of the vital function of Csk, it is not known how the activity of this important enzyme can be regulated.

It has now been found by the inventors that protein kinase A phosphorylates Csk on serine 364 and that cAMP/protein kinase A-mediated phosphorylation of this residue leads to a 2 to 3 -fold increase in Csk kinase activity.

In vivo, the increased activity of Csk following treatment with cAMP leads to a decreased activity of Lck and to decreased Lck-mediated phosphorylation of TCR ζ- chain and Zap-70 which are key events in T cell activation. It has also now been demonstrated by the inventors that transfection of a mutated Lck gene, Lck- Y505F, which can no longer be phosphorylated by Csk, abolishes the inhibitory effect of cAMP/protein kinase A on TCR ζ-chain phosphorylation. Mutation of the human gene encoding Csk (accession number X60114) in nucleotides 1502 to 1504 that encodes serine 364 of the gene product, to change serine to alanine, cysteine, aspartic acid or glutamic acid (Csk-S364A, Csk-S364C,

Csk-S364D or Csk-S364E, respectively) abolishes protein kinase A-mediated phosphorylation of Csk. Use of the mutated gene will protect cells from the effect of protein kinase A on Csk. Furthermore, use of mutated genes of the Src family of kinases (Lck, Fyn, Src, Yes, Fgr, Lyn, Hck, Blk, Yrk) , mutated at the C-terminal tyrosine phosphorylated by Csk (eg. Lck-Y505F) , will protect them from regulation by Csk and thereby from regulation by protein kinase A and cAMP.

Vav is expressed specifically in T cells and other hematopoietic cells and is a prominent and early substrate of antigen receptor-coupled tyrosine kinases. Vav is a 95-kDa signaling protein which was first identified in its oncogenic form during fibroblast transformation with human tumor DNA (Katzav et al . , 1989, EMBO J., 8, p2283-2290). Vav is expressed exclusively in hematopoietic and trophoblast cells, and becomes rapidly phosphorylated on tyrosine in response to a variety of stimuli, including stimulation of TCR, B cell antigen receptor (BCR) , and various cytokine receptors (Romero & Fischer, 1996, Cell. Signaling, 8, p545-553; Collins et al . , 1997, Immunol. Today, 18, p221-225) . Although its precise function remains elusive, Vav displays several structural motifs encountered in signaling proteins, including a Dbl- homology (DH) domain present in guanine nucleotide exchange proteins specific for the Rho family of small GTPases, a pleckstrin-homology (PH) domain, a cysteine- rich domain, two Src-homology 3 (SH3) and one Src- homology 2 (SH2) domain. These domains mediate protein- protein interactions with an array of various membrane, cytoskeletal, cytoplasmic and nuclear proteins (Romero & Fischer, 1996, supra; Collins et al . , 1997, supra).

Recent gene ablation studies demonstrated that Vav function is important in lymphocyte development and activation. Chimeric mice derived from blastocysts lacking the recombinase-activating gene (RAG-2) and reconstituted with vav^'1' embryonic stem cells display a dramatic reduction in the number of thymocytes and peripheral T and B cells (Zhang et al . , 1995, Nature, 374, p470-473; Fischer et al . , 1995, Nature, 374, p474- 477; and Tarakhovsky et al . , 1995, Nature, 374, p467- 470) . The mature T and B cells of these mice display a profound defect in proliferation and cytokine production in response to TCR and BCR stimulation, respectively. The involvement of Vav in T cell signal transduction pathways was also shown by overexpression of Vav in human leukemic Jurkat T cells, which resulted in increased basal or TCR-stimulated activity of a reporter gene driven by the nuclear factor of activated T cells (NFAT) of the IL-2 promoter (Wu & Weiss, 1995, Mol. Cell. Biol., 15, p4337-4346; Holsinger et al . , 1995, Proc. Natl. Acad. Sci. USA, 92, p9810-9814; and Deckert et al . , 1996, Immunity, 5, p591-604) . This effect could be blocked by dominant-negative Ras (Wu & Weiss, 1995, supra) . Overall, current evidence suggests that Vav may couple Ras- and Rho-dependent signals in hematopoietic cells, and have a dual function as an adaptor that mediates protein-protein interactions, and as a guanine nucleotide exchange factor for small GTPases (Collins et al. , 1997, supra) .

Although it is known that Vav becomes phosphorylated, regulation of Vav function by serine/threonine phosphorylation is not known in the prior art . The inventors have however now demonstrated that Vav is phosphorylated by protein kinase A and that protein kinase A-mediated inhibition of T cell IL-2 production can be inhibited via Vav as forskolin/cAMP inhibits the Vav-mediated activation of the NFAT element of the IL-2 promoter. It has further been demonstrated that a change of the Vav proto-oncogene peptide sequence in residue 440 from serine to alanine (Vav-S440A) will remove the protein kinase A phosphorylation site. This is accomplished by mutation of the human composite gene sequence encoding the full length Vav proto-oncogene (peptide sequence accession numbers 586213 and P15498; composite nucleotide sequence from accession numbers X16316 and M59834 with corrections as published in Adams, J. et al, Oncogene 7:611-618) in nucleotides +1318 to +1320, corresponding to nucleotides 1281 to 1283 of accession number X16316, that encodes serine 440 of the respective gene product. The correct nucleotide and amino acid sequences appear in Accession numbers AH006196 and AAC25011, respectively. The inventors demonstrate that the gene product from the gene with this mutation is not phosphorylated by protein kinase A. Furthermore, transfection of the mutated gene into Jurkat T cells abolished the inhibitory effect of cAMP on Vav-mediated activation of the IL-2 promoter. The present invention thus relates to the identification of substrates for protein kinase A. It is demonstrated by the inventors that protein kinase A- mediated phosphorylation of two substrates believed to be involved in signaling through antigen receptors in T cells, regulates their function and has a major impact on different aspects of lymphocyte function. For both of these substrates it is surprisingly demonstrated that mutation of the wild type gene to remove the amino acid serine which is subject to phosphorylation, protects the signaling molecule from the negative regulation exerted by protein kinase A-mediated phosphorylation.

As mentioned above, the present invention relates to a method of altering the activity of the PKA signaling pathway in a cell by altering the extent of phosphorylation of one or more PKA substrates, or kinase substrates downstream in the PKA signaling pathway.

As will be appreciated, an alteration in the extent of phosphorylation of endogenous proteins may be achieved in a number of different ways . Broadly speaking, the endogenous protein may be altered to affect its susceptibility to phosphorylation, or an exogenous substrate (or fragment, derivative or functionally equivalent variant) may be added but - In ^¬ appropriately modified to alters its susceptibility to phosphorylation relative to the wild-type protein, or an exogenous fragment or derivative or variant may be added which alters, e.g. inhibits, phosphorylation of the endogenous substrate .

Furthermore, regulation may be achieved by affecting the kinase activity of the kinase responsible for phosphorylation of the substrate. This may be achieved as described herein when the activity of that kinase is itself controlled by phosphorylation.

Alternatively, the enzymatic activity of the kinase may be altered by other means, e.g. by use of inhibitors to its active site, inactivation or hyperactivation of the catalytic site through modification, or introduction of equivalent exogenous kinases which are overexpressed and which have been modified to affect, e.g. remove or impair, full kinase function. Thus the invention also extends to alteration of the PKA signaling pathway by modifying the activity of PKA or a downstream kinase. Preferably this is achieved by modifying or altering the phosphorylation of PKA or a downstream kinase where such phosphorylation exerts an influence on the activity of that kinase. Alternatively this may be achieved by manipulating the wild-type gene endogenously encoding the kinase or by manipulating its expression or by manipulating the expressed product.

Altering the susceptibility of the endogenous protein to phosphorylation may for example be achieved by altering expression of the endogenous gene, e.g. by reducing expression thereby reducing the number of phosphorylated substrates. The invention thus extends to altering the extent of phosphorylation by manipulation of the wild-type gene endogenously encoding PKA or the downstream kinase or by manipulation of expression of said gene or by manipulation of the expressed product . This could for example be achieved using antisense oligonucleotides, ribozymes or antibodies and the invention extends to such molecules. Alteration of the extent of phosphorylation by introducing an exogenous fragment or derivative might be achieved for example by use of an inhibitory molecule which interferes with phosphorylation of a substrate by mimicking the phosphorylation site of the substrate or binding to, or associating with, the substrate.

To identify or assess if an exogenous molecule is capable of successfully altering the extent of phosphorylation of a substrate or fragment, derivative or functionally equivalent variant thereof, initial studies may be performed in vi tro, particularly using isolated substrates, fragments, derivatives or functionally equivalent variants thereof in a cell-free system with the kinase responsible for its phosphorylation. Thus for example, purified or recombinant PKA signaling pathway kinases, or functionally active parts thereof may be incubated with the substrate, fragment, derivative or functionally equivalent variant thereof in the presence of test molecules to examine their effect on phosphorylation of the substrate. Alternatively, the substrate may be modified to examine the extent of phosphorylation remaining after modification compared to the unmodified substrate. Conveniently, for this purpose, to allow high-throughput screening, recombinant PKA signalling pathway kinases may be used. As an example, Vang et al . , 1998, Biochemica et Biophysica Acta, 1384, p285- 293, describes the expression of Csk as a fusion protein with glutathione-S-transferase, which may be used in kinase assays using different substrates.

This method of expression is described in Example 2 herein and this method and the Csk protein thus formed form further aspects of the present invention. Thus in a further aspect the present invention provides a method of producing a fully active Csk molecule comprising cloning human Csk by reverse transcriptase PCR from human T-cell RNA, sub-cloning into pGEX-KG expression vector, expressing in E. coli BL21 and isolating said expressed protein.

Viewed from a further aspect the present invention provides a method of identifying i) a modified PKA substrate or downstream kinase substrate, or fragment, precursor or functionally equivalent variant thereof which exhibits altered phosphorylation by PKA or the downstream kinase relative to the endogenous substrate; or ii) a molecule which alters, preferably inhibits, the phosphorylation of the PKA substrate, or downstream kinase substrate by PKA or the kinase, wherein the extent of phosphorylation of the PKA substrate or downstream kinase substrate or modified PKA substrate or modified downstream kinase substrate, or fragment, precursor or functionally equivalent variant thereof, by said kinase, optionally in the presence of a molecule to alter said phosphorylation, is assessed. This method may also be used for assessing the effectiveness of test modified PKA substrate or downstream kinase substrates, or fragments, precursors or functionally equivalent variants thereof or test molecules which alter the phosphorylation of the PKA substrate of downstream kinase substrate.

Conveniently, as mentioned above, the invention extends to introducing a modified PKA substrate, downstream kinase substrate, or fragment, derivative or functionally equivalent variant thereof into the cell. This may be achieved for example by manipulating the endogenously occurring substrate, e.g. by manipulation of the wild-type gene, by manipulating expression of the gene or by manipulating the expressed product. For example, to manipulate the endogenous gene, this could be performed for example by somatic cell gene therapy with homologous recombination to for example remove the phosphorylation sites. This could be performed on for example hematopoietic stem cells or on blood cells ex vivo or in vivo.

Preferably however, the invention extends to the introduction of an exogenous nucleic acid molecule (or the amino acid sequence encoded by it) containing a sequence encoding a modified substrate, or fragment, derivative or functionally equivalent variant thereof.

Nucleic acid molecules which may be used according to the invention may be single or double stranded DNA, cDNA or RNA, preferably DNA and include degenerate, substantially homologous and hybridizing sequences as described before. Ideally however genomic DNA or cDNA is employed.

Such exogenous molecules may be introduced in the case of nucleic acid molecules by any appropriate means. Suitable transformation or transfection techniques are well described in the literature. The nucleic acid molecules described above may be operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule. In particular, appropriate nucleic acid molecules may be introduced into vectors for appropriate expression in the cell . Alternatively, the naked DNA molecule may be injected directly into the cell. Appropriate expression vectors include appropriate control sequences such as for example translational (e.g. start and stop codons, ribosomal binding sites) and transcriptional control elements (e.g. promoter- operator regions, termination stop sequences) linked in matching reading frame with the nucleic acid molecules required for performance of the method of the invention. Appropriate vectors may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) . Suitable viral vectors include baculovirus and also adenovirus, adeno-associated virus, herpes and vaccinia/pox viruses . Many other viral vectors are described in the art . A variety of techniques are known and may be used to introduce the vectors into cells for expression.

Exogenous peptides or proteins may be introduced by any suitable technique known in the art such as in a liposome, niosome or nanoparticle or attached to a carrier or targetting molecule (see hereinafter) . It will be appreciated that the level of exogeneous substrate introduced into a cell will need to be controlled to avoid adverse effects of overexpression, e.g. hyperactivation by the effects of the introduced substrate, albeit modified. This has particularly been found to be the case for Csk and in such cases the use of inhibitory molecules as described hereinafter may be preferred. Preferably the substrate to be modified is a direct PKA substrate. As mentioned above, two such substrates have been identified, namely Csk and Vav.

Thus, preferably the method of the invention is achieved by causing an alteration in the extent of phosphorylation of a protein in the Csk family, preferably Csk and homologous kinases (such as Chk, Lsk, Hyl , Matk or proteins of the Csk type, e.g. Ctk, Bhk, Ntk or fragment, precursor or functionally equivalent variant thereof. Especially preferred are substrates in the mammalian Csk family, such as from humans, dogs, cats, horses, sheep, goats, cows, rats and mice.

Conveniently, preferred substrates of this type may be defined as peptides or proteins encoded by a nucleic acid molecule comprising the sequence:

1 tccggggcgg cccccggcag ccagcgcgac gttccaaaat cgaacctcag 51 tggcggcgct cggaagcgga actctgccgg ggccgcgccg gctacattgt 101 ttcctccccc cgactccctc ccgccccctt cccccgcctt tcttccctcc 151 gcgacccggg ccgtgcgtcc gtccccctgc ctctgcctgg cggtccctcc 201 tcccctctcc ttgcacccat acctctttgt accgcacccc ctggggaccc 251 ctgcgcccct cccctccccc ctgaccgcat ggaccgtccc gcaggccgct 301 gatgccgccc gcggcgaggt ggcccggacc gcagtgcccc aagagagctc 351 taatggtacc aagtgacagg ttggctttac tgtgactcgg ggacgccaga

401 gctcctgaga agatgtcagc aatacaggcc gcctggccat ccggtacaga

451 atgtattgcc aagtacaact tccacggcac tgccgagcag gacctgccct

501 tctgcaaagg agacgtgctc accattgtgg ccgtcaccaa ggaccccaac 551 tggtacaaag ccaaaaacaa ggtgggccgt gagggcatca tcccagccaa

601 ctacgtccag aagcgggagg gcgtgaaggc gggtaccaaa ctcagcctca

651 tgccttggtt ccacggcaag atcacacggg agcaggctga gcggcttctg

701 tacccgccgg agacaggcct gttcctggtg cgggagagca ccaactaccc

751 cggagactac acgctgtgcg tgagctgcga cggcaaggtg gagcactacc 801 gcatcatgta ccatgccagc aagctcagca tcgacgagga ggtgtacttt

851 gagaacctca tgcagctggt ggagcactac acctcagacg cagatggact

901 ctgtacgcgc ctcattaaac caaaggtcat ggagggcaca gtggcggccc

951 aggatgagtt ctaccgcagc ggctgggccc tgaacatgaa ggagctgaag

1001 ctgctgcaga ccatcgggaa gggggagttc ggagacgtga tgctgggcga 1051 ttaccgaggg aacaaagtcg ccgtcaagtg cattaagaac gacgccactg

1101 cccaggcctt cctggctgaa gcctcagtca tgacgcaact gcggcatagc

1151 aacctggtgc agctcctggg cgtgatcgtg gaggagaagg gcgggctcta

1201 catcgtcact gagtacatgg ccaaggggag ccttgtggac tacctgcggt

1251 ctaggggtcg gtcagtgctg ggcggagact gtctcctcaa gttctcgcta 1301 gatgtctgcg aggccatgga atacctggag ggcaacaatt tcgtgcatcg

1351 agacctggct gcccgcaatg tgctggtgtc tgaggacaac gtggccaagg

1401 tcagcgactt tggtctcacc aaggaggcgt ccagcaccca ggacacgggc

1451 aagctgccag tcaagtggac agcccctgag gccctgagag agaagaaatt

1501 ctccactaag tctgacgtgt ggagtttcgg aatccttctc tgggaaatct 1551 actcctttgg gcgagtgcct tatccaagaa ttcccctgaa ggacgtcgtc

1601 cctcgggtgg agaagggcta caagatggat gcccccgacg gctgcccgcc

1651 cgcagtctat gaagtcatga agaactgctg gcacctggac gccgccatgc

1701 ggccctcctt cctacagctc cgagagcagc ttgagcacat caaaacccac

1751 gagctgcacc tgtgacggct ggcctccgcc tgggtcatgg gcctgtgggg 1801 actgaacctg gaagatcatg gacctggtgc ccctgctcac tgggcccgag

1851 cctgaactga gccccagcgg gctggcgggc ctttttcctg cgtcccagcc

1901 tgcacccctc cggccccgtc tctcttggac ccacctgtgg ggcctgggga

1951 gcccactgag gggccaggga ggaaggaggc cacggagcgg gcggcagcgc

2001 cccaccacgt cgggcttccc tggcctcccg ccactcgcct tcttagagtt 2051 ttattccttt ccttttttga gatttttttt ccgtgtgttt attttttatt

2101 atttttcaag ataaggagaa agaaagtacc cagcaaatgg gcattttaca

2151 agaagtacga atcttatttt tcctgtcctg cccgtgaggt gggggggacc 2201 gggcccctct ctagggaccc ctcgccccag cctcattccc cattctgtgt

2251 cccatgtccc gtgtctcctc ggtcgccccg tgtttgcgct tgaccatgtt

2301 gcactgtttg catgcgcccg aggcagacgt ctgtcagggg cttggatttc

2351 gtgtgccgct gccacccgcc cacccgcctt gtgagatgga atcgtaataa 2401 accacgccat gaggaaaaaa

or a sequence which hybridizes to said sequence under non- stringent binding conditions of 6 x SSC/50% formamide at room temperature and washing under conditions of low stringency (2 x SSC, room temperature, more preferably 2 x SSC, 42°C) or conditions of higher stringency, e.g. 2 x SSC, 65°C, where SSC = 0.15 M NaCl, 0.015M sodium citrate, pH 7.2, or a sequence which exhibits at least 60%, preferably at least 70 or 80%, e.g. at least 90% sequence homology (as determined by, e.g. FASTA Search using GCG packages, with default values and a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0 with a window of 6 nucleotides) or a sequence complementary to any of the aforesaid sequences, or a fragment of any of the aforesaid sequences containing the region encoding or complementary to at least the PKA phosphorylation site.

In the above defined nucleotide sequence, the coding region is from residues 413 to 1765. Thus in an alternative form the invention extends to performance of the method of the invention in which the substrate comprises the amino acid sequence:

1 Met Ser Ala lie Gin Ala Ala Trp Pro Ser Gly Thr Glu

14 Cys He Ala Lys Tyr Asn Phe His Gly Thr Ala Glu Gin

27 Asp Leu Pro Phe Cys Lys Gly Asp Val Leu Thr He Val

40 Ala Val Thr Lys Asp Pro Asn Trp Tyr Lys Ala Lys Asn

53 Lys Val Gly Arg Glu Gly He He Pro Ala Asn Tyr Val 66 Gin Lys Arg Glu Gly Val Lys Ala Gly Thr Lys Leu Ser

79 Leu Met Pro Trp Phe His Gly Lys He Thr Arg Glu Gin

92 Ala Glu Arg Leu Leu Tyr Pro Pro Glu Thr Gly Leu Phe 105 Leu Val Arg Glu Ser Thr Asn Tyr Pro Gly Asp Tyr Thr

118 Leu Cys Val Ser Cys Asp Gly Lys Val Glu His Tyr Arg

131 He Met Tyr His Ala Ser Lys Leu Ser He Asp Glu Glu

144 Val Tyr Phe Glu Asn Leu Met Gin Leu Val Glu His Tyr 157 Thr Ser Asp Ala Asp Gly Leu Cys Thr Arg Leu He Lys

170 Pro Lys Val Met Glu Gly Thr Val Ala Ala Gin Asp Glu

183 Phe Tyr Arg Ser Gly Trp Ala Leu Asn Met Lys Glu Leu

196 Lys Leu Leu Gin Thr He Gly Lys Gly Glu Phe Gly Asp

209 Val Met Leu Gly Asp Tyr Arg Gly Asn Lys Val Ala Val 222 Lys Cys He Lys Asn Asp Ala Thr Ala Gin Ala Phe Leu

235 Ala Glu Ala Ser Val Met Thr Gin Leu Arg His Ser Asn

248 Leu Val Gin Leu Leu Gly Val He Val Glu Glu Lys Gly

261 Gly Leu Tyr He Val Thr Glu Tyr Met Ala Lys Gly Ser

274 Leu Val Asp Tyr Leu Arg Ser Arg Gly Arg Ser Val Leu 287 Gly Gly Asp Cys Leu Leu Lys Phe Ser Leu Asp Val Cys

300 Glu Ala Met Glu Tyr Leu Glu Gly Asn Asn Phe Val His

313 Arg Asp Leu Ala Ala Arg Asn Val Leu Val Ser Glu Asp

326 Asn Val Ala Lys Val Ser Asp Phe Gly Leu Thr Lys Glu

339 Ala Ser Ser Thr Gin Asp Thr Gly Lys Leu Pro Val Lys 352 Trp Thr Ala Pro Glu Ala Leu Arg Glu Lys Lys Phe Ser

365 Thr Lys Ser Asp Val Trp Ser Phe Gly He Leu Leu Trp

378 Glu He Tyr Ser Phe Gly Arg Val Pro Tyr Pro Arg He

391 Pro Leu Lys Asp Val Val Pro Arg Val Glu Lys Gly Tyr

404 Lys Met Asp Ala Pro Asp Gly Cys Pro Pro Ala Val Tyr 417 Glu Val Met Lys Asn Cys Trp His Leu Asp Ala Ala Met

430 Arg Pro Ser Phe Leu Gin Leu Arg Glu Gin Leu Glu His

443 He Lys Thr His Glu Leu His Leu

or a sequence which has more than 70 or 80%, preferably more than 90% (e.g. more than 95%) sequence homology thereto (as determined by, e.g. using the SWISS-PROT protein sequence databank using FASTA pep-cmp with a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0, and a window of 2 amino acids) , or a fragment of any of the aforesaid sequences containing at least the PKA phosphorylation site. As mentioned above, substrates which may be modified include fragments, precursors and functionally equivalent variants. "Functionally-equivalent" is used herein to define proteins related to or derived from the native protein, where the amino acid sequence has been modified by single or multiple amino acid substitution, addition and/or deletion and also sequences where the amino acids have been chemically modified, including by deglycosylation or glycosylation, but which nonetheless retain the same function, ie . are capable of being phosphorylated by a kinase in the PKA signaling pathway, e.g. by PKA, prior to their modification to affect phosphorylation .

