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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to the control of glycogen metabolism and protein synthesis, in particular through the use of insulin.
• glycogen synthase kinase-3 (Embi et al., 1980) is implicated in the regulation of several physiological processes, including the control of glycogen (Parker et al., 1983) and protein (Welsh et al., 1993) synthesis by insulin, modulation of the transcription factors AP-1 and CREB (Nikolaki et al, de Groot et al., 1993 and Fiol et al 1994) , the specification of cell fate in Drosophila (Siegfied et al., 1992) and dorsoventral patterning in Xenopus embryos (He et al., 1995).
• GSK3 is inhibited by serine phosphorylation in response to insulin or growth factors (Welsh et al., 1993, Hughes et al., 1994, Cross et al., 1994 and Saito et al., 1994) and in vitro by either MAP kinase-activated protein (MAPKAP) kinase-1 (also known as p90 rslc ) or P70 ribosomal S6 kinase (p70 S6k ) (Sutherland et al., 1993 and Sutherland et al., 1994).
• MAPKAP MAP kinase-activated protein
• p70 S6k P70 ribosomal S6 kinase
• GSK3 is inhibited in response to insulin with a half time of two min, slightly slower than the half time for activation of PKB ⁇ (one min) . Inhibition of GSK3 by insulin results in its phosphorylation at the same serine residue (serine 21) which is targeted by PKB ⁇ in vitro. Like the activation of PKB ⁇ , the inhibition of GSK3 by insulin is prevented by phosphatidyl inositol (PI-3) kinase inhibitors wortmannin and LY 294002. The inhibition of GSK3 is likely to contribute to the increase in the rate of glycogen synthesis (Cross et al., 1994) and translation of certain mRNAs by insulin (Welsh et al., 1994) .
• PKB ⁇ Coffer & Woodgett, 1991
• 3 Choeng et al. , 1992
• PKB ⁇ Konishi et al., 1995
• the catalytic domain is most similar to cyclic AMP-dependent protein kinase (PKA, 65% similarity) and to protein kinase C (PKC, 75% similarity) findings that gave rise to two of its names, namely PKB (i.e. between PKA and PKC) and RAC (Related to A and C kinase) .
• PKA cyclic AMP-dependent protein kinase
• PKC protein kinase C
• PI 3-kinase the enzyme which converts PI 4,5 bisphosphate (PIP2) to the putative second messenger PI 3,4,5 trisphosphate (PIP3)
• PKB lies downstream of PI 3-kinase
• PKB ⁇ is converted from an inactive to an active form with a half time of about one minute when cells are stimulated with PDGF (Franke et al., 1995), EGF or basic FGF (Burgering & Coffer, 1995) or insulin (Cross et al., 1995 and Kohn et al., 1995) or perpervanadate (Andjelkovic et al., 1996).
• PDGF Factort al., 1995
• EGF or basic FGF Bovine growth factor
• insulin Cross et al., 1995 and Kohn et al., 1995
• perpervanadate Andjelkovic et al., 1996.
• Activation of PKB by insulin or growth factors is prevented if the cells are preincubated with inhibitors of PI 3-kinase (wortmannin or LY 294002) or by overexpression of a dominant negative mutant of PI 3-kinase (Burgering & Coffer 1995) .
• the present invention thus provides the use of PKB, its analogues, isoforms, inhibitors, activators and/or the functional equivalents thereof to regulate glycogen metabolism and/or protein synthesis, in particular in disease states where glycogen metabolism and/or protein synthesis exhibits abnormality, for example in the treatment of type II diabetes; also in the treatment of cancer, such as ovarian, breast and pancreatic cancer.
• a composition comprising such agents is also covered by the present invention, and the use of such a composition for treatment of disease states where glycogen metabolism and/or protein synthesis exhibit abnormality.
• the present invention also provides a novel peptide comprising the amino acid sequence Arg-Xaa-Arg-Yaa-Zaa- Ser/Thr-Hyd, where Xaa is any amino acid, Yaa and Zaa are any amino acid (preferably not glycine) , and Hyd is a large hydrophobic residue such as Phe or Leu, or a functional equivalent thereof.
