MXPA96002798A - Modulators of the acnl protein function - Google Patents

Abstract

The present invention provides a composition and methods useful for isolating calcineurin as well as for inhibiting the activity of calcineurin. The compositions are peptides that contain regions that are homologous to the calcineurin binding region of AKAP 79. Methods are also provided to determine whether a cell contains a calcineurin binding anchor protein and PKA binding that are useful for identifying proteins. additional drugs that bind to both calcineurin and PKA. Another aspect of the present invention is that of methods for improving the expression of interleukin-2 by cells

Description

"MODULATORS OF ANCHORAGE PROTEIN FUNCTION" This application is a continuation in part of the Copending US Patent Application Serial No. 08 / 503,226, filed July 17, 1995, which, in turn, is Application 08 / 404,731, filed on March 15, 1995, which, in turn, is a continuation in part of the Copending US Patent Application Number 08 / 344,227, filed on November 23, 1994.

FIELD OF THE INVENTION The present invention relates generally to the regulation of the enzymatic activity of calcineurin phosphatase and the modulation of interleukin-2 expression by T cells. More particularly, the present invention relates to inhibition of calcineurin phosphatase activity by certain peptides and improving the expression of the interleukin 2 T cell by treating the cells with certain other peptides.

BACKGROUND OF THE INVENTION Calcineurin is a protein phosphatase dependent on Ca2 + / calmodulin and is a participant in many lifetimes of intracellular signals. Guerini and Klee, Proc. Nati Acad. Sci. USA 86: 9183-9187 (1989). The enzyme has been identified in eukaryotic cells that vary from yeast to mammals. Cyert and Thorner. J. Cell. Biol. , 107: 841a (1989) and Klee et al., Adv. Enzymol. , 61: 149-200 (1984). Because calcineurin can participate in many signal pathways in the same cell, there must be means for specific targeting of calcineurin activity. A cellular means to specifically guide the activity of the enzyme is by compartmentalization. Compartmentalization secretes signal pathways and contributes to the specificity of cellular responses to different stimuli. The compartmentation of certain enzymes occurs through the interaction of enzymes with specific anchoring proteins. For example, the cAMP-dependent protein kinase (PKA) is anchored at specific intracellular sites by binding to the A-kinase anchor proteins (AKAPs). Because AKAPs have been shown to bind proteins other than PKA, the family of proteins is generally referred to herein as anchoring proteins. Hirsch et al., J. Biol. Chem., 267: 2131-2134 (1992). cAMP activates PKA by binding to the regulatory subunits (R) of the inactive PKA holoenzyme and causes the release of the active catalytic subunit (C). There are two kinds of subunit R; Rl and RII that form the PKA holoenzymes of type I and type II, respectively. The subcellular distributions of these PKA isoforms appear to be different. The Rl isoforms (RIalpha and RIbeta) are reported to be predominantly cytoplasmic and exclude from the nuclear compartment, while up to 75 percent of the RII isoforms (Rllalfa or Rubeta) are particulate and associated with either the memebrane plasma, the cytoskeletal components, the secretory granules, the Golgi apparatus, microcenters or possibly nuclei. Anchorage proteins have been identified in a variety of organisms. At least, proteins that bind the regulatory subunit of PKA have been identified in Aplysia californica, a marine invertebrate. Cheley et al., J. Bi ol. Chem. , 269: 2911-2920 (1994). One of these proteins is enriched in fractions of the crude membrane and microtubules stabilized with taxol and, therefore, can anchor the microtubules in the cell membrane, as well as bind PKA. A mammalian anchor protein has been identified as being related to microtubules; the protein associated with microtubule 2 (MAP2) binds PKA to the cytoskeleton. Threurkauf and Vallee, J. Biol. Chem., 257: 3284-3290 (1982) and De Camilli et al., J. Cell Biol. , 103: 189-203 (1986). The binding site of PKA in MAP2 is a peptide of 31 residues in the amino terminal region of the molecule. Rubino et al., Neuron, 3: 631-638 (1989) and Obar et al., Neuron, 3,639-645 (1989). Another anchor protein that is associated with microtubules, AKAP 150, is accumulated in dendrites in close association with microtubules. Glantz et al., Mol. Biol. Cell, 3: 1215-1228 (1992). AKAP 150 is present in several types of neuronal cell and is a member of a family of anchor proteins that are the main anchor proteins in the mammalian brain. Other members of this family include AKAP 75 which is found in bovine brain and AKAP 79 which is found in the human brain. Glantz et al., J. Biol. Chem., 268: 12796-12804 (1993). AKAP 75 apparently binds the cytoskeletal elements through two non-contiguous regions near the N-terminus of AKAP 75. AKAP 79 is predominantly present in post-synoptic densities (PSDs) in the human forebrain. Carr et al., J. Bi ol. Chem., 267: 16816-16823 (1992). Other anchor proteins have also been characterized. The exposure of granulosa cells to the follicle stimulating hormone and estradiol has been shown to regulate the expression of an 80 kDa AKAP. Carr et al., J. Biol. Chem., 268: 20729-20732 (1993). Another AKAP, Ht31 has been cloned from a human thyroid cDNA library. Carr et al., J. Biol. Chem., 267: 13376-13382 (1992). Another anchor protein, AKAP 95, changes its intracellular location during the cell cycle. AKAP 95 is an integral nuclear protein during the interphase but is associated with cytoplasmic PKA when the nuclear membrane disintegrates during mitosis. This suggests that AKAP 95 might have a role in the targeting activity of certain PKA isoforms during cAMP-responsive events linked to the cell cycle. Coghian et al., J. Biol. Chem., 269: 7658-7665 (1994). Other known anchor proteins include an 85 kDa AKAP that binds PKA to the Golgi apparatus (Rios et al., EMBO J., 11: 1723-1731 (1992)) and a 350 kDa AKAP that binds PKA to microcenters (Keryer and others., Exp. Cell Res., 204: 230-240 (1993)). Known anchor proteins link PKA by a common mechanism. Even though the primary structure of anchoring proteins is not conserved, each has a secondary structure motif that includes a region of antipathetic helices. Scott and McCartney, Mol. Endo. , 8: 5-11 (1994). The binding of the anchor proteins to the regulatory subunit of PKA is blocked by a peptide that mimics this helical structure of the PKA binding region of the anchor proteins. Breaking the helical structure of the peptide by an amino acid substitution suppresses the anchor protein-PKA binding block (Carr et al., J. Biol. Chem., 266: 14188-14192 (1991)), demonstrating that the Bonding occurs in the amphipathic helix of the anchor proteins and is governed by the secondary structure of the anchor protein molecules. This intracellular binding and localization of PKA by anchoring proteins provides a means for segregating a kinase that, like calcineurin, is common to many signal pathways and can nevertheless act in a specific manner with respect to the pathway. . PKA works in many intracellular ways.

For example, the inhibition of the alloy between AKAP 79 and PKA in hippocampal neurons has been shown to inhibit the alpha-amino-3-hydroxy-5-methyl-4-isoxasol propionic acid / kainate receptors. Rosenmund et al., Na ture, 368: 853-856 (1994). This indicates that PKA regulates these receptors. PKa also regulates the activity of glycogen phosphorylase by reversibly phosphorylating the enzyme in response to hormonally induced increases in intracellular cAMP. Walsh et al., J. Biol Chem., 243: 3763-3765 (1969). CAMP has also been shown to inhibit signals through the MAP kinase pathway. Wu et al., Sci ence, 262: 1065-1072 (1993). This inhibition is mediated by activation of PKA which inhibits the activation of Raf-1 by Ras, thus blocking the Kinaase MAP pathway. Vojtek et al., Cell, 74: 205-214 (1993) and Hafner et al., Mol. Cell Biol. , 14: 6696-6703 (1994). These pathways are important in many cell types and have been implicated in many cell functions, such as the transcriptional activation of the interleukin 2 gene that is important in the activation of T cells. Weiss an Littman, Cell, 76: 263-274 (1994); Owaki et al., EMBO J., 12: 4367-4373 (1993). Like PKA, calcineurin is associated with the activation of the T cell. Clipstone and Crabtree, Na ture, 357: 695-697 (1992); O'Keefe et al., Na ture, 357: 692-694 (1992). In T cells, calcineurin participates in the regulation of IL-2 expression after cell stimulation T. Weiss and Littman, supra. The nuclear factor of activated T cells (NFATp) has been shown to be a substrate for the activity of calcineurin phosphatase. It has been suggested that, after stimulation of the T cell, the dephosphorylation of NFATp mediated by calcineurin allows the translocation of NFATp from the cytoplasm to the nucleus where NFATp interacts with Fos and Jun to induce the expression of the IL-2 gene. Jain et al., Nature, 365: 352-355 (1993). The role of calcineurin in the activation of the T cell provides a target for therapeutic intervention in disorders mediated with the T cell and several drugs have been developed that inhibit calcineurin. Two drugs that inhibit calcineurin, cyclosporin A (cyclosporine) and FK506, have been used in the clinic. Thomson and Starzl, Immunol. Rev., 136: 71-98) 1993). Both ciclosporin and FK506 inhibit calcineurin only after binding to the different intracellular proteins known as immunophilins (cyclophilin and FKBP 12, respectively). Schreiber and Crabtree, Immunology Today, 13: 136-142 (1992). Therefore, ciclosporin and FK506 act as prodrugs. After binding to the respective immunophilins, the drug / immunophilin complexes bind calcineurin, thereby inhibiting phosphatase activity. The inhibition of calcineurin has been exploited in a very effective way in the treatment of graft rejection after organ transplantation. Cyclosporin and FK506 have been used after kidney, liver, heart, lung and bone marrow transplants. The Canadian Multicentre Transplant Study Group, N. Engl. J. Med., 314: 1219-1225 (1986): Oyer et al., Transplant Proc. , 15: Suppl 1: 2546-2552 (1983); Starzl et al., N. Engl. J. Med., 305: 266-269 (1981); The Toronto Lung Transplant Group, JAMA, 259: 2258-2262 (1988); and Deeg et al., Blood, 65: 1325-1334 (1985). The use of these medications has significantly prolonged graft survival and reduced morbidity after transplantation. Najarian and others., Ann. Surg. , 201: 142-157 (1985) and Showstack et al., N. Engl. J. Med., 321: 1086-1092 (1989). Cyclosporine has also been used in a variety of autoimmune related diseases. Uveitis usually gets better within a few weeks of therapy, but relapses quickly after ciclosporin is discontinued. Nussenblatt et al., Am J. Ophthalmol. 96: 275-282 (1983). In a similar way. Psoriasis usually improves with ciclosporin therapies but relapses quickly after treatment. Ellis and others., JAMA, 256: 3110-3116 (1986). The "honeymoon" periods of insulin independence can be induced and prolonged both in the initiation of Type I and Type II diabetes mellitus when ciclosporin is administered within two months of insulin therapy. Feutren et al., Lancet, 2: 119-124 (1986) and Bougneres et al., N. Engl. J. Med., 318: 663-670 (1988). A variety of nephropathies, including focal minimal and segmental nephropathies, membranous and mediated with IgA, may also be sensitive to cyclosporin even when the reductions observed in proteinuria may be due to a dysfunction in the glomerular filtration regimen and not scarring of the base membrane. Tej ani et al., Kidney Intl. , 29: 206 (1986). The administration of ciclosporin also has a dose-dependent effect in remumatoid arthritis, even though this treatment is associated with a high incidence of nephrotoxicity. Forre and others., Arthri ti s Rheum. , 30: 88-92 (1987). As mentioned above, cyclosporine has been associated with nephrotoxicity. Mason, Pharmacol. Rev., 42: 423-434 (1989). Depressed renal function occurs in virtually all patients treated with ciclosporin. Kahan, N. Engl. J. Med., 321: 1725-1738 (1989). This can usually be reversed by cessation of ciclosporin therapy. Unfortunately, in recipients of organ grafts the substitution of other immunosuppressants commonly used for cyclosporine carries a great risk of graft rejection. In renal transplant patients, this may require dialysis reinstitution. In patients who have received hearts, lungs or liver, rejection of the graft can be fatal. Although it is less common than nephrotoxicity, neurotoxicity and hepatotoxicity are also associated with ciclosporin therapy. de Groen et al., N. Engl. J. Med., 317: 861-866 (1989) and Kahan et al., Transplantation, 43: 197-204 (1987). Significant toxicity has also been evident in the use of FK506. Like ciclosporin, FK506 is associated with nephrotoxicity. Peters et al., Drugs, 4: 746-794 (1993). The clinical presentation, lesion morphology and incidence are approximately equivalent to those of cyclosporine. McCauley., Curr. Op. Nephrol. Hyperten. , 2: 662-669 (1993). Neurotoxicity has also been associated with FK506. Eidelman and others., Transplant. Proc. , 23: 3175-3178 (1991) and Fung et al., Transplant. Proc. , 23: 3105-3108 (1991). In contrast to cyclosporine, FK506 has a hepatotrophic effect instead of a hepatotoxic effect. Peters and others., Supra. In view of the significant potential toxicity of immunosuppressive agents, such as cyclosporin and FK506, it is clear that there is a need in the art for additional agents that inhibit calcineurin. These agents would preferably be associated with fewer toxic side effects than the agents currently obtainable and, therefore, would provide an advance in immunosuppressive therapy. In addition, there is a need for agents that inhibit PKA in T cells allowing improved expression of interleukin 2 by cells.

SUMMARY OF THE INVENTION The present invention is based, in part, on the discovery that calcineurin binds to AKAP 79. By linking both PKA and calcineurin, AKAP 79 co-locates a kinase and a phosphatase which can regulate the flow through a specific signal path. The present invention therefore provides compositions and methods for isolating calcineurin as well as for inhibiting the activity of calcineurin in a cell. Isolation methods comprise contacting a cell fraction with AKAP 79 or a calcineurin binding fragment thereof that has been immobilized on a solid substrate and then eluting calcineurin therefrom. The calcineurin inhibition method comprises contacting the cells with AKAP 79 or with a peptide of the calcineurin binding fragment thereof. Preferably, the calcineurin binding peptide does not bind PKA either. Preferred peptides comprise the following amino acid subsequence.

Arg-Arg-Lys-Arg-Ser-Gln-Ser-Ser-Lys-Glu-Glu-Lys-Pro (SEQ ID N0: 1).

Alternative peptides useful in the practice of the calcineurin inhibition methods of the present invention include: Arg-Arg-Lys-Arg-Ser-Gln-Ser-Ser-Lys-Glu-Glu-Lys-Pro-Leu-Gln ( SEQ ID N0: 2) and Arg-Arg-Lys-Arg-Ser-Gln-Ser-Ser-Lys-Glu-Glu-Lys-Pro-Phe-Lys (SEQ ID N0: 3) These peptides are homologous to the sequence of AKAP 79 amino acids that bind calcineurin. Although the peptides are similar to the calcineurin binding region of FKBP12, unlike the inhibition of calcineurin by the FK506 / FKBP12 complex, the peptides inhibit the activity of calcineurin without requiring interaction with another molecule. The peptides can be modified to facilitate their passage into the cell, such as by conjugation with a lipid soluble residue. For example, the peptides can be conjugated in myristic acid. Alternatively, the peptides can be packaged in liposomes that can fuse with the membranes of the cell and release the peptides into the cells.