Within the meaning of "addition" variants are included amino and/or carboxy terminal fusion proteins or polypeptides, comprising an additional protein or polypeptide fused to the substrate. Such functionally- equivalent variants mentioned above include natural biological variations (e.g. allelic variants or geographical variations within a species) and derivatives prepared using known techniques. For example, functionally-equivalent proteins may be prepared either by chemical peptide synthesis or in recombinant form using the known techniques of site- directed mutagenesis including deletion, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids. In particular functionally equivalent variants of the substrates of methods of the invention extend to analogues in different genera or species than the specific substrates mentioned herein.

Variants also include derivatives of the substrates which may be prepared by post-synthesis/isolation modification of the substrate without affecting functionality, e.g. certain glycosylation, methylation etc. of particular residues.

Derivatives particularly include peptidomimetics which may be prepared using techniques known in the art . For example, non-standard amino acids such as α- aminobutyric acid, penicillamine, pyroglutamic acid or conformationally restricted analogs, e.g. such as Tic (to replace Phe) , Aib (to replace Ala) or pipecolic acid (to replace Pro) may be used. Other alterations may be made when the substrate, or modified substrate (or more particularly a fragment thereof) , is to be used in the method of the invention. In such cases, the stability of the peptide may be enhanced, e.g. by the use of D- amino acids, or amide isosteres (such as N-methyl amide, retro-inverse amid, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, (E) -vinyl, methyleneamino, methylenethio or alkane) which protect the peptides against proteolytic degradation. Di (oligo) peptidomimetics may also be prepared.

"Precursors" of the substrates may be larger proteins which would be processed, e.g. by proteolysis to yield the substrate. Such precursors may take the form of zymogens, ie . inactive precursors of enzymes, activated by proteolytic cleavage. Necessarily however such precursors even if inactive must be capable of phosphorylation in the PKA signaling pathway. Alternatively precursors may for example be polymeric structures . "Fragments" comprise portions of the substrates (or the variants thereof) which behave as substrates to the kinase in question, ie . are capable of being phosphorylated, prior to modification if this is to be performed in accordance with the invention. Thus such fragments will comprise at least the phosphorylation site of the kinase of the PKA signaling pathway, and possibly also additional flanking regions to support the correct conformation of the phosphorylation site. Conveniently, appropriate fragments are 5-250, e.g. 10- 100, preferably 15-30 amino acids in length. Reference herein to fragments of the amino acid or nucleic acid sequences defined herein refers to fragments having for example the size mentioned above (or the corresponding length in nucleotides) . Preferably these fragments satisfy the homology (relative to a comparable region) or hybridizing conditions mentioned herein. Conveniently, when using the program mentioned herein to determine homology, sequences scoring over 100 (nucleic acid sequences) or 200 (amino acid sequences) are selected.

As mentioned above, the invention may be performed by altering the activity of the kinase responsible for phosphorylating the substrate. In the case of the substrate Csk (described above) or Vav (described hereinafter) , the activity of PKA may be altered as described above. In the case of the substrate Lck described hereinafter, the activity of Csk may be altered, e.g. by overexpression of a mutant of Csk which has been altered, e.g. which has impaired kinase function, e.g. Csk-K222R. However, a preferred way to perform the invention is to modify the exogenous substrate which is introduced into the cell. This may be achieved by any convenient means which alters the ability of that substrate to become or remain phosphorylated by the kinase in question. Whilst conveniently the modification may be made to the phosphorylation site(s) of the substrate, this is not essential and modification of sites in the remainder of the molecule which influences phosphorylation, e.g. by reducing accessability of the phosphorylation site(s) to the kinase, is also contemplated. Thus for example, one or more residues at at least one of the phosphorylation sites or elsewhere in the molecule may be chemically modified, e.g. by the addition of a bulky group preventing access by the kinase and hence phosphorylation . In a preferred embodiment, one or more residues in at least one of the phosphorylation sites (or affecting phosphorylation at the phosphorylation site) are mutated. Particularly preferred is the mutation of the serine or threonine residue which become phosphorylated. Conveniently, a conservative mutation is made, e.g. by the introduction of amino acids with uncharged polar side chains, such as glycine, aspartic acid, glutamic acid or cysteine. Alternatively, non-conservative substitutions may be made, such as alanine or glutamic acid. Preferably however one or more residues are converted to an alanine, glycine, aspartic acid, glutamic acid or cysteine residue. Where appropriate, e.g. when proteins are produced by chemical synthesis, non-naturally occurring amino acids may be used. Conveniently, modification may be made in the wild-type gene encoding a PKA substrate or downstream kinase substrate.

In the above described sequence of Csk (Accession No. X60114, e.g. from the EMBL database), preferably a modification is made in the phosphorylation site which spans amino acid residues 361 to 364 (KKFS) in the above sequence (or 1502 to 1504 in the nucleic acid sequence) . Modification may be for example by derivatization of particular residues, but especially preferably, this is by way of mutation and a preferred mutation is by replacement of the serine at position 364 (or alteration of nucleic acid residues in the region 1502 to 1504) in the human sequence, or a corresponding position in another organism or derivative or variant with an alanine, cysteine or glutamic acid residue. Alternatively, a modification may be made in the putative PKA site of KEASST at residues 336 to 341. In other organism or variant sequences, e.g. other members of the Csk-family, equivalent sites may be modified, particularly those exhibiting the same PKA phosphorylation site consensus sequence to those mentioned above, or related sequences, e.g. RFS or KFT. The method of the invention may also be achieved by causing an alteration in the extent of phosphorylation of a protein in the Vav family, preferably guanine release factors with GTPase activity that activates small G proteins, specifically Vav, Vav2 , Vav-3, Vav-3 β, Vav transforming protein and Vav-2 oncogene, or fragment, precursor or functionally equivalent variant thereof. Especially preferred are substrates in the mammalian Vav family, such as from humans, dogs, cats, horses, sheep, goats, cows, rats and mice.

Conveniently, preferred substrates of this type may be defined as peptides or proteins encoded by a nucleic acid molecule comprising the sequence :

1 actagctgtc gctccacagg cgagcagggc aggcgtgcgg gcgggtgggt

51 ggtggaggct gcgagggtgc acggccggcc ctgggcaggc ggtagccatg 101 gagctgtggc gccaatgcac ccactggctc atccagtgcc gggtgctgcc

151 gcccagccac cgcgtgacct gggatggggc tcaggtgtgt gaactggccc

201 aggccctccg ggatggtgtc cttctgtgtc agctgcttaa caacctgcta

251 ccccatgcca tcaacctgcg tgaggtcaac ctgcgccccc agatgtccca

301 gttcctgtgc cttaagaaca ttagaacctt cctgtccacc tgctgtgaga 351 agttcggcct caagcggagc gagctcttcg aagcctttga cctcttcgat

401 gtgcaggatt ttggcaaggt catctacacc ctgtctgctc tgtcctggac

451 cccgatcgcc cagaacaggg ggatcatgcc cttccccacc gaggaggaga

501 gtgtaggtga tgaagacatc tacagtggcc tgtccgacca gatcgacgac

551 acggtggagg aggatgagga cctgtatgac tgcgtggaga atgaggaggc 601 ggaaggcgac gagatctatg aggacctcat gcgctcggag cccgtgtcca

651 tgccgcccaa gatgacagag tatgacaagc gctgctgctg cctgcgggag

701 atccagcaga cggaggagaa gtacactgac acgctgggct ccatccagca

751 gcatttcttg aagcccctgc aacggttcct gaaacctcaa gacattgaga

801 tcatctttat caacattgag gacctgcttc gtgttcatac tcacttccta 851 aaggagatga aggaagccct gggcacccct ggcgcagcca atctctacca

901 ggtcttcatc aaatacaagg agaggttcct cgtctatggc cgctactgca

951 gccaggtgga gtcagccagc aaacacctgg accgtgtggc cgcagcccgg

1001 gaggacgtgc agatgaagct ggaggaatgt tctcagagag ccaacaacgg

1051 gaggttcacc ctgcgggacc tgctgatggt gcctatgcag cgagttctca 1101 aatatcacct ccttctccag gagctggtga aacacacgca ggaggcgatg

1151 gagaaggaga acctgcggct ggccctggat gccatgaggg acctggctca

1201 gtgcgtgaac gaggtcaagc gagacaacga gacactgcga cagatcacca 1251 atttccagct gtccattgag aacctggacc agtctctggc tcactatggc

1301 cggcccaaga tcgacgggga actcaagatc acctcggtgg aacggcgctc

1351 caagatggac aggtatgcct tcctgctcga caaagctcta ctcatctgta

1401 agcgcagggg agactcctat gacctcaagg actttgtaaa cctgcacagc 1451 ttccaggttc gggatgactc ttcaggagac cgagacaaca agaagtggag

1501 ccacatgttc ctcctgatcg aggaccaagg tgcccagggc tatgagctgt

1551 tcttcaagac aagagaattg aagaagaagt ggatggagca gtttgagatg

1601 gccatctcca acatctatcc ggagaatgcc accgccaacg ggcatgactt

1651 ccagatgttc tcctttgagg agaccacatc ctgcaaggcc tgtcagatgc 1701 tgcttagagg taccttctat cagggctacc gctgccatcg gtgccgggca

1751 tctgcacaca aggagtgtct ggggagggtc cctccatgtg gccgacatgg

1801 gcaagatttc ccaggaacta tgaagaagga caaactacat cgcagggctc

1851 aggacaaaaa gaggaatgag ctgggtctgc ccaagatgga ggtgtttcag

1901 gaatactacg ggcttcctcc accccctgga gccattggac cctttctacg 1951 gctcaaccct ggagacattg tggagctcac gaaggctgag gctgaacaga

2001 actggtggga gggcagaaat acatctacta atgaaattgg ctggtttcct

2051 tgtaacaggg tgaagcccta tgtccatggc cctcctcagg acctgtctgt

2101 tcatctctgg tacgcaggcc ccatggagcg ggcaggggca gagagcatcc

2151 tggccaaccg ctcggacggg actttcttgg tgcggcagag ggtgaaggat 2201 gcagcagaat ttgccatcag cattaaatat aacgtcgagg tcaagcacat

2251 taaaatcatg acagcagaag gactgtaccg gatcacagag aaaaaggctt

2301 tccgggggct tacggagctg gtggagtttt accagcagaa ctctctaaag

2351 gattgcttca agtctctgga caccaccttg cagttcccct tcaaggagcc

2401 tgaaaagaga accatcagca ggccagcagt gggaagcaca aagtattttg 2451 gcacagccaa agcccgctat gacttctgcg cccgagaccg atcagagctg

2501 tcgctcaagg agggtgacat catcaagatc cttaacaaga agggacagca

2551 aggctggtgg cgaggggaga tctatggccg ggttggctgg ttccctgcca

2601 actacgtgga ggaagattat tctgaatact gctgagccct ggtgccttgg

2651 cagagagacg agaaactcca ggctctgagc ccggcgtggg caggcagcgg 2701 agccaggggc tgtgacagct cccggcgggt ggagactttg ggatggactg

2751 gaggagcgca gcgtccagct ggcggtgctc ccgggatgtg ccctgacatg

2801 gttaatttat aacaccccga tttcctcttg ggtcccctca agcagacggg

2851 gctcaagggg gttacattta ataaaaggat gaagatgg

or a sequence which hybridizes to said sequence under non-stringent binding conditions of 6 x SSC/50% formamide at room temperature and washing under conditions of low stringency (2 x SSC, room temperature, more preferably 2 x SSC, 42 °C) or conditions of higher stringency, e.g. 2 x SSC, 65°C, where SSC = 0.15 M NaCl, 0.015M sodium citrate, pH 7.2 , or a sequence which exhibits at least 60%, preferably at least 70 or 80%, e.g. at least 90% sequence homology (as determined by, e.g. FASTA Search using GCG packages, with default values and a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0 with a window of 6 nucleotides) or a sequence complementary to any of the aforesaid sequences, or a fragment of any of the aforesaid sequences containing the region encoding or complementary to at least the PKA phosphorylation site. In the above defined nucleotide sequence, the coding region is from residues 98 to 2635. Thus in an alternative form the invention extends to performance of the method of the invention in which the substrate comprises the amino acid sequence:

1 melwrqcthw liqcrvlpps hrvtwdgaqv celaqalrdg vllcqllnnl 51 lphainlrev nlrpqmsqfl clknirtfls tccekfglkr selfeafdlf 101 dvqdfgkviy tlsalswtpi aqnrgimpfp teeesvgded iysglsdqid 151 dtveededly dcveneeaeg deiyedlmrs epvsmppkmt eydkrccclr 201 eiqqteekyt dtlgsiqqhf lkplqrflkp qdieiifini edllrvhthf 251 lkemkealgt pgaanlyqvf ikykerflvy grycsqvesa skhldrvaaa 301 redvqmklee csqranngrf tlrdllmvpm qrvlkyhlll qelvkhtqea 351 mekenlrlal damrdlaqcv nevkrdnetl rqitnfqlsi enldqslahy 401 grpkidgelk itsverrskm dryaflldka llickrrgds ydlkdfvnlh 451 sfqvrddssg drdnkkwshm flliedqgaq gyelffktre lkkkwmeqfe 501 maisniypen atanghdfqm fsfeettsck acqmllrgtf yqgyrchrcr 551 asahkeclgr vppcgrhgqd fpgtmkkdkl hrraqdkkrn elglpkmevf 601 qeyyglpppp gaigpflrln pgdiveltka eaeqnwwegr ntstneigwf 651 pcnrvkpyvh gppqdlsvhl wyagpmerag aesilanrsd gtflvrqrvk 701 daaefaisik ynvevkhiki mtaeglyrit ekkafrglte lvefyqqnsl 751 kdcfksldtt lqfpfkepek rtisrpavgs tkyfgtakar ydfcardrse 801 lslkegdiik ilnkkgqqgw wrgeiygrvg wfpanyveed yseyc or a sequence which has more than 70 or 80%, preferably more than 90% (e.g. more than 95%) sequence homology thereto (as determined by, e.g. using the SWISS-PROT protein sequence databank using FASTA pep-cmp with a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0, and a window of 2 amino acids) , or a fragment of any of the aforesaid sequences containing at least the PKA phosphorylation site.

The above sequences are taken from Accession number AH006196 and AAC25011 for the nucleotide and amino acid sequences respectively. Modification of these sequences for performance of the method of the invention is appropriately performed as described above for the Csk family proteins. In particular, in the above described sequence of Vav (Accession No. AAC25011) , preferably a modification is made in the phosphorylation site which spans amino acid residues 436 to 441 (RRGDSY) in the above sequence (or 1403 to 1420 in the nucleic acid sequence) . Modification may be for example by derivatization of particular residues, but especially preferably this is by way of mutation and a preferred mutation is by replacement of the serine at position 440 (or alteration of nucleic acid residues in the region

1415 to 1417) in the human sequence, or a corresponding position in another organism or derivative or variant with an alanine, cysteine, aspartic acid, glutamic acid or glycine residue. In other organism or variant sequences, e.g. other members of the Vav-family, equivalent sites may be modified, particularly those exhibiting the same PKA phosphorylation site consensus sequence to that mentioned above, or related sequences (e.g. RKGYSY) . The method of the invention may also be performed using substrates (or modified forms thereof) which are phosphorylated by kinases downstream in the signaling pathway, ie . indirect PKA substrates. A preferred example in the connection is members of the Src-family which have intact the residue corresponding to tyrosine residue 505 in Lck, preferably Lck, Fyn, Src, Yes, Fgr, Lyn, Hck, Blk, Yrk, C-tkl, Fyk, Src-1 or Src-2, or fragments, precursors or functionally equivalent variants thereof. Especially preferred are substrates in the mammalian Src family, such as from humans, dogs, cats, horses, sheep, goats, cows, rats and mice. It has been shown by the inventors that modification of the indirect PKA substrate Lck by mutation of the tyrosine at position 505 to a phenylalanine residue prevented cAMP-mediated inhibition of TCR/CD3 -induced ζ -chain phosphorylation . Thus, in a preferred aspect, the method of the invention may be directed to substrates conveniently defined as peptides or proteins encoded by a nucleic acid molecule comprising the sequence:

1 cgcctggacc atgtgaatgg ggccagaggg ctcccgggct gggcagggac

51 catgggctgt ggctgcagct cacacccgga agatgactgg atggaaaaca

101 tcgatgtgtg tgagaactgc cattatccca tagtccgact ggatgggaag

151 ggcaggctgc tcatccgaaa tggctctgag gtgcgggacc cactggttac

201 ctacgaaggc tccaatccgc cggcttcccc actgcaagac aacctggtta 251 tcgctctgca cagctatgag ccctctcacg acggagatct gggctttgag

301 aagggggaac cactccgcat cctggagcag agcggcgagt ggtggaaggc

351 gcagtccctg accacgggcc aggaaggctt catccccttc aattttgtgg

401 ccaaagcgaa cagcctggag cccgaaccct ggttcttcaa gaacctgagc

451 cgcaaggacg cggagcggca gctcctggcg cccgggaaca ctcacggctc 501 cttcctcatc cgggagagcg agagcaccgc cgggtccttt tcactgtcgg

551 tccgggactt cgaccaaaac cagggagagg tggtgaaaca ttacaagatc

601 cgtaatctgg acaacggtgg cttctacatc tcccctcgaa tcacttttcc

651 cggcctgcat gaactggtcc gccattacac caatgcttca gatgggctgt

701 gcacacggtt gagccgcccc tgccagaccc agaagcccca gaagccgtgg 751 tgggaggacg agtgggaggt tcccagggag acgctgaagc tggtggagcg

801 gctgggggct gcacagttcg gggaggtgtg gatggggtac tacaacgggc

851 acacgaaggt ggcggtgaag agcctgaagc agggcagcat gtccccggac 901 gccttcctgg ccgaggccaa cctcatgaag cagctgcaac accagcggct

951 ggttcggctc tacgctgtgg tcacccagga gcccatctac atcatcactg

1001 aatacatgga gaatgggagt ctagtggatt ttctcaagac cccttcaggc

1051 atcaagttga ccatcaacaa actcctggac atggcagccc aaattgcaga 1101 aggcatggca ttcattgaag agcggaatta tattcatcgt gaccttcggg

1151 ctgccaacat tctggtgtct gacaccctga gctgcaagat tgcagacttt

1201 ggcctagcac gcctcattga ggacaacgag tacacagcca gggagggggc 1251 caagtttccc attaagtgga cagcgccaga agccattaac tacgggacat

1301 tcaccatcaa gtcagatgtg tggtcttttg ggatcctgct gacggaaatt 1351 gtcacccacg gccgcatccc ttacccaggg atgaccaacc cggaggtgat

1401 tcagaacctg gagcgaggct accgcatggt gcgccctgac aactgtccag

1451 aggagctgta ccaactcatg aggctgtgct ggaaggagcg cccagaggac

1501 cggcccacct ttgactacct gcgcagtgtg ctggaggact tcttcacggc

1551 cacagagggc cagtaccagc ctcagccttg agaggaggcc ttgagaggcc 1601 ctggggttct ccccctttct ctccagcctg acttggggag atggagttct

1651 tgtgccatag tcacatggcc tatgcacata tggactctgc acatgaatcc

1701 cacccacatg tgacacatat gcaccttgtg tctgtacacg tgtcctgtag

1751 ttgcgtggac tctgcacatg tcttgtgcat gtgtagcctg tgcatgtatg

1801 tcttggacac tgtacaaggt acccctttct ggctctccca tttcctgaga 1851 ccaccagaga gaggggagaa gcctgggatt gacagaagct tctgcccacc

1901 tacttttctt tcctcagatc atccagaagt tcctgaaggg ccaggacttt

1951 atctaatacc tctgtgtgct cctccttggt gcctggcctg gcacacatca

2001 ggagttcaat aaatgtctgt tgatgactgc cg

or a sequence which hybridizes to said sequence under non- stringent binding conditions of 6 x SSC/50% formamide at room temperature and washing under conditions of low stringency (2 x SSC, room temperature, more preferably 2 x SSC, 42 °C) or conditions of higher stringency, e.g. 2 x SSC, 65°C, where SSC = 0.15 M NaCl, 0.015M sodium citrate, pH 7.2, or a sequence which exhibits at least 60%, preferably at least 70 or 80%, e.g. at least 90% sequence homology (as determined by, e.g. FASTA Search using GCG packages, with default values and a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0 with a window of 6 nucleotides) or a sequence complementary to any of the aforesaid sequences, or a fragment of any of the aforesaid sequences containing the region encoding or complementary to at least the phosphorylation site.