• a suitable peptide would be RXRX'X'S/TF/L, where X' can be any amino acid, but is preferably not glycine; glycine can in fact be used, but other amino acids are preferred.
• Typical peptides include GRPRTSSFAEG, RPRAATC or functional equivalents thereof.
• the peptide is a substrate for measuring PKB activity.
• the invention also provides a method for screening for substances which inhibit the activation of PKB in vivo by preventing its interaction with PIP3 or PI3,4-bisP.
• the invention also provides a method of determining the ability of a substance to affect the activity or activation of PKB, the method comprising exposing the substance to PKB and phosphatidyl inositol polyphosphate (ie PIP3 , PI3,4-bisP etc) and determining the interaction between PKB and the phosphatidyl inositol polyphosphate.
• PKB and phosphatidyl inositol polyphosphate ie PIP3 , PI3,4-bisP etc
• the interaction between PKB and the phosphatidyl inositol polyphosphate can conveniently be measured by assessing the phosphorylation state of PKB (preferably at T308 and/or S473) , eg by measuring transfer of radiolabelled 3 P from the PIP3 (for example) to the PKB and/or by SDS- PAGE.
• the method of the invention can also be used for identifying activators or inhibitors of GSK3 , such a method can comprise exposing the substance to be tested to GSK3, and (optionally) a source of phosphorylation, and determining the state of activation of GSK3 (optionally by determining the state of its phosphorylation.
• This aspect of the invention can be useful for determining the suitability of a test substance for use in combatting diabetes, cancer, or any disorder which involves irregularity of protein synthesis or glycogen metabolism.
• the invention also provides a method for screening for inhibitors or activators of enzymes that catalyse the phosphorylation of PKB, the method comprising exposing the substance to be tested to - one or more enzymes upstream of PKB; - PKB; and (optionally) - nucleoside triphosphate and determining whether (and optionally to what extent) the PKB has been phosphorylated on T308 and/or S473.
• test substance may be an analogue, isoform, inhibitor, or activator of PKB, and the above method may be modified to identify those agents which stimulate or inhibit PKB itself.
• a method may comprise the following steps:
• the method of the invention can be used to determine whether the substance being tested acts on PKB or directly on GSK3. This can be done by comparing the phosphorylation states of the peptide and PKB; if the phosphorylation state of GSK3 is changed but that of PKB is not then the substance being tested acts directly on GSK3 without acting on PKB.
• the present invention provides a method of treatment of the human or non-human (preferably mammalian) animal body, said method comprising administering PKB, its analogues, inhibitors, stimulators or functional equivalents thereof to said body. Said method affects the regulation of glycogen metabolism in the treated body.
• the method of treatment of the present invention may be of particular use in the treatment of type II diabetes (where desirably an activator of PKB is used, so that the down-regulation of GSK3 activity due to the action of PKB is enhanced) .
• the method of treatment of the present invention may alternatively be of particular use in the treatment of cancer such as ovarian cancer (where desirably an inhibitor of PKB is used, so that the down-regulation of GSK3 activity due to the action of PKB is depressed) .
• cancers such as ovarian cancer (where desirably an inhibitor of PKB is used, so that the down-regulation of GSK3 activity due to the action of PKB is depressed) .
• Other cancers associated with irregularities in the activity of PKB and/or GSK3 may also be treated by the method, such as pancreatic cancer, and breast cancer.
• PKB transiently transfected into cells can be activated 20-fold in response to insulin and 46-fold in response to IGF-1 and also became phosphorylated at Thr-308 and Ser-473.
• the activation of PKB and its phosphorylation at both Thr-308 and Ser-473 can be prevented by the phosphatidylinositol (Pl) 3-kinase inhibitor wortmannin.
• Pl phosphatidylinositol
• MAPKAP kinase-2-phosphorylated PKB at Ser-473 in vitro increases activity seven-fold, an effect that can be mimicked (fivefold activation) by mutating Ser-473 to Asp.