Another aspect of the present invention are methods for determining whether a cell contains a calcineurin binding anchor protein and PKA binding. The methods usually comprise lysing the cell to form a lysate; cover the lysate with a solid support, whose solid support has calcineurin molecules immobilized therein; wash the solid support lysate; contacting the solid support with a regulatory subunit of irradiated PKA, washing the unbound regulatory subunit of the solid support; detect the radiation remaining in the solid support; and determining therein the presence of a calcineurin binding anchor protein and PKA binding in the cell. Alternatively the regulatory subunit of PKA can be immobilized on the solid support and the calcineurin can be the irradiated molecule. In general, the regulatory subunit of PKA will be an RII subunit. These methods are useful for identifying additional proteins that bind to both PKA and calcineurin. The identification of other of these proteins can provide tissue-specific targets for therapeutic intervention. Methods for identifying compounds that modulate the binding between calcineurin and a calcineurin anchoring protein are also encompassed by the present invention. Any calcineurin or anchoring protein can be bound to a solid substrate. The non-binding binding partner irradiates detectably. The binding partners incubate in the presence of a test compound. The effect of the test compound on the binding between calcineurin and the calcineurin anchoring protein is determined by observing the amount of irradiation that binds to the immobilized binding partner. A reduction in the amount of the bound irradiation in the presence of a test compound compared to the amount of the bound irradiation in the absence of the test compound, indicates that the test compound is a binding inhibitor between calcineurin and the calcineurin anchoring protein. Other assays may also be employed, such as scintillation proximity assays. A further aspect of the present invention includes methods for improving the expression of interleukin 2 by T cells. The inhibition of PKA kinase activity or the localization of PKA in T cells improves the expression of proteins under the control of the promoter elements that regulate the transcription of the interleukin 2 gene. These methods usually comprise contacting the T lymphocyte with one of the following amino acid sequences.

Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile (SEQ ID N0: 5) Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala Val-Ile-Glu-Gln-Val-Lys-Ala-Ala-Gly-Ala-Tyr (SEQ ID NO: 9) The peptide of SEQ ID NO: 5 is a peptide that inhibits the activity of PKA kinase. The peptide of SEQ ID NO: 9 is a peptide that is homologous to the PKA binding region of the HT31 anchor protein. These peptides can be modified to facilitate passage into cells or packed into liposomes as described above. The invention proposes the variety of uses for the methods employing the peptides. For example, the methods can be used to stimulate the immune response to stimulate the activated T cells for the selected clonal expansion or to improve the T cell responses of the experimental stimuli for the evaluation of the early events in the biology of the cell. T and activation of the immune response.

BRIEF DESCRIPTION OF THE FIGURES Figures IA to IB illustrate the inhibition of calcineurin phosphatase activity by full-length AKAP 79 and a calcineurin binding fragment of AKAP 79. Figures 2A to 2C illustrate a subcellular localization of PKA type II and calcineurin as well as the co-localization of PKA type II and calcineurin. Figure 3 illustrates the homology between clone 11.1 and isoform 11.1 of human calcineurin. Figure 4 illustrates the increase in intracellular concentration of cAMP induced by treatment of Jurkat cells with forskolin and IBMX. Figures 5A to 5H illustrate the FACS chunks demonstrating the PKA inhibition effect and delocalization in the transcription of proteins controlled by the interleukin 2 promoter.

DETAILED DESCRIPTION OF THE INVENTION The peptides employed in the methods of the present invention can be synthesized in solution or in a solid support in accordance with conventional techniques as described in the Stewart and Young article, Solid Phase Peptide Synthesis, second edition., Pierce Chemical Company (1984 ) or Tam et al., J. Am. Chem. Soc., 105: 6442 (1983), both of which are incorporated herein by reference. Peptides can be miristolized by standard techniques as described in the Eichholtz et al., J. Biol. Chem, 268: 1982-1986 (1993), incorporated herein by reference. The encapsulation of the peptides in liposomes can be carried out by standard techniques as generally described in US Patent Nos. 4,766,046, 5,169,637, 5,180,713, 5,185,154, 5,204,112 and 5,252,263 and PCT Patent Application Number 92/02244, each of the which is incorporated herein by reference. The following examples are offered by way of illustration and not limitation. Example 1 describes the association of calcineurin with AKAP 79 and PKA. Example 2 is related to inhibition of calcineurin activity using peptides derived from AKAP 79 amino acid sequences. Example 3 is directed to the subcellular distribution of PKA type II and calcineurin. Example 4 describes a dihydride assay demonstrating the physiological linkage between AKAP 79 and calcineurin. Example 5 is directed to the analysis of the binding of AKAP 79 and calcineurin. Example 6 describes the use of calcineurin mutants to define an AKAP binding site 79. Example 7 relates to the interaction between the Rl subunit of AKAP 79 and PKA. Example 8 describes a method for selecting inhibitors of PKA compartmentation. Example 9 describes the involvement of the anchor protein in the modulation of IL-2 expression. Example 10 relates to the identification of other AKAP 79 binding proteins. Example 11 describes the interaction between AKAP 79 and PKC. Example 12 relates to the potential therapeutic application of the anchor proteins.

Example 1 This example demonstrates the association that occurs naturally from calcineurin with AKAP 79 and PKA. AKAP 79 therefore functions to co-localize both an omnipresent kinase and an omnipresent phosphatase. These co-localizations can provide the specific regulation of enzymes in signal pathways through the phosphorylation or dephosphorylation of the enzymes. Immunoprecipitation of calcineurin (CaN) from a bovine brain purified with calmodulin-agarose was achieved using specific affinity-purified antibodies for either CaN A or CaN B as generally described in the Harlowe and Lane article., Antibodi is: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1988), except for a final wash using a stabilizer A (10 mM HEPES pH 7.9, 1.5 mM MgCl, 10 mM KCl, 1 mM PMSF and 10 uM IBMX) + 0.4 M NaCl that was included. The activity of PKA was measured as described in the article by Scott et al., Proc. Nat. Acad. Sci. USA, 82: 4379-4383 (1985), incorporated herein by reference, after elution of the immunoprecipitate with 0.1 mM cAMP. Phosphorylation of the immunoprecipitated proteins was initiated by the addition of 0.1 mM of ^ P-TP (i.5 x? O ^ cpm / nmol) and after 30 minutes at 30 ° C the reactions were terminated by adding the SDS charge stabilizer and subjected to SDS-PAGE. The R subunit of PKA was purified from 30 percent to 60 percent (NH4) 2S? 4 of the brain extract fraction using cAMP-agarose by the methods described in the article by Coghlan et al., J. Biol. Chem , 269: 7658-7665 (1994) (which is incorporated herein by reference) except that the protein was eluted with 0.5 mM of the peptide Ht31 (SEQ ID NO: 4). Western blots and PKA RII tests were carried out as described in the article by Coghlan et al., Supra. The kinase activity was detected in the extract purified with calmodulin and enriched at 123 + 3.6 fold (+ standard deviation, n = 3) in the immunoprecipitate of CaN and specifically inhibited by a peptide that inhibits the activity of PKA kinase, the PKI peptide (SEQ ID NO: 5) indicating that the catalytic subunit (C) of PKA was a component of the isolated complex. A bovine homolog of AKAP 79 (AKAP 75) and RII, both substrates for the C subunit, were also present in the immunoprecipitate and phosphorylated during the addition of cAMP and 32p-tp. In complementary experiments, the R subunits of PKA were isolated from crude bovine brain extracts by affinity chromatography on cAMP-agarose. The treatment of the affinity column with the Ht31 peptide specifically eluded the AKI 75 of RII bound with cAMP and also released the subunits both CaN A and B. Approximately 5 percent of the total CaN present in the material subjected to smoothing was found to be associated with AKAP 75 and RII as detected in "western blots". Combined, these results suggest the simultaneous association of PKA and CaN with the anchor protein.

Example 2 This example demonstrates the inhibition of calcineurin phosphatase activity by AKAP 79 peptides. To determine whether the binding of the peptide AKAP 79 was inhibitory, calcineurin (CaN) activity was assayed in the presence of recombinant AKAP 79. Briefly, recombinant AKAP 79 was expressed in E. coli as described in the article by Carr et al., J. Biol. Chem., 267: 16816-16823 (1992), incorporated herein by reference. CaN and the constitutively active truncated mutant CaN420 (a constitutively active independent form of Ca2 + / truncated calmodulin from CaN (Perrino et al., J. Biol. Chem., In press)) was expressed on Sf9 cells and purified on calmodulin. - Sepharose as described in the article by Perrino et al., J. Bi ol. Chem. , 267: 15965-15969 (1992), which is incorporated herein by reference. The activity of the phosphatase towards the RII 32p peptide substrate was measured as described in the article by Perrino et al., Supra. CaN (30 nM), calmodulin (100 nM) and peptide RII 32P (22 microns) were incubated with the protein AKAP 79 and the peptide AKAP 79 (SEQ ID NO: 1-amino acids 81-102) through a scale of concentrations indicated in Figure IB. Calmodulin was omitted from the CaN420 assays The "P" released from the substrate was measured in triplicate samples in three separate experiments by scintillation counting.The inhibition constant (K) of recombinant AKAP 79 for CaN was determined by analysis of linear regression of data The K_ values of the AKAP 79 peptide were calculated by determining the IC50 using a fixed substrate concentration as Km (42 microns).

Figure IA illustrates a Lineweaver-Burk trace of the inhibition of AKAP 79 of CaN (Ca2 + / calmodulin-dependent) full-length (circuios) as of CaN420 (frames) in a non-competitive manner with respect to the phosphorylated RII peptide substrate . The open symbols represent the phosphatase activity in the absence of AKAP 79 and the filled symbols represent the phosphatase activity in the presence of AKAP 79. The synthetic peptide corresponding to the peptide AKAP 79 inhibited both full length CaN (filled circles) and CaN42? while the Ht31 peptide was not a CaN inhibitor (Figure IB). The observed inhition was specific for calcineurin; the AKAP 79 peptide did not significantly affect the activity of protein phosphatases 1 (open diamonds) or 2A (crossings) of peptide concentrations as high as 0.4 mM. Even though the binding sites of CaN in AKAP 79 and FKBP-12 are similar, their differences may have functional importance: FK506 (2 micrometers) did not affect the inhibition potency and recombinant AKAP 79 did not exhibit prolyl isomerase activity of peptiril towards a fluorescent peptide substrate. In addition, the CaN B subunit that is required for the interaction of FK506 / FKBP with the CaN A subunit, is not required for AKAP 79 interaction with the CaN subunit A. Also, while the interaction of FK506 / FKBP with CaN A depends on calcium / calmodulin, the AKAP 79 inhibition of calcineurin activity is independent of calcium / calmodulin. Together, these findings suggest that CaN in its inactive state is localized by AKAP 79 in a manner analogous to PKA bound by the anchor protein.

Example 3 This example demonstrates the subcellular distribution of PKA type II and calcineurin in different tissues. The subcellular localization of many protein kinases and protein phosphatases is defined by association with the target subunits. AKAP 79 represents a novel member of this class of regulatory proteins since it serves a bifunctional role in the localization of both PKA and CaN. The cells were cultured, fixed with formalin and immunostained as described in the Rosenmund et al. Article., Nature, 368: 853-856 (1994). The secondary anti-goat antiserum conjugated with FITC was used to stain RII. The secondary antiserum of biotinylated anti-rabbit and streptavidin-Texas-Red (Jackson) were used to stain CaN. Images were obtained using a Biorad MRC-600 confocal laser scanning system (Al and A2 filters) with a Nikon optiphot 2 microscope equipped with 60x planoppo chromat oil immersion lenses (1.6 NA). The confocal sections were between 1.5 and 2 micrometers in absolute thickness. The AKAP 79 homologs were observed in bovine, porcine, rabbit and murine brain. This indicates that the co-localization of PKA and CaN may be a universal phenomenon that adapts neurons to specific signal transduction events. Using the immunocytochemical methods, the subcellular distribution of PKA and CaN type II was examined in cultured hippocampal neurons. The staining patterns for RII (green irradiation in Figure 2A) and CaN (red irradiation in Figure 2B) were regionally dispersed and overlapped in the neurites (RII is red and CaN is green in Figure 2C). These findings are compatible with the co-localization of PKA and type II CaN by anchoring protein and suggest a role for the ternary complex in regulating synaptic transmission. This is compatible with the experiments demonstrating the co-localization of RII and AKAP 79 in these cells and by studies showing that AKAP 79, PKA type II and CaN are components of post-synaptic densities. Potential substrates for the localized ternary transduction complex may include AMPA / kinetic receptors, which were modulated by PKA targeting the anchor protein.