In the above defined nucleotide sequence, the coding region is from residues 52 to 1581. Thus in an alternative form the invention extends to performance of the method of the invention in which the substrate comprises the amino acid sequence :

1 mgcgcsshpe ddwmenidvc enchypivrl dgkgrllirn gsevrdplvt

51 yegsnppasp lqdnlvialh syepshdgdl gfekgeplri leqsgewwka

101 qslttgqegf ipfnfvakan slepepwffk nlsrkdaerq llapgnthgs

151 fliresesta gsfslsvrdf dqnqgewkh ykirnldngg fyispritfp 201 glhelvrhyt nasdglctrl srpcqtqkpq kpwwedewev pretlklver

251 lgaaqfgevw mgyynghtkv avkslkqgsm spdaflaean Imkqlqhqrl

301 vrlyawtqe piyiiteyme ngslvdflkt psgikltink lldmaaqiae

351 gmafieerny ihrdlraani lvsdtlscki adfglarlie dneytarega

401 kfpikwtape ainygtftik sdvwsfgill teivthgrip ypgmtnpevi 451 qnlergyrmv rpdncpeely qlmrlcwker pedrptfdyl rsvledffta

501 tegqyqpqp

or a sequence which has more than 70 or 80%, preferably more than 90% (e.g. more than 95%) sequence homology thereto (as determined by, e.g. using the SWISS-PROT protein sequence databank using FASTA pep-cmp with a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0, and a window of 2 amino acids) , or a fragment of any of the aforesaid sequences containing at least the phosphorylation site. The above sequences are taken from Accession numbers M36881 and AAA59502, respectively.

Modification of these sequences for performance of the method of the invention is appropriately performed as described above for the Csk and Vav family proteins. In particular, in the above described sequence of Lck, preferably a modification is made in the phosphorylation site which spans amino acid residues 476 to 509 in the above sequence (or 1477 to 1578 in the nucleic acid sequence) . Modification may be for example by derivatization of particular residues, but especially preferably this is by way of mutation and a preferred mutation is by replacement of the tyrosine at position 505 (or alteration of nucleic acid residues in the region 1564 to 1566) in the human sequence, or a corresponding position in another organism or derivative or variant with a phenylalanine residue.

In other organism or variant sequences, e.g. other members of the Src-family, equivalent sites may be modified, particularly those exhibiting the same phosphorylation site consensus sequence to that mentioned above, or related sequences, e.g. the equivalent phosphorylation site which is phosphorylated and regulated by Csk in the entire Src kinase family may be modified.

As well as the use of modified PKA substrates or downstream kinase substrates or fragments, precursors or functionally equivalent variants thereof, the method of the invention may also be performed using a different molecule which alters the phosphorylation of the endogenous substrate. As mentioned previously, molecules which modify the expression of the endogenous substrate gene (e.g. antisense oligonucleotides, ribozymes and antibodies) may be used. Similarly, molecules which affect the expression and/or activity of PKA or the downstream kinase may be used. However, in a preferred aspect, agonists or antagonists of phosphorylation are used. Thus, for example, molecules which bind to the phosphorylation site of the substrate thereby influencing the extent of phosphorylation may be used. These could for example be molecules which specifically recognize the phosphorylation site, such as antibodies (or fragments thereof) , or proteins or peptides which associate with that region or sufficiently close to affect accessability by the kinase active site. Alternatively, peptides which mimic the phosphorylation site may be used to provide excess phosphorylation sites to reduce the extent of phosphorylation on the endogenous substrates .

Appropriate molecules may for example be proteins or peptides or other molecules which can affect phosphorylation at the phosphorylation site, or a nucleic molecule which encodes such a product . Although the above description mainly describes processes for reducing phosphorylation, it will be appreciated that molecules which for example aid access to the phosphorylation site may be used if the desired outcome is increased phosphorylation. Thus, view from a further aspect, the present invention provides a method as defined above wherein the extent of phosphorylation is altered by introducing a molecule which alters, preferably inhibits, the phosphorylation of the PKA substrate, or downstream kinase substrate, into said cell.

In a preferred aspect, the inhibitory (or activatory) molecule is a nucleic acid molecule comprising a nucleotide sequence encoding a protein or peptide which interferes with phosphorylation of a substrate, or fragment, precursor or functionally equivalent variant thereof containing at least the phosphorylation site, as defined above. Alternatively, the inhibitory (or activatory) molecule is a protein or peptide encoded by said nucleic acid molecule. Preferably, such a protein or peptide mimics the phosphorylation site of the substrate or binds to, or associates with, the substrate, or fragment, precursor or functionally equivalent variant thereof, thereby affecting phosphorylation of said phosphorylation site. Such molecules may be introduced into the cell using the techniques described above .

The above described method of the invention may be used in vi tro, for example in cell or organ culture, particularly for affecting PKA signaling pathways which have been activated or to reduce the extent of endogenous signaling. The method may also be used ex vivo, on animal parts or products, for example organs or collected blood, cells or tissues, particularly when it is contemplated that these will be reintroduced into the body from which they are derived. In particular, in samples in which abnormal levels of PKA signaling are occurring, levels may be normalized, e.g. by inhibiting the activity of the PKA signaling pathway. The method may also be used in vivo for the treatment of diseases in which abnormal PKA signaling occurs and this will be discussed in more detail below. The present invention also extends to novel modified, e.g. mutated PKA substrates or downstream substrates or fragments, precursors or functionally equivalent precursors thereof and the nucleic acid molecules which encode them. The invention also extends to other inhibitory or activatory molecules which interfere with phosphorylation of a PKA substrate or downstream kinase substrate. Particularly, the invention extends to gene sequences (or amino acid sequences) encoding (or comprising) a product with an altered susceptibility to (e.g. protection from) phosphorylation by PKA or a downstream kinase.

Thus, viewed from a yet further aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a PKA substrate, or fragment, precursor or functionally equivalent variant thereof, as defined above, wherein said sequence is modified as defined above to alter its susceptibility to phosphorylation by PKA.

In particular there is provided a mutated gene sequence of the wild type gene which encodes a substrate phosphorylated by protein kinase A during lymphocyte activation (or which encodes a gene product which is phosphorylated by a kinase downstream of PKA) , wherein the phosphorylation site of the wild type gene is mutated such that the inhibition of the lymphocyte activation mediated by protein kinase A is reduced or abolished.

Conveniently for use in methods of the invention the above nucleic acid molecules may be provided in a cloning or expression vector, as described previously. Preferred vectors include pGEX-KG, pEF-neo and pEF-HA. The nucleic acid molecule may conveniently be fused with DNA encoding an additional polypeptide, e.g. glutathione-S-transferase, to produce a fusion protein on expression. Thus viewed from a further aspect, the present invention provides a vector comprising a nucleic acid molecule as defined above.

Other aspects of the invention include methods for preparing recombinant nucleic acid molecules according to the invention, comprising inserting nucleotide sequences encoding the modified substrate into vector nucleic acid.

A variety of techniques are known and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression. Preferred host cells for this purpose include insect cell lines, eukaryotic cell lines or E. coli , such as strain BL21/DE3. The invention also extends to transformed or transfected prokaryotic or eukaryotic host cells containing a nucleic acid molecule, particularly a vector as defined above.

A further aspect of the invention provides a method of preparing a modified substrate of the invention as hereinbefore defined, which comprises culturing a host cell containing a nucleic acid molecule as defined above, under conditions whereby said substrate is expressed and recovering said substrate thus produced. The expressed protein product forms a further aspect of the invention.

The invention also extends to a modified protein or peptide encoded by a nucleic acid molecule as hereinbefore described. This may be produced by expression of a host cell as described above, or may be prepared by chemical means, such as the well known Merrifield solid phase synthesis procedure. Preferably such products are substantially purified, e.g. pyrogen- free, e.g. more than 70%, especially preferably more than 90% pure (as assessed for example, in the case of peptides or proteins, by an appropriate technique such as peptide mapping, sequencing or chromatography) . Purification may be performed for example by chromatography (e.g. HPLC, size-exclusion, ion-exchange, affinity, hydrophobic interaction, reverse-phase) or capillary electrophoresis. As mentioned previously, the substrates or modified forms thereof may be used in clinical situations in which abnormal PKA signaling is exhibited. In particular since PKA is a key negative regulator of T cell function, diseases which exhibit lymphocyte dysfunction are particular targets for this treatment. Thus, the substrates or modified forms thereof may be used to treat immunosuppressive disorders (such as HIV infection, AIDS or common variable immunodeficiency) or proliferative diseases (such as diseases where Src kinase has been implicated and thus regulation via Csk would be relevant, e.g. cancers such as colorectal carcinoma, pancreatic carcinoma, hepatocellular carcinoma, cancer mamma, ovarian cancer and non-small cell carcinoma of the lung) . Conditions in which upregulation of the PKA pathway is required, such as autoimmune diseases, e.g. systemic lupus erythematosus, may also be treated. The substrates and modified forms thereof may therefore be formulated as pharmaceutical compositions in which the substrates, modified substrates or other molecules affecting the extent of phosphorylation may be provided as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts may be readily prepared using counterions and techniques well known in the art.

The invention thus further extends to pharmaceutical compositions comprising one or more nucleic acid molecules, peptides or proteins, encoding or comprising a PKA substrate,, downstream kinase substrate, or modified form thereof, or fragment, precursor or functionally equivalent variant thereof, or other molecule (such as an antisense oligonucleotide, ribozyme or antibody, nucleic acid molecule or peptide/protein) which alters, preferably inhibits, the phosphorylation of the PKA substrate, or downstream kinase substrate as defined above and one or more pharmaceutically acceptable excipients and/or diluents. By "pharmaceutically acceptable" is meant that the ingredient must be compatible with other ingredients in the composition as well as physiologically acceptable to the recipient .

The active ingredient for administration may be appropriately modified for use in a pharmaceutical composition. For example when peptides are used these may be stabilized against proteolytic degradation by the use of derivatives such as peptidomimetics as described hereinbefore. The active ingredient may also be stabilized for example by the use of appropriate additives such as salts or non-electrolytes, acetate, SDS, EDTA, citrate or acetate buffers, mannitol, glycine, HSA or polysorbate .

Conjugates may be formulated to provide improved lipophilicity, increase cellular transport, increase solubility or allow targeting. Conjugates may be made terminally or on side portion of the molecules, e.g. on side chains of amino acids. These conjugates may be cleavable such that the conjugate behaves as a pro-drug. Stability may also be conferred by use of appropriate metal complexes, e.g. with Zn, Ca or Fe .

The active ingredient may be formulated in an appropriate vehicle for delivery or for targeting particular cells, organs or tissues. Thus the pharmaceutical compositions may take the form of microemulsions, liposomes, niosomes or nanoparticles with which the active ingredient may be absorbed, adsorbed, incorporated or bound. This can effectively convert the product to an insoluble form. These particulate forms have utility for transfer of nucleic acid molecules and/or protein/peptides and may overcome both stability (e.g. enzymatic degradation) and delivery problems .

These particles may carry appropriate surface molecules to improve circulation time (e.g. serum components, surfactants, polyoxamine908 , PEG etc.) or moieties for site-specific targeting, such as ligands to particular cell borne receptors. Appropriate techniques for drug delivery and for targeting are well known in the art, but see for example Kreuter, 1994, Eur. J. Drug Metab. Pharmacokinet . , 3, p253-256; Shen, 1997, J. Drug Targeting, 5(1), pll-13; Mrsny, 1997, J. Drug Targeting, 5(1), p5-9; Pettit & Gombotz, 1998, TIBTECH, 16, p343- 349; and Duncan, 1997, J. Drug Targeting, 5(1), pl-4 regarding drug targeting and Simari & Nabel, 1996, Semin. Intervent . Cardiol . , 1, p77-83; Torchilin, 1998, J. Microencapsulation, 15(1), pl-19; Klyashchitsky &

Owen, 1998, J. Drug Targeting, 5(6), p443-458; Kreuter, 1996, J. Anat., 189, p503-505; Fasano, 1998, TIBTECH, 16, pl52-157; Kataoka et al . , 1993, 24, pll9-132; Anderson, 1998, Nature, 392 (suppl) , p25-30; Langer, 1998, Nature, 392 (suppl) , p5-10; Gregoriadis, 1995,

TIBTECH, 13, p527-536; Gregoriadis et al . , 1997, FEBS Lett., 402, pl07-110; Rolland, 1998, Critical Reviews in Therapeutic Drug Carrier Systems, 15(2), pl43-198; Hope et al., 1998, Molec . Memb. Biol., 15, pl-14; and Scherman et al . , 1998, Curr. Opinion Biotech., 9(5), p480-485 regarding peptide and nucleic acid molecule delivery. For an example of specific site directed targeting, see for example Schafer et al . , 1992, Pharm. Res., 9, p541-546 in which nanoparticles can be accumulated in HIV-infected macrophages . Clearly such methods have particular applications in the methods of the invention described herein.

Such derivatized or conjugated active ingredients are intended to fall within the definition of nucleic acid molecules, peptide/proteins or activatory or inhibitory molecules which form aspects of this invention.

Pharmaceutical compositions for use according to the invention may be formulated in conventional manner using readily available ingredients. Thus, the active ingredient may be incorporated, optionally together with other active substances as a combined preparation, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like. Biodegradable polymers (such as polyesters, polyanhydrides, polylactic acid, or polyglycolic acid) may also be used for solid implants. The compositions may be stabilized by use of freeze- drying, undercooling or Permazyme .

Suitable excipients, carriers or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, aglinates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol , water/glycol, water/polyethylene, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat- or suitable mixtures thereof. The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, adsorption enhancers, e.g. for nasal delivery (bile salts, lecithins, surfactants, fatty acids, chelators) and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration of the patient by employing procedures well known in the art.

The active ingredient in such compositions may comprise from about 0.01% to about 99% by weight of the formulation, preferably from about 0.1 to about 50%, for example 10%. The invention also extends to pharmaceutical compositions as described above for use as a medicament . Preferably the pharmaceutical composition is formulated in a unit dosage form, e.g. with each dosage containing from about 0.1 to 500mg of the active ingredient. The precise dosage of the active compound to be administered and the length of the course of treatment will of course, depend on a number of factors including for example, the age and weight of the patient, the specific condition requiring treatment and its severity, and the route of administration. Generally however, an effective dose may lie in the range of from about O.Olmg/kg to 20mg/kg, depending on the animal to be treated, and the substance being administered (e.g 0.1 to 7mg/kg for antisense oligonucleotides) , taken as a single dose.

The administration may be by any suitable method known in the medicinal arts, including for example oral, parenteral (e.g. intramuscular, subcutaneous, intraperitoneal or intravenous) percutaneous, buccal, rectal or topical administration or administration by inhalation. As will be appreciated oral administration has its limitations if the active ingredient is digestible. To overcome such problems, ingredients may be stabilized as mentioned previously and see also the review by Bernkop-Schnύrch, 1998, J. Controlled Release, 52, pl-16. It will be appreciated that since the active ingredient for performance of the invention takes a variety of forms, e.g. oligonucleotide, antibody, ribozyme, nucleic acid molecule (which may be in a vector or host cell) or peptide/protein, the form of the composition and route of delivery will vary. Preferably however liquid solutions or suspensions would be employed, particularly e.g. for nasal delivery and administration will be systemic.

As mentioned above, these pharmaceutical compositions may be used for treating conditions in which the PKA signaling pathway is abnormal, in particular when the activity of this pathway is elevated. Thus, viewed from a further aspect the present invention provides a method of treating disorders exhibiting abnormal PKA signaling activity, preferably immunosuppressive disorders or proliferative diseases, in a human or non-human animal wherein a pharmaceutical composition as described hereinbefore is administered to said animal. Alternatively stated, the present invention provides the use of a pharmaceutical composition as defined above for the preparation of a medicament for the treatment of immunosuppressive disorders or proliferative diseases.

In the following the invention is described in further detail by the non-limiting examples and illustrations, wherein the figures depict:

Figure 1. Anti-CD3 stimulation induces increased Ser/Thr phosphorylation of Vav. Phosphate-starved Jurkat T cells were metabolically labeled with 0.5 mCi³²Pi/l0⁷ cells/sample for 4 hrs, and left unstimulated (-) or stimulated (+) with 10 μg/ml OKT3 for the final 90 sec. Control normal rabbit serum (NRS) or Vav IPs were prepared, resolved by SDS-PAGE, and transferred electrophoretically to Immobilon membranes. (A), autoradiography of the resolved proteins . Molecular weight standards are shown on the right. WCL, whole cell lysate from Jurkat cells (1 x 10⁶ cells) . (B) , PAA analysis of immunoprecipitated Vav. The phosphorylated Vav band was excised, washed with water, and subjected to PAA analysis as described in Materials and Methods of Example 1. The positions of phosphoamino acid standards comprising phosphoserine (PS) , phosphothreonine (PT) and phosphotyrosine (PY) , detected by ninhydrin staining, are indicated. This experiment is representative of two experiments that gave similar results.

Figure 2. PKA is involved in the increased phosphorylation of Vav in T cells .

(A) , effects of PKA agonist or antagonist on the basal or anti-CD3- induced phosphorylation of Vav. Jurkat cells were metabolically labeled with ³²Pi as described for Figure 1, and subjected to treatment with 100 μM forskolin (a PKA agonist) or 100 nM KT5720 (a PKA antagonist) for the final 15 min of the incubation where indicated. Some groups were left unstimulated (-), and others were stimulated with 0KT3 (10 μg/ml; +) for the final 90 sec of culture. The cells were lysed, subjected to anti-Vav immunoprecipitation, and the labeled Vav band was detected by autoradiography following SDS-PAGE. The position of Vav is indicated.

(B) , in vi tro phosphorylation of the Vav PH domain by purified PKA. Samples of glutathione-Sepharose- immobilized purified GST or GST-Vav-PH (5 μg) were subjected to an in vi tro kinase reaction in the absence (-) or presence (+) of purified PKA catalytic subunit. The reaction was stopped after 30 min, and the products were resolved by 12% SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography (left panel) or by immunoblotting with an anti-Vav mAb which recognizes the Vav PH domain (right panel) . The positions of the GST-Vav-PH substrate or immunoreactive Vav are indicated by arrows. The positions of molecular weight standards are also indicated. Similar results were obtained in one additional experiment. Figure 3. Coimmunoprecipitation of PKA-I subunits with Vav. (A) and (B) , Jurkat cells (1 x 10 /sample) were either left untreated (lanes 1, 2) or stimulated with anti-CD3 (SpvT3d ascites; 1/200 dilution; lane 3) , anti- CD3 plus 8-CPT-cAMP (1 mM; lane 4) , or 8-CPT-cAMP alone (lane 5) for 5 min at 37°C. Thereafter the cells were lysed in a 1% Brij-96 lysis buffer, and immunoprecipitated (IP) with an anti-RIα mAb (lane 1) or with a polyclonal anti-Vav (lanes 2-5) antibody. Immunoprecipitated proteins were resolved by 10% SDS- PAGE and transferred to nitrocellulose. The membrane was probed with protein A-purified anti-Cα (B) or biotinylated anti-RIα (A) antibodies, and signals were visualized by ECL.

(C) and (D) , photoaffinity labeling of the regulatory subunits of PKA. (C) , Cells (5 x 10⁷/sample) were stimulated, lysed and subjected to anti-RIα (lane 1) or -Vav (lanes 2-5) immunoprecipitation as described above. Following lysis and prior to immunoprecipitation, the cellular proteins were photoaffinity labeled with 8- azido- [³²P] cAMP. IPs were subjected to 10% SDS-PAGE, and 8-azido- t³²P] cAMP-labeled proteins were visualized by autoradiography.

(D) shows a similar experiment to the one shown in (C) , with the addition of a normal rabbit Ig (NRIg) IP as a specificity control. No 8-azido- [³²P] cAMP-labeled proteins are present in this control IP. The positions of Rlα (A, C, D) or Cα (B) are indicated by the arrows. These experiments were repeated three or more times with similar results . Figure 4. Coimmunoprecipitation of Vav with PKA. Rlα (A,

B, C) , Cα (C) or control (NMIg, normal mouse Ig; A, C) IPs were prepared from lysed Jurkat cells (1 x lOVsample) which were left unstimulated (-) or stimulated (+) for 90 sec with OKT3 (10 μg/ml) in the absence (-) or presence (+) of a 15-min forskolin (100 μM) pretreatment . The IPs, as well as whole cell lysates (WCL; 1 x 10⁶ cells), were resolved by 8.5% SDS- PAGE and, following transfer to nitrocellulose, the resolved proteins were immunoblotted with an anti-Vav mAb (A, B, C) or, as a control for the expression level of Rlα, with an anti-RIα mAb (B) . The Vav and Rlα bands are indicated, as are the molecular weight standards. These results are representative of three similar experiments .

Figure 5. PKA, but not PKC, activation causes a reduction in the TCR/CD3-induced tyrosine phosphorylation of Vav. (A) , control (NRS; lanes 1-4) or Vav (lanes 9-12) IPs were prepared from Jurkat cells which were either left untreated (-) or stimulated (+) with the indicated combinations of OKT3 (10 μg/ml; 90 sec) and/or forskolin (100 μM; 15 min pretreatment) . The proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an anti-PTyr mAb (4G10; top panel) . The membrane was stripped, washed and reprobed with an anti-Vav mAb (bottom panel) . As a control, whole cell lysates (WCL) from 1 x 10⁶ Jurkat cells were similarly resolved and immunoblotted (lanes 5-8) . The arrow on the right (top panel) indicates a phosphoprotein whose tyrosine phosphorylation was not inhibited by forskolin pretreatment.