• Mutation of Thr-308 to Asp also increases PKB activity five-fold and subsequent phosphorylation of Ser-473 by MAPKAP kinase-2 stimulates activity a further fivefold, an effect mimicked (18-fold activation) by mutating both Thr-308 and Ser-473 to Asp.
• the activity of the Asp-308/Asp-473 double mutant was similar to that of the fully phosphorylated enzyme and could not be activated further by insulin.
• Mutation of Thr-308 to Ala did not prevent the phosphorylation of transfected PKB at Ser-473 after stimulation of 293 cells with insulin or IGF-1, but abolished the activation of PKB.
• mutation of Ser-473 to Ala did not prevent the phosphorylation of transfected PKB at Thr-308 but greatly reduced the activation of transfected PKB. This demonstrates that the activation of PKB by insulin or IGF-1 results from the phosphorylation of Thr-308 and Ser-473 and that phosphorylation of both residues is preferred to generate a high level of PKB activity in vitro or in vivo.
• phosphorylation of Thr-308 in vivo is not dependent on the phosphorylation of Ser-473 or vice versa, that the phosphorylation of Thr-308 and Ser-473 are both dependent on PI 3-kinase activity and suggest that neither Thr-308 nor Ser-473 phosphorylation is catalysed by PKB itself.
• the present invention incorporates the use of any agent which affects phosphorylation of PKB at amino acids 308 and/or 473, for example insulin, inhibitors of PI 3-kinase such as wortmannin or the like.
• agent which affects phosphorylation of PKB at amino acids 308 and/or 473 for example insulin, inhibitors of PI 3-kinase such as wortmannin or the like.
• PKB itself altered at amino acids 308 and/or 473 (eg by phosphorylation and/or mutation) is also suitable.
• stimulation or inhibition of PKB may be assessed by monitoring the phosphorylation states of amino acids 308 and/or 473 on PKB itself.
• PKB may be used or targeted in the present invention; for example PKB ⁇ , ⁇ or ⁇ .
• Fig 1 L6 myotubes were incubated for 15 min with 2 mM 8-bromocyclic-AMP (8Br-cAMP) and then with 0.1 ⁇ M insulin (5 min) .
• L6 myotubes were stimulated with insulin for the times indicated with (filled triangle) or without (filled circles) a 15 min preincubation with LY 294002, and GSK3 measured as in a.
• the open circles show experiments from insulin-stimulated cells where GSK3 was assayed after reactivation with PP2A (Cross et al., 1994) .
• rapamycin triangles
• rapamycin plus PD 98059 circles
• GSK3 activity measured as in a, before (filled symbols) and after (open symbols) pretreatment with PP2A.
• FIG. 2 Identification of PKB as the insulin- stimulated .
• a. Cells were incubated with 50 ⁇ M PD 98059 (for 1 hour) and 0.1 ⁇ M rapamycin (10 min), then stimulated with 0.1 ⁇ M insulin (5 min) and lysed (Cross et al., 1994) .
• the lysates (0.3 g protein) were chromatographed on Mono Q (5 x 0.16cm) and fractions (0.05ml) were assayed for Crosstide kinase (filled circles) .
• insulin was omitted (open circles) or wortmannin (0.1 ⁇ M) added 10 min before the insulin (filled triangles) .
• the broken line shows the NaCl gradient. Similar results were obtained in six experiments.
• L6 myotubes were stimulated with 0.1 ⁇ M insulin and PKB immunoprecipitated from the lysates (50 ⁇ g protein) essentially as described previously (Lazar et al., 1995) , using the anti-PH domain antibody and assayed for Crosstide kinase (open circles) .
• myotubes were incubated with 0.1 ⁇ M rapamycin plus 50 ⁇ M PD 98059 (open triangles) or 2 mM 8Br-cAMP (open squares) , or 0.1 ⁇ M wortmannin (filled circles) or 100 ⁇ M LY 294002 (filled triangles) before stimulation with insulin.