Example 4 This example demonstrates the interaction between AKAP 79 and calcineurin in a yeast dihibited assay. Employing AKAP 79 as the "bait" calcineurin encoded by cDNA from a murine T-cell library was found to bind to AKAP 79. The assay was carried out as generally described in the Durfee et al. Article. and Development, 7: 555-567 (1993), incorporated herein by reference. The "white" and the "bait" were two plasmids, each containing part of the transcription factor Gal-4. The "bait" plasmid (pASI) was a 2-micron base plasmid with an ADH promoter linked to the DNA binding subunit, Gal-4 [amino acids 1-147 as described in the article by Keegan et al. , Sci ence, 231: 699-704 (1986), incorporated herein by reference] followed by a polyclonal site of the hemagglutin tag (HA) and an ADH terminator. The selection was maintained using SC-Trp media. The "blank" construct was a leu 2 -based plasmid, 2 micron containing an ADH promoter and a terminator with Gal-4 transcription activation domain 2 [amino acids 768-881 as described in the article of Ma and Ptashne, Cell, 48: 847-853 (1987), incorporated herein by reference] followed by a multiple cloning site. This pACT vector was used in the construction of a mouse T cell cDNA fusion library. Saccharomyces cerevisiae yl90 used in the selection was designated with two genes integrated into its genome. The genes are under the control of a Gal-1 promoter that contains Gal-4 binding sites. If the proteins encoded by the bait plasmid and the associated target plasmid, the transcription factor Gal-4 units come together and function to initiate the transcription of the genes. A 1.3 Kb NcoI / BamHI fragment containing the coding region of AKAP 79 was isolated from a basic structure of pETlld and ligated to pASI to act as the "bait" for selection. A microgram of this construction was transformed into yl90 MATa and yl90 MATalfa using a normal transformation protocol of lithium acetate-PEG. Four isolates of each matching type (yl90A pASI AKAP 79 1-4 yl90alpha pASl AKAP 79 1-4) were tested for their ability to interact with a fusion construct of pACT-RII containing the regulatory subunit (RII amino acids 1-89). ) of PAK. This was achieved by matching the YEPD strains (1 percent Bacto-yeast extract, 2 percent Bacto-peptone, 2 percent dextrose and 2 percent Bacto-agar) overnight at 30 ° C and then selecting the diploids on SC-Leu-Trp plates. The lac Z gene of E. coli that acts as a reporter could then be assayed for beta-galactosidase activity. The resulting strains that were duplicated on SC-Leu-Trp plates that had been placed on the Hybond-N (Amersham) filters and had been allowed to grow overnight. The filters were placed in liquid nitrogen for one minute to open the yeast. A 3 mm paper disk was saturated with about 3 milliliters of 0.1 percent X-gal in 60 mM NaH0, 40 mM NaH2P04, 10 mM KCl and 10 mM MgS ?4. The yeast filter subjected to the lysate was placed on top of the disk and allowed to develop at 30 ° C for about 1 to 2 hours. Diploid strains containing both pASl AKAP 79 and pACT RII fusions that were positive for beta-gal activity were indicated by converting the yeast patch to a blue color. As a control, the AKAP 79 bait plasmid remained white when it was matched to an empty pACT control. Detection of the AKAP 79 fusion protein of Gal-4 was achieved by growing yl90A AKAP 79 (isolates 1 and 2) and yl90a AKAP 79 (isolates 1 and 2) to a density of 2x10 ^ cells / milliliter in 50 milliliters of medium of SC-Trp. Cells were pelleted at 3000 x g for 10 minutes and lysed with 200 microliter glass beads (size approximately 425 to 600 microns) in 25 mM Tris pH8, 5 mM EDTA, 5 mM EGTA, 2 mM O-phenanthroline, 1 mM DTT, 25 micrometers of 4- (2-aminoethyl) -benzenesulfonyl-HCl fluoride, molecular weight 239.5 (AEBSF), 1 mM benzanidine, 1 microgram per milliliter of PLACC (pepstatin, leupeptin, aprotinin, calpain I and II) and 20 micrograms per milliliter of a bestantin lysis stabilizer. The cells were alternately vortexed for one minute and frozen for one minute for a total of 24 minutes (12 cycles). Protein concentrations were determined and 30 micrograms of the total protein was loaded on a 10 percent SDS-PAGE gel. The gel was wet transferred to Immobilon-P (Millipore) and detected by normal procedures using a monoclonal antibody of anti-HA 12 CA5 (Bab Co., Berkeley, CA) and secondary antiserum conjugated with alkaline phosphatase of anti-IgG. goat mouse (Biorad, Hercules, CA). A fusion protein of AKAP 79 of Gal-4 of approximately 100 kDa was easily detectable indicating that the product of correct size was present within these strains.

Isolate 1 of yl90A pASl AKAP 79 was selected to select a murine T cell cDNA library from pACT. A culture of 500 milliliters of SC-Trp (ODgQO = 0-6-0-8) was harvested and washed with 100 milliliters of distilled water and re-granulated. The granule was brought up to 50 milliliters of LiSORB (100 mM lithium acetate, 10 mM Tris pH8, 1 mM EDTA pH8 and 1 M Sorbitol) was transferred to a one-liter capacity flask and stirred at 220 rpm. Minute for a 30 minute incubation at 30 ° C. The cells were then pelleted and resuspended with 625 microliters of LiSORB and kept on ice, while the DNA was prepared. The DNA was prepared for transformation by boiling 400 microliters and 10 milligrams / milliliter of salmon sperm DNA for 10 minutes, after which 500 microliters of LiSORB were added and allowed to cool slowly to room temperature. The DNA of the Mu T cell library was added (40 to 50 micrograms) of a material of 1 milligram / milliliter. The frozen yeast culture was distributed to the ten Eppendorf tubes with 120 milliliters of the prepared DNA. The tubes were incubated at 30 ° C at 220 Revolutions Per Minute. After 30 minutes, 900 microliters of 40 percent PEG3350 in 100 mM Li acetate, 10 mM Tris pH8 and 1 mM EDTA pH8 were mixed with each culture and returned to incubate for an additional 30 minutes. The samples were then collected and a small aliquot (5 microliters) was removed to test for transformation efficiency and placed on the SC-Leu-Trp plates, the rest of the cells were added to 100 milliliters of the SC-Leu medium. Trp-His and were grown for 1 hour at 30 ° C with shaking and 220 Revolutions Per Minute. The harvested cells were resuspended in 5.5 milliliters of a medium of SC-Leu-Trp-His plus 50 mM of 3AT (3-aminotriazole) and aliquots of 300 microliters were placed in 150-millimeter plates of SC-Leu-Trp-His + 50 mM 3 AT and they were allowed to grow for one week at 30 ° C. After four days, the evaluation plates were counted and 1.1x10 ^ colonies were selected. Large-scale beta-gal assays were carried out on library plates and 10 positive clones were isolated for individual colonies. One of these colonies grew considerably larger than the rest and was named clone 11.1. Total yeast DNA was prepared from these strains and the plasmid leu2 was isolated from the DNA. The "recovered" plasmid was used to retransform the original bait strain yl90A pASl AKAP 79 e yl90a. Only clone 11.1 remained positive for beta-galactosidase activity in yl90 pASl AKAP 79. The yl90a containing pACT clone 11.1 remained white serving as a negative control. Restriction digestion with the XhoI endonuclease released a 2.3 Kb insert and the plasmid was sequenced in the forward and reverse directions. The reactions of the Cycle Sequence Case of the Deoxy Dye Terminator (Applied Biosystems, Inc. Foster City, CA) using symmetric polymerase chain reaction (PCR) in double chain templates were analyzed in an automated ABI 373A sequence apparatus ( Applied Biosystems, Inc.). The sequence of clone 11.1 revealed an open reading frame of 487 aa long (SEQ ID NO: 6) that correctly fused with the Gal-4 activation domain of pACT. The basis of the NIH sequence data was searched and the sequence was found to be closely homologous to the protein phosphatase dependent on human calmodulin, calcineurin. The computer analysis between clone 11.1 and the human Isoform Al showed an identity of 80 percent at the level of nucleic acid and 93 percent identity at the amino acid level (Figure 3). The first lOaa and an 18aa insert in the human sequence were not present in the mouse sequence 11.1. Clone 11.1 is closely related to the A beta sequence of mouse calcineurin, but is distinctly different at the carboxy terminus. Likewise, human calcineurin Al and human calcineurin A2 isoforms are closely homologous but are distinct from one another at their 3 'terminals. The specificity of the AKAP interaction 79-calcineurin was shown by matching the strain containing calcineurin pACT with other unrelated bait strains. The crosses were carried out as described above with strains containing pASl fused with RII (1-89), casein kinase 1, phosphodiesterase 32 (HDUN2) and AKAP Ht31. Beta-galactosidase activity was negative in all diploid strains.

Example 5 In order to further evaluate the nature of the interaction of AKAP 79 with clone 11.1, a series of calcineurin deletion mutants 11.1 was constructed and each plasmid was tested in the inhibited system. Using the same 5 '-oligo (MH47) and four 3'-oligos (MH48, MH49, MH50 and MH51), PCR reactions were established to amplify the calcineurin 11.1 regions encoding amino acids 1-104, 1-204, 1-312 and 1-400, respectively. These fragments were digested with BglII and cloned into pACT. The orientation was confirmed by restriction digestion map and PCR errors determined by automated sequence. The plasmids determined to appropriately encode the desired deletion mutant were transformed into yM90MATa and yl90MATalfa. The yeast strains were matched with yl90apASl and yl90apASl of AKAP 79 together with the original clone pACT 11.1 encoding amino acids 1-487 in SEQ ID NO: 6. The resulting matching plate was tested in a filter as described above and observed that only protein fusions that encoded either amino acids 1-400 or amino acids 1-487 were able to initiate transcription of the reporter gene. The observation that a fusion protein containing amino acids 1-312 was unable to initiate transcription indicated that in the binding of AKAP 79 requires residues between amino acids 313 and 400. This region has previously shown that it includes the binding domain of FKBP / FK506 as well as the calcineurin B binding region [Husi et al., J. Biol. Chem., 269: 14199-14204 (1994)]. In order to more accurately define the calcineurin amino acid sequences required for AKAP 79 binding, additional deletion mutants were constructed and tested for the binding of AKAP 79. The expression constructs were generated using the pACT coding domains. of calcineurin 11.1 332-441, 332-487 and 442-487. As above, each construct was subjected to sequence and determined to express the correct mutant prior to transformation into the yeast strain pASl AKAP 79. During transformation, however, no expression of the reporter gene was detected indicating that the mutants were incapable of interacting with AKAP 79. A possible explanation for the lack or lack of binding of AKAP 79 is that the secondary protein structure necessary for binding was lost with these truncated clones or that a certain amino-terminal sequence may be required for binding. The above observations have indicated that the interaction between the immunophilin complex FKBP / FK506 with calcineurin A requires calcineurin B [Haddy et al., FEBS 314: 37-40 (1992)]. In order to determine if calcineurin B expressed endogenously in yeast strain yl90 participated in the observed binding of AKAP 79 / calcineurin A, a designated calcineurin B strain yl53b (Mat a gall4 gal80 his3 trpl-901 ade2- 101 ura3-52 leu2-3-112 + URA:: GAL -> lacZ, LYS2:: GAL -> HIS3cnbl / \ l:: ADE2) to eliminate the possibility of the participation of calcineurin B in the binding of calcineurin A / AKAP 79. Initially yl53b was transformed with pASl and AKAP 79 from pASl was assayed for beta-gal activity in the absence of a plasmid. No expression of the reporter gene was detected indicating that expression of the reporter gene after transformation with clone 11.1 would necessarily result from the binding of AKAP 79 / 11.1. Plasmids pACT calcineurin 11.1 and pACT calcineurin 1-400 were then separately introduced into AKAP 79 of pASI, and 533bl through normal procedures. Beta-gal activity was observed in the strains transformed with each plasmid, indicating that the interaction between AKAP 79 and calcineurin A does not require calcineurin B. The result also suggests that the binding of the immunophilin complex FKBP / FK506 with respect to calcineurin A it is different from the AKAP linkage 79.

Example 6 In order to try to define more precisely the binding region of AKAP 79 in calcineurin 11.1, an additional series of plasmids coding for deletion mutations was constructed, singular for those described above or dot mutations.

A. Terminal Suppressions. This example demonstrates that the interaction between AKAP 79 and calcineurin 11.1 requires residues 30 to 336 of calcineurin. Abbreviating, primers were designed to various regions of calcineurin 11.1 to be used in the PCR reactions to create specific N-terminal and C-terminal deletions, as described in Table 1. The PCR products were generated by mixing 1 microgram or each one 3'- and 5'-primer with 200 micrograms each of dNTPS and 1 ng of the plasmid template with PCR stabilizer # 2 (containing 20 mM Tris-HCl, pH 8.75, 10 mM KCl, 10 mM of (NH) 2S0, 2 mM of MgS? 4, 0.1 percent of Triton X-100 and 100 micrograms per milliliter of BSA) (Stratagene) and 2.5 units of DNA polymerase Pyrococus furiosus (Pfu) (Stratagene ) in 10 microliters of reaction volume. 30 cycles were carried out, each one minute at 95 ° C, two minutes at 50 ° C and four minutes at 72 ° C. The amplification products were purified and cloned into a BglII site of pACT. The resulting constructs were analyzed to determine PCR orientation and errors by sequential order as described above. Each construct was individually transformed into yeast and olfactory strains, and 90a for APAK79 and yl53b for AKAP 79 each described above in Example 4A, and beta-galactosidase filter assays were also carried out as described above. The results using the first set of vectors encoding the C-terminal deletions defined an area between the amino acid 312-400 required for the binding of AKAP 79. The positive filter assays of the transformants of yl53b pASI APAK 79 also confirmed that no Calcineurin B was required for the binding of AKAP 79. Previous studies have indicated that the binding of calcineurin B requires amino acids 348, 349, 355 and 356 [Watanabe et al., J. Biol. Chem., 270: 456-460 (1995)], the autoinhibitory domain of calcineurin includes amino acids 442-487 and the binding of FKBP / FK506 requires amino acids 350, 353 and 359 [Kawamura and Su., J. Biol. Chem., 270: 15463-15466 (1995)]. Additional 11.1 calcineurin constructs encoding additional deletions from the C-terminus indicated that the binding of calcineurin 11.1 / AKAP 79 required amino acids 1 to 336. These deletions demonstrate that the binding domain of calmodulin [WHERE IS THIS DOMAIN ?], the autoinhibitory domain and the binding domain of calcineurin B are not required for AKAP 79 and calcineurin A to form a complex.

The results of the binding of all deletions are presented in Table 1. Amino suppressions indicated that at least one area required for the binding of AKAP 79 lies between residues 30 and 99. As above, transformants yl53b pASl AKAP 79 expressing N-terminal deletions did not require calcineurin B for binding.

Table 1 AKAP 79 / Immunophilin Ligand to Calcmeurin Suppression Mutants * Primers used to construct the expression plasmids MH S (SEQ ID NO: 10) 5 'GTATTAGCAGG AGATCTTCCT ACTTC-3' MH49 (SEQ ID NO: 11) c '- rrGTGTcrrAGATcrrGG "rGAAAGTcc-3' MH50 ID NO: 12; 5 -ATTGTAGAGATCTAAGTAATTAGGTGCCG-3 'MH51 (SEQ D Nü. 13) 5 -GCCAA'rTGCTCAGATCrTGTTTCTTATG-3 * MH52 (SEQ ID NO: 14) 5 • -GGAATTCGGATCCrCGAGAGATCrCGCCG-3 'MH57 (SEQ ID NO: 15) 5 --CCACTlTG? S? TCTCTACCsrCCTCCAGCC-3 - MH58 (SEQ ID NO: 16) 5 '-CCCTGA GATCTrCACCTGCl'AAGAC-3' MH59 (SEQ ID MO.17) 5 '-GGCGGAGATCTGGCAGACCTTGCAAAGTGG-3' MH66 (SEQ ID HO. 18) 5 • -srcATGAAGATCT AC? YesTTAITrGCTcrcc-a 'MH74 rSHQ IV NO. 19) 5"-TGC CACA'I CT TGG AAGGACCATC-3 'MH75 (SEQ ID NO: 20) 5' -CACCTTCTGT? GATCR?" C? TC? TCAGAAC-3 'MH76 (SEQ ID NO. 21) .V' - CATCGGCAG? TCTCTG? AGAAGTG 3 'MH77 (SOQ? NO.22) 5' • -CCATGGCCAATNTAGATCTCÜATGAAAC-3 'MH93 (SEQ ID NO.23) 5' - -GG ACC ATG? .G ATCTAATCCAT? AAATTGGG-3 'H94 (SEQ ID NO 24) fl '-AAATCGG? GATCTAATAAGG? TGTGGAGAGC-3' MH95 (SFQ ID No. 25) i '' -G? A (ÍAGCAAT A. "VAGATCTAAATGTGCATCAC-3 'MH96 (ST.Q m NO 26) 5 - - RATTCATAGAT TATACA? GCAGCTTt-3 'MH1C7 ÍSFQ ID NO 27)' .CAACC? GATCTAATGTGGAGAGCAATTAAACTGTCG-3 'MH108 (SEQ ID NO. 2.8) 5 - CC AAT AA GAG AT CT? AGAG CA? TTAA ACTGTCG-3' MH üT »(SQ ID NO.29) r - '-TG1 G AGATCr AATTAAA' TGTCGA? Tt7rTCATCAC-3 'MH? CKSP.Q IO NO. . 30) 5 - GGA? AGCAG TCTACrrrrCGA? TGTTCATC? C-3 'MH1U (SEQ; D O 31) 5 -AAGGAT ^ O ^ TG GAGCA.'VTJ-AAACTVGI GAATGTTCATCAC B. Point Mutations. In order to accurately assess which of the amino acids are involved in the AKAP '7 ligation, the 11.1 calcineuin point mutations were created using a PCR-based strategy. Three mutants of alanines Cys335 ~ > Ala Ser336-- > Ala and Pro339- > Ala were generated and assayed for modulation of AKAP 79 binding in the inhibited system. None of these mutants have prevented AKAP 79 from binding to calcineurin, indicating that the modification of these residues is insufficient, ... ^ breaking the AKAP binding 79.