(B) , a similar experiment was conducted, except that the cells were left untreated, or stimulated with the indicated combinations of anti-CD3 (10 μg/ml) and/or PMA (10 ng/ml) as indicated. Immunoprecipitation, SDS-PAGE separation, and immunoblotting with anti-PTyr (top panel) or -Vav (bottom panel) antibodies were performed as described above .

(C) , transient PKA Cα subunit overexpression inhibits the tyrosine phosphorylation of Vav. Jurkat-TAg cells were transiently cotransfected with a c-Myc-tagged Vav plus a Cα or control (empty SRα) expression vectors (15 μg plasmid DNA each) , cultured for 40 hrs in the absence of stimulation, or stimulated for the final 14 hrs of culture with anti-CD3. The tyrosine phosphorylation of the transfected Vav immunoprecipitated by an anti-c-Myc mAb was assessed by anti-PTyr immunoblotting (top panel) . The membrane was then stripped and reprobed with the anti-c-Myc antibody in order to assess the expression level of the immunoprecipitated Vav. Similar results were obtained in two additional experiments. Figure 6. PKA activation reduces the association of S P- 76 with Vav. Jurkat cells (10 /sample) were either left unstimulated or stimulated with the indicated combination of OKT3 (90 sec) , forskolin and/or PMA (15 min each) prior to lysis and Vav immunoprecipitation. The expression of SLP-76 in the Vav IPs (lanes 1-6) or in whole cells lysates from 1 x 10⁶ cells (WCL; lanes 7- 12) was determined by immunoblotting with an anti-SLP-76 mAb (top panel) . The same membrane was stripped and reprobed with anti-Vav antibody (bottom panel) . The positions of SLP-76 and Vav are indicated.

Figure 7. The effect of mutating Ser-440 in Vav on its association with endogenous PKA-I.

(A) , COS-1 cells were transfected with 10 μg empty pEF (vector) , mutated Vav (VavS440A) or wild-type Vav (WT- Vav) plasmid DNA as indicated, and cultured for 48 hrs. The cells were either left untreated (-) or treated (+) with 100 μM forskolin for the final 15 min of culture. Vav was immunoprecipitated from cell lysates and, following SDS-PAGE and transfer to nitrocellulose, the resolved proteins in the Vav IPs (lanes 1-6; 10⁷ cells/sample) or in whole cell lysates (WCL; lanes 7-12; 10⁶ cells/sample) were immunoblotted with anti-Vav (top panel) or anti-RIα (bottom panel) mAbs . The corresponding blots for a Jurkat whole cell lysate (JK WCL; 10⁶ cells) are shown in lane 13 for reference. (B) , a similar experiment showing the association of immunoprecipitated endogenous Cα with transfected Vav in COS-1 cells. This experiment is representative of two similar experiments.

Figure 8. Mutation of Ser-440 in Vav confers resistance to forskolin-induced inhibition of Vav-mediated NFAT activation. Jurkat cells were transiently cotransfected with empty vector or the same Vav expression vectors as in Figure 7 (15 μg plasmid DNA) plus 5 μg NFAT-Luc and 20 μg carrier DNA, and cultured for 40 hrs. The cells were either left untreated, or stimulated for the final 6 hrs of culture with the indicated combinations of OKT3 and/or forskolin. Luciferase activity in cell extracts was measured as described in Materials and Methods in Example 1. Luciferase activity is expressed as fold activation relative to the activity in unstimulated cells transfected with NFAT-Luc plus empty vector (140 relative luciferase units) . Similar results were obtained in two additional experiments. Figure 9. Effect of cAMP on Zap-70 and TCR-ζ phosphorylation activated through the T cell receptor. Jurkat T cells were either pretreated with 8-CPT-cAMP

(300 μM, 30 min, lanes 7 to 12) or left untreated (lanes 1 to 6) , and subsequently stimulated with anti-CD3 antibody (OKT-3) for the indicated time (0 to 15 minutes) . Cells were then lysed and immunoprecipitation with anti-Zap-70 antibodies was conducted. Anti- phosphotyrosine blots of Zap-70 and ζ-chain (upper and middle panel, respectively) and anti-Zap-70 blot (lower panel) of immunoprecipitates, are shown. Figure 10. Effects of cAMP treatment of Jurkat T cells on endogenous Csk and Lck phosphotransferase activities. Tyrosine kinase activities of Csk and Lck were assessed in immunoprecipitates of Jurkat cells either treated with 8-CPT-cAMP (300 μM) for 20 min (solid bars, +) or untreated cells (open bars, -) (means ± s.e.m.) . Subsequently, cells were lysed and immunoprecipitations with anti-Csk antibodies and anti-Lck antibodies were conducted separately. Phosphotransferase activities were then examined as described using p(E;Y) as a substrate .

Figure 11. Cyclic AMP-mediated inhibition of TCR/CD3- induced ζ-chain phosphorylation is dependent on inactivation of Lck by phosphorylation at Y505. Lck- deficient JCaMl cells were transfected with vector alone (pEF = vector) , with wildtype (wt) Lck, or with mutant Lck where the C-terminal Y505-regulating site is mutated to resist tyrosine phosphorylation and inactivation by Csk. The transfected cells were incubated (30 min) in the absence or presence of 8-CPT-cAMP (300 μM) followed by incubation in the absence or presence of anti-CD3 antibody (5 min) . P0₃-ζ chains were then fished with GST-Zap-70- (SH2) ₂ from cell lysates and detected by phosphotyrosine immunoblotting (Tailor et al . , 1996,

Eur. J. Biochem., 237, p736-742) . ( Upper panel ) Anti- phosphotyrosine blot of P0₃-ζ (arrow) . (Lower panel ) Anti-Lck blot. All lanes contain the endogenous truncated Lck present in JCaMl cells (catalytically inactive; lower bands) whereas full length Lck is present in equal amounts in transfected cells (arrow, lanes 5-12) .

Figure 12. Phosphorylation of Csk by C subunit of PKA. (A) Csk (200 ng at lOng/μl) was incubated with native Cα (100 ng active at 5ng/μl) (lane 1) and heat-inactivated (65°C, 10 min) Cα (lane 2) . Native Cα alone (lane 3) , heat-inactivated Cα alone (lane 4) , and Csk incubated alone (lane 5) were used as controls. Incubations were at 30°C for 30 min in 20 μl buffer containing 5 μM γ- [³²P] -ATP followed by SDS-PAGE as described in Materials and Methods in Example 2. Arrows indicate phosphorylated Csk (50 kDa) and autophosphorylated Cα ( 40 kDa ) .

(B) Time course of phosphorylation of Csk by PKA. Csk (240 ng at 12ng/μl) was incubated with GST-Cβ (23 ng active at 1 ng/μl) at 30°C for the time indicated, followed by SDS-PAGE as described in Materials and

Methods in Example 2. Arrows indicate phosphorylated Csk (50 kDa) and autophosphorylated GST-Cβ (66 kDa) .

(C) Phosphoamino acid analysis of Csk phosphorylated by PKA. Csk (170 ng at 3ng/μl) was incubated with GST-Cβ (70 ng active at 1.5ng/μl) at 30°C for 30 min in 50 μl buffer with 3 μM γ~ [³²P] -ATP (320 Ci/mmol) . Phosphoamino acid analysis was carried out as described in Materials and Methods in Example 2. Figure 13. PKA mediated phosphorylation increases the tyrosine kinase activity of Csk.

(A) Evaluation of the tyrosine kinase activity of Csk (50 ng at lng/μl) when incubated alone (Bar 1) , in the presence of native (Bar 2) or heat-inactivated (65°C, 10 min) (Bar 3) Cα (at 2ng/μl corresponding to 100 ng active enzyme) (means ± SD, n=5) . Coincubation of Csk and PKI (85 μM) with (Bar 4) or without (Bar 5) native Cα are also shown. The reaction mixtures (50 μl) were incubated for 15 min at 30°C in a buffer containing 200 μM Y- [³²P] -ATP, and phosphate incorporation into p(E;Y) was assessed as described in Materials and Methods in

Example 2. Each combination was run in five parallels, and the three median values are presented.

(B) Time-dependent incorporation of phosphate into p(E;Y) by Csk in the presence of native (open circles) and heat-inactivated (65°C, 10 min) (filled circles) Cα. Csk (250 ng at lng/μl) was incubated in the presence of native (500 ng active at 2ng/μl) or heat-inactivated (same amount) Cα at 30°C in 250 μl of buffer containing 200 μM γ-[³²P]-ATP. Aliquots (35 μl) of the reaction mixture were withdrawn and spotted, at the time points indicated, onto Whatman paper and processed as described in Materials and Methods . Time course was performed with single points measurements, and one representative of a total of seven assays is shown. (C) The effects of different amounts (0-100 ng, ie . 0-2ng/μl) of native (open circles) and heat-inactivated (filled circles) Cα on Csk-catalyzed phosphate transfer to p(E;Y) . Csk (50 ng at lng/μl) was incubated with the indicated amounts of Cα at 30°C for 12 min in a total volume of 50 μl buffer containing 200 μM γ~ [³²P] -ATP, and phosphate incorporation into p(E;Y) was assessed as described in Materials and Methods . Duplicate measurements were performed, and one representative assay of a total of four is shown. Dotted lines represent curve-fit analyses (SigmaPlot) . Error bars (half range) are shown. Where error bars are not visible they are within the point .

(D) Concentration-dependent tyrosine kinase activity of Csk in the presence of a constant amount of native (100 ng active at 2ng/μl) (open circles) or heat-inactivated (filled circles) Cα. Csk (0-100 ng, ie. 0-2ng/μl) was incubated for 12 min at 30°C in the presence of Cα in a total volume of 50 μl buffer containing 200 μM γ-ATP, and phosphate incorporation into p(E;Y) was assessed as described in Materials and Methods in Example 2. All samples were assayed in duplicate and error bars (half range) are shown. Where not visible, they are within the point One representative experiment of four is presented. Figure 14. Csk is phosphorylated on S364 by PKA.

(A) Tryptic peptide mapping of wild type Csk phosphorylated by Cα in vi tro as described elsewhere.

(B) As in (A) , but with mutant Csk-S364A.

Figure 15. PKA phosphorylation of Csk-S364 is necessary for the regulatory effect of cAMP on Csk. (A) HA-tagged Csk-wild type (panel A) was expressed in Jurkat T-Ag T cells in the presence or absence of PKA Cβ subunit inserted in pEFneo in sense or reverse orientation, . subjected to immunoprecipitation with anti- HA antibodies, and precipitates were assayed for phosphotransferase activity towards poly(E;Y) (8 min assay; 200 μM ATP) to determine Csk kinase activity. Triplicate immunoprecipitations were analysed from each experiment. The levels of Csk expression were examined by an i-HA immunoblotting of precipitates and the kinase activities were normalized for the levels of HA-Csk (density units, du) as analyzed by densitometric scanning of immunoblots (means ± s.e.m.). Expression of Cβ was also verified by immunoblotting. The data are representative of 3 independent experiments. (B) Csk activity was assessed in anti-HA immunoprecipitates of Jurkat TAg T cells transfected with HA-Csk-wt or mutant HA-Csk-S364C (means ± s.e.m.) . Cells were incubated in the presence or absence of 8CPT- cAMP (300μl, 40 min) , and immunoprecipitations and kinase assays were performed as in (A) . The relative increase in activity following cAMP treatment is shown. Western blot analyses confirmed equal levels of expression of Csk.

Example 1: Binding and Phosphorylation of Vav by cAMP- Dependent Protein Kinase Type I Regulates its Tyrosine Phosphorylation and NFAT Activation in T Cells.

In this Example the functional and physical interactions between Vav and PKA were investigated. We show that PKA- I associates with Vav in intact T cells, and that this association is accompanied by increased serine phosphorylation of Vav. Furthermore, pharmacological activation of PKA or transient overexpression of its catalytic subunit (Cα) inhibited the TCR-induced increase in the tyrosine phosphorylation of Vav and its association with the adaptor protein SLP- 76, as well as Vav-dependent transcriptional activation of NFAT. In contrast, a Vav mutant in which a putative PKA phosphorylation site has been replaced was largely resistant to this inhibitory effect. MATERIALS AND METHODS Cell culture, Stimulation and Lysis- -Human leukemic Jurkat T cells were grown in RPMI-1640 medium (Irvine Scientific, Irvine, CA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA) , 10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES; pH 7.3), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 mM non-essential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin and 50 μM β- mercaptoethanol . Jurkat-TAg, a derivative of Jurkat cell line stably transfected with the SV40 large T antigen (Clipstone et al . , 1992, Nature, 357, p695-697) , was maintained in the above media containing 400 μg/ml G418. COS-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) with the above supplements except β-mercaptoethanol . Jurkat cells were washed once with serum-free RPMI-1640 medium, resuspended in 90 μl of the same medium and activated at 37°C with anti-human CD3 mAbs, OKT3 (10 μg/ml) or SpvT3d (1/200 dilution of ascites fluid; kindly provided by Dr. H. Spits, The Netherlands Cancer Institute) , forskolin (cAMP activator) (100 μM; Calbiochem, La Jolla, CA) , 8- (4- chlorophenyl) thio-cAMP (8-CPT-cAMP; 1 mM; Sigma Chemical Co., St. Louis, MO), or their combinations for the indicated times. 8-CPT-cAMP was dissolved to a concentration of 10 mM in PBS, and its exact concentration was calculated using the extinction coefficient and absorption maximum indicated by the manufacturer. In some experiments, the cells were also stimulated with phorbol myristate acetate (PMA; Sigma) . Stimulation was terminated by adding 1 ml lysis buffer A (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP- 40, 10 μg/ml each aprotinin and leupeptin, 100 μg/ml soybean trypsin inhibitor and 1 mM Na₃V0₄) . After incubating for 10 min on ice, nuclei and cell debris were removed by centrifugation at 4°C (15 min at 13,000 x g) .

Plasmids--Vav cDNA was amplified by PCR using pSK115 (Katzav et al . , 1991, Mol. Cell. Biol., 11, pl912-1920) as a template and oligonucleotide primers carrying EcoRI site at the 5' end, and encoding a Myc tag epitope for in-frame fusion at the 3' end of Vav. The PCR product was cloned into a mammalian expression vector pEFneo (Liu et al., 1993, Proc. Natl. Acad. Sci., USA 90, p8957-8961) . An EcoRI-BstXI fragment from the resulting construct was then replaced with the corresponding fragment from pSK115 to ensure that the cloned Vav cDNA was free of PCR errors . The ends of the Vav cDNA cloned in pEFneo were sequenced to confirm the correct sequence. A point mutation resulting in serine to alanine substitution (S440A) of Vav was generated in pEF-Vav-Myc using a site-directed mutagenesis kit (Clontech, Palo Alto, CA) and confirmed by sequencing. To generate a glutathione S-transferase (GST) fusion protein encoding the Vav pleckstrin-homology (PH) domain (GST-Vav-PH) , the Vav PH domain was amplified by PCR from pSK115 and subcloned into pGEX-3T. A mammalian expression vector encoding the catalytic subunit of human PKA (pSRαneo-Cα) was generated by inserting a 1,302-bp Nael - PvuI I fragment of the human Cα cDΝA (Beebe et al., 1990, Mol. Endocrinol . , 4, p465-475) in the Clal site of pSRα-neo using Clal linkers (K. Tasken, unpublished) . pSRαneo carries the strong, constitutive SRα promoter inserted in the Sail site of pRSVneoI (Takebe et al . , 1988, Mol. Cell. Biol., 8, p466-472) . ΝFAT-Luc has previously been described (Northrop et al . , 1993, J. Biol. Chem., 268, p2917-2923).

Transfection and Luciferase Assay- -COS cells were transfected by the method of Chen and Okayama (Chen & Okayama, 1988, BioTechniques, 6, p632-638) . Jurkat-TAg cells in 300 μl serum-free RPMI-1640 were electroporated with 40 μg total plasmid DNA per 10-15 x 10⁶ cells using a Bio-Rad (Richmond, CA) electroporator set at 960 μF, 240 volts. For luciferase assays, cells were grown for 24-40 hrs after electroporation, and were then either left unstimulated or treated with OKT3 (10 μg/ml) , forskolin, or a combination of both, for 4-8 hrs. Cells were harvested, washed once in phosphate-buffered saline, pH 7.2 (PBS), and lysed in 100 μl lysis buffer B (0.2% Triton X-100, 100 mM potassium phosphate, pH 7.4, 1 mM dithiothreitol) . Lysates were centrifuged and the supernatant was used for luciferase assay as described previously (Liu et al . , 1997, J. Biol. Chem., 272, pl68- 173) . For other assays, transfected cells were grown in culture for up to 48 hrs, washed once with serum-free RPMI-1640, resuspended in 1 ml of the same medium and stimulated with various agonists as indicated.

Immunoprecipitation and Immunoblotting- -Cell lysates were precleared by incubation with normal mouse Ig or preimmune rabbit serum and Protein G-Sepharose beads for 1 hr at 4°C, and then subjected to immunoprecipitation with Vav- or PKA subunit-specific antibodies. For Vav, 8 μl of a rabbit polyclonal antiserum against a recombinant MBP- fusion protein containing the DH, PH and Cys-rich domains of Vav, was used. For PKA, 5 μg of anti-RIα (Tasken et al . , 1993, J. Biol. Chem., 268, p21276-21283) or -Cα mAb (Transduction Laboratories, Lexington, KY) , were used. After incubating for 2 hr- overnight at 4°C, 25 μl of Protein G-Sepharose beads

(Pharmacia Biotech, Piscataway, NJ) was added, and the incubation continued at 4°C for 1 hr. Immune complexes were pelleted, washed 5 times in lysis buffer, resuspended in SDS sample buffer (60 mM Tris/HCl, pH 6.8, 2.3% SDS, 10% glycerol , 5% β-mercaptoethanol) , resolved by 8.5% SDS-PAGE, and transferred electrophoretically to nitrocellulose membranes. Immunoblotting was performed by probing the membranes with 1 μg/ml anti-Vav, anti-phosphotyrosine (PTyr) mAbs (both from Upstate Biotechnology Inc., Lake Placid, NY), anti-SLP-76 (kindly provided by Drs . R. Lahesmaa and P. Findell, Roche Biosciences, Palo Alto, CA) , anti-c-Myc (9E10) or 10 ng/ml biotinylated anti-RIα (30) mAbs, or with an affinity-purified polyclonal anti-Cα antibody (100 ng/ml; Santa Cruz) . Protein A-purified anti-RIα mAb was biotinylated by mixing with a 10-fold molar excess of Biotin-X-NHS (Sigma) in 0.1 M borate buffer, pH 8.0. The solution was incubated overnight at 4°C, ethanolamine, pH 8.0, was added to a final concentration of 0.1 M, and the incubation continued for 2 hrs at room temperature. Finally, the biotinylated antibody was dialyzed against PBS containing 0.1% NaN₃ overnight. Blots were developed by the enhanced chemiluminescence technique (ECL kit, Amersham, Arlington Heights, IL) according to the manufacturer's instructions. The biotinylated antibody was detected by incubation with streptavidin-coupled horseradish peroxidase (Amersham) prior to ECL detection. Where indicated, membranes were stripped by incubation in 62.5 mM Tris-HCl, pH 6.7/100 mM 2-mercaptoethanol/2% SDS for 1 hr at 70°C with constant agitation, washed, and then reprobed with other antibodies. Photoaffinity Labeling of PKA with 8-Azido- [³²P] cAMP--

Jurkat cells were either left untreated or treated with the indicated combinations of anti-CD3 and/or 8-CPT-cAMP for 5 min, and cells (5 x 10⁷ in 1 ml/sample) were lysed in a 1% Brij -96-containing buffer. The regulatory RI subunit of PKA (Rlα) was photoaffinity-labeled with 8- azido- [³²P] cAMP prior to immunoprecipitation as described (Tasken et al . , 1993, supra), and subsequently immunoprecipitated with anti-Vav or -Rlα antibodies as described above. Following 10% SDS-PAGE, labeled proteins were visualized by autoradiography. Phosphoamino Acid Analysis (PAA) --Cells were starved overnight in phosphate-free medium, washed, resuspended in 0.5 ml phosphate-free medium (10 x 10⁶ cells/group), and labeled with 0.5-1.0 mCi ³²Pi (9120 Ci/mmol, DuPont

NEN, Boston, MA) for 4 hrs. Vav immunoprecipitates were prepared, resolved by SDS-PAGE, and transferred electrophoretically to Immobilon membranes (Millipore, Bedford, MA) . Following autoradiography, the phosphorylated Vav band was excised, washed extensively with distilled water, and subjected to hydrolysis in 100 μl of 6N HCl at 110°C for 1 hr. The samples were lyophilized, resuspended in 10 μl electrophoresis buffer (pH 1.9) containing 1 μg each of phosphoamino acid standards comprising phosphoserine, phosphothreonine and phosphotyrosine . Samples were spotted on thin-layer cellulose plates and analyzed by two-dimensional electrophoresis at pH 1.9 followed by pH 3.5. Nonradioactive standards were detected by staining with 0.25% ninhydrin in acetone, and radiolabeled phosphoamino acids by autoradiography.