• Mono Q chromatography was performed as described (Burgering et al., 1995) , except that the buffer also contained 1 mM EGTA, 0.1 M sodium orthovanadate and 0.5% (w/v) Triton X-100.
• Two PKB ⁇ antibodies were raised in rabbits against the C-terminal peptide FPQFSYSASSTA and bacterially expressed PH domain of PKB ⁇ .
• the C-terminal antibody was affinity purified (Jones et al. , 1991) .
• the activity of PKB towards Crosstide is threefold higher than its activity towards histone H2B and 11-fold higher than its activity towards myelin basic protein, the substrates used previously to assay PKB.
• FIG. 3 GSK3 is inactivated by PKB from insulin- stimulated L6 myotubes.
• a Cells were stimulated for 5 min with 0.1 ⁇ M insulin, and PKB immunoprecipitated from 100 ⁇ g of cell lysate and used to inactivate GSK3 isoforms essentially as described previously (Sutherland et al., 1993 and Sutherland et al., 1994).
• the black bars show GSK3 activity measured after incubation with MgATP and PKB as a percentage of the activity obtained in control incubations where PKB was omitted. In the absence of PKB, GSK3 activity was stable throughout the experiment.
• the white bars show the activity obtained after reactivation of GSK3 with PP2A (E bi et al., 1980) .
• the results ( ⁇ s.e.m.) are for three experiments (each carried out in triplicate) .
• FIG. 4 Identification of the residues in GSK3 phosphorylated bv PKB in vitro and in response to insulin in 6 myotubes.
• GSK3-/3 was maximally inactivated by incubation with PKB and Mg-[ 7 - 32 P]ATP and after SDS-PAGE, the 32 P- labelled GSK3-/3 (M r 47K) was digested with trypsin 11 and chromatographed on a C 18 column (Sutherland et al., 1993) . Fractions (0.8 ml) were analysed for 32 P- radioactivity (open circles) , and the diagonal line shows the acetonitrile gradient.
• GSK3- ⁇ and GSK3-/3 were co-immunoprecipitated from the lysates of 32 P-labelled cells, denatured in SDS, subjected to SDS-PAGE, transferred to nitrocellulose and autoradiographed (Saito et al., 1994) .
• GSK3 isoforms from c. were digested with trypsin, and the resulting phosphopeptides separated by isoelectric focusing (Saito et al., 1994) and identified by auto-radiography.
• Lanes 1 and 4 show the major phosphopeptide resulting from in vi tro phosphorylation of GSK3-0 by PKB and MAPKAP kinase-1, respectively; lanes 2 and 5, the phosphopeptides obtained from GSK3-/3 and GSK3- ⁇ , immunoprecipitated from unstimulated cells; lanes 3 and 6, the phosphopeptides obtained from GSK3-/3 and GSK3- ⁇ immunoprecipitated from cells stimulated for 5 min with 0.1 ⁇ M insulin; the arrow denotes the peptides whose phosphorylation is increased by insulin.
• the pi values of two markers, Patent Blue (2.4) and azurin (5.7) are indicated.
• the protein kinases PKB- ⁇ , PKB-3 and PKB ⁇ are Ser/Thr- specific and cellular homologues of the viral oncogene v-akt (Coffer et al., 1991, Jones et al 1991, Ahmed et al 1995 and Cheng et al., 1992) . These enzymes have recently been shown to be activated in NIH 3T3 , Rat-1 or Swiss 3T3 cells in response to growth factors or insulin, activation being suppressed by blocking the activation of PI 3-kinase in different ways (Franke et al., 1995 and Burgering et al., 1995).
• Insulin stimulation of L6 myotubes increased PKB activity by more than tenfold (Fig 2c) , and activation was blocked by wortmannin or LY 294002, but was essentially unaffected by 8-bromo-cyclic AMP or rapamycin plus PD 98059 (Fig 2c) .