Example 7 Further selection using DNA from the T cell Mu cell pACT and the AKAP 79 bait strain pASl was carried out in order to identify other AKAP binding proteins 79 by the protocol described above. The results of the selection were approximately 211,000 colonies that provided a positive clone designated pACT 2-1 that remained positive after being rescued and retransformation. The library sequence was removed from the plasmid with Xhol digestion and shown to be an insertion of 1,200 bp. The sequence and search of the subsequent database indicated that the clone had 91 percent identity with a regulatory subunit of the lalfa-like rat protein kinase A (Rl). The library was selected again using the same bait of AKAP 79 and fifteen positives were detected from approximately 520,000 transformants. Of these fifteen, eleven were found to be homologous to the regulatory subunit of rat type 1 PKA. Each of these isolated products was fused with the 5'-untranslated region of R1 and remained open through the initiation methionine. Based on the analysis of restriction digestion and sequence data, nine individual clones were isolated, including the original pACT 2-1 isolate. These results are the first demonstration that an anchor protein binds both regulatory subunits RII and RL of PKA, which is unexpected in view of the structurally different primary structures between the two subunits. In order to further define the sequence of interaction between Rl and AKAP 79 and to determine if the interaction is unique to AKAP 79, new yeast strains were developed. Using a BglII site within the first bp of Rl, a fragment encoding amino acids 1-80 of pACT72 was isolated and ligated to pASl and pACT. The orientation was confirmed by restriction digestion analysis. Using normal yeast transformation procedures, the DNA plasmid was introduced into MATa yl90 and the transformed yeast was assayed for beta-gal activity. The truncated fusion product Rl was determined to be incapable of activating the expression of the reporter gene. Transformed strains were subsequently used in a series of experiments to determine whether the truncated form of Rl interacts with AKAP 79. The expression of the reporter gene was observed in the double transformation yeast strain indicating that the RI / AKAP 79 binding was effected through the first 80 amino acids of Rl. Finally, in an effort to determine whether the ability to bind both Rl and RII subunits was unique to AKAP 79, a human AKAP thyroid [Carr et al., J. Biol. Chem. 267: 133376-133382 (1992)] the product of the Ht31 pACT gene was tested by the dihybrid selection with the truncated Rl peptide described above containing amino acids 1 to 80 and encoded in plasmid pASI (1-80). The binding of Ht31 / RI observed in combination with the previous observation that Ht31 binds to RII indicated that the binding of the anchor protein, both with Rl and with RII, is not unique to AKAP.

Example 8 In view of the fact that AKAP 79 was shown to bind the RI and RII subunits of PKA, a scintillation proximity screening technique was developed to identify specific inhibitors that break the localization of PKA and interfere with the binding of AKAP 79 to PKA. Initially, a thioredoxin fusion protein (TRX) -AKAP 79 expression plasmid was constructed. See generally, by LaVallie et al., BIO / TECHNOLOGY 11: 187-193 (1993). In short, a fragment of thioredoxin Xbal / HindIII was subcloned into pUC19 containing a lac Z gene and a tacZ promoter. The resulting plasmid was designated TRX F / S pUC19. In order to insert an AKAP coding sequence 79 in TRX F / S pUC19, an Ncol site was created with an oligonucleotide (SEQ ID? O: 32) having the terminal sequences Spel and Hindl l. After digestion of Spel / ífindIII, the oligonucleotide was inserted into the vector-Ncol / Xhol fragment encoding AKAP 79 was ligated with the thioredoxin gene. The fusion protein was expressed in E. coli and immobilized on the 96-well ScintiStrip plates (Wallac, Turbu, Finland) containing a scintillator embedded in the solid support. The plates were pre-coated with a rabbit anti-mouse antibody that was used to immobilize the immunospecific mouse monoclonal antibody for TRX. The TRX-AKAP 79 fusion protein was then captured on the plates through the anti-TRX antibody, and was added to the H-HRII plates in the presence or absence of a reference inhibitor, for example, unirradiated RII. When H-RII was bound to AKAP 79, the irradiation was brought sufficiently close to the scintillator embedded in the support resulting in an emission detected in a MicroBeta scintillation counter.

The results of this test indicated that irradiated RII and the peptide of Ht31 described above were able to inhibit the binding of AKAP 79 / RII with an IC50 of 1 mM and 50 nM, respectively. These results are similar to the values reported for other anchor proteins [Carr et al., J. Biol. Chem. 267: 13376-13382 (1992)]. The Ht31 peptide substituted with proline, also described above, did not block the binding of AKAP 79 / RII. Because these results are compatible with those observed in previous "Western blotting" and overlapping trials, it is assumed that this technique will allow the rapid selection of potential AKAP 79 / RII binding inhibitors as well as AKAP 79 binding inhibitors. other known physiological partners, for example, calcineurin and protein kinase C.

Example 9 This example demonstrates that the association of PKA with an anchoring protein in T cells modulates the activity of PKA in the activation of NFAT thereby modulating the production of interleukin 2. IL-2 gene expression is tightly bound to T cell activation. Transcription of IL-2 was studied after activation with PMA and ionomycin. These two agents are known respectively to enhance the responses of the kinase C protein and the second calcium messenger (including the activation of CaN). Protein kinase C activates the Ras-Raf-1-Mek-MAP kinase pathway that participates in the induction of the nuclear component of NFAT. The increased calcium concentration activates calcineurin which in turn activates the cytoplasmic component of NFAT and allows translocation to the nucleus. This activation of the NFAT components includes the expression of the IL-2 gene. To quantitate the transcript, a Jurkat T cell line (NFATZ) was stably transfected with a vector containing 3 tandem copies of the binding site of NFAT and the minimal promoter of molten IL-2 coding for the beta-lacZ gene. galactosidase (beta-gal). The quantification of transcription of IL-2 was achieved through a fluorescence-activated cell sorting (FACS) analysis of beta-gal activity. Typically, lxlO6 NFATZ cells and one milliliter of culture medium were pre-incubated for 60 minutes at 37 ° C with varying concentrations of cyclosporin, and myristylated peptides, including amino acids 81-108 of AKAP 75 (SEQ ID NO: 8 which described in the article by Glantz et al., J. Biol. Chem., 268: 12796-12804 (1993), which is incorporated herein by reference), PKI (a PKA inhibitor peptide (GRRNAIHDI-SEQ ID N0: 5)) and an Ht31 peptide (SEQ ID N0: 9; amino acids 493 to 515 of the full-length Ht31 protein described in Carr et al., J. Biol. Chem., 5 267: 13376-13382 ( 1992), incorporated herein by reference which blocks the interaction of the anchor protein with the RII subunit of PKA). Each of the peptides was miristilized as described in the article by Eichholtz et al., J. Biol. Chem., 268: 1982-1986 (1993). In the experiments with cyclosporin, PKI (SEQ ID NO.5) and a peptide Ht31 (SEQ ID NO: 9), incubation with cyclosporin or the respective peptides was followed by an incubation of an additional 30 minutes with forskolin (25). micrometers) and iso-butyl-methyl-xanthine (IBMX, 0.1 mM). Incubation with forskolin / IBMX raises the concentrations ,., intracellular cAMP (Figure 4), activating in this way PKA. Finally, 12-myristate phorbol acetate (PMA) (lOng / milliliter) and ionomycin (2) were added. microns) and the incubations continued for 4 hours. The controls were incubated with PMA / ionomycin alone or forscolin / IBMX and PMA / ionomycin, under the conditions described above. During the last 20 minutes of the PMA / ionomycin incubation, chloroquine (300 microns) was added to inhibit the activity of lysosomal beta-gal ingested The cells were pulled out and resuspended in 50 microliters of the culture medium to which 50 microliters of fluorescent di-beta-D-galactopyranoside (FDG) (final concentration of 0.1 mM: Molecular Probes) was added. This osmotic shock procedure continued for 75 seconds before returning the cells to isotonic conditions by the addition of 1 milliliter of the cold FACS stabilizer (including chloroquine). The activity of beta-gal lacZ was measured by flow cytometry configured for fluorocein analysis. Figures 5A-5H illustrated the results of this experiment. Figures 5A and 5B are FACS traces showing the background fluoroscence of the assay with and without an added dye. Figure 5C shows that PMA / ionomycin treatment of NFATZ Jurkat cells induced a 6 to 7 fold increase in beta-gal activity. Cyclosporin (CsA) completely abolished this activity as expected for the important signaling role of CaN in the transcription of IL-2 (Figure 5D). The myristylated AKAP 75 peptide (SEQ ID NO: 8) when used at 10 micrometers in the medium was found to resize the PMA / ionomycin activity induced by beta-gal by 40 percent to 50 percent.

Figure 5E shows that forskolin and IBMX reduced beta-gal activity induced by PMA / ionomycin by approximately 50 percent. This blockade was completely reversed by both 100 microns of the myristylated PKA21 peptide (SEQ ID NO: 5) and 100 microns of the myristylated Ht31 peptide (SEQ ID NO: 9) (Figures 5F and 5G). Figure 5H shows that a myristylated Ht31 peptide with a proline substitution that is known to inactivate the peptide in blocking the PKA anchor did not affect forskolin / IBMX blocking. These results demonstrate the importance of PKA and its localization through an anchor protein to regulate the expression of the IL-2 gene. As described above, interference with PKA activity with localization can be used to improve the immune response by activating T cells for selective clonal expansion or investigation of the early events of T cell activation.

Example 10 Two additional singular isolates pACT 59 and pACT 74 were identified that were coded for the same region for another protein. The sequences for these clones are indicated in SEQ ID NOs: 33 and 34, respectively.

The results of the blasto search indicated significant amino acid homologies with three gene products of unknown function: C. elegants (a protein of amino acid 319 designated U00032 number in the database state), the sequence label expressed human fetal brain (an amino acid protein 97 which was designated T08697) and in expressed sequence label HL60 (a 90 amino acid protein designated D20731). The homology was also found between a product of S. pombe gene designated as PAD 1+ (an amino acid protein 308 designated D31731) which has been shown to be a positive regulator of PAP1 +, a similar transcription factor AP-1. In addition, two positive clones were detected in this selection; pACT 36 that encoded an open reading frame of amino acid 143 fused correctly in Gal4, and pACT 60 that encoded a slightly shorter region resulting from apparent suppression. The sequences for these clones are indicated in SEQ ID NOs: 35 and 36, respectively. Two isolates were unique to each other and showed no identity with any known sequence in the NIH data base.

EXAMPLE 11 Previous research suggests that AKAP 79 is a multifunctional anchoring protein that is capable of associating with at least two signal enzymes; PKA and Ca2 + / calmodulin-dependent phosphatase calcineurin, (CaN). Each signal enzyme binds to a different region of the anchor protein and each enzyme is inhibited when it is anchored. Furthermore, it has been shown that the Ca2 + / phospholipid C-dependent protein kinase (PKC) is linked to AKAP 79 also in a region other than that of PKA and CaN. Like PKA and CaN, PKC activity is measured by its association with the anchor protein. The PKC binding site is contained within the first 75 residues of the anchor protein and peptide studies have shown that a fragment containing residues 31-52 of AKAP 79 inhibits PKC activity. In addition, evidence suggests that the binding of calmodulin (CaM) to the anchor protein may release PKC activity suggesting a competition for the AKAP 79 sequence. In order to more fully characterize the interaction of PKC with AKAP 79, they took carried out experiments to characterize the PKC binding site, isolate the PKC / AKAP complexp from the bovine brain and determine whether CaM is a physiological regulator of the interaction of PKC / AKAP 79.