In Vitro Kinase Assay- -Five μg of purified GST or GST- Vav-PH was immobilized by binding to glutathione- Sepharose beads (Pharmacia Biotech) for 1 hr at 4°C and washed once in kinase buffer (20 mM tris/HCl, pH 7.6 , 10 mM magnesium acetate, 2.5 mM β-mercaptoethanol) . Kinase reactions were carried out for 30 min at 30°C in 100 μl kinase buffer supplemented with 10 μM ATP and 10 μCi [γ- ³²P]ATP (DuPont NEN, Boston, MA; 7,000 Ci/mmol) in the presence or absence of 1 μg purified PKA catalytic subunit from bovine heart (Sigma) . The reaction was stopped by washing the beads twice with cold kinase buffer. Bound proteins were eluted into SDS sample buffer, resolved by 12% SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography. RESULTS TCR/CD3-Induced Ser/Thr Phosphorylation of Vav

In addition to the early increase in the PTyr content of Vav in different types of activated cells, Vav has also been found to be phosphorylated on Ser/Thr residues in B cells or in Vav-transfected fibroblasts (Margolis et al . , 1992, supra; and Bustelo & Barbacid, 1992, supra) .

In order to examine whether T cell activation causes increased Ser/Thr phosphorylation of Vav, we analyzed the phosphorylation status of immunoprecipitated Vav in ³²P-metabolically labeled resting or anti-CD3 -activated Jurkat T cells. Vav, but not control (normal rabbit serum) , immunoprecipitates (IPs) from resting cells contained a 95-kDa phosphoprotein (Figure 1A,- lane 3) which comigrated with authentic Vav detected by immunoblot analysis of whole cell lysates (lane 5) . The phosphorylation of this protein increased by ~2-fold following 0KT3 stimulation (lane 4) . The ³²P-labeled Vav band was eluted from the membrane and subjected to PAA analysis. As shown in Figure IB, Vav from resting cells exhibited low basal phosphorylation on serine and even lower phosphorylation on tyrosine residues (left panel) . Anti-CD3 stimulation resulted in increased phosphorylation of Vav on tyrosine, serine and threonine residues (right panel) . Based on four independent experiments, OKT3 stimulation increased the phosphorylation of Vav by an average of five- and two-fold on tyrosine or serine residues, respectively. These results indicate that, in addition to tyrosine phosphorylation of Vav, ligation of the TCR/CD3 complex also induces phosphorylation of Vav on Ser/Thr residues. PKA is Involved in TCR/CD3-Induced Ser/Thr Phosphorylation of Vav

We investigated whether PKA may play a role in the phosphorylation of Vav. The phosphorylation status of Vav was evaluated as before in ³²P-labeled Jurkat cells which were left unstimulated, or treated with combinations of OKT3 , a specific inhibitor of the catalytic subunit of PKA (KT5720) , or a direct PKA activator (forskolin) . As shown in Figure 2A, Vav from resting cells exhibited basal phosphorylation (lane 1) which was increased following OKT3 stimulation (lane 2) . Pretreatment with forskolin alone caused a minimal increase, which was not consistently observed, in Vav phosphorylation (lane 3), while KT5720 (lane 5) did not seem to affect Vav phosphorylation. However, pretreatment of cells with KT5720 prior to 0KT3 stimulation largely prevented the 0KT3-induced increased phosphorylation of Vav, and Vav phosphorylation (lane 4) was comparable to that seen in resting cells (lane 1) . A potential phosphorylation site for PKA exists in residues 437 to 441 (RGDSY) of Vav. Since this motif lies in the PH domain of Vav, we generated a GST-Vav-PH fusion protein and ascertained its in vi tro phosphorylation by purified Cα. GST was used in parallel as a negative control. The in vi tro kinase assay products were resolved by SDS-PAGE, transferred to nitrocellulose and subjected to autoradiography or anti- Vav immunoblotting. As shown in Figure 2B, a 38 -kDa protein corresponding to the GST-Vav-PH fusion protein was the major substrate of phosphorylation by PKA (left panel, lane 1) . No phosphorylation of the control 26- kDa GST protein was seen under the same conditions (left panel, lane 3). Similarly, no phosphorylated proteins were detected in the absence of added PKA (left panel, lanes 2, 4) . Immunoblot analysis (right panel) demonstrated that equivalent amounts of the GST-Vav-PH protein were present in the kinase assay (lanes 1, 2) . These results indicate that the PH domain of Vav is an in vi tro substrate of PKA. Association of Vav with PKA in Intact T cells

The above findings prompted us to determine whether Vav can associate with PKA in intact T cells. First, Vav IPs from control or activated Jurkat cells were resolved by SDS-PAGE and probed for the presence of PKA either by immunoblotting with purified anti-RIα or -Cα antibodies, or by photoaffinity labeling of Rα with 8- azido- [³²P] cAMP . Rlα IPs served as positive controls. Vav IPs from resting or stimulated Jurkat cells contained immunoreactive Rlα (Figure 3A) or Cα (Figure 3B) subunits (lanes 2-5) which comigrated with the authentic PKA subunits immunoprecipitated by the corresponding specific antibodies (lane 1) . Although there were variations in the amount of Rlα or Cα coimmunoprecipitating with Vav under different stimulation conditions, these differences were not reproducibly observed (data not shown) . The Vav- associated Rlα subunit could also be detected by photoaffinity labeling with 8-azido- [³²P] cAMP (Figures

3C, lanes 2, 3; Figure 3D, lanes 1, 2) . The absence of signal in Figure 3 , lanes 4 and 5, reflects the competition between «cold» 8-CPT-cAMP used for stimulation and 8-azido- [³²P] cAMP used for affinity labeling. The specificity of this association is evident from the finding that no labeled protein was detected in control (NRS) IPs (Figure 3D, lanes 3, 4) . In all cases, the membranes were reprobed with an anti- Vav antibody, revealing that very similar levels of Vav were immunoprecipitated in each experiment (data not shown) . The specificity of this interaction with regard to PKA isoforms could not be evaluated in this system since Jurkat cells do not express the type II isoform of PKA (Skalhegg et al . , 1998, J. Cell. Physiol., 177, p85- 93) . Densitometric scanning of the antibody- or 8- azido- [³²P] cAMP-labeled Rlα bands precipitated by anti- Rlα or -Vav antibodies, respectively, from unstimulated Jurkat cells (Figures 3A and 3C, lanes 1, 2) revealed that -10-15% of the total immunoprecipitable Rlα was associated with Vav. The -90% reduction in Vav- associated Cα in 8 -CPT-cAMP-treated cells (Figure 3B, lanes 4, 5) is consistent with the dissociation of Cα from Rlα (and its subsequent activation) induced by this PKA agonist .

In order to further explore this association, we conducted the reverse experiments, i.e., by probing Rlα or Cα IPs from unstimulated or activated Jurkat cells with anti-Vav antibodies, and using Vav immunoblots of total lysates from the same cells as a positive control (Figure 4) . The anti-RIα mAb (Figure 4A, lanes 3, 4) , but not normal mouse Ig (lanes 5, 6), immunoprecipitated a 95-kDa protein recognized by the anti-Vav antibody. This protein comigrated with Vav detected by anti-Vav immunoblotting of whole cell lysates (lanes 1, 2) . Densitometry analysis indicated that -1% of cellular Vav was associated with the immunoprecipitable Rlα. Anti- CD3 stimulation did not modulate significantly the level of Rlα-associated Vav (compare lanes 3 vs . 4) . Similar amounts of Rlα were present in the Rlα IPs from resting or stimulated cells (data not shown; see also Figure 4B) . TCR/CD3 ligation has previously been shown to induce colocalization of Rlα with the TCR/CD3 complex (Skalhegg et al . , 1994, Science, 263, p84-87) . Vav appears to be involved in proximal TCR signaling events, and is known to associate with TCR-coupled PTKs (Collins et al., 1997, supra; Deckert et al . , 1996, supra). Therefore, we extended the analysis of the Vav-PKA association in order to assess the effect of a PKA agonist (forskolin) on this association, as well as its specificity. Rlα IPs from resting Jurkat cells, or from cells treated with the indicated combinations of 0KT3 and/or forskolin, were subjected to anti-Vav immunoblotting. Figure 4B (top panel, lanes 5-8) demonstrates the coimmunoprecipitation of Vav with Rlα. When normalized to the expression levels of Rlα present in the same immunoprecipitates (bottom panel, lanes 5- 8), it can be concluded that stimulation with 0KT3 and/or forskolin did not affect the level of Rlα- associated Vav to a significant degree. Taken together, these results indicate that Vav is associated constitutively with the Rlα subunit of PKA in intact T cells and, consistent with our earlier findings (Figure 3) , this association does not appear to be affected by anti-CD3 stimulation. Furthermore, PKA activation by forskolin treatment also does not affect this association.

We next ascertained whether Vav also coimmunoprecipitates with the catalytic subunit (Cα) of PKA by probing PKA IPs with anti-Vav (and, for comparison, with anti-RIα) antibodies. To assess the effects of PKA activation, some groups were additionally pretreated with forskolin. As shown in Figure 4C, Vav was specifically coimmunoprecipitated by the anti-Cα

(lanes 4-6) and anti-RIα (lanes 1-3) antibodies, but not by a control mouse Ig (lanes 7, 8) . The PKA-associated Vav band comigrated with the immunoreactive Vav protein observed in Jurkat cell lysates (lanes 9-11) . As shown above, the association of Rlα with Vav was constitutive and exhibited little change following OKT3 or forskolin treatment (compare lanes 1-3) . Similarly, Vav was also found to be constitutively associated with Cα, and no significant changes in the amount of Vav associated with Cα were observed following treatment of cells with either OKT3 (lane 5) or forskolin (lane 6) . The lower amount of Vav seen in lane 4 was due to a loading artifact and the presence of Vav in Cα IPs in resting cells was confirmed in several other experiments. These results indicate that Vav constitutively associates in vivo with both Rlα and Cα subunits of PKA. TCR/CD3-Induced Tyrosine Phosphorylation of Vav is Inhibited by Activated PKA

We next wished to address the functional implications of the interaction between Vav and PKA. In T cells, elevation in intracellular cAMP levels and the subsequent activation of PKA lead to a block in cell proliferation, and some of the targets that are negatively regulated by PKA include PLCγl (Granja et al . , 1991, J. Biol. Chem., 266, pl6277-16280 ; and Park et al., 1992, J. Biol. Chem., 267, pl496-1501) , Raf-1 and mitogen-activated protein kinases (Cook & McCormick, 1993, Science, 262, pl069-1072; Mischak et al . , 1996, Mol. Cell. Biol., 16, p5409-5418; Graves et al . , 1993, Proc. Natl. Acad. Sci. USA 90, pl0300-10304 ; Burgering et al., 1993, EMBO J. , 12, p4211-4220; Wu et al . , 1993, Science, 262, pl065-1069; Hafner et al . , 1994, Mol.

Cell. Biol., 14, p6696-703; and Tamir et al . , 1996, J. Immunol., 157, pl514-1522) . Moreover, PKA-mediated phosphorylation of PLCγl was found to inhibit its TCR/CD3 -induced tyrosine phosphorylation (Granja et al . , 1991, supra; and Park et al . , supra). Therefore, we decided to examine first whether PKA activation can affect the increased tyrosine phosphorylation of Vav induced by TCR/CD3 ligation. The PTyr content of immunoprecipitated Vav was evaluated in unstimulated or anti-CD3 -stimulated Jurkat cells which were pretreated with forskolin or with a vehicle control .

Immunoprecipitated Vav from unstimulated Jurkat cells exhibited basal tyrosine phosphorylation (Figure 5A, top panel, lane 12) which was increased following OKT3 stimulation (lane 11) . Forskolin pretreatment reduced both the basal (top panel, lane 10) and 0KT3- induced (top panel, lane 9) tyrosine phosphorylation of Vav. No phosphoproteins were observed in the control (NRS) IPs (lanes 1-4) . Examination of PTyr-containing proteins in total lysates from the same cells revealed that the inhibitory effect of forskolin was selective since the tyrosine phosphorylation of other proteins

( e . g. , pl20) was not reduced by forskolin pretreatment (top panel, lanes 5-8) . Anti-Vav immunoblot analysis revealed similar levels of Vav in all IPs (bottom panel, lanes 9-12) . The effect of forskolin on the tyrosine phosphorylation of Vav was confirmed by probing Myc tag IPs from Myc-pEF-Vav-transfected Jurkat-TAg cells with an anti-PTyr mAb (data not shown) . Thus, forskolin treatment appears to result in selective inhibition of tyrosine phosphorylation of Vav. In order to further assess the specificity of the forskolin-mediated inhibitory effect on the anti-CD3- induced tyrosine phosphorylation of Vav, we ascertained whether activation of PKC by phorbol ester treatment can cause a similar inhibition. When Jurkat cells were pretreated with PMA, no reduction in the PTyr content of immunoprecipitated Vav was detected under either basal or anti-CD3-induced conditions (Figure 5B, top panel, lanes 1-4) . Vav immunoblotting of the same immunoprecipitates confirmed the similar expression levels of Vav in all groups (bottom panel, lanes 1-4) . These results confirm that inhibition of the tyrosine phosphorylation of Vav is not a general outcome of the activation of cellular Ser/Thr kinases but, rather, represents a selective effect of PKA activation. In order to directly correlate this observed inhibitory effect with activated PKA, Jurkat-TAg cells were cotransfected with an expression plasmid encoding the Cα subunit of PKA plus a Myc epitope-tagged Vav expression vector (or with the corresponding empty vector as a negative control) , and tyrosine phosphorylation of Vav in Myc IPs was examined under basal or anti-CD3-stimulated conditions. As shown in Figure 5C, Cα subunit cotransfection (top panel, lanes 2, 4) caused reduced tyrosine phosphorylation of Vav by comparison with the empty vector (top panel, lanes 1, 3) in both resting (lane 2) and 0KT3 -activated (lane 4) cells. The expression levels of Vav were similar in all groups as shown by anti-Myc immunoblotting (bottom panel, lanes 1-4) . Although the inhibitory effect of Cα cotransfection on tyrosine phosphorylation of Vav did not appear to be as strong as that observed in forskolin-treated cells, this may partly be due to the lower expression of Cα when cotransfected with Vav. Similar inefficient effect of Cα cotransfection compared to forskolin treatment has also been reported for v-Mos phosphorylation (Yang et al . , 1996, Mol. Cell. Biol., 16, p800-809) . Thus, tyrosine phosphorylation of Vav in resting or TCR/CD3 -activated T cells is reduced by the activation of PKA, suggesting a potential role for PKA- mediated phosphorylation in the regulation of Vav function. PKA Activation Inhibits Vav and SLP-76 Association It has recently been reported that Vav forms a signaling complex with SLP-76 (Deckert et al . , 1996, supra; Raab et al . , 1997, Immunity, 6, pl55-164; Fang et al., 1996, J. Immunol., 157, 9, p3769-3773; Tuosto et al., 1996, J. Exp . Med. , 184, pll61-1166; and Wu et al . 1996, Immunity, 4, p593-602), an adaptor protein which, like Vav, becomes rapidly phosphorylated on tyrosine upon T cell activation (Jackman et al . , 1995, J. Biol. Chem., 270, p7029-7033). This complex formation is mediated by binding of the Vav SH2 domain to inducibly phosphorylated tyrosine residues in SLP-76. Furthermore, cotransfection with Vav and SLP-76 can lead to synergistic stimulation of NFAT activity in T cells (Fang et al . , 1996, supra; Wu et al . , 1996). Thus, we next investigated whether PKA activation affects the

Vav-SLP-76 complex in T cells. Jurkat cells were left unstimulated or treated with OKT3 in the absence or presence of forskolin. As a control, the cells were also pretreated with PMA. The cells were lysed, and the presence of SLP-76 in Vav IPs was determined by anti- SLP-76 immunoblot analysis. As seen in Figure 6 (top panel) , immunoprecipitated Vav from unstimulated T cells contained a small amount of SLP-76 (lane 1) which increased considerably following 0KT3 stimulation (lane 6) , in agreement with previous data (Wu et al . , 1996, supra; and Jackman et al., 1995, supra) . When the cells were pretreated with forskolin, the amount of SLP-76 coimmunoprecipitating with Vav decreased significantly in both resting (lane 2) or 0KT3 -stimulated (lane 4) cells. In contrast, PMA pretreatment did not significantly reduce the association of SLP-76 with Vav in resting cells (lanes 3 vs . 1) . Although cells treated with PMA plus OKT3 did exhibit a slight reduction in the amount of Vav- associated SLP-76 (lanes 5 vs . 6), this reduction was not as pronounced as that seen in forskolin-pretreated cells (lane 4) . The overall expression of SLP-76 was not altered following the various treatment regimens as evidenced by anti-SLP-76 immunoblot analysis of the cell lysates (lanes 7-12) . Furthermore, anti-Vav immunoblotting demonstrated that similar amounts of Vav were present in all IPs or lysates (bottom panel) , confirming that the above effects on the association of SLP-76 with Vav were not due to differential expression of either SLP-76 or Vav. These results indicate that in addition to inhibiting the tyrosine phosphorylation of Vav itself, PKA activation also results in reduced association between Vav and SLP-76.

Ser-440 of Vav is a Likely Regulatory Target for the Inhibitory Effect of PKA

The ability of purified Cα to phosphorylate the PH domain of Vav in vi tro (Figure 2B) indicated that Ser/Thr residues within the PH domain of Vav may represent a physiological target for PKA in T cells . Serine-440 (Ser-440) in an RGDS*Y motif within this domain may represent a PKA phosphorylation site. We therefore examined the potential role of this site in the PKA-mediated regulation of the function of Vav. Since ligands of the Vav PH domain, or its function in general, are not known, we used the well established Vav-mediated activation of NFAT (Wu et al . , 1995, supra; Holsinger et al . , 1995, supra; and Deckert et al . , 1996, supra) as a read-out for potential modulation of its function. Using site-directed mutagenesis, a Vav mutant (S440A) was generated in which Ser-440 was replaced by alanine .

We examined first whether mutation of Ser-440 influences the association between Vav and PKA. COS cells were transfected with (WT) or mutated (S440A) Vav expression vectors, the endogenous Rlα or Cα subunits were immunoprecipitated with the respective antibodies, and the association of Vav was assessed by immunoblotting with a Vav-specific mAb. As shown in Figure 1A, expression of S440A was equivalent to WT Vav as evidenced by Vav immunoblotting of total cell lysates (top panel, lanes 8, 9, 11, 12), and the transfected gene product comigrated with Vav from a Jurkat cell lysate (lane 13) . Both S440A and WT Vav coimmunoprecipitated with Rlα to a similar extent (top panel, lanes 2, 3, 4, 6) . In agreement with our earlier findings (Figures 3, 4), forskolin pretreatment did not affect the association of Rlα with either WT Vav (lanes 3 vs . 6) or S440A (lanes 2 vs . 5) . An anti-RIα blot confirmed that similar amounts of Rlα were precipitated from cells transfected with WT (bottom panel, lanes 3, 6) vs . S440A Vav (lanes 2, 5) . No Vav was detectable in control cells which were transfected with the empty vector (top panel, lanes 1, 4), although Rlα was present in these IPs (bottom panel, lanes 1, 4) .

Similar results were obtained using Cα IPs from the transfected COS cells (Figure 7B) . In this case, too, similar amounts of WT and S440A Vav were detected in association with Cα (lanes 6, 7), while no Vav was detectable in Cα IPs from control cells transfected with the empty vector alone (lane 5) . The association was constitutive, and was not modulated by forskolin pretreatment (data not shown) . Thus, the S440A mutation does not impede the association of Vav with PKA subunits .

We next evaluated the ability of WT or S440A Vav to activate an NFAT-luciferase reporter gene under different stimulation conditions in transiently transfected Jurkat-TAg cells. The cells were treated with OKT3 alone or in combination with forskolin, harvested, and luciferase activity in the cell extracts was determined. As seen in Figure 8, Vav overexpression resulted in constitutive activation of NFAT-driven luciferase activity, which was 24 -fold higher than that seen in control cells transfected with the empty vector alone. Anti-CD3 stimulation augmented the Vav-induced NFAT activity by an additional 6-fold, as previously reported (Wu et al . , 1995, supra; Holsinger et al . , 1995, supra; and Deckert et al . , 1996, supra). Forskolin pretreatment reduced by -40% and -60%, respectively, the constitutive or OKT3 -stimulated activation of NFAT by Vav compared to untreated cells. In control cells transfected with empty vector alone, forskolin treatment did not result in any significant inhibition of basal NFAT activity, but caused a -70% inhibition of NFAT activity induced by OKT3 stimulation, consistent with the well established inhibitory effect of PKA on T cell activation (Isakov et al . , 1994, supra; Kammer, 1988, Immunol. Today, 9, p222-229; Isakov & Altman, 1985, J. Immunol., 135, p3674-3680) .