• the half-time (t 05 ) or activation of PKB (1 min) was slightly faster than that for inhibition of GSK3 (2 min) (Cross et al., 1994) .
• the activation of MAPKAP kinase-1 (Fig 2d) and p70 s6k was slower (t 05 > 5 min) .
• MAPKAP kinase-1 Activation of MAPKAP kinase-1 was prevented by 8-bromo- cyclic AMP or PD 98059 (Fig 2d) , and activation of p70 s6k by rapamycin (Cross et al., 1994) .
• Akt/RAC phosphorylated the Ser in the Crosstide equivalent to Ser 21 in GSK3- ⁇ and Ser 9 in GSK3-3 (data not shown).
• PKC-5, ⁇ and ⁇ are reported to be activated by mitogens, and PKC-f activity is stimulated in vitro by several inositol phospholipids, including PI (3,4, 5)P 3 the product of the PI 3-kinase reaction (Andjelkovic et al., 1995) .
• PI (3,4, 5)P 3 the product of the PI 3-kinase reaction
• GSK3- ⁇ is unaffected, while their downregulation in L6 myotubes by prolonged incubation with phorbol esters abolishes the activation of MAPKAP kinase-l in response to subsequent challenge with phorbol esters, but has no effect on the inhibition of GSK3 by insulin (not shown) .
• Insulin induces the activation and phosphorylation of PKB ⁇ in L6 myotubes.
• Three 10 cm dishes of L6 myotubes were 32 P-labelled and treated for 10 min with or without 100 nM wortmannin and then for 5 min with or without 100 nM insulin.
• PKB ⁇ was immunoprecipitated from the lysates and an aliquot (15%) assayed for PKB ⁇ activity (Fig 5A) .
• the activities are plotted + SEM for 3 experiments relative to PKB ⁇ derived from unstimulated cells which was 10 mU/mg.
• the remaining 85% of the immunoprecipitated PKB ⁇ was alkylated with 4-vinylpyridine, electrophoresed on a 10% polyacrylamide gel (prepared without SDS to enhance the phosphorylation-induced decrease in mobility) and autoradiographed.
• the positions of the molecular mass markers glycogen phosphorylase (97 kDa) , bovine serum albumin (66 kDa) and ovalbumin (43 kDa) are marked.
• FIG. 6 Insulin stimulation of L ⁇ myotubes induces the phosphorylation of two peptides in PKB ⁇ .
• Bands corresponding to 32 P-labelled PKB ⁇ from Fig 5B were excised from the gel, treated with 4-vinylpyridine to alkylate cysteine residues, digested with trypsin and chromatographed on a Vydac 218TP54 C18 column (Separations Group, Hesperia, CA) equilibrated with 0.1% (by vol) trifluoroacetic acid (TFA) , and the columns developed with a linear acetonitrile gradient (diagonal line) .
• TFA trifluoroacetic acid
• the flow rate was 0.8 ml / min and fractions of 0.4 ml were collected (A) tryptic peptide map of 32 P-labelled PKB ⁇ from unstimulated L6 myotubes; (B) tryptic peptide map of 32 P-labelled PKB ⁇ from insulin-stimulated L6 myotubes; (C) tryptic peptide map of 32 P-labelled PKB ⁇ from L6 myotubes treated with wortmannin prior to insulin.
• Peptide A and Peptide B The two major 32 P-labelled peptides eluting at 23.7% and 28% acetonitrile are named Peptide A and Peptide B, respectively. Similar results were obtained in four (A and B) and two (C) experiments.
• Fig 7. Identification of the phosphorylation sites in peptides A and B.
• Peptides A and B from Fig5B (lOOOcpm) were incubated for 90min at 110°C in 6M HCl, electrophoresed on thin layer cellulose at pH 3.5 to resolve orthophosphate (Pi) , phosphoserine (pS) , phosphthreonine (pT) and phosphotyrosine (pY) and autoradiographed.
• Peptide A was phosphorylated predominantly on serine while peptide B was labelled on threonine (Fig 7A) .