An overlapped PKC was initially carried out in Used from the bovine brain using PKC from the rabbit brain as a probe. The binding of PKC was detected with a monoclonal antibody (M7) that recognizes the isoforms of PKCalfa and beta. Several PKC binding proteins ranging in size from 50 to 300 kDa were detected and included a migrating protein with similar mobility as a prominent 75 kDa RII binding protein. The control experiments confirmed that the PKC binding was specific and could be detected only in the presence of 1.2 mM CaCl2 and 20 micrograms per milliliter of phosphatidylserine and when PKC was added to the reaction mixture. In order to determine if the identified 75 kDa protein can be the bovine homologue of AKAP 79, the PKC assay was used to test AKAP 79 'and the related fragments. Abbreviating, the proteins were separated by SDS polyacrylamide electrophoresis (SDS-PAGE) and bound to the nitrocellulose following the normal protocols. The samples were blocked in Blotto [1 microgram / milliliter of bovine serum albumin (BSA), 5% of dry milk in saline stabilized with Tris.(TBS)] and incubated for one hour at room temperature in the test stabilizer [TBS containing 1 milligram per milliliter of BSA, 1.2 mM calcium, 1 mM EGTA, 20 micrograms per milliliter of phosphatidylserine (PS), 2 micrograms per milliliter of leupeptin, 2 micrograms per milliliter of pepstatin and 3 micrograms per milliliter of the partially purified rabbit PKC brain]. The PKC- binding was detected with the monoclonal antibody M7 that recognizes both PKCalfa and beta after normal chemiluminescent detection methods. PKC bound to the full-length recombinant AKAP 79 protein and the recombinant fragments spanning the first 75 PKC residues bound to the protein, but the C-terminal fragments that cover the CaN and RII binding regions did not. Control experiments demonstrated that the 32 P-irradiated RII was ligated to both full-length AKAP 79 and the C-terminal fragments. These results demonstrate that AKAP 79 is a PKC binding protein and that the principle of the binding site receives within the first 75 amino acids of the protein. Previous studies on PKC binding proteins have suggested that the basic and hydrophobic regions of the PKC binding sites are involved in the formation of a phospholipid bridge with the enzyme. The first 75 residues of AKAP 79 contain a basic hydrophobic region between positions 31 and 52 and several lines of evidence suggest that this region is a primary contact site with PKC. A synthetic peptide towards residues 31-52 blocked the interaction of PKC / AKAP 79 as assessed by an assay. In order to enhance the ability of these peptides to modulate PKC activity, the following assay was carried out in the presence and absence of the AKAP 79 peptide fragments. PKC [50nM dissolved in 50 mM tris-HC1 (pH 7.4 ), 5 M MgCl 2, 1.2 mM CaCl 2, 1 mM DTT, 1 mM EGTA and 100 micrograms per milliliter of PS] were incubated with the substrate of the EGF receptor peptide (5 microns) at 30 ° C for 5 minutes. The phosphorylation reaction was initiated by the addition of 100 micrometres of 32p-ATP (500 cpm / pmol) and the reaction allowed to proceed for 10 minutes at 30 ° C. The aliquots of the reaction mixture were removed and placed on P81 filter paper and the reaction was terminated by washing the filter paper with an excess of 75 mM phosphoric acid (three washes for three minutes each). After a final ethanol wash, the P81 filters were dried and the radioactivity was measured by liquid scintillation counting. The peptide containing residues 31-52 as well as a recombinant fragment for the first 75 amino acids of AKAP 79 were potent inhibitors of PKC activity with IC50 of 2 microns and 25 nM, respectively. The more detailed kinetic analysis showed that the AKAP peptide 79, 31-52 exhibited mixed inhibition of PKC activity with Kj of 1411 + 0.28 micrometer using the epidermal growth factor receptor (EGF) peptide as a substrate. In addition, this region also resembles a binding domain of CaM, and incubation of recombinant fragment 1-75 or peptide 31-52 with CaM (15 micrometers) prevented the inhibition of PKC in the presence of an excess of Ca2"1" Since AKAP 79 is a binding protein of CaM, these findings suggest that Ca2 + / CaM can regulate the binding of PKC to the anchor protein. Combined, these results suggest that PKC associates with AKAP 79 in vi tro, the binding site of PKC is contained within the first 75 AKAP residues 79 and the peptides spanning residues 31-52 inhibit PKC activity. The results also suggest that the interaction of PKC / AKAP 79 can be regulated by CaM, since incubation with an excess of Ca2 + / CaM prevents the inhibition of PKC by a peptide 31-52 (Figure 3). In order to understand more fully the nature of the AKAP 79 / PKC interaction, experiments were designed to 1) identify the residues important for the binding of PKC to AKAP 79, 2) isolate a complex of PKC / AKAP 79 from the cells and 3) establish whether CaM regulates the interaction of PKC / AKAP 79. The analysis Sequence sequence of several PKC binding proteins has suggested that a highly positive surface loading may be required for association with PKC. Compatible with this hypothesis are the previous results where a fragment of the AKAP 79 peptide of amino acids 31 to 52 which covers a group of basic and hydrophobic residues inhibits the PKC activity (K¿ 1.4 + _ 0.28 micrometer) and a fragment Recombinant to this region is an even more potent inhibitor of this kinase (IC50 = 25 + 5 nM). In order to evaluate the role of the basic secondary chains placed between residues 31-52 of AKAP 79 as determinants for inhibition of PKC, a family of AKAP 79 mutants is generated in a recombinant AKAP 79 polypeptide containing amino acids 1-75 and PKC binding properties of each mutant tested by the method and for changes in inhibitory potency towards PKC betai. Five AKAP 79 mutants are constructed where the basic residue groups are replaced with alanine. Given the high density of the positive charge, it is possible that simultaneous substitution of several basic secondary chains is necessary before significant changes in the binding affinity of PKC are recorded. Therefore, multiple basic waste is replaced. Known mutants in the AKAP sequence 79 are created by alanine scanning mutagenesis using the methods described by Hausken et al., [J. Biol. Chem., 269: 24245-24251 (1994)]. Each AKAP 79 protein is expressed as a His-tag fusion protein and purified to homogeneity by nickel affinity chromatography. The mutant alanine peptides are shown below. SEQ ID NO.37 is the sequence of native AKAP 79.

AKAP 79 (37-50) FXRRKKAAKALAPK (SEQ ID NO: 37) AKAP 79 AA38.39 FAARKKAAKALAPK (SEQ ID NO: 38) AKAP 79 AAA40-42 FKRAAAAAKALAPK (SEQ ID NO: 39) AKAP 79 4A38-42 FAAAAAAAKALAPK (SEQ ID NO: 40) AKAP 79 AA45.50 FKRRKKAAAALAPA (SEQ ID NO: 41) AKAP 79 A37-50 FAAAAAAAAALAPA (SEQ ID NO: 42) The protein of PKC betai is expressed in baculovirus and monoclonal antibodies M4 and M7 are used to detect the isoforms of PCK alpha and beta, by the following method. In addition, each mutant of the AKAP mutant fragment 79 is assayed for its ability to inhibit PKC by the method described above.

Because the preliminary data suggests that PKC and AKAP 79 are associated in vi tro, it should be possible to isolate the AKAP 79 / PKC complex from the cells that occurs in vivo in an equal or similar bond. In order to try to isolate the binary complex of PKC / AKAP 79 with the ternary complex of PCK / AKAP 79 / CaN from the bovine brain, two independent biochemical approaches are used which were previously successful in isolating an AKAP complex in vi vo 79 / Dog. The techniques are briefly described below. Initial studies involve the immunoprecipitation of the APAK 79, AKAP 75 homolog of the bovine brain using the monoclonal antibody MCI6 generated against AKAP 79. The co-purification of PKC in the immunoprecipitates is detected by "Western blot" with rabbit polyclonal antisera that recognizes the predominant brain PKC isoforms, alpha betai, betalll and gamma. Alternatively, PKC is immunoprecipitated from the bovine brain extracts with the monoclonal M7 antibody that recognizes the PKC brain isoforms of alpha and beta and co-purifying AKAP 75 which is detected by RII or "Western blot". Finally, the identical samples are immunoprecipitated with the anti-PKC antibodies are tested for CaN with the monoclonal antibody C24 which recognizes the bovine CaN A subunit. These experiments can establish if a ternary complex of APAK 79 / PKC and CaN is formed. Alternatively, an affinity purification is carried out in order to isolate a ternary complex of RII, AKAP 79 and PKC from the bovine brain. The R subunit of PKA is purified by affinity chromatography on cAMP-agarose and the eluate is selected for the presence of PKC and AKAP by "Western blots" with the monoclonal antibodies M7 and MC16, respectively. Since recombinant AKAP 79 and PKC do not bind to cAMP-agarose, the detection of any protein in the cAMP eluate confirms the formation of a complex between both kinases and the anchor protein. Confirmation of a ternary complex is achieved by elution of PKC and AKAP 79 from cAMP-agarose with an excess of the anchor inhibitor peptide. This peptide has been shown above to displace the immobilized RAP AKA / CaN complex in cAMP-agarose.