The S440A Vav mutant largely retained the ability to stimulate NFAT activity in both unstimulated (20-fold stimulation vs . control) or anti-CD3 -treated (108-fold vs . control) cells, although it was slightly less efficient than WT Vav in this regard. However, in contrast to WT Vav, forskolin treatment of the S440A Vav-transfected cells resulted in no inhibition of NFAT activity in unstimulated cells, and a minimal (15%) inhibition in 0KT3 -activated cells, respectively. Thus, mutation of Ser-440 confers upon Vav relative resistance to the inhibitory effect of forskolin with regard to the activation of NFAT. This result implies a physiological role for this site in Vav-mediated NFAT activation. DISCUSSION

Vav has previously been reported to undergo a transient increase in tyrosine phosphorylation upon ligation of multiple receptors, including the TCR/CD3 complex, in hematopoietic cells. In addition several studies have reported that Vav is phosphorylated constitutively on Ser/Thr residues in B cells (Bustelo & Barbacid, 1992, supra) and in Vav-transfected fibroblasts (Margolis et al . , 1992, supra) and, furthermore, that cell stimulation does not increase its Ser/Thr phosphorylation. On the other hand, rapid phosphorylation of Vav on Ser/Thr residues has been reported to occur in response to prolactin stimulation in a rat T cell lymphoma line (Clevenger et al . , 1995, J. Biol. Chem., 270, pl3246-13253) . Our results demonstrate that anti-CD3 stimulation of Jurkat T cells leads to increased phosphorylation of Vav on Ser/Thr residues, in addition to the increased tyrosine phosphorylation (Figures 1, 2) . This phosphorylation occurred rapidly (within 90 sec) . Thus, Vav is a substrate for both PTKs and Ser/Thr kinases which are stimulated upon T cell activation, and phosphorylation by these kinases may potentially regulate Vav function. Since the increased phosphorylation of Vav induced by anti-CD3 stimulation was partially inhibited by pretreatment with a PKA antagonist (Figure 2) , PKA is a candidate Ser/Thr kinase to regulate Vav phosphorylation. However, it is quite likely that PKA is not the only, or even the major, Ser/Thr kinase that phosphorylates Vav. This may account for our findings that, first, forskolin caused only a minimal and inconsistent increase in Vav phosphorylation (Figure 2A) and, second, no difference in the phosphorylation status of immunoprecipitated wild-type (WT) vs . mutated Vav (S440A; a potential PKA phosphorylation site) could be detected under different stimulation conditions (i.e., -/+ anti-CD3 and/or forskolin) in transiently transfected, ³²P metabolically labeled cells (data not shown) . Thus, PKA-mediated phosphorylation of Vav appears to account for only a small portion of its total Ser/Thr phosphorylation. In addition, it is possible that Vav is already nearly maximally phosphorylated by the associated PKA in resting T cells. Either case would make it difficult to detect any additional, forskolin-stimulated Vav phosphorylation .

Our findings indicate that both the regulatory and catalytic subunits of PKA-I can be associated with Vav. The decreased coimmunoprecipitation of Cα with Vav following stimulation with the PKA agonist 8-CPT-cAMP (Figure 3B) is consistent with a dissociation of Cα from Rlα (and, hence, its catalytic activation) expected to be triggered by the agonist. However, we have not observed the corresponding decrease in Vav association in Cα IPs from forskolin-stimulated cells (Figure 4C) . The reasons for this apparent discrepancy are unclear, but may reflect the different sensitivities of detecting the association in Vav vs . Cα IPs. Thus, whereas 10-15% of the total immunoprecipitable PKA- I is present in Vav IPs (Figure 3) , only -1% of cellular Vav was found in a complex with immunoprecipitable Rlα (Figure 4) , thereby making it more difficult to detect any agonist-induced dissociation in the latter situation. In addition, our experiments do not reveal the molecular basis for the observed associations. Thus, it remains to be determined whether Cα associates directly with Vav, or indirectly via its association with Rlα which then forms a complex with Vav. A recent study demonstrated that the catalytic subunit of PKA forms a direct complex with IKB proteins in the absence of the regulatory subunit and, furthermore, that the catalytic activity of PKA in this complex is blocked and can be regulated in a cAMP- independent manner (Zhong et al . , 1997, Cell, 89, p413- 424) . Although our findings differ from this study in that we could also detect the presence of Rlα in a complex with Vav, this does not rule out the interesting possibility that Cα is maintained in an inactive state in the Vav complex. This would be consistent with our failure to detect increased PKA-mediated phosphotransferase activity in Vav IPs (data not shown) . Agents that elevate the intracellular concentration of cAMP, thereby causing activation of PKA, are known to modulate, generally in a negative manner, immune responses (Kammer, 1988, Immunol. Today, 9, p222-229) . Several studies have demonstrated that elevation of cAMP inhibits the proliferation of T cells (Isakov et al . , 1994, supra; Isakov & Altman, 1985, supra), and this inhibition has been shown to be mediated predominantly by the type I isoform of PKA (Skalhegg et al . , 1992, supra). PLCγl may represent one target for the inhibitory effect of PKA activation (Granja et al . , 1991, supra; Park et al . , 1992, supra) . However, the precise mechanism (s) which underlie this inhibitory action, and the targets of PKA in T cells are largely unknown. Our results demonstrate that Vav associates with PKA- I (Figures 3, 4). However, the stoichiometry of the association between Vav and PKA was low and varied among experiments, suggesting that Vav may interact with PKA through an intermediate molecule (s) . Vav was also a potential target for PKA mediated phosphorylation (Figure 2) and, furthermore, PKA activation modulated the function of Vav as indicated by the reduction in its tyrosine phosphorylation (Figure 5) and association with SLP-76 (Figure 6) . The reduced SLP-76 association most likely also reflects deficient tyrosine phosphorylation of this adaptor protein since PTyr residues in SLP-76 mediate its binding to the Vav SH2 domain (Fang et al . , 1996 supra) . It is not clear whether the reduced tyrosine phosphorylation of Vav reflects a direct effect on Vav or an indirect effect due to the PKA-mediated inhibition of PTKs which phosphorylate Vav. However, the effect is relatively selective since forskolin pretreatment did not reduce the tyrosine phosphorylation of all substrates in T cells .

Although our studies have not determined the precise site(s) in Vav for PKA-mediated phosphorylation, several findings suggest that Ser-440, which is located within the PH domain of Vav, is a likely target. First, a GST-Vav-PH fusion protein was efficiently phosphorylated by PKA in vi tro . Second, whereas NFAT activation by wild-type Vav was sensitive to inhibition by forskolin pretreatment, a Vav mutant in which Ser-440 has been replaced was markedly less sensitive. Although this result does not exclude a role for other potential PKA phosphorylation sites, it strongly suggests that PKA-mediated phosphorylation of Ser-440 regulates the function of Vav in T cell signal transduction pathways. It would be interesting to assess the effect of this phosphorylation event on the function and ligand association of the PH domain of Vav, once these are elucidated.

Distinct PKA isoforms most likely mediate specific and different cellular functions. This specificity may be achieved by differential subcellular localization of the PKA isoforms which, in turn, can be determined by anchoring proteins such as members of the AKAP family (Faux & Scott, 1996, Cell, 85, p9-12; Mochly-Rosen, 1995, Science, 268, p247-251) . Indeed, it has been shown that PKA- II activity in lymphocytes is exclusively particulate and associated with the Golgi-centrosomal compartments (Keryer et al . , 1993, Exp . Cell. Res., 204, p230-240) , whereas PKA-I activity is cytosolic and redistributes to the membrane where it colocalizes with the TCR/CD3 complex following T cell activation (Skalhegg et al . , 1992, supra; Skalhegg et al . , 1994, Science, 263, p84-87). Furthermore, T cell activation by either anti-CD3 plus IL-1 or by a combination of phorbol ester and ionomycin was recently found to selectively stimulate PKA-I, and not PKA-II activity, leading to phosphorylation of several membrane- associated proteins (Laxminarayana & Kammer, 1996, supra) . Upon PKA activation, the catalytic subunits dissociate from the regulatory subunits and phosphorylate particulate, cytosolic, and nuclear proteins. While the association of Vav with PKA subunits (Figures 3, 4) suggests that Vav is a likely substrate for PKA in vivo, the precise intracellular location of such complexes remains to be determined. It will be interesting to determine whether Vav is present in the PKA- I complex that inducibly translocates to the ligated TCR/CD3 complex (Skalhegg et al . , 1994, Science, 263, p84-87) . Our results show that the stimulation of PKA phosphotransferase activity is likely to be responsible for at least some of the increased serine/threonine phosphorylation of Vav following TCR/CD3 ligation and, moreover, implicates PKA as a negative regulator of the ability of Vav to cause transcriptional activation of

NFAT. Since the rapid activation of T cell non-receptor PTKs which leads, among other events, to PKC activation and Ca²⁺ mobilization, appears to precede PKA- I activation (Laxminarayana & Kammer, 1996, supra), it is likely that PKA- I activation serves as a negative feedback mechanism to dampen and terminate T cell activation. This is in general agreement with the known inhibitory effect of PKA agonists on T cell activation (Kammer, 1988, supra; Ledbetter et al . , 1986, supra; Isakov & Altman, 1985, supra), including the inhibition of activation of enzymes such as PLC_γl (Granja et al . , 1991, supra; Park et al . , 1992, supra), Raf (Cook &

McCormick, 1993, supra; Wu et al . , 1993, supra; Hafner et al., 1994, supra), PKC (Patel et al . , 1987, J. Biol. Chem., 262, p5831-5838; and Klausner et al . , 1987, J. Biol. Chem., 262, pl2654-12659) , and MAP kinases (Gravers et al . , 1993, supra; Tamir et al . , 1996, supra; Hsueh & Lai, 1995, J. Biol. Chem., 270, pl8094-18098 ; and Pillinger et al . , 1996, J. Biol. Chem., 271, pl2049- 12056) , as well as transcription factors (Chen & Rothenberg, 1994, J. Exp . Med. , 179, p931-942; and Tamir Sc Isakov, 1994, J. Immunol., 152, p3391-3397) . The findings reported herein add Vav to the list of potential PKA-regulated targets in lymphocytes and, moreover, imply that Vav is regulated by both PTKs and Ser/Thr kinases .

Example 2. Activation of Csk by cAMP-dependent protein kinase inhibits signaling through the T-cell receptor.

Introduction In order to elucidate the molecular mechanisms for the inhibitory effects of cAMP on TCR-mediated signaling, we examined the role of cAMP/PKA on the initial tyrosine phosphorylation following engagement of TCR/CD3. We demonstrate that activation of PKA strongly reduces TCR/CD3-induced tyrosine phosphorylation of ζ- chain and Zap-70, and that this effect is due to PKA- mediated phosphorylation and activation of Csk with subsequent inactivation of Lck.

MATERIALS AND METHODS

Cell culture, stimulation and transfection. The human leukemic T cell line Jurkat, Jurkat T-Ag, a derivate of the Jurkat cell line stably transfected with the SV40 large T antigen (Clipstone & Crabtree, 1992, Nature, 357, p695-697) , and the Lck-deficient JCaMl cell line (Straus and Weiss, 1992, Cell, 70, p585-593) were kept in logarithmic growth in RPMI 1640 medium supplemented with 10% fetal bovine serum, 25 μg/ml gentamycin, 1 mM sodium pyruvate, non essential amino acids (BioWhittaker, Walkersville, Maryland) , and monothioglycerol (Sigma, St. Louis, MO) at 37°C in an humidified atmosphere containing 5% C0₂. For activation of PKA, cells were incubated with 50-500 μM 8- (4- chlorophenylthio) -adenosine 3' :5' -cyclic mono phosphate (8-CPT cAMP, Sigma) for 20 or 30 min prior to T cell activation by addition of 5-10 μg/ml of anti-CD3 ε mAb OKT 3. For transfections, Jurkat T-Ag cells (2 x 10⁷) in 0.4 ml serum free RPMI 1640 were layered on top of 2-10 μg of each DNA construct in electroporation cuvettes with 0.4 cm electrode gap (BioRad) and subjected to an electric field of 240V/cm with 960 μF capacitance. The cells were expanded in supplemented medium and harvested after 48 hours.

Immunoprecipitations . Immunoprecipitation of Zap-70, Lck and Csk was as described before (Couture et al . , 1994, Proc. Natl. Acad. Sci. U.S.A., 91, p5301-5305) . For immunoprecipitation of HA-tagged Csk, transfected cells were washed twice in ice cold PBS and disrupted in lysis buffer (50 mM Tris, 2 mM EDTA, 2 mM DTT, 0.5 % Nonidet P-40, 1 mM Na₃V0₄ , 50 mM NaF, 10 μg/ml leupeptin, 10 μg/ml antipain, 10 μg/ml pepstatin A and 10 μg/ml chymostatin, pH 7.4) . Cell lysates were precleared by incubation with 50 μl of protein G-Sepharose (Sigma) for 1 hour at 4°C, and subjected to immunoprecipitation with 20 μg anti-HA mAb (Babco, Richmond, CA) to immunoprecipitate HA-tagged Csk. After overnight incubation at 4°C, 40 μl of protein G-Sepharose was added, and the incubation continued for 2 hours. Immune complexes were washed once in lysis buffer, twice in lysis buffer with 150 mM NaCl, and twice in Csk kinase assay buffer (50 mM Hepes, 5 mM MgCl₂, pH 7.4), resuspended in 160 μl Csk kinase assay buffer and split into four samples; one for Western blot analysis and three for Csk kinase activity.

SDS-polyacrylamide gel electrophoresis and Western blot analysis. Immune complexes were suspended in SDS sample buffer and boiled for 3 min. Proteins were resolved by SDS-polyacrylamide gel electrophoresis using 10% separating gels and transferred onto immobilon-P membranes (Millipore, Bedford, MA) . Detection of phosphotyrosine was accomplished by blocking membranes in 5% bovine serum albumin (BSA) and incubating with anti-PTyr mAb (4G10, Upstate Biotechnology, Lake Placid, NY) . Immunoblotting with anti-Zap-70 and anti-Lck antibodies was as before (Couture et al . , 1994, supra) . For immunoblotting with anti-HA (Babco) and anti-Cγ (sc 905, Santa Cruz) , membranes were blocked in 5% dry milk, incubated with the indicated antibodies and developed by enhanced chemiluminescense technique (ECL, Pierce,

Rockford, IL) . Anti-Cγ antibody was demonstrated to be reactive with all human C subunits (Cα, Cβ, Cy, using recombinant proteins, K. Tasken, unpublished data) . Zap-70 (SH2)₂ precipitations of tyrosine phosphorylated TCR ζ-chain. As described previously (Tailor et al . , 1996, supra) .

Plasmid constructs. Constructs directing the expression of Lck wild type and Lck-Y505F have been described previously (Couture et al . , 1996, J. Biol. Chem., 271, p24880-24884) . The gene encoding the human Csk was subcloned from pCRII (Invitrogen, Leek, Netherlands) into the expression vector pEF/HA at Nhel-Xbal sites. Csk-S364A and Csk-S364C mutants were made by PCR or using a site directed mutagenesis kit (Quick Change, Stratagene, La Jolla, CA) , primers 5'-

TGAGAGAGAAGAAATTCTCCACTAAGTCTG-3 ' and 5 ' - CAGACTTAGTGGAGAATTTCTTCTCTCTCA-3 ' , covering nucleotides 1485 to 1514 on both strands of the published human Csk sequence with mutations of interest introduced, and pCRIICsk wild type as a template. Mutations were verified by sequencing. A 350-bp Bsu36I-XbaI fragment from each of these mutants were substituted with the corresponding fragment in pEF/HA Csk wt to generate mutated constructs that were used for transfection. The open reading frame of human Cβ was subcloned from pCRBlunt (Invitrogen) into an EcoRI-site in pEFneo. Expression of recombinant enzymes. Cloning, expression and purification of human Csk has been reported previously (Vang et al . , 1998, supra). The full-length open reading frame of human Csk was cloned by reverse transcriptase PCR from human T-cell RNA, using primers A (5' -GGATC CATGT CAGCA ATACA GGCCG C-3 ' , upper primer) and B (5 ' -TCTAG AGTCC ATGAT CTTCC AGGTT C-3', lower primer) . Underlined sequences represent non-homologous sequences added to generate convenient cloning sites. The PCR product was sequenced to ascertain absence of PCR-generated mutations, sub-cloned into the pGEX-KG expression vector (a gift from J.E. Dixon, Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI) and expressed in E. coli BL21 as a fusion protein with glutathione-S-transferase (GST) using methods described previously (Tasken et al . , 1993, J. Biol. Chem., 268, p21276-21283 and Solberg et al . , 1994, Exp. Cell Res., 214, p595-605) . The fusion protein was purified on glutathione-agarose beads and Csk was isolated by cleavage from the GST with thrombin as described previously. Milligram amounts of expressed, purified proteins were stored for several months with no loss of activity at -20°C in 20mM Tris- HC1, pH 7.4, ImM dithiothreithol , ImM EDTA, 50% glycerol . This yielded an enzyme with a specific activity in the range of that of the native purified enzyme [217 + 38 and 105 pmol/μg Csk/min, respectively, both assayed with Mn²⁺ (2 mM) as divalent cation (Vang et al., 1998, supra; Okada & Nakagawa, 1989, J. Biol. Chem., 264, p20886-20893 ) . Recombinant purified Cα was a kind gift from Friedrich Heiberg (Herberg & Taylor, 1993, Biochemistry, 32, pl4015-14022) . GST-Cβ fusion protein was prepared as follows: Human Cβ was amplified from cloned cDNA (Beebe et al . , 1992, J. Biol. Chem., 267, p25505-25512) by PCR with primers A (5 ' -GGATC CATGG GGAAC GCGGC GACC-3 ' , upper primer) and B (5 ' -TCTAG ACACG GATGA TGGCA ATAAA GACCT-3', lower primer) in order to generate a full-length ORF with convenient cloning sites

(underlined nucleotides) , and subcloned, expressed and purified as described above for Csk. Milligram amounts of expressed, purified proteins were stored for several months with no loss of activity at -20°C in 20 mM Tris- HCl, pH 7.4, 1 mM dithiothreithol , 1 mM EDTA, 50% glycerol . The activities of the different batches of C subunit were determined using a standard Kemptide kinase assay (Roskoski, 1983, Methods Enzymol., 99, p3-6). Phosphorylation of Csk. Csk was incubated with different C subunits of PKA at 30°C for the indicated time periods in 50 mM Hepes, pH 7.4,5 mM MgCl₂, 3 to 5 μM ATP, and 5 μCi y- [³²P] -ATP (50-320 Ci/mmol) . All reactions were stopped by boiling samples in SDS-sample buffer, followed by SDS-PAGE. Gels were stained with Coomassie brilliant blue, dried and subjected to autoradiography.

In vitro tyrosine kinase assays. The tyrosine kinase activity of human Csk was measured as incorporation of phosphate into the synthetic polyamino acid poly (Glu, Tyr) 4:1 (Sigma), abbreviated poly(E;Y). A standard protocol was followed with reaction volumes of 50 μl containing Hepes buffer pH 7.4 , 5 mM MgCl₂, 200 μM ATP, 1.5 μCi γ~ [³²P] -ATP, 200 μg/ml poly(E;Y), and different amounts of purified Csk. Native or heat- inactivated (65°C, 10 min) C subunit and/or protein kinase inhibitor peptide (PKI 6-22 amide, Sigma, cat. no. P-6062) was added where indicated. The incubation temperature was 30°C, and the incubation times were as indicated in the figure legends. At the end of the incubations, aliquots (35 μl) of each reaction mixture were withdrawn and spotted onto 10 x 35 mm strips of Whatman 3MM chromatography paper, which were washed as described (Bougeret et al . , 1993, Oncogene, 8, pl241- 1247) and counted with 4 ml Opti-Fluor (Packard) scintillation fluid in a Liquid Scintillation Counter. Activity in the absence of poly(E;Y) (generally less than 4%) was subtracted from all samples.

Phosphoamino acid analysis. Csk was phosphorylated by PKA for 30 min as indicated above and subjected to SDS- PAGE. The band corresponding to phosphorylated Csk was cut from the dried gel and subjected to partial acid hydrolysis in 6 M HCl at 110 °C for 2 h. The acid was evaporated under vacuum and the hydrolysed sample was dissolved in 30 μl H₂0. Ten μl of sample (approx. 1000 cpm of [³²P] ) was separated in two dimensions as described, together with 10 μg each of PSer, PThr and PTyr. Phosphoamino acid standards were stained with ninhydrin and [³²P] -labeled amino acids were detected by autoradiography using Hyperfilm MP (Amersham) and SuperRapid intensifying screens (Kodak Eastman, Rochester, NY) (exposure 24-48 h at -70°C) . Protein measurements. Proteins were quantified by the method of Bradford (Bradford, 1976, Anal. Biochem., 72, p248-254) , using BSA as a standard.

Tryptic peptide mapping. As described previously (von Willebrand et al . , 1998, J. Biol. Chem., 273, p3994- 4000) . RESULTS

To assess the effect of cAMP through protein kinase A type I in the proximal signaling from the T cell antigen receptor, we examined the effect of cAMP on anti-CD3 -induced tyrosine phosphorylation of cellular proteins. Figure 9 shows the tyrosine phosphorylation of Zap-70 and TCR-ζ after addition of anti-CD3 (OKT3) to untreated Jurkat T cells (lanes 1-6, upper and middle panels, respectively) or to cells pretreated with the cAMP analog 8-CPT-cAMP (lanes 7-12) . As can be seen from the figure, there was a clear reduction in tyrosine phosphorylation of both Zap-70 and ζ-chain when cells were pretreated with 8-CPT-cAMP. In addition, the tyrosine phosphorylation of both Zap-70 and TCR-ζ were delayed in cells pretreated with 8-CPT-cAMP (compare lanes 3 and 9) . The amount of immunoreactive Zap-70 was the same in all the lanes and indicates equal levels of immunoprecipitated Zap-70 (lower panel) .

We next examined the activity of Lck and the C- terminal Src kinase (Csk) that regulates Lck. Untreated and 8-CPT-cAMP-treated Jurkat T cells were subjected to immunoprecipitation with antibodies to Csk and Lck, and their phosphotransferase activities were assayed (Figure 10) . The tyrosine kinase activity of immunoprecipitated Csk was increased 2-3 fold in cells treated with 8-CPT- cAMP, while a 50 % reduction in the activity of Lck was observed. Similar amounts of Lck were present in the assays, as verified by the anti-Lck immunoblotting (data not shown ) . Csk co-migrated with IgG heavy chain in immunoblotting as a doublet band and the amount of Csk appeared equal in all immunoprecipitates (data not shown) . These results were obtained in several independent experiments .