• Amino acid sequencing established that peptide A commenced at residue 465. Only a single burst of 32 P-radioactivity was observed after the eighth cycle of Edman degradation (Fig 7B) , demonstrating that insulin stimulation of L6 myotubes had triggered the phosphorylation of PKB ⁇ at Ser-473, which is located 9 residues from the C-terminus of the protein.
• Phosphopeptide B was only recovered in significant amounts if 32 P-labelled PKB ⁇ was treated with 4-vinylpyridine prior to digestion with trypsin, indicating that this peptide contained a cysteine residue(s) , and a single burst of 32p radioactivity was observed after the first cycle of Edman degradation (Fig 7C) .
• Fig 8 Mapping the phosphorylation sites of PKB ⁇ in transiently transfected 293 cells.
• 293 cells were transiently transfected with DNA constructs expressing wild type PKB ⁇ , or a haemagglutonin epitope-tagged PKB ⁇ encoding the human protein, such as HA-KD PKB ⁇ , HA-473A PKB ⁇ or HA-308A PKB ⁇ . After treatment for 10 min with or without 100 nM wortmannin, the cells were stimulated for 10 min with or without 100 nM insulin or 50 ng/ml IGF- 1 in the continued presence of wortmannin.
• PKB ⁇ was immunoprecipitated from the lysates and assayed, and activities corrected for the relative levels of expression of each HA-PKB ⁇ . The results are expressed relative to the specific activity of wild type HA-PKB ⁇ from unstimulated 293 cells (2.5 + 0.5 U/mg).
• B 20 ⁇ g of protein from each lysate was electrophoresed on a 10 % SDS/polyacrylamide gel and immunoblotted using monoclonal HA-antibody. The molecular markers are those used in Fig 5B.
• Fig 9 IGF-i stimulation of 293 cells induces the phosphorylation of two peptides in transfected HA-PKB ⁇ .
• 293 cells transiently transfected with wild type HAPKB ⁇ DNA constructs were 32 P-labelled, treated for 10 min without (A,B) or with (C) 100 nM wortmannin and then for 10 min without (A) or with (B, C) 50 ng/ml IGF-1.
• the 32p labelled HA-PKB ⁇ was immunoprecipitated from the lysates, treated with 4-vinylpyridine, electrophoresed on a 10% polyacrylamide gel, excised from the gel and digested with trypsin. Subsequent chromatography on a C 18 column resolved four major phosphopeptides termed C, D, E and F. Similar results were obtained in 6 separate experiments for (A) and (B) , and in two experiments for (C) .
• Peptide C usually eluted as a doublet at 20-21% acetonitrile while the other, termed Peptide F, eluted at 29.7% acetonitrile.
• Peptides C, D, E and F were further purified by re- chromatography on the C18 column at pH 6.5 and sequenced. Peptides C gave rise to three separate (but closely eluting) 32 P-labelled peptides (data not shown) . Amino acid sequencing revealed that all three commenced at residue 122 of PKB ⁇ and that Ser-124 was the site of phosphorylation (Fig 10A) . Peptide D only contained phosphoserine and, as expected, corresponded to the PKB ⁇ tryptic peptide commencing at residue 465 that was phosphorylated at Ser-473 (Fig 10B) .
• Peptide E only contained phosphothreonine and amino acid sequencing demonstrated that it corresponded to residues 308-325, the phosphorylation site being Thr-308 (Fig 10C) .
• Peptide F only contained phosphothreonine and corresponded to the peptide commencing at residue 437 of PKB ⁇ phosphorylated at Thr-450 (Fig 10D) .
• PKB ⁇ In the presence of phosphatidylserine, PKB ⁇ binds to PIP3 with sub icromolar affinity (James et al., 1996, Freeh et al.,1996) . Phosphatidyl , 5-bisphosphate and phosphatidyl 3,4 bisphosphate bind to PKB ⁇ with lower affinities and PI 3,5 bisphosphate and PI 3 phosphate did not bind at all under these conditions (James et al., 1996) .