Example 12 The previous demonstration that AKAP 79 binds to calcineurin is important in view of the fact that calcineurin is the target of two clinically useful and potent immunosuppressants, cyclosporin and FK506 both of which inhibit the activity of calcineurin. As will be described below, both cyclosporin and FK506 are useful in the treatment of a variety of diseases but have significant limiting side effects. Supposedly, the factors that modulate the binding of the anchor / calcineurin protein can finally modulate the activity of calcineurin in a similar way to the activities of ciclosporin or FK506. The identification of this modulator particularly with collateral effects lower than those observed with other immunosuppressants, possibly has extensive therapeutic use of a multitude of diseases that are currently treated with ciclosporin or FK506. Numerous clinical indications of ciclosporin and FK506 have been reported. For example, cyclosporine has defined the norm for post-transplant immunosuppression, making possible transplants of the liver, lung, intestine and pancreas even when FK506 is generally believed to be a stronger immunosuppressant. Transplant patients who can not tolerate or fail either due to ciclosporin or FK506 can sometimes successfully switch to any other drug. As another example, inflammatory bowel disease (IBD) is a common term for two diseases that have different clinical appearance. Crohn's disease and ulcerative colitis (UC). Cyclosporins have been used successfully to treat Crohn's disease, with statistically significant treatment results that have been demonstrated in at least one Index of disease activity [Brynskov, Dan. Med. Bull. 41: 332-344 (1994)]. Other indices, however, that correlate better with the resolution of acute exacerbations showed non-significant tendencies toward improvement. Cyclosporine has also demonstrated activity in UC resistant to severe acute steroid (data are not significant since the test was stopped for ethical reasons). Another trial of patients with sclerosis cholangitis and UC demonstrated importance towards a smoother UC course. Relapse was common after the withdrawal and that the treatment had been limited by concern for toxicity [Choi and Tragan., Dig. Dis. and Sci. , 39: 1885-1892 (1994)]. In addition, other immunosuppressants have been used successfully in IBD, such as methotrexate, azathioprine and 6-MP. As another example, cyclosporins have been shown to be effective in the treatment of rheumatoid arthritis in several tests when used as a second or third line therapy of the disease, ie, in patients who have failed in other established therapies and who They have a serious illness. In these tests, cyclosporine was found to be generally as effective and as toxic as other second-line agents, such as gold, antimalarial agents, azathioprine. D-penicillamine and methotrexate [Wells and Tug ell., Br. J. Rheum. , 32 (suppl 1): 51-56 (1993); Forre and others., Arth. Rheum. , 30: 88-92 (1987)]. The tests alone disclose the treatment of "very serious refractory active RA" due to the "potentially irreversible toxicity" of cyclosporin [Dougados and Torley., Br. J. Rheum. , 32 (suppl 1): 57-59 (1993)]. Renal toxicity is believed to have been mediated primarily through renal vasoconstriction texacerbates the nephrotoxicity NSAID and the renal disease inherent in rheumatoid arthritis [Leaker and Cairns., Br. J. Hosp. Med. 52: 520-534 (1994); Sturrock et al., Nephrol. Dial. Transplant. 9: 1149-1156 (1994); Ludwin and Alexopolulou., Br. J. Rheum. , 32 (suppl 1): 60-64 (1993)]. Approximately 10 percent of renal biopsies from RA patients treated with cyclosporine showed morphological features of cyclosporin toxicity [International Kidney Biopsy Registry of Cyclosporin in Autoimmune Diseases, Br. J. Rheum. , 32 (suppl 1): 65-71 (1993)]. As yet another example, cyclosporine has been reported to be effective for the treatment of asthma that depends on steroids. In one trial, a small number of patients were randomized to ciclosporin or placebo and the ciclosporin group exhibited increased airflow and FVC as well as lower prednisolone rescue courses. As another example, cyclosporine was shown to be effective in the treatment of minimal change disease nephrotic syndrome that depends on steroirdes. Patients in this trial showed that they had lower steroid requirements in a low dose of ciclosporin but all relapsed when ciclosporin was discontinued. Steroid-resistant forms of the nephrotic syn- drome only have a response rate of 20 percent to 30 percent for cyclosporine [Meyrier., Nephrol. Dial. Transplant. , 9: 596-598 (1994); Hulton et al., Pedia tr. Nephrol. , 8: 401-403 (1994)]. With respect to the treatment of systemic lupus erythematosus (SLE), one study reported a significant decrease in SLE activity indices in a non-randomized prospective non-randomized study [Tokuda et al., Arthr. Rheumat. , 37: 551-558 (1994)]. However, other studies have not shown efficacy in SLE. As another example, cyclosporine has been shown to induce remission in diabetes mellitus that depends on insulin when it is instituted previously, after the initial presentation. The remissions averaged approximately one year even though some were extended up to 850 days [Jenner et al., Diabetol ogía. , 35: 884-888 (1992); Bougneres et al., Diabetes. , 39: 1264-1272 (1990)]. A non-durable effect of ciclosporin was observed in a subsequent extended study [Martin et al., Diabetol ogía. , 34: 429-434 (1991)]. In other studies, however, renal function deteriorated during treatment for 12 to 18 months and did not return completely to the placebo level indicating that chronic kidney damage may have occurred [Feldt-Rasmussen et al., Diabetes Medicine, 7: 429-433 (1990)]. Early intervention would be needed to improve the effect of immunosuppressive therapy in the course of insulin-dependent diabetes mellitus. Certain investigators are selecting relatives in the first degree and successfully treating those with diabetic markers successfully [Elliot and Chase., Diabetología. , 34: 362-365 (1991)]. In yet another example, psoriasis has been effectively treated with cyclosporine [Cuellar et al., Balli ere 's Clin. Rheum. , 8: 483-498 (1994); Ellis and others., JAMA. , 256: 3110-3116 (1986 ().) A high-dose therapy was effective for the treatment of psoriatic arthritis, a particularly serious form of destructive arthritis and the discontinuation of therapy was usually followed by exacerbation of a skin disease and of joints In view of the potential side effects and the need for long-term continuous treatment, cyclosporine has only been indicated for refractory psoriatic arthritis that is not adequately treated by other means.Furthermore, cyclosporine has been shown to be effective for the treatment of serious atopic dermatitis in double blind and placebo controlled studies [Van Jóos et al., Br. J. Derm., 130: 634-640 (1994); Cooper., J. Invest. Derm., 102: 128-137 (1994).] The side effects of nausea, abdominal discomfort, paresthesias, cholestasis, and renal failure of the drug are preferred by patients to their untreated disease. randomized double-blind placebo found that treatment with cyclosporine significantly increased quality of life for patients with serious atopic dermatitis [Salek et al., Br. J. Derm. , 129: 422-430 (1993)]. The skin lesions relapse quickly after the administration of cyclosporine ceased, but the quality of life remained improved. As yet another example, cyclosporine has been used in the treatment of chronic dermatitis of the hands, a prevalent disease reported from 4 percent to 22 percent and which is typically treated with topical steroids to which, however, many patients do not respond. Low dose cyclosporin has been shown in 6 to 7 patients treated effectively in an open study [Reitamo and Granlund., Br. J. Derm. 130: 75-78 (1994)]. Approximately half of the patients relapsed after ciclosporin was discontinued. As yet another example, cyclosporin has been used in the treatment of urticaria and angioedema, idiopathic skin diseases that present subcutaneous swelling and urticaria. The pathology is related to the master cells and the treatment is often ineffective. In one case, three patients with refractory urticaria and angioedema were treated with cyclosporine and all symptoms resolved within one week [Fradin et al., J. Am. Acad. Derm. , 25: 1065-1067 (1991)]. All patients had to stop therapy due to side effects and the symptoms relapsed after therapy was discontinued. With respect to other rheumatological diseases, studies disclose effective cyclosporine treatment of other less common autoimmune diseases, including Behcet's Disease [Pacor et al., Clin. Rheum. , 13: 224-227 (1994), Wegner's Granulomatosis [Alien et al., Cyclosporin A Therapy for Wegner's Granul omatosis in ANCA-Associated Vasculitides: Immunological and Clinical Aspects, Gross ed. Plenum Press (1993)] and immune-mediated thrombocytopenia [Schultz et al., Blood. , 85: 1406-1408 (1995)]. In many of the tests described above, the use of cyclosporin or FK506 was associated with many unwanted side effects. In general, the increased risk of infection and malignancy are associated with general immunosuppression, and it is unlikely that an immunosuppressive agent related to an anchoring protein would not have similar risks. Other side effects can be avoided or reduced, however, by tissue specificity of the anchoring protein. The most common serious side effect of both cyclosporin and FK506 is nephrotoxicity that is at least to some degree related to the dose and occurs in most patients generally in the form of a decrease in the glomerular filtration rate during the treatment. This side effect, however, is at least partially reversible when the drug is discontinued [Leaker and Cairns, supra]. Typically, progressive renal failure does not develop even when it needs to be followed for definitive evaluation. Chronic damage has also been observed in patients receiving a low dose of ciclosporin (3 to 4 milligrams per kilogram per day), approximately 40 percent of the biopsies of these patients showed changes of interstitial fibrosis, tubular atrophy and arteriolopathy [ Svarstad et al., Nephrol. Dial. Transplant. , 9: 1462-1467 (1994); Young and others., Kidney International. , 46: 1216-1222 (1994)]. Changes in endothelial cells were also apparent in histological sections [Kahan., N. Engl. J. Med., 321: 1725-1748 (1989)]. Nephrotoxicity was assumed to have resulted primarily due to arteriolar vasoconstriction and chronic low-grade ischemia [Leaker and Cairns, supra], even though the drugs have also been shown to be toxic directly to tubular cells and vascular interstitial cells [Platz et al. , Transplanta ti on. , 58: 170-178 (1994)]. Some reports indicate that the incidence and severity of nephrotoxicity may be slightly higher with FK506 [Platz et al., Supra]. Another significant toxicity reported from both cyclosporine and FK506 was neurotoxicity, with clinical manifestations including seizures, confusion, blindness, coma, headache, ataxia, Parkinson's syndrome, paresthesias, psychosis, focal deficits, akinetic mutism, tremors, neuropathy and sleep disorders (Shimizu et al., Pediatr Nephrol., 8: 483-385 (1994); Wilson et al., Muscle and Nerve., 17: 528-532 (1994); Reece et al., Bone Marrow Transpl ., 8: 393-401 (1991), Eidelman et al., Transpl Proc. 23: 3175-3178 (1991), de Groen et al., N. Engl. J. Med. 317: 861-566 (1987). )] After transplantation of the liver, moderate to severe neurotoxicity has been shown to occur in 10 percent to 20 percent of patients treated with FK506 and 3 percent to 12 percent of patients treated with cyclosporine. Neurotoxicity has also been associated with abnormalities of serum lipid and defective liver function Other side effects of cyclosporin and / or FK506 include hepatotoxicity, glucose intolerance, hypertension, hirsutism, gastrointestinal symptoms, venous thrombosis, pancreatitis and gingival hyperplasia [Morris., J. Heart Lung Transplant. , 12: S275-S286 (1993); Fung et al., Transpl. Proc. 23: 3105-3108 (1991); Mason., Pharmacol. Rev., 42: 423: 434 (1989); Kahan., N. Engl. J. Med., 321: 1725-1738 (1989); Thomason et al., Renal Failure. , 16: 731-745 (1994)]. Therefore, in view of the extensive use of ciclosporin and FK506, and the inherent side effects of its use, the development of alternative immunosuppressive agents could be extremely beneficial. For example, it is possible that the delocalization of calcineurin from a putative T cell anchoring protein could inhibit the activity of calcineurin on T cell activation and thus provide a specific immunosuppressive agent to the T cell that has the usefulness of cyclosporine or FK506, but less collateral effects. The previous observation that the delocalization of PKA from the T cell anchoring protein improved the expression of IL-2 in stimulated cells indicated that the PKA localized in the anchor Protein somehow contributes to the regulatory role in the expression of IL -2 during the activation of the T cell. The specific delocalization of the T cell of PKA can, therefore, provide a means to improve the secretion in vi vo of IL-2, thus mimicking the administration of IL-2. recombinant and possibly reducing the toxicity disclosed above of the IL-2 treatment as described below. IL-2 has been tested for treatment of metastatic renal carcinoma and approximately 15 percent to 20 percent of patients with metastatic renal cell carcinoma or malignant melanoma respond to IL-2 therapy. Some of these responses are durable and effective for more than 66 months [Dillman., Cancer Biotherapy. , 9: 183-209 (1994); Whittington and Faulds., Drugs. , 46: 446-514 (1993)]. Although high-dose large-dose pill therapy has been associated with several serious side effects (as will be described below), low dose continuous or subcutaneous infusion therapy produced a modest response regimen (12 percent), while reduces toxicity [Vogelzang et al., J. Clin. Oncol. , 11: 1809-1816 (1993)]. IL-2 therapy (with and without interferon-alpha and other agents) has been investigated in the treatment of other malignancies. For example, sustained clinical responses but not cures have been obtained in direct application of IL-2 to tumor beds after glioma resection [Merchant et al., J. Neuro. , 8: 173-188 (1990)]. In still other routes, limited efficacy has been reported in lymphoma [Dillman, supra], colorectal carcinoma [Whittington and Faulds, supra], AML limited [Bruton and Koeller, Pharmacotherapy. , 14: 635-656 (1994)], ovarian cancer and early gallbladder cancer [Whittington and Faulds, supra]. The number of participants in each of these studies was too small to allow meaningful conclusions regarding its effectiveness, however. IL-2 has also been used in combination with adoptive immunotherapy and has been shown to be effective for the treatment of metastatic renal carcinoma [Pierce et al., Sem. Oncol. , 22: 74-80 (1995); Belldegrun et al., J. Urol. , 150: 1384-1390 (1993)]. In addition IL-2 may also be effective for the treatment of certain infectious diseases by decreasing the bacterial load of the skin and antigen levels in patients with leprosy following an intradermal injection [Kaplan., J. Infect. 5 Dis. , 167 (suppl l): Sl8-22 (1993)]. It has also been observed that compared to healthy controls with positive PPD, lymphocytes from patients with tuberculosis produce lower levels of IL-2 [Sánchez et al., Inf. - Immun. , 62: 5673-5678 (1994)], suggesting that therapy with IL-2 may be valuable in the treatment of mycobacterial infections. Despite the potential therapeutic value of IL-2 the cytokine is also associated with significant toxicity [unless otherwise stated, the sources are Whittington and Faulds, Dillman and Bruton and Koeller, supra]. The main limiting side effects of treatment are the capillary leak syndrome. The administration of IL-2 increases vascular permeability causing pulmonary and interstitial edema, with patients developing hypotension with a considerable number that requires depressant agents. Resuscitation of vigorous fluid can cause life-threatening pulmonary edema. Up to 20 percent of patients may require intubation and mechanical ventilation. The administration of the large high-dose pill causes more serious leakage than low dose or slow continuous infusions and in some regulations. One hundred percent of patients require ICU support during treatment with IL-2. Myocarditis, cardiomyopathies and cardiac arrhythmias have also been observed. Acute renal failure can occur as a result of the sipotension induced by capillary leak syndrome. IL-2 can also cause serious diarrhea and electrolyte imbalances, cholestasis, thyroid abnormalities, and acute pancreatitis. Anemia that requires transfusions occur in 15 percent to 20 percent of treated patients [MacFarlane et al., Cancer., 75: 1030-1037 (1995)]. Thrombocytopenia with hemorrhage can occur and the effects of the clotting pathway are common. More than 70 percent of patients experience changes in their current mental state including paranoid delusions, hallucinations, loss of interest, sleep disturbances, and drowsiness. Coma, visual defects, transient ischemic attacks and paresthesias have also been reported. These drawbacks associated with exogenous IL-2 suggest that alternatives, where for example, the production of endogenous IL-2 can be modulated and therefore the requirement for treatment with exogenous IL-2 that can be explored as therapeutics can be eliminated. potential In addition, to provide possible means to identify the immunosuppressive drugs and the modulators of IL-2 production, the identification of anchoring proteins makes it possible to regulate another cellular activity in view of the diverse metabolic pathways in which it has been shown that the anchor proteins participate. For example, AKAP 79 is important in the regulation of ion channels regulated with a glutamate receptor in the post-synaptic density of neurons, presumably through PKA, PKC and binding calcineurin. PKA regulates the activity of regulated channels with the AMPA receptor and the delocalisation or inhibition of PKA attenuates the activity of the AMPA ion channel. PKC regulates the activity of regulated channels with the NMDA receptor and calcineurin has been shown to desensitize the NMDA receptor to stimuli. These observations indicate that localized kinases (PKA and PKC) can regulate the activity of glutamate receptors in neurons. Dephosphorylation by calcineurin is a counterregulatory mechanism of NMDA receptors. This model agrees physiologically with the evidence of attacks induced by ciclosporin or FK506. In addition, glutamate receptors have been implicated in many neurological diseases. Glutamate and other excitatory amino acids can produce excitotoxicity of neurons and the excessive stimulation of synaptic glutamate receptors has been shown to be toxic to neurons, causing acute neuronal degeneration. Hypoxia, such as after an attack or cardiac arrest, and CNS trauma have been shown to cause a remarkable outflow of glutamate into the extracellular space that then interacts with the glutamate receptors and activates the excitotoxic cascade. It has been shown that anti-excitatory agents protect against brain damage in animal models [Olney, Neurobiology of Aging. , 15: 259-260 (1994)]. Interestingly, NMDA antagonists are toxic to certain types of neurons indicating that glutamate can inhibit other excitatory pathways in those cells. Macrolide antibodies, such as FK506 have also been shown to protect against NMDA, but not kainate, excitotoxicity in cultured neurons [Manev et al., Brain Res. 624: 331-335 (1993)]. Glutamate has also been implicated in Parkinson's Disease. NMDA antagonists protect dopaminergic neurons in substantia nigra in monkeys exposed to MPTP, a chemical substance that induces Parkinson's syndrome in humans and other primates. Amantadine and memantine are NMDA antagonists and have been used in Europe to treat Parkinson's disease, however, both have been shown to cause psychosis in some patients. There is also some evidence that glutamatergic neurons may be overactive in Parkinson's disease and inhibition would decrease in the motor symptoms of the disease [Lange and Riederer., Life Sciences. , 55: 2067: 2075 (1994)]. Glutamate also plays an important role in attack disorders by participating in the initiation, dissemination and maintenance of attack activity.