Next, we examined if the observed changes in the catalytic activities of Lck and Csk could account for the cAMP-mediated inhibition of ζ-chain phosphorylation. No direct down-regulation of Lck or Fyn activity by PKA could be demonstrated (data not shown) . For this reason, we examined the possibility that PKA regulates Lck through Csk. JCaMl cells which lack Lck, were transfected with wild-type Lck or Lck-Y505F, mutated at the Csk-phosphorylation site and therefore not inactivated by Csk. As expected, Lck reconstituted TCR- induced signaling in these cells as evidenced by OKT-3 induced ζ-chain phosphorylation. Cells transfected with wild-type Lck showed a distinct reduction in 0KT3- induced phosphorylation of TCR- ζ-chain when pretreated with 8-CPT-cAMP (upper panel, lanes 6 versus 8). In contrast, cells transfected with Lck-Y505F revealed higher levels of ζ-chain phosphorylation in the absence of 0KT3 -stimulation (lanes 9 and 11) and, importantly, 0KT3-induced ζ-chain phosphorylation was not inhibited by pretreatment with 8-CPT-cAMP (lane 10 versus lane 12) . The anti-Lck reactive band at -50 kDa in the lower panel of Figure 11 is the catalytically inactive endogenous truncated Lck present in JCaMl cells. We conclude that Y505 of Lck is required for cAMP-mediated inhibition of TCR- ζ -phosphorylation. We next examined the possibility of direct phosphorylation-dependent regulation of Csk by PKA. For this purpose, we used a fully active and well characterized human Csk expressed in E. coli (Vang et al . , 1998, supra) in an in vi tro experiment. We found that recombinant Csk was readily phosphorylated by the Cα catalytic subunit of PKA, as shown in Figure 12A (lane 1, arrow) , whereas no phosphorylation of Csk was detected when incubated with heat-inactivated (65°C, 10 min) Cα (lane 2) . Under optimal conditions this phosphorylation reached 0.50 mol phosphate incorporated per mol Csk (data not shown) . A very low-level autophosphorylation of Csk on tyrosine was also observed, but could only be seen after prolonged exposures. Figure 12B depicts the time-dependent phosphorylation of Csk mediated by PKA Cβ subunit. A time-dependent autophosphorylation of the Cβ fusion protein was also observed. Phosphoamino acid analysis of Csk phosphorylated by GST-Cβ demonstrated strong labeling on phosphoserine (Figure 12C) . Identical results were obtained when Csk was phosphorylated with purified bovine C subunit of PKA (data not shown) . Next, the effect of the PKA-mediated phosphorylation on the catalytic activity of Csk was examined (Figure 13) . Coincubation of recombinant Csk with recombinant catalytic subunit of PKA (Cα) more than doubled the Csk-catalyzed tyrosine phosphorylation of poly (E;Y) compared to Csk incubated alone (Figure 13A, bar 2 versus bar 1) . This effect was not seen with heat -inactivated Cα . Furthermore, the increase in Csk activity in the presence of native Cα was strongly reduced by addition of PKI (protein kinase inhibitor) , a specific inhibitor of PKA (bar 4) . Figure 13B shows time kinetics of Csk activity in the presence of either native or heat-inactivated Cα. In the presence of heat- inactivated Cα, Csk activity was linear the first 10 minutes and then declined. The curve of accumulated activity was much steeper in the presence of native Cα, and activities were approximately two-fold higher at each time point. The decrease in Csk activity by time was smaller and appeared later, perhaps due to ongoing PKA-mediated phosphorylation and activation of Csk compensating for the decline in Csk activity by time. PKA-mediated activation of Csk also depended on the stoichiometry of phosphorylation, as inferred from Figure 13C, where increasing concentrations of C subunit led to a saturable increase in activation of Csk reaching a maximum around a 2-fold molar excess of C- subunit over Csk. Incubation of increasing concentrations of Csk in the presence of a constant amount of recombinant Cα led to a concentration dependent increase in phosphotransfer which was approximately 2-fold higher at all concentrations compared to the activity of unphosphorylated Csk (incubated in the presence of heat-inactivated Cα) (Figure 13D) .

We next mapped the PKA phosphorylation site in Csk. Tryptic peptide mapping of Csk phosphorylated by PKA revealed two major radioactive spots both of which contained PSer. The human Csk amino acid sequence contains one putative phosphorylation site that fits the motif preferred by PKA, at amino acids 361-364 in the sequence KKFS. Another site at the activation loop of Csk in the sequence KEASST (amino acids 336-341) , could also potentially be phosphorylated by PKA, although not fully consistent with the motif preferred by PKA. This last region is often the site of kinase activation by autophosphorylation (Mustelin & Burn, 1993, Trends Biochem. Sci., 18, p215-220) or by transphosphorylation by another kinase, for example MAP kinase activation by Mek (Cobb et al . , 1994, Cell. Mol. Biol. Res., 40, p253- 256) . Two Csk mutants, S364A and S339A/S340A/T341A, were generated, expressed in E. coli and purified. The S364A mutant was only weakly phosphorylated by PKA (Figure 14 and data not shown) , and when subjected to tryptic peptide mapping, both major peptides were missing compared to wild-type Csk phosphorylated by PKA (Figure 14) . The observation that two phosphorylated peptides disappeared by mutation of a single residue is probably due to partial proteolysis. The extent of PKA- mediated phosphorylation of the S339A/S340A/T341A mutant was comparable to that of wild type Csk. Furthermore, its tryptic peptide map was identical to that of wild- type, and we also observed a similar 2-fold PKA-mediated increase in the kinase activity of this mutant (data not shown) .

The effect of PKA on wild type and mutant Csk activity in T cells, was examined by cotransfection of constructs directing the expression of HA-tagged Csk and Cβ subunit of PKA. When Csk wild type was cotransfected with PKA, a 1.8 fold increase in activity was observed compared to Jurkat TAg T cells transfected with the Csk construct together with a vector with Cβ in the reverse orientation (Figure 15A) . The Csk-S364A mutant was not catalytically active when expressed and purified from E. coli , nor was it active when transfected into Jurkat TAg cells. Another mutant Csk-S364C was catalytically active when expressed in Jurkat TAg cells (Figure 15B) . Perhaps Cys, but not Ala, in position 364 permits a normal folding of Csk. However, in contrast to the 1.8- fold increase in activity of wild-type Csk by treatment of Jurkat T cells with cAMP, the activity of the mutant Csk-S364C enzyme was not affected by cAMP (Figure 15B) . In contrast, cotransfection of PKA with mutant Csk-S364C had no effect on the activity compared to control transfected cells (Figure 15B) . DISCUSSION

The molecular mechanisms for cAMP-dependent inhibition of T-cell antigen receptor signaling has so far not been elucidated. We demonstrate that PKA- dependent phosphorylation on S364 of Csk leads to an increased activity of Csk. Next, this PKA-mediated up- regulation of Csk activity leads to a significantly reduced Lck activity following T cell activation, and almost abolishes Lck-mediated ζ-chain phosphorylation which in turn will inhibit recruitment of Zap-70 to TCR/CD3.

Cyclic AMP and PKA will have downstream targets involved in cell-cycle regulation and common to a number of cell types including lymphocytes, e . g. the PKA- dependent regulation of the Ras-Raf interaction and PKA- mediated regulation of PLC-γl/2 (Cook & McCormick, 1993, Science, 262, pl069-1072; andGraves et al . , 1993, Proc. Natl. Acad. Sci. U.S.A., 90, pl0300-10304) . However, several lines of evidence indicate that inhibition of lymphocyte activation by cAMP and PKA, at least in part, involves targets in the proximal signaling pathways from antigen receptors. We have previously shown that cAMP- dependent inhibition of lymphocyte proliferation is dependent on PKA type I both in T cells and B cells and that PKA type I is redistributed to and colocalized with antigen receptors in these cells (Skalhegg et al . , 1992, supra; Skalhegg et al . , 1994, Science, 263, p84-87; Levy et al., 1996, Eur. J. Immunol., 26, pl290-1296) . This indicates targets for PKA type I in the proximal signaling events in B and T cells. Furthermore, cAMP- dependent inhibition of the acute cytotoxicity of NK cells is dependent on PKA type I (Torgersen et al . , 1997, J. Biol. Chem., 272, p5495-5500). In addition, it has been demonstrated that when T or B cells are stimulated to proliferation by direct activation of PKC using a combination of phorbol ester and calcium ionophore, lymphocyte proliferation is no longer sensitive to inhibition by cAMP (Skalhegg et al . , 1994, Scand. J. Immunol., 40, p201-208; and Whisler et al . , 1992, Cell. Immunol., 142, p398-415) . This indicates that major targets for PKA-mediated inhibition of lymphocyte activation are located upstream of PKC activation and is further supported by the fact that NK cell cytotoxicity becomes insensitive to inhibition by cAMP when examined in the presence of phorbol ester and calcium ionophore (Torgersen et al . , 1997, supra) . Finally, previous observations indicate that cAMP has inhibitory effects on the rapid tyrosine phosphorylation following T cell activation (Skalhegg et al . , 1994, Scand. Immunol., 40, p201-208) .

We examined the acute effects of cAMP on the initial tyrosine phosphorylation following T cell activation and show that cAMP has a distinct effect on activation-dependent tyrosine phosphorylation both of Zap-70 and TCR-ζ, and in the case of ζ-chain phosphorylation, this is almost abolished by incubation with cAMP. The cAMP-mediated inhibition of phosphorylation of ζ-chain and Zap-70 is explained by the observed reduction in Lck activity. To our knowledge no report has demonstrated any effect of PKA on Lck. One report has examined Src and showed a change in K_m towards an artificial substrate for Src following coincubation with PKA (Budde, 1993, J. Biol. Chem., 268, p24868-24872) , whereas two other reports have not been able to demonstrate any regulation of Src by PKA (Hirota et al., 1988, Mol. Cell Biol., 8, pl826-1830; and Yaciuk et al., 1989, Mol. Cell, 9, p2453-2463).

However, we have not been able to demonstrate any phosphorylation of Lck by PKA or direct PKA-induced changes in Lck activity. For this reason we explored the possibility that PKA could regulate Lck through Csk, and demonstrate that whereas T cell activation in Lck- deficient JCaMl cells was reconstituted by transfection with wild type Lck and clearly inhibited by cAMP, mutant Lck-Y505F also restored T cell activation, but was completely insensitive to the effect of cAMP . This shows clearly that the effect of cAMP is mediated through regulation of Csk activity, and is supported by the observation of increased activity in Csk immunoprecipitates from cAMP-treated cells. Biochemical studies on recombinant Csk demonstrated a specific Csk phosphorylation on S364 which was accompanied by a twofold increase in Csk activity as analyzed by kinetic studies . Phosphate incorporated into Csk following incubation with PKA was maximally 0.5 mol per mol Csk, such phosphate to protein ratios are common for phosphorylation of recombinant proteins produced in bacteria. This may indicate that the magnitude of regulation in Csk activity may be higher when fully phosphorylated.

Sun et al recently reported phosphorylation and inactivation of Csk by PKA in vi tro (Sun et al . , 1997, Arch. Biochem. Biophys., 343, pl94-200) . They did not map the phosphorylation site, and their data may be obscured by the non-physiological phosphorylation of several sites. Furthermore, their observations indicate a regulation of Csk which is opposite from our observations. In that study a 40-fold molar excess of PKA over Csk was used, whereas a 2-fold molar excess of PKA was employed in the present study. Furthermore, time-dependent incorporation of phosphate into Csk in that study does not correspond to the time-dependent decrease in Csk activity. In the present study mapping of the phosphorylation site to S364 and transfection of HA-tagged wild-type and mutant Csk-S364C clearly demonstrates a 2-fold increase in Csk activity when cotransfected with PKA whereas no such increase was detected in mutant Csk-S364C.

Csk is present in all human cells (Partanen et al . , 1991, supra) as a key regulator of Src kinases. The fact that the presence of Y505 in Lck is essential for the inhibitory effect of cAMP on ζ-chain phosphorylation indicate that the PKA-mediated phosphorylation of Csk may be a major mechanism by which cAMP inhibits TCR- mediated T cell activation. We have recently reported that the T cell dysfunction in HIV can be reversed by inhibition of the increased activity of PKA type I (Aandahl et al . , 1998, supra), indicating that immunomodulation through cAMP/PKA contributes to the pathogenesis of this immunodeficiency. Inhibition of Lck through activation of Csk may provide a molecular mechanism for this effect. Furthermore, a recent report demonstrates that suppression of T cell IL-2 production and proliferation in rats with sepsis is associated with a decrease in Fyn activity and elevated levels of prostaglandin E₂ (PGE₂) (Choudhry et al . , 1998, J. Immunol., 160, p929-935) . The sepsis or PGE₂-related suppression of Fyn kinase activity was prevented by treatment with the cyclooxygenase inhibitor, indomethacin, indicating that the suppression is mediated by PGE₂. We have also obtained results which show that PGE₂ causes activation of Csk. PGE₂ is produced by T cells, and in large amounts by monocytes and macrophages and could thus be involved in affecting lymphocyte dysfunction as well as by TCR/CD3 triggering. PKA-regulation of Csk activity provides a molecular explanation for these observations since PGE₂ through cAMP activates PKA and Fyn is controlled by Csk. In addition, various members of the Src kinase family are implicated in the proximal signaling following activation of B cells through the B cell antigen receptor or activation of NK cells through specific receptors (Burkhardt et al . , 1991, Proc. Natl. Acad. Sci. U.S.A. 88, p7410-7414; Yamanashi et al . , 1992,

Proc. Natl. Acad. Sci. U.S.A., 89, plll8-1122; Campbell & Sefton, 1992, Mol. Cell Biol., 12, p2315-2321; Azzoni et al., 1992, J. Exp . Med. , 176, pl745-1750; Bottino et al., 1994, Eur. J. Immunol., 24, p2527-2534; and Cerny et al., 1997, Immunogenetics , 46, p231-236). Cyclic

AMP-mediated regulation of Src family kinase activities by regulation of Csk provide a molecular mechanism for regulation of both B cell activation and NK cell activation. Finally, Csk and Src kinases are expressed in a number of other tissues including neuronal tissues (Bolen, 1993, Oncogene, 8, p2025-2031) , and the impact of cAMP-regulation of Csk in these tissues will be interesting to pursue. The PKA phosphorylation site in Csk is conserved between mammalian species which indicates that the site for regulation by PKA may have been subject to selection pressure. In addition to Csk, the family of C-terminal Src kinases consists of other kinases like Csk homologous kinase (Chk/Lsk/Hyl/Matk) and Csk-type protein kinase (Ctk/Bhk/Ntk) (Grgurevich et al., 1997, Growth Factors, 14, pl03-115; and Ershler et al., 1995, FEBS Lett., 375, p50-52). Homology alignment of these kinases demonstrates that the PKA phosphorylation site in Csk (KKFS) is partially conserved in these other kinases as either RFS or KFT. These sequences are atypical PKA sites, and may be phosphorylated by PKA.

In conclusion, we report the mapping of a PKA phosphorylation site on Csk and regulation of Csk activity by cAMP/PKA. PKA-mediated activation of Csk provides a molecular mechanism for cAMP-dependent inhibition of lymphocyte activation and Csk-S364 and/or Lck-Y505F offer targets for immunomodulating therapies.

Claims

Claims :

1. A method of altering the activity of the PKA signaling pathway in a cell wherein the extent of phosphorylation of one or more PKA substrates, or kinase substrates downstream in the PKA signaling pathway, is altered.

2. A method as claimed in claim 1 wherein said cell is a T lymphocyte.

3. A method as claimed in claim 1 or 2 wherein said PKA enzyme is PKA Type I .

4. A method as claimed in claim 1 to 3 wherein the extent of phosphorylation is altered by manipulation of the wild-type gene endogenously encoding PKA or the downstream kinase or by manipulation of expression of said gene or by manipulation of the expressed product.

5. A method as claimed in any one of claims 1 to 4 wherein the extent of phosphorylation is altered by modifying the activity of PKA or a downstream kinase.

6. A method as claimed in any one of claims 1 to 4 wherein the extent of phosphorylation is altered by introducing a modified, preferably mutated PKA substrate, downstream kinase substrate, or fragment, precursor or functionally equivalent variant thereof, into said cell.

7. A method as claimed in claim 6 wherein said modified PKA substrate, downstream kinase substrate, or fragment, precursor or functionally equivalent variant thereof, is introduced into said cell by manipulation of the wild-type gene endogenously encoding said substrate or fragment, precursor or functionally equivalent variant thereof, by manipulation of expression of said gene or by manipulation of the expressed product.

8. A method as claimed in claim 6 wherein said modified PKA substrate, downstream kinase substrate, or fragment, precursor or functionally equivalent variant thereof, is introduced into said cell by the introduction of an exogenous nucleic acid molecule containing a sequence encoding said modified substrate, or fragment, precursor or functionally equivalent variant thereof, or an exogenous peptide or protein containing an amino acid sequence corresponding to said modified substrate, or fragment, precursor or functionally equivalent variant thereof.

9. A method as claimed in any one of claims 1 to 8 wherein the substrate is a direct PKA substrate.

10. A method as claimed in claim 9 wherein the substrate is in the Csk-family, preferably Csk, Chk, Lsk, Hyl, Matk, Ctk, Bhk or Ntk, or is a fragment, precursor or functionally equivalent variant thereof.

11. A method as claimed in claim 10 wherein the substrate is encoded by a nucleic acid molecule comprising the sequence:

1 tccggggcgg cccccggcag ccagcgcgac gttccaaaat cgaacctcag

51 tggcggcgct cggaagcgga actctgccgg ggccgcgccg gctacattgt 101 ttcctccccc cgactccctc ccgccccctt cccccgcctt tcttccctcc

151 gcgacccggg ccgtgcgtcc gtccccctgc ctctgcctgg cggtccctcc

201 tcccctctcc ttgcacccat acctctttgt accgcacccc ctggggaccc

251 ctgcgcccct cccctccccc ctgaccgcat ggaccgtccc gcaggccgct

301 gatgccgccc gcggcgaggt ggcccggacc gcagtgcccc aagagagctc 351 taatggtacc aagtgacagg ttggctttac tgtgactcgg ggacgccaga

401 gctcctgaga agatgtcagc aatacaggcc gcctggccat ccggtacaga

451 atgtattgcc aagtacaact tccacggcac tgccgagcag gacctgccct 501 tctgcaaagg agacgtgctc accattgtgg ccgtcaccaa ggaccccaac

551 tggtacaaag ccaaaaacaa ggtgggccgt gagggcatca tcccagccaa

601 ctacgtccag aagcgggagg gcgtgaaggc gggtaccaaa ctcagcctca

651 tgccttggtt ccacggcaag atcacacggg agcaggctga gcggcttctg 701 tacccgccgg agacaggcct gttcctggtg cgggagagca ccaactaccc

751 cggagactac acgctgtgcg tgagctgcga cggcaaggtg gagcactacc

801 gcatcatgta ccatgccagc aagctcagca tcgacgagga ggtgtacttt

851 gagaacctca tgcagctggt ggagcactac acctcagacg cagatggact

901 ctgtacgcgc ctcattaaac caaaggtcat ggagggcaca gtggcggccc 951 aggatgagtt ctaccgcagc ggctgggccc tgaacatgaa ggagctgaag

1001 ctgctgcaga ccatcgggaa gggggagttc ggagacgtga tgctgggcga

1051 ttaccgaggg aacaaagtcg ccgtcaagtg cattaagaac gacgccactg

1101 cccaggcctt cctggctgaa gcctcagtca tgacgcaact gcggcatagc

1151 aacctggtgc agctcctggg cgtgatcgtg gaggagaagg gcgggctcta 1201 catcgtcact gagtacatgg ccaaggggag ccttgtggac tacctgcggt

1251 ctaggggtcg gtcagtgctg ggcggagact gtctcctcaa gttctcgcta

1301 gatgtctgcg aggccatgga atacctggag ggcaacaatt tcgtgcatcg

1351 agacctggct gcccgcaatg tgctggtgtc tgaggacaac gtggccaagg

1401 tcagcgactt tggtctcacc aaggaggcgt ccagcaccca ggacacgggc 1451 aagctgccag tcaagtggac agcccctgag gccctgagag agaagaaatt

1501 ctccactaag tctgacgtgt ggagtttcgg aatccttctc tgggaaatct

1551 actcctttgg gcgagtgcct tatccaagaa ttcccctgaa ggacgtcgtc

1601 cctcgggtgg agaagggcta caagatggat gcccccgacg gctgcccgcc

1651 cgcagtctat gaagtcatga agaactgctg gcacctggac gccgccatgc 1701 ggccctcctt cctacagctc cgagagcagc ttgagcacat caaaacccac

1751 gagctgcacc tgtgacggct ggcctccgcc tgggtcatgg gcctgtgggg

1801 actgaacctg gaagatcatg gacctggtgc ccctgctcac tgggcccgag

1851 cctgaactga gccccagcgg gctggcgggc ctttttcctg cgtcccagcc

1901 tgcacccctc cggccccgtc tctcttggac ccacctgtgg ggcctgggga 1951 gcccactgag gggccaggga ggaaggaggc cacggagcgg gcggcagcgc

2001 cccaccacgt cgggcttccc tggcctcccg ccactcgcct tcttagagtt

2051 ttattccttt ccttttttga gatttttttt ccgtgtgttt attttttatt

2101 atttttcaag ataaggagaa agaaagtacc cagcaaatgg gcattttaca

2151 agaagtacga atcttatttt tcctgtcctg cccgtgaggt gggggggacc 2201 gggcccctct ctagggaccc ctcgccccag cctcattccc cattctgtgt

2251 cccatgtccc gtgtctcctc ggtcgccccg tgtttgcgct tgaccatgtt

2301 gcactgtttg catgcgcccg aggcagacgt ctgtcagggg cttggatttc 2351 gtgtgccgct gccacccgcc cacccgcctt gtgagatgga atcgtaataa 2401 accacgccat gaggaaaaaa

or a sequence which hybridizes to said sequence under binding conditions of 6 x SSC/50% formamide at room temperature and washing at 2 x SSC, 65┬░C, where SSC = 0.15 M NaCl, 0.015M sodium citrate, pH 7.2 , or a sequence which exhibits at least 80% sequence homology or a sequence complementary to any of the aforesaid sequences, or a fragment of any of the aforesaid sequences containing the region encoding or complementary to at least the PKA phosphorylation site.