NMDA antagonists and not NMDA antagonists are potent anticonvulsants [Meldrum., Neurology. , 44 (suppl 8): S14-S23 (1994)]. AMPA receptors have also been implicated in ALS and a receptor antagonist test is currently being carried out.49 In view of the total of these observations, it is not surprising that numerous other immunosuppressive agents are involved in clinical trials. The following information related to tests was obtained from Haydon and Haynes, Balliere 's Clin. Gastroentero, 8: 455-464 (1994); Thomason and Starzi., Immunol. Rev. 1993, 71-98 (1993); and Morris., J. Heart Lung Transplant. , 12: S275-S286 (1993). For example, the azaspirano is a compound of SKB that suppresses the cellular infiltrates of graft and the induction of IL-2R and also avoids the production of IL-2 and of IFN-gama. Apparently, the azaspirano induces a certain type of suppressor cell and there is some evidence of synergistic effects with ciclosporin. As another example, mycophenolate mofetial is a Syntex compound that inhibits purine synthesis and has a selective antiproliferative effect for T and B cells. It depletes the antibody. Mycophenolate mofetial can also deplete adhesion molecules from cell surfaces. Even though the drug evidently has little toxicity, it can cause leukopenia and has been used to treat psoriasis for 20 years. As another example, mizoribin in a Sumitomo compound that inhibits DNA synthesis. The mechanism of action is identical to mycophenolate. As another example, brequinar is a DuPont-Merck compound that inhibits the synthesis of pyrimidine by blocking the dihydrogenase dehydrogenase. Full reports of clinical trials are expected. The drug has been reported to act synergistically with cyclosporine but may cause thrombocytopenia, dermatitis and mucositis. As yet another example, 15-Deoxiespergualin is a Nippon-Kayaku compound that predominantly affects monocyte / macrophage function including the inhibition of oxidative metabolism, lysosomal enzyme synthesis. The production of IL-1 and the cell surface expression of MHC class II antigens. It is effective 70 percent to 90 percent in refractory kidney rejection but toxicity to the bone marrow can occur at higher doses. As another example, leflunomide is a compound of Hoechst that inhibits the action of cytokine, blocks the activation of the T cell and the synthesis of the antibody. It is not toxic to the kidneys or to the bone marrow. As another example, rapamycin is a Wyeth-Ayerst compound that is related to FK506. It is a prodrug that must bind an immunophilin to be active and does not inhibit the production of calcineurin or block the cytokine production of the T cell. By an unknown mechanism, rapamycin blocks the transition from Gl to S. Numerous modifications and variations of the invention as noted in the foregoing illustrative examples is expected to occur to those skilled in the art. Accordingly, only the limitations appearing in the appended claims should be included in the invention.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Lockerbie, Robert Owen, and others. (ii) TITLE OF THE INVENTION: Calcineurin Inhibitory Compounds and Anchor Proteins, (iii) SEQUENCE NUMBER: 42 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Marshall, O 'Toóle, Gerstein, Murray & Borun. (B) STREET: 233 South Wacker Drive, 6300 Sears Tower. (C) CITY: Chicago. (D) STATE: Illinois. (E) COUNTRY: United States of America. (F) POSTAL CODE: 60606 (v) COMPUTER READING FORM: (A) INTERMEDIATE TYPE: Diskette (B) COMPUTER: IBM PC compatible. (C) OPERATING SYSTEM: PC-DOS / MS- DOS. (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.25. (vi) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 08/404, 731. (B) DATE OF SUBMISSION: 15-MAR-1995. (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 08 / 344,227 (B) SUBMISSION DATE: 23-NOV-1994. (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Williams Jr. / Joseph A. (B) REGISTRATION NUMBER: 38,659. (C) ATTORNEY'S NUMBER OF REFERENCE / TOCA: 27866/32861. (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 312-474-6300 (B) TELEFAX: 312-4740448 (2) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: l: Arg Arg Lys Arg Ser Gln Ser Ser Lys Glu Glu Lys Pro 1 5 10 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: Arg Arg Lys Arg Ser Gln Ser Ser Lys Glu Glu Lys Pro Leu Gln 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: Arg Arg Lys Arg Ser Gln Ser Ser Lys Glu Glu Lys Pro Phe Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 4: (i ) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Asp Leu lie Glu Glu Ala Ala Val Ser Arg lie Val Asp Ala Val 1 5 10 15 He Glu Glu Val Lys Ala Wing Gly Wing 20 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: Gly Arg Arg Asn Ala He His Asp He 1 5 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF. THE SEQUENCE: (A) LENGTH: 2257 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) FEATURES: (A) NAME / KEY: CDS (B) LOCATION: 1..1 61 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: CCG CCC CCG CCC CCG CCC CCA CCG CCr CCT CTC GGG CCC Cf C CGC GTC Pro Pro Pro Pro Pro Pro Pro Pro Leu Gi A..a Aap Ara Va 1 5 10 15 GTC AAA GCT CTT CCT TTT CCC CCA ACT CAI CGG CTC ACÁ TCT GA¿ G. Va. b Wing Val Prc Pne Pr ^ Pio Thr Hl. Are; Leu Th: »c: CU C,: or 2 p1 G1G TTT GAT ATG GAT GGG ATA CCC AGG GTT GAT GH CTG PAG AAC CA. li. Val Phe Asp Mei Aßp Gly He Pro Arg Val Asp Val Leu "y. Asn H-. 35"40 45 TTG GTA AAA GAA GGG CGG GTG GAT GAA GAA ATT GCA CTA AGA ATT ATC 19- .. Leu Val Lys Glu Gl> Arg Val A30 Glu Glu He Wing .. Arg He Ho 50 55 60 AAT GAG GGT GCT GCC ATA CTG CGG GAG AAA ACC ATG ATA GAA GTA 24C Aar.Glu Gly Ala Wing Leu Ara Ary Glu Lvs Thr Mei He Glv, Val 65 70"75 80 GAA GCT CCA ATT ACÁ GTG TGT GGT GAC ATC C? T GGC CAA TT1 TTT G? ~ 2ti Glu Aia Pro He Thr Va. Cvs Gl \ Aso He Hiß Glv Gln Phe Phe Aet 65 90 '95 CTG ATG AAA CTT TTT GAA GTA GGA GGA TCA CCT GCT AAT ACÁ CGA TA- 3 i Le Mei Lvs Lcu Pne Glu Val Gis Clv Ser Pro id Asn Th: Arq T. 100 '105 110 CTT TTT CTT GGT GAT TAT GTG GAC AGA GGT TAI TTT AGT nTA CAG Tu "' 8- Leu Phe Leu Gly Asp Tvr Val Aßr> Ar¡ Gl \ Tyr Phe Ser lie Glu Cvs 115 120"?: S GTC TTA TAT TTA TGG GTC TTG AAG ATT CTA TAC CCA AGC ACÁ TTA TC 32 Val Leu Tyr Leu Trp Val Leu Lvs He Leu Tyr Pro Ser Tnr Leu Phe 130 135 140 CTT CTG AGA GGC AAC CAT GAA TGC AGA CAC CTT ACT GA? TAT TTT ACC 48C Leu Leu Arg Gxy Asn Kxs Giu Cvs Arg h ß Leu Thr Glu Tyr Pne? «- 145 150" 155 Ibf .; TTT AAG CAG GAA TGT AAA ATT AAA TAT TCA GAA AGA GTC TAT GAA CCT = 6 Pne Lys Gin Glu Cvs Lvs He Ly-. T > r Ser G ^ u Arg Val Ivr Glu A. »a leb * 170 '175 TGT ATG GAG GCT TTP GAC AGC TTG CCC CTT GCT GCA CTT CTA AAC C'? 5"?? Cys Met. Glu Ala Pne Asp Ser Leu Pro Leu Ala Ala Leu Leu Aan Gin ltíO. _ _ 1S5_. "_. _ 19C CA TT TT TGT GTT CAT GGT GGA CTT TCA CCA GAA ATA CAC AC CTG 61 -. Gln Phe Leu Cye Val His Gly Gly Leu Ser Pro Glu He KIB Thr Leu 195 200 20 GAT GAT ATT AGG AG? TTA GAT AGA TTT AAA GAG CCA CCT GCA TTT GGA ~ ~. Aso Aflp He Arq Are Leu Aso Arg Phe Lys Glu Prc Prc A.a Phe G. >; * 210 215 22C CCA ATG TGT CAC TTG CTA TGG TCT GAT CCT TCT GAA GAC TTT GGA AAT Pro Met CVB Asp Leu Leu Irp Ser Aßp Pro Ser Glu Asp Pne G _. \ Aßr. 225": 30 235 240 GAA AAA TCA CAA GAA CAT TTT AGT CAT AAT ACÁ GTT CGA GG? TGT TCT" 6 = Glu Lvs Ser Gln Glu His Phe Ser Hxß Aßn Thr Val Arg Gly Cye Ser 245 250 255 TAT TTT TAT AAC TAT CCA GCA GTG TGT GAA TTT TTG CAA AAC AAT AAT Sir Tyr Phe Tyr Acn Tyr Pro Wing Val Cys Glu Pbe Leu Gln Aßr. ? sp Even 260 265 270 T G TTA TAT AGA So, Leu Leu Tv: A ATG TAC AGA A? A AGT CAA ACT ACÁ GGG TTT CCT TCA TTA ATA ATÁ 911 Met Tyr Arg Lys Ser Gln Tnr Thr Gly Phe Pro Ser Leu II. Tnr He 290 295 20C TTT TCG GCA CCT AAT TAC TTA GAT GTC TAC AAT AAT AAA GCT GCT GTA 960 Phe Ser Ala Pro Asn Tvr Leu Asp Val Tyr As Asn Lys Ala Ala Val 305 3Í0 315"320 CTA AAG TAT GAA AAT AAT GTG ATG AAC A1T CGA CAG T-TT AA1 TGC TCT i 005 Leu Lye Tyr Glu Asn Aen Val Met Asn He Arg Gln Phe Asn Cvs Ser 325 330 335 CCA CAT CC TAT TGG TTÜ CCC AAT TTT? TG GAT GTC TTT ACÁ TCG TCC 1056 Pro Hiß Pro Tvr 1 rp Leu Pro Asn Phe Kct Aso Val P te Thr Trp Se; 34C '345"350 TTA CCA TTT GTT GGA GAA AAA GTG ACA GAA ATG TTG GTA AAT GTT CTG 1104 Leu Pro Phe Val Gi \ Glu LVB VaJ Thr Glu Met Leu Val Aon Val Leu 355 '360 365 AGT ATT TGT TCT GAT G? T GAA CTA ATG ACA GAA GGT GAA GAC CAG TTT 1152 Ser He Cys S »Asp Aep Glu Leu Met Thr Glu Glv Glu Aop Cln Phe 370 375 38C GAT GTA GGT TCA CCT GCA GCC CGG AAA GAA ATC ATA AC A? C AAC AY 200 Aap Val Gly Be Ala Wing Wing Ring Lye Glu He He Arg Aon Lys 1 1 c 335 390 3 ^ < J00 CGA GCA ATT GGC AAG A "G GCA AGA GTC TTC TCT GTT CTC ACG GAG GAG 124 ° Arg Ala He Gl> L> s Mei A. a Are Val Phe Ser Val Leu, g Giu Glu 40? 41C lh AGT GAA AGC GTG CTG ACÁ CTC AAG GGC CTG ACT CCC ACÁ GGG ATG TT &1? Q-Ser Glu Ser Va. ' Leu Tnr Leu lys Glv Leu Thr Pro Thr &_ _ Met Leu 420 42 4"o CCT AGT GGA GTG TTG GCT GGA GGA CGG CAG ACC TTG CAA AGT GGr AAT 1344 Pro Sei Gly Val Leu Aia Gly Gly Arg Gln Thr Leu Gln £ c »r G 'Aßr. 435 440 44S GAT GTT? TG CAA CTT GCT CTG CCT CAG ATG GAC TGG CGC ACA ACT CAC Aßp Val Mei Gln Leu Wing Val Pro Gln Met Atsp Trp Gly Thr Tr.r Hit 450 455 460 TCT TTT GCT AAC AAT ACÁ CAT AAT GCA TGC AGG GAA CTC CTT CTG CTT 1: Ser Pne? The Aen Asn Thr His? Sn Ala Cys Arg Glu Leu Leu Leu Le. 465 470 475 •? S. -TT AGT TCC TGT CTT AGC AGC TGACATATGC? GGGTATTAT CTGATAGGCA 149. Pne Be Cye Leu Ser Ser 4Ü5 TCTGATTAGT ACCTGGCCAG GGCATAAT? T TGATAGAACA AG TGTCTTT TAACTGAAAA 1551 TAACAATCAG TTTCCCAGAT TTTCATAAGG TGATATGGGG AGC? GCTCAT GTCATAATIC 1 ...

CGA? ATATTT ATTCATTTCT TTA? TGCACC CCTTTC? TTC AAA? GCCTCA GTCAAGA? TG 1 or ".

TGAATCAGCG ATATATCTAT ATATCTATTT ACACACATAC ATAA? T? TAT AT? ACTAAAA *. ~: TGGAAATGT? ATTCCGAGTT TCTTACTTTT AAAATTTACC TAATTGTATT? CAI TVCCT l "" - TATGTTTT A AGTATTTATT TTTI'GACTTA AAATTCTGCT TAGGCCCCAA AACTTCCTTT 1S3I ATGCACTCAT TTGCCAAAAG ATTTATCCTA AATTTTG? AC CCTSGTA? AT ATTACAGTT 1 H TGTTTTCTGT GGTGTTTGTC AAACGTTCTA TGTATAATTC- ACTGT TGTA ACATGC TGTT 19"1 TCCTTCCTCT GCAGATATAG CTCCTTTCCT AAATCTGTC? G? CTVTCTTT AGGATA'-CTG 2031 TATGTCTGTA AATATATGTT C? A 1 AAY ACTCTATC? G ACGCTiÜTCT GTCTTTTu? T C ^ > CTAGAACCAA CTTTGT? GCA CCTTG? TTTT ACGTTTGCTG C? TTTGTTGC TGCACTTGCT 215 ,: TCACTCTGAA JY.TCAATGTA Í.CATTAGATA TTGAGCTATT GTTATAAACG GTTGAATTTA 2211 ATCATGTAA GTC? AAATTG AAAGGGTGTG? TAAAGTC? G CCTTTA - ^ ~; (2) INFORMATION FOR SEQ ID NO: 7 i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 487 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7 Pío Pro Pro Pro Prr F: c Pro Pro Prc Pro Leu Gly Aa Aap Arg Val 5 1C 15 Val Lvs Ala Val Pro h Pr- Prc Thr HJ.? Arg Leu Thr Ser Glu Glu 20 25 30 Val Phe Aac Met ABG G1- »He Pro Arg Val Asp Va» leu Lys AB? H-B 3 ~ G '40 45 Leu Val Lys Glu G¿. A-'J Val Asp Glu Glu He Wing 13μ Arg He He 50"55 60 Asn Glu Glv Ala Ala lie ^ Arg Arq Glu lys Thr Met He Glu Va. • 65" 70 5 H C Glu Ala Pro He Thr Val Cvs Glv Asp He Ha.e G; Gl-Pne Phe ABG 85 90? - Leu Met Lys Leu Pre Glu Val Glv Glv Ser Pro Wing Apn Thr Are 1 \: loo ios n: - "Leu Phe Leu Glv Aap Tvr Val Aßp Arg Gl Tyr Phe Ser He GU Cyr. 11: '' 120 1-5 Val Leu Tyr Le. Trp Val Leu Lye He Leu Tyr Pro Ser Thr Lou Pho 130 13e I40 Leu Leu Arg Gly Asn Hie G¿ Cys Arg Hiß Leu Thr Glu Tyr Phe Th "145 '150 155 lóC Phe Lys Gln Glu "CVB Lys He Lye Tyr Ser Glu Aro Val Tyr Glu? 165 170 175 ' "Cyß Met Glu Ala Phe Aep Ser Leu Pro Leu Ala Ala Leu Leu Aßn G 180 180 190 Gln Phe Leu Cys Val His Glv Gly Leu Ser Pro Glu He Hie Thr Leu 195 '200 205 Aßp Aßp He Arg Aig Leu Asp Arg Phe Lye Glu Pro Pro Wing Phe Glv 210 .215 220 Pro Met CVB Aep Lau Leu Tro Ser Asp Pro Ser Glu Aßp Phe Gly Aan 225"230 '235 240 Glu LVB Ser Glr G. HIS Phe Ser his Aßn Thr Val Arg Glv Cya Ser 245 250 '255 Tyr Phe Tyr Aßn Tvr Pro Wing Val Cys Glu Phe Leu Gln Aan Aßn Aon 260 255 270 Leu Leu Ser lie He Ara Ala His Glu Ala Gln Asp Ala Glv Tyr Arg 275 260 2CS Met lyr Arg L s Ser Gin Thr Thr Gly Phe Pro Ser Leu He Thr He 290 295 300 Phe 3rd Pro Aßn Tvr Leu Asp Val Tyr Aan Asn Lvs Wing Ala Val 305 310 315 320 Leu Lys Tvr Glu Asn Asn Val Met Asn He Arg Gln Phe Aßn Cyß Ser 325 330 335 Pro ms Pro T \ r Tr Le_ Pro Asn Phe Met Asp Val Phe Thr Trp Ser 340 345 350 Leu Pro Phe Val Gly Glu Lys Val Thr Glu Met Leu Val Aon Val Leu 355 36. "3 5 Ser lie Cvs Ser Ast) ADD I ^ J Leu Met Thr Giu Cly Clu Aar Gln Phe 370 '' '3'5 380 Asp Val Gly Ser Ala Ala Ala Arg Lys Glu He- He Arg Aar, Lys He 385 390 395 400 Arg Ala He Gly Lvß Met Ala Arg Val Phe Ser Val Leu Are Glu GH 405 41C '415 Ser Giu Ser Val Leu Thr Leu LVR Glv Leu Tnr Prc Thr G s- Met Le. 420' 425 43C Pro Ser Giy Val Leu Wing C-ly Gly Ary Gln Thr Leu Glr Ser Glv Aßr 435 440 445 Aßp Val Met Glr. Leu Ala Val Pro Gln Met Aep Trp Glv Tnr Thr Kie 450 455 - 460 Ser Phe Ala Aen Asn Thr Hiß Aßn Ala Cvß Arg Glu Leu Le; Le-, Leu 465 470 4? 5 460 Pne Ser Ser Cys Leu Ser Ser 485 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 amino acids (D) TYPE: amino acid (C) TYPE OF CHAIN: imple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 8: Ser lie Lys Arg Leu Val Thr Arg Arg Lys Arg Ser Glu Ser Ser 1 5 10 15 (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 9: Asp Leu lie Glu Glu Ala Ala Ser Arg lie Val Asp Ala Val lie 1 5 10 15 Glu Gln Val Lys Wing Wing Gly Wing Tyr 20 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: GTATTAGCAG GAGATCTTCC TACTTC (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: GTGTGTGTAG ATCTGGTGAA AGTCC (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: ATTGTAGAGA TCTAAGTAAT TAGGTGCCG (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 13 GCCAATTGCT CAGATCTTGT TTCTTATG (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 pairs of base (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 14: GGAATTCGGA TCCTCGAGAG ATCTCGCCG (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: CCACTTTGAG ATCTCTACCG TCCTCCAGCC (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16 CCCTGAGATC TTCAGCTGCT AAGAC (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: GGCTGAGATC TGGCAGACCT TGCAAAGTGG (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: GTGATGAAGA TCTTACAGTT TAATTGCTGT CC (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRING TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 19: TTCTCCAGAT CTTGGTAAGG ACCATG (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20: CACCTTCTGT AGATCTTTCA TCATCAGAAC (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21 CATCGGCAGA TCTCTGAAGA AGTG (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22 CCATGGCCAA TTTTAGATCT CGATGAAAC (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 23: GGACCATGAG ATCTAATCCA TAAAATTGGG (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 pairs base (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (Ü) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 24 AAATGGGAGA TCTAATAAGG ATGTGGAGAG C (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO.25: GGAGAGCAAT TAAAGATCTA AATGTTCATC AC (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26: TTTTCATAGA TCTATACAAG CAGCTTT (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27: CAACCAGATC TAATGTGGAC AGCAATTAAA CTGTCG (2) INFORMATION FOR SEQ ID NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 28: CCAATAAGAG ATCTAAGAGC AATTAAACTG TCG (2) INFORMATION FOR SEQ ID NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRING TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 29: TGTGAGATCT AATTAAACTG TCGAATGTTC ATCAC (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( Ü) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 30: GGAGAGCAGA TCTACTGTCG AATGTTCATC AC (2) INFORMATION FOR SEQ ID NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) TYPE OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 31: AAGGATAGAT CTAGCAATTA AACTGTCGAA TGTTCATCAC (2) INFORMATION FOR SEQ ID NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 32: TACAACTAGT ACCATGGTCG ATGGTCGACA GATCTCTCGA GAAGCTTAGC TAGC (2) INFORMATION FOR SEQ ID NO: 33: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 981 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 33: (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 33: r »C? GTATCG ATGAAATCTA CAAATATGAC AAAAAACAAC AACAAGAAAT CCTGGCGGCG c." AAACCCTGGA CTAACGATCA CCACTACTTT AAATACTGCA AAATC? CAGC ATTGGCTCTñ Hí CTGAAAATGG TG? TGCATGC CAGCTCACGA GGCAACTTGG AAGTGATGGG T1TGATGCTC 15. " GGGAAAGTCG ACGGCÍAGAC CATGATCATC ATGGACAGTT TCGCTTTGAC TCTAGAGGCC 2 1 ACAG? A? CTC GAGT? P.ATCC TCAAGCTGCT GCGTATGACT A7? TGGCTCC ATACATAGA-. .