12. A method as claimed in claim 10 wherein the substrate comprises the amino acid sequence :

1 Met Ser Ala He Gin Ala Ala Trp Pro Ser Gly Thr Glu 14 Cys He Ala Lys Tyr Asn Phe His Gly Thr Ala Glu Gin

27 Asp Leu Pro Phe Cys Lys Gly Asp Val Leu Thr He Val 40 Ala Val Thr Lys Asp Pro Asn Trp Tyr Lys Ala Lys Asn

53 Lys Val Gly Arg Glu Gly He He Pro Ala Asn Tyr Val

66 Gin Lys Arg Glu Gly Val Lys Ala Gly Thr Lys Leu Ser

79 Leu Met Pro Trp Phe His Gly Lys He Thr Arg Glu Gin

92 Ala Glu Arg Leu Leu Tyr Pro Pro Glu Thr Gly Leu Phe 105 Leu Val Arg Glu Ser Thr Asn Tyr Pro Gly Asp Tyr Thr

118 Leu Cys Val Ser Cys Asp Gly Lys Val Glu His Tyr Arg

131 He Met Tyr His Ala Ser Lys Leu Ser He Asp Glu Glu

144 Val Tyr Phe Glu Asn Leu Met Gin Leu Val Glu His Tyr

157 Thr Ser Asp Ala Asp Gly Leu Cys Thr Arg Leu He Lys 170 Pro Lys Val Met Glu Gly Thr Val Ala Ala Gin Asp Glu

183 Phe Tyr Arg Ser Gly Trp Ala Leu Asn Met Lys Glu Leu

196 Lys Leu Leu Gin Thr He Gly Lys Gly Glu Phe Gly Asp

209 Val Met Leu Gly Asp Tyr Arg Gly Asn Lys Val Ala Val

222 Lys Cys He Lys Asn Asp Ala Thr Ala Gin Ala Phe Leu 235 Ala Glu Ala Ser Val Met Thr Gin Leu Arg His Ser Asn

248 Leu Val Gin Leu Leu Gly Val He Val Glu Glu Lys Gly

261 Gly Leu Tyr He Val Thr Glu Tyr Met Ala Lys Gly Ser 274 Leu Val Asp Tyr Leu Arg Ser Arg Gly Arg Ser Val Leu 287 Gly Gly Asp Cys Leu Leu Lys Phe Ser Leu Asp Val Cys 300 Glu Ala Met Glu Tyr Leu Glu Gly Asn Asn Phe Val His 313 Arg Asp Leu Ala Ala Arg Asn Val Leu Val Ser Glu Asp 326 Asn Val Ala Lys Val Ser Asp Phe Gly Leu Thr Lys Glu 339 Ala Ser Ser Thr Gin Asp Thr Gly Lys Leu Pro Val Lys 352 Trp Thr Ala Pro Glu Ala Leu Arg Glu Lys Lys Phe Ser 365 Thr Lys Ser Asp Val Trp Ser Phe Gly He Leu Leu Trp 378 Glu He Tyr Ser Phe Gly Arg Val Pro Tyr Pro Arg He 391 Pro Leu Lys Asp Val Val Pro Arg Val Glu Lys Gly Tyr 404 Lys Met Asp Ala Pro Asp Gly Cys Pro Pro Ala Val Tyr 417 Glu Val Met Lys Asn Cys Trp His Leu Asp Ala Ala Met 430 Arg Pro Ser Phe Leu Gin Leu Arg Glu Gin Leu Glu His 443 He Lys Thr His Glu Leu His Leu or a sequence which has more than 90% sequence homology thereto, or a fragment of any of the aforesaid sequences containing at least the PKA phosphorylation site.

13. A method as claimed in any one of claims 10 to 12 wherein the substrate is Csk, or a fragment, precursor or functionally equivalent variant thereof, in which one or more residues in at least one of the PKA phosphorylation sites in said substrate is modified, preferably at the residue which is phosphorylated by PKA.

14. A method as claimed in claim 13 in which the serine at position 364 in the human sequence, or corresponding position in another organism or precursor or variant, is altered to an alanine, cysteine, glutamic acid or aspartic acid residue.

15. A method as claimed in claim 9 wherein the substrate is in the Vav-family, preferably Vav, Vav2 , Vav-3, Vav-3 ╬▓, Vav transforming protein and Vav-2 oncogene, or is a fragment, precursor or functionally equivalent Variant thereof .

16. A method as claimed in claim 15 wherein the substrate is encoded by a nucleic acid molecule comprising the sequence :

1 actagctgtc gctccacagg cgagcagggc aggcgtgcgg gcgggtgggt

51 ggtggaggct gcgagggtgc acggccggcc ctgggcaggc ggtagccatg

101 gagctgtggc gccaatgcac ccactggctc atccagtgcc gggtgctgcc

151 gcccagccac cgcgtgacct gggatggggc tcaggtgtgt gaactggccc

201 aggccctccg ggatggtgtc cttctgtgtc agctgcttaa caacctgcta 251 ccccatgcca tcaacctgcg tgaggtcaac ctgcgccccc agatgtccca

301 gttcctgtgc cttaagaaca ttagaacctt cctgtccacc tgctgtgaga

351 agttcggcct caagcggagc gagctcttcg aagcctttga cctcttcgat

401 gtgcaggatt ttggcaaggt catctacacc ctgtctgctc tgtcctggac

451 cccgatcgcc cagaacaggg ggatcatgcc cttccccacc gaggaggaga 501 gtgtaggtga tgaagacatc tacagtggcc tgtccgacca gatcgacgac

551 acggtggagg aggatgagga cctgtatgac tgcgtggaga atgaggaggc

601 ggaaggcgac gagatctatg aggacctcat gcgctcggag cccgtgtcca

651 tgccgcccaa gatgacagag tatgacaagc gctgctgctg cctgcgggag

701 atccagcaga cggaggagaa gtacactgac acgctgggct ccatccagca 751 gcatttcttg aagcccctgc aacggttcct gaaacctcaa gacattgaga

801 tcatctttat caacattgag gacctgcttc gtgttcatac tcacttccta

851 aaggagatga aggaagccct gggcacccct ggcgcagcca atctctacca

901 ggtcttcatc aaatacaagg agaggttcct cgtctatggc cgctactgca

951 gccaggtgga gtcagccagc aaacacctgg accgtgtggc cgcagcccgg 1001 gaggacgtgc agatgaagct ggaggaatgt tctcagagag ccaacaacgg

1051 gaggttcacc ctgcgggacc tgctgatggt gcctatgcag cgagttctca

1101 aatatcacct ccttctccag gagctggtga aacacacgca ggaggcgatg

1151 gagaaggaga acctgcggct ggccctggat gccatgaggg acctggctca

1201 gtgcgtgaac gaggtcaagc gagacaacga gacactgcga cagatcacca 1251 atttccagct gtccattgag aacctggacc agtctctggc tcactatggc

1301 cggcccaaga tcgacgggga actcaagatc acctcggtgg aacggcgctc

1351 caagatggac aggtatgcct tcctgctcga caaagctcta ctcatctgta

1401 agcgcagggg agactcctat gacctcaagg actttgtaaa cctgcacagc

1451 ttccaggttc gggatgactc ttcaggagac cgagacaaca agaagtggag 1501 ccacatgttc ctcctgatcg aggaccaagg tgcccagggc tatgagctgt

1551 tcttcaagac aagagaattg aagaagaagt ggatggagca gtttgagatg

1601 gccatctcca acatctatcc ggagaatgcc accgccaacg ggcatgactt 1651 ccagatgttc tcctttgagg agaccacatc ctgcaaggcc tgtcagatgc

1701 tgcttagagg taccttctat cagggctacc gctgccatcg gtgccgggca

1751 tctgcacaca aggagtgtct ggggagggtc cctccatgtg gccgacatgg

1801 gcaagatttc ccaggaacta tgaagaagga caaactacat cgcagggctc 1851 aggacaaaaa gaggaatgag ctgggtctgc ccaagatgga ggtgtttcag

1901 gaatactacg ggcttcctcc accccctgga gccattggac cctttctacg

1951 gctcaaccct ggagacattg tggagctcac gaaggctgag gctgaacaga

2001 actggtggga gggcagaaat acatctacta atgaaattgg ctggtttcct

2051 tgtaacaggg tgaagcccta tgtccatggc cctcctcagg acctgtctgt 2101 tcatctctgg tacgcaggcc ccatggagcg ggcaggggca gagagcatcc

2151 tggccaaccg ctcggacggg actttcttgg tgcggcagag ggtgaaggat

2201 gcagcagaat ttgccatcag cattaaatat aacgtcgagg tcaagcacat

2251 taaaatcatg acagcagaag gactgtaccg gatcacagag aaaaaggctt

2301 tccgggggct tacggagctg gtggagtttt accagcagaa ctctctaaag 2351 gattgcttca agtctctgga caccaccttg cagttcccct tcaaggagcc

2401 tgaaaagaga accatcagca ggccagcagt gggaagcaca aagtattttg

2451 gcacagccaa agcccgctat gacttctgcg cccgagaccg atcagagctg

2501 tcgctcaagg agggtgacat catcaagatc cttaacaaga agggacagca

2551 aggctggtgg cgaggggaga tctatggccg ggttggctgg ttccctgcca 2601 actacgtgga ggaagattat tctgaatact gctgagccct ggtgccttgg

2651 cagagagacg agaaactcca ggctctgagc ccggcgtggg caggcagcgg

2701 agccaggggc tgtgacagct cccggcgggt ggagactttg ggatggactg

2751 gaggagcgca gcgtccagct ggcggtgctc ccgggatgtg ccctgacatg

2801 gttaatttat aacaccccga tttcctcttg ggtcccctca agcagacggg 2851 gctcaagggg gttacattta ataaaaggat gaagatgg

or a sequence which hybridizes to said sequence under binding conditions of 6 x SSC/50% formamide at room temperature and washing at 2 x SSC, 65┬░C, where SSC = 0.15 M NaCl, 0.015M sodium citrate, pH 7.2 , or a sequence which exhibits at least 80% sequence homology or a sequence complementary to any of the aforesaid sequences, or a fragment of any of the aforesaid sequences containing the region encoding or complementary to at least the PKA phosphorylation site.

17. A method as claimed in claim 15 wherein the substrate comprises the amino acid sequence:

1 melwrqcthw liqcrvlpps hrvtwdgaqv celaqalrdg vllcqllnnl

51 lphainlrev nlrpqmsqfl clknirtfls tccekfglkr selfeafdlf 101 dvqdfgkviy tlsalswtpi aqnrgimpfp teeesvgded iysglsdqid

151 dtveededly dcveneeaeg deiyedlmrs epvsmppkmt eydkrccclr

201 eiqqteekyt dtlgsiqqhf lkplqrflkp qdieiifini edllrvhthf

251 lkemkealgt pgaanlyqvf ikykerflvy grycsqvesa skhldrvaaa

301 redvqmklee csqranngrf tlrdllmvpm qrvlkyhlll qelvkhtqea 351 mekenlrlal damrdlaqcv nevkrdnetl rqitnfqlsi enldqslahy

401 grpkidgelk itsverrskm dryaflldka llickrrgds ydlkdfvnlh

451 sfqvrddssg drdnkkwshm flliedqgaq gyelffktre lkkkwmeqfe

501 aisniypen atanghdfqm fsfeettsck acqmllrgtf yqgyrchrcr

551 asahkeclgr vppcgrhgqd fpgtmkkdkl hrraqdkkrn elglpkmevf 601 qeyyglpppp gaigpflrln pgdiveltka eaeqnwwegr ntstneigwf

651 pcnrvkpyvh gppqdlsvhl wyagpmerag aesilanrsd gtflvrqrvk

701 daaefaisik ynvevkhiki mtaeglyrit ekkafrglte lvefyqqnsl

751 kdcfksldtt lqfpfkepek rtisrpavgs tkyfgtakar ydfcardrse

801 lslkegdiik ilnkkgqqgw wrgeiygrvg wfpanyveed yseyc

or a sequence which has more than 90% sequence homology thereto, or a fragment of any of the aforesaid sequences containing at least the PKA phosphorylation site.

18. A method as claimed in any one of claims 15 to 17 wherein the substrate is Vav, or a fragment, precursor or functionally equivalent variant thereof, in which one or more residues in at least one of the PKA phosphorylation sites in said substrate is modified, preferably at the residue which is phosphorylated by PKA.

19. A method as claimed in claim 18 in which the serine at position 440 in the human sequence, or corresponding position in another organism or precursor or variant, is altered to an alanine, cysteine, aspartic acid, glutamic acid or glycine residue.

20. A method as claimed in any one of claims 1 to 8 wherein the substrate is a indirect PKA substrate.

21. A method as claimed in claim 20 wherein the substrate is in the Src-family, preferably Lck, Fyn,

Src, Yes, Fgr, Lyn, Hck, Blk, Yrk, c-tkl, Fyk, Src-1 or Src-2, or is a fragment, precursor or functionally equivalent variant thereof.

22. A method as claimed in claim 21 wherein the substrate is encoded by a nucleic acid molecule comprising the sequence:

1 cgcctggacc atgtgaatgg ggccagaggg ctcccgggct gggcagggac 51 catgggctgt ggctgcagct cacacccgga agatgactgg atggaaaaca

101 tcgatgtgtg tgagaactgc cattatccca tagtccgact ggatgggaag

151 ggcaggctgc tcatccgaaa tggctctgag gtgcgggacc cactggttac

201 ctacgaaggc tccaatccgc cggcttcccc actgcaagac aacctggtta

251 tcgctctgca cagctatgag ccctctcacg acggagatct gggctttgag 301 aagggggaac cactccgcat cctggagcag agcggcgagt ggtggaaggc

351 gcagtccctg accacgggcc aggaaggctt catccccttc aattttgtgg

401 ccaaagcgaa cagcctggag cccgaaccct ggttcttcaa gaacctgagc

451 cgcaaggacg cggagcggca gctcctggcg cccgggaaca ctcacggctc

501 cttcctcatc cgggagagcg agagcaccgc cgggtccttt tcactgtcgg 551 tccgggactt cgaccaaaac cagggagagg tggtgaaaca ttacaagatc

601 cgtaatctgg acaacggtgg cttctacatc tcccctcgaa tcacttttcc

651 cggcctgcat gaactggtcc gccattacac caatgcttca gatgggctgt

701 gcacacggtt gagccgcccc tgccagaccc agaagcccca gaagccgtgg

751 tgggaggacg agtgggaggt tcccagggag acgctgaagc tggtggagcg 801 gctgggggct gcacagttcg gggaggtgtg gatggggtac tacaacgggc

851 acacgaaggt ggcggtgaag agcctgaagc agggcagcat gtccccggac

901 gccttcctgg ccgaggccaa cctcatgaag cagctgcaac accagcggct

951 ggttcggctc tacgctgtgg tcacccagga gcccatctac atcatcactg

1001 aatacatgga gaatgggagt ctagtggatt ttctcaagac cccttcaggc 1051 atcaagttga ccatcaacaa actcctggac atggcagccc aaattgcaga

1101 aggcatggca ttcattgaag agcggaatta tattcatcgt gaccttcggg

1151 ctgccaacat tctggtgtct gacaccctga gctgcaagat tgcagacttt 1201 ggcctagcac gcctcattga ggacaacgag tacacagcca gggagggggc

1251 caagtttccc attaagtgga cagcgccaga agccattaac tacgggacat

1301 tcaccatcaa gtcagatgtg tggtcttttg ggatcctgct gacggaaatt

1351 gtcacccacg gccgcatccc ttacccaggg atgaccaacc cggaggtgat 1401 tcagaacctg gagcgaggct accgcatggt gcgccctgac aactgtccag

1451 aggagctgta ccaactcatg aggctgtgct ggaaggagcg cccagaggac

1501 cggcccacct ttgactacct gcgcagtgtg ctggaggact tcttcacggc

1551 cacagagggc cagtaccagc ctcagccttg agaggaggcc ttgagaggcc

1601 ctggggttct ccccctttct ctccagcctg acttggggag atggagttct 1651 tgtgccatag tcacatggcc tatgcacata tggactctgc acatgaatcc

1701 cacccacatg tgacacatat gcaccttgtg tctgtacacg tgtcctgtag

1751 ttgcgtggac tctgcacatg tcttgtgcat gtgtagcctg tgcatgtatg

1801 tcttggacac tgtacaaggt acccctttct ggctctccca tttcctgaga

1851 ccaccagaga gaggggagaa gcctgggatt gacagaagct tctgcccacc 1901 tacttttctt tcctcagatc atccagaagt tcctgaaggg ccaggacttt

1951 atctaatacc tctgtgtgct cctccttggt gcctggcctg gcacacatca

2001 ggagttcaat aaatgtctgt tgatgactgc cg

or a sequence which hybridizes to said sequence under binding conditions of 6 x SSC/50% formamide at room temperature and washing at 2 x SSC, 65┬░C, where SSC = 0.15 M NaCl, 0.015M sodium citrate, pH 7.2 , or a sequence which exhibits at least 80% sequence homology or a sequence complementary to any of the aforesaid sequences, or a fragment of any of the aforesaid sequences containing the region encoding or complementary to at least the phosphorylation site.

23. A method as claimed in claim 21 wherein the substrate comprises the amino acid sequence:

1 mgcgcsshpe ddwmenidvc enchypivrl dgkgrllirn gsevrdplvt

51 yegsnppasp lqdnlvialh syepshdgdl gfekgeplri leqsgewwka

101 qslttgqegf ipfnfvakan slepepwffk nlsrkdaerq llapgnthgs 151 fliresesta gsfslsvrdf dqnqgewkh ykirnldngg fyispritfp

201 glhelvrhyt nasdglctrl srpcqtqkpq kpwwedewev pretlklver

251 lgaaqfgevw mgyynghtkv avkslkqgsm spdaflaean lmkqlqhqrl 301 vrlyawtqe piyiiteyme ngslvdflkt psgikltink lldmaaqiae

351 gmafieerny ihrdlraani lvsdtlscki adfglarlie dneytarega

401 kfpikwtape ainygtftik sdvwsfgill teivthgrip ypgmtnpevi

451 qnlergyrmv rpdncpeely qlmrlcwker pedrptfdyl rsvledffta

501 tegqyqpqp

or a sequence which has more than 90% sequence homology thereto, or a fragment of any of the aforesaid sequences containing at least the phosphorylation site.

24. A method as claimed in any one of claims 21 to 23 wherein the substrate is Lck, or a fragment, precursor or functionally equivalent variant thereof, in which one or more residues in at least one of the Csk phosphorylation sites in said substrate is modified, preferably at the residue which is phosphorylated by Csk.

25. A method as claimed in claim 24 in which the tyrosine at position 505 in the human sequence, or corresponding position in another organism or precursor or variant, is altered to an phenylalanine residue.

26. A method as claimed in any one of claims 1 to 5 wherein the extent of phosphorylation is altered by introducing a molecule which alters, preferably inhibits the phosphorylation of the PKA substrate, or downstream kinase substrate, into said cell.

27. A method as claimed in claim 26 wherein said inhibitory molecule is a nucleic acid molecule comprising a nucleotide sequence encoding a protein or peptide which interferes with phosphorylation of a substrate, or fragment, precursor or functionally equivalent variant thereof containing at least the phosphorylation site, as defined in any one of claims 1 to 26, or the inhibitory molecule is a protein or peptide encoded by said nucleic acid molecule.

28. A method as claimed in claim 27 wherein said protein or peptide mimics said phosphorylation site of said substrate or binds to, or associates with, said substrate, or fragment, precursor or functionally equivalent variant thereof, thereby affecting phosphorylation of said phosphorylation site.

29. A nucleic acid molecule comprising a nucleic acid sequence encoding a PKA substrate, or fragment, precursor or functionally equivalent variant thereof, as defined in any one of claims 1 to 18, wherein said sequence is modified as defined in any one of claims 6 to 18 to alter its susceptibility to phosphorylation by PKA.

30. A vector comprising a nucleic acid molecule as claimed in claim 29.

31. A host cell containing a vector as claimed in claim 30.

32. A protein or peptide encoded by a nucleic acid molecule as claimed in claim 29.

33. A pharmaceutical composition comprising one or more nucleic acid molecules, peptides or proteins, encoding or comprising a PKA substrate, downstream kinase substrate, or modified form thereof, or fragment, precursor or functionally equivalent variant thereof, or other molecule which alters, preferably inhibits, the phosphorylation of the PKA substrate, or downstream kinase substrate as defined in any one of claims 1 to 32 and one or more pharmaceutically acceptable excipients and/or diluents.

34. A pharmaceutical composition as claimed in claim 33 wherein the nucleic acid molecule, peptide, protein or other molecule is derivatized or conjugated to improve cellular transport, increase solubility or lipophilicity or allow targeting, preferably to effect drug delivery and/or site-specific targeting.

35. A pharaceutical composition as claimed in claim 33 or 34 for use as a medicament.

36. A pharmaceutical composition as claimed in claim 33 or 34 for use in treating a disorder exhibiting abnormal PKA signaling activity.

37. A pharmaceutical composition as claimed in claim 33 or 34 for use in treating immunosuppressive disorders or proliferative diseases.

38. Use of a pharmaceutical composition as claimed in claim 33 or 34 for the preparation of a medicament for the treatment of immunosuppressive disorders or proliferative diseases.