AATGCCAAAC AGGTTGGCCT CCTTGAGAAT GCAATCGGTT GGTATCATAG C ACCCTCG. * 3o0 TATGGCTGCT GGCTCTCCGG GATTGATGTT AGTACACAGA TG TGAACCA CCAGTTTCAA 42V GAACCATTTG TAGCAGTGGT GATTGATCCA ACCAGAACAA TCTCTGCAIG AAAAGTGAAT 430 CTTGGCGCCT TTAGGACATA TCCAAAGGGC TACAAACCTC CTGATGAAGG? CCTTCTGAG 540 TACCAGACTA TCCCACCTTA ATAAAATAGA AGATTTGGGC GTGCACTGAA ACAATATTAT 60 GCCTTAGAAC TCTCATATTT CAA? TCATCT TGGATCGTAA ACTACTTGAG CTTTGGTGGA 6? 0 ATAAATÍCTG CGTGAAT? C CTGA TCCTC TAGCTTGCTT ACTAATGCAG ACTACñCC C ".'2C AGGCCAG TG TTGATTTGTC TGAÜAAGTTA GAGCAGTCGG AAGCCCAACT GGG? CCTGGC 780 AGTTTCATGT TGGGCTTAGA AACACATGAC CGCAAGTCGG AAGACAAACT TGCC? AAGCT 64 C ACTAGAGACA CCTGTAAAAC CACCATAGAA CCCACCATGC ACTGATGTCT CAGGTTATTA 3 í AGGATAAACT GTTTAATCAG ATTAACG TG T? AGTTACCA CCACGTACTT CTCAAAGTGG? 0 TGTGTGGAAG G? AAAGAGCT C 98; (2) INFORMATION FOR SEQ ID NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 919 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear 'H.-, 1oo (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO.34: AAACCCTGGA CTAAGGAT A CCACTACT7T AAAr CTCCA AAATCTCAGC AÍTGGCTCTA 60 CTGAAAATGG TGATGCAT C CAGGTCAGGA GGCAACTTGG AAGTGATGGG TTTGATCCTC 120 GGGAAAG CG ACGGCGAGAC CATGATCATC ATGGACAGTT TCGCTTTGCT CTAGAGGGC? ISO CAGAAACTCG AGTAAATGCT CAAGCTGCTG CGTATGAGTA TATGGCTGCA TAC? TAGAAA 24.T ATGCCAAACA GGTTGGCCGC CTTGAGAATG CAATCGGTTG GTATCATAGC CACCCTGGTT 30: ATGGCTGCTG GCTCTCCGGG ATTCATGTTA GTACACAGAT GCTGAACCAG CAGTTTCAAC- 362 AACCATTTGT AGCAGTGGTG ATTGATCCAA CCAGAACAAT CTCTGCAGGA AAAGTGA? TC 420 TTGGCGCCTT TAGGACATAT CCAAAGGGCT ACAAACCTCC GATGAAGGAC CTTCTGAGTA 4S2 CCAGACTATC CCACCTTAAT AAAAT? GAAG ATTTGGGCGT GCACTGAAAC AATATTATGC 54C CTTAGAAGTC TC? TATTTCA AATCATCTTG GATCGTAAAC TACTTGAGCT TTGGTGG ^ AT 600 AAATACTGGG TGAATACCCT GAGTCCTCTA GCTTGCTTAC TAATGCAGA 'TACACCACAG f6T GCCAGGTGTT GATTTGTCTG AG? AGTTAGA GCAGTCGGAA GCCCAACTGG GACGTGGCAG 720 TTTCATGTTG GGCTTAGAAA CACATGACCC- CAAGTCGGAA GACAAACTTG CCAAAG T? C 7SC TAGAGACAGC TGTAAAACCA CCATAGAAGC CACCATGGAC TGATGTCTCA GGTTATTAAC 840 GATAAACTGT TTAATCAGAT TAACGTTGTT AGTTACCACC ACGTACTTCT CAAAGTGGTG 0C TGTGGAAGGA AAAGAGCTC 915 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 541 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 35: GACCACCGAG ATGCCAATTC CAGTGTCATG AGATTTCTGC GAGACCTCAT CCACACAGGA 60 GTAGCCAATG ATTTATCTGT TTTCTTACAG CATGAAGA? G ATTTTGTTGC GGAAGGAACT 120 AATTGGACAG GTGATGAGCC AGCTTGGGCA CCAACTTGTC AGCCAGCTGC TCCACACATG 180 I I ctGc rttt »i, TTCUCCCCTA CACCCTAGCC C-ACGTGGTTG AAGTGCTCTC GGAGATGATC 240 CAGGT1CAC? OR? CGTICTT "C7.CTCGG7CG CTAC.?GAATT CCTTGA? AGG TTGC'AAAA 300 GAGACCACAG TGCGAGCTGT CACAO "CACA CATAAACAAC TTACAGATTT CCACAACCAA 360 GTCACTAGTG CCGAGG A ~ G TAAGCAAGT. ' TGrTCCGCC "TGAGAGACT" "CACCAGGTT-- 420 TTTCGATAGC TCAAGCTCA ACTCCTCCAC 7GTGCCTGTC ATCCAGCAAT GTCTTTT TT 480 ATTAGAAGAC AGGAAGAAAA C? ACCOAGAC TGTCTCCCAC AATCAGAAAC CTCTGTTÓT. "540 G 541 (2) INFORMATION FOR SEQ ID NO: 36: 'i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 51 base pairs (B1 TYPE: nucl ico acid (C) PE TYPE CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 36: CGAGATGCCA ATTCCAGTGT CATGAGATTT CTGCGAGACC TCATCCACAC ACGAGTACC '=. "F_nuftA rt- .. -. CTTGGCCAGC AACTTG'CA-. C AGCTGCTC CACACATGCT G TTTTGTC7 TCCCCCTAf., 15" CCCTACCCGA CGTG3TTGAA GTGCTCTGGG AGATCATGCA GGTTGACAGA CCGACTTTCT 240 CTCGGTGGCT AGAGAATTCC TTGAAAGGTT TGCCAAAAGA GACCACAGTG CGAGCTG-C "3 '-.GTGAC? Cr-TA? ACAAC"; ? C? GATT'I CC To AAGC? AGT CACTAGTGCC CAGCAATGrA 3c »: AGCAAGTTTG CTGGCCCTTi AGAGACTTCA CCAGGTTGTT TCGATAGCTC AAGC1CACAC 420 • XCCTGCAC-; TGCC7GTCAT CCAGGAAtGT CTTTTTTTTAT TAGAAGACAG GAAGAAAACA 480 ACCCAGACTG TGTCCCACAA TCAGAAACCT CTGTGTCG tic (2) INFORMATION FOR SEQ ID NO: 37: ii) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (Cl CHAIN TYPE: simple • (2) INFORMATION FOR SEQ ID NO: 38: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 38: Phe Wing Wing Arg Lys Lys Wing Wing Lys Wing Wing Leu Pro Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 39: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 39: Phe Lys Arg Ala Ala Ala Ala Ala Ala Lys Ala Leu Ala Pro Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 40: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 0: Phe Ala Ala Ala Ala Ala Ala Ala Ala Lys Ala Leu Ala Pro Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 41: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 41: Phe Lys Arg Arg Lys Lys Ala Ala Ala Ala Ala Ala Ala Pro Ala 1 5 (2) INFORMATION FOR SEQ IN NO: 42: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 42 Phe Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Pro Ala 1 5 10

Claims (29)

R E I V I N D I C A C I O N E S:

1. A purified and isolated polynucleotide encoding a pACT 59 polypeptide having the sequence set forth in SEQ ID NO: 33.

2. A pACT 59 polypeptide encoded by the polynucleotide of claim 1.

3. A purified and isolated polynucleotide encoding a pACT polypeptide 74 having the sequence set forth in SEQ IS NO: 34.

4. A pACT 74 polypeptide encoded by the polynucleotide of claim 3.

5. A purified and isolated polynucleotide encoding a pACT 36 polypeptide having the sequence set forth in SEQ ID NO. : 35.

6. A pACT 36 polypeptide encoded by the polynucleotide of claim 5.

7. A purified and isolated polynucleotide encoding a pACT 60 polypeptide having the sequence set forth in SEQ ID NO: 36.

8. A pACT 36 polypeptide encoded by the polynucleotide of claim 7.

9. A method for identifying a putative inhibitor compound that inhibits binding between a protein of anchor and a binding partner, comprising: incubating the anchor protein and an irradiated binding partner in the presence and absence of the putative inhibitor compound under conditions appropriate for binding between the anchor protein and the binding partner, wherein the protein anchor is immobilized on a solid support; washing the non-retained binding partner of the solid support; determining the amount of the binding partner retained in the immobilized anchor protein; comparing the amount of the binding partner retained in the anchor protein in the presence of the compound with respect to the amount of the binding partner that binds the anchor protein in the absence of the compound; and determine that this if the compound inhibits the binding between the anchor protein and the binding partner.

10. The method according to claim 9, wherein the binding partner is irradiated.

11. The method according to claim 9, wherein the binding partner is irradiated with a fluorophore.

The method according to claim 9, wherein the binding partner is a regulatory subunit of PKA of type I.

13. The method according to claim 9, wherein the binding partner is a regulatory subunit of PKA. of type II.

The method according to claim 9, wherein the anchoring protein is AKAP 79.

15. The method according to claim 9, wherein the binding partner is a calcineurin polypeptide.

16. The method according to claim 15, wherein the calcineurin polypeptide is a deletion mutant that is selected from the group of calcineurin polypeptides consisting of amino acids 1-487, 1-400, 1-312, 1- 204, 1-104, 332-487, 441-487, 332-441, 1-375, 1-354, 30-375, 98-375, 1-347, 1-340, 1-330, 1-320, 1-338, 1-336, 1-334, 1-332 and 1-335 of SEQ ID NO: 7.

17. A calcineurin deletion mutant that is selected from the group of calcineurin polypeptides consisting of amino acids 1- 487, 1-400, 1-312, 1-204, 1-104, 332-487, 441-487, 332-441, 1-375, 1-354, 30-375, 98-375, 1-347, 1-340, 1-330, 1-320, 1-338, 1-336, 1-334, 1332 and 1-335 of SEQ ID NO: 7.

18. A method for improving the expression of interleukin-2 by a lymphocyte T comprising contacting the T lymphocyte with one of the following amino acid sequences: Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile or Asp-Leu-Ile-Glu-Glu-Ala-Ala Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-V al-Lys-Ala-Ala-Gly-Ala

19. A method according to claim 18, wherein the amino acid sequence is: Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile

20. A method according to claim 19, wherein the amino acid sequence is myristylated.

21. A method according to claim 18, wherein the amino acid sequence is: Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys-Ala-Ala-Gly-Ala

22. A method according to claim 21, wherein the amino acid sequence is myristylated.

23. A method according to claim 18, further comprising activating the T cell with phorbol, 12-myristate, 13-acetate and ionomycin.

24. A method for isolating calcineurin from a cell fraction containing the same which comprises contacting the cell fraction with AKAP 79 or a calcineurin binding fragment thereof, immobilized to a solid substrate and eluting the calcineurin thereof.

25. A method for inhibiting calcineurin activity in a cell comprising contacting the cell with a calcineurin binding peptide comprising the following amino acid sequence: Arg-Arg-Lys-Arg-Ser-Gln-Ser-Ser -Lys-Glu-Glu-Lys-Pro

26. A method according to claim 25, wherein the peptide is Arg-Arg-Lys-Arg-Ser-Gln-Ser-Ser-Lys-Glu-Glu-Lys-Pro-Leu-Gln

27. A method according to claim 25, wherein the peptide is Arg-Arg-Lys-Arg-Ser-Gln-Ser-Ser-Lys-Glu-Glu-Lys-Pro-Phe-Lys

28. A method according to claim 25, wherein the peptide does not bind PKA.

29. A method for determining whether a cell contains a calcineurin binding anchor protein and PKA binding comprising: using the cell to form a lysate; incubating the lysate with a solid support whose solid support has calcineurin molecule immobilized therein; wash the isolate of the solid support; contacting the solid support with an irradiated PKA regulatory subunit that binds an anchor protein; wash the regulatory subunit of the solid support; detect the radiation remaining in the solid support; determine the presence of a binding protein of calcineurin binding ligand of PKA, in the cell.