• the region of PKB ⁇ that interacts with PIP3 is almost certainly the PH domain, because the isolated PH domain binds PIP3 with similar affinity to PKB ⁇ itself (Freeh et al., 1996) and because the PH domain of several other proteins, such as the PH-domains of, -spectrin and phospholipase Cl, are known to interact specifically with other phosphoinositides (Hyvonen et al., 1995 and Lem on et al., 1995).
• Example 3 MAPKAP kinase-2 phosphorylates Ser-473 of PKB ⁇ causing partial activation.
• Ser-473 of PKBa lies in a consensus sequence Phe-x-x-Phe/Tyr-Ser/Thr-Phe/Tyr found to be conserved in a number of protein kinases that participate in signal transduction pathways (Pearson et al. 1995).
• Ser-473 kinase(s) we therefore chromatographed rabbit skeletal muscle extracts on CM-Sephadex, and assayed the fractions for protein kinases capable of phosphorylating a synthetic peptide corresponding to residues 465 to 478 of PKB ⁇ .
• the Ser-473 kinase continued to cofractionate with MAPKAPkinase-2 through pheny1-Sepharose, heparin-Sepharose, Mono S and Mono Q and was immunoprecipitated quantitatively by an anti-MAPKAP kinase-2 antibody (Gould et al, 1995) demonstrating that MAPKAP kinase-2 was indeed the Ser-473 kinase we had purified.
• HA-PKB ⁇ was immunoprecipitated from the lysates of unstimulated COS-1 cells expressing these constructs.
• HA-PKB ⁇ phosphorylated for 1 h with MAPKAP kinase-2 and 32 P- ⁇ -ATP as in (A) was digested with trypsin and chromatographed on a C18 column as described in the legend for Fig 2.
• C The major 32 P-labelled peptide from (B) was analysed on the 470A sequencer as in Fig 3 to identify the site of phosphorylation.
• HA-PKB ⁇ that had been maximally phosphorylated with MAPKAP kinase-2 was digested with trypsin and C18 chromatography revealed a single major 32 P-labelled phosphoserine-containing peptide (Fig 11B) .
• This peptide eluted at the same acetonitrile concentration (Fig 11B) and had the same isoelectric point of 7.2 (data not shown) as the 32p labelled tryptic peptide containing Ser-473 (compare Figs 11B and 6B) .
• Solid phase sequencing gave a burst of 32 P-radioactivity after the eighth cycle of Edman degradation (Fig 11C) , establishing that Ser-473 was the site of phosphorylation.
• the same 32 P-peptide was obtained following tryptic digestion of catalytically inactive HA-KD PKB ⁇ that had been phosphorylated with MAPKAP kinase-2 (data not shown) .
• Example 4 Phosphorylation of Thr-308 and ser-473 causes synergistic activation of PKB ⁇ .
• the experiments described above demonstrated that phosphorylation of Ser-473 activates PKB ⁇ in vitro but did not address the role of phosphorylation of Thr-308, or how phosphorylation of Thr-308 might influence the effect of Ser-473 phosphorylation on activity, or vice versa.
• HA hae agglutonin
• HA-PKB ⁇ mutants in vitro by MAPKAP kinase-2.
• A Wild type and mutant HA-PKB ⁇ proteins were immunoprecipitated from the lysates of unstimulated COS-1 cells expressing these constructs and incubated for 60 min with MgATP in the absence (-, filled bars) or presence (+, hatched bars) of MAPKAP kinase-2 and MgATP (50 U/ml) .
• the PKB ⁇ protein was expressed as similar levels in each construct and specific activities are presented relative to wild type HA-PKB ⁇ incubated in the absence of MAPKAP kinase-2 (0.03 U/mg) . The results are shown as the average + SEM for 3 experiments.
• B 20 ⁇ g of protein from each lysate was electrophoresed on a 10 % SDS/polyacrylamide gel and immunoblotted using monoclonal HA-antibody.
• HA-473D PKB ⁇ was five-fold to six-fold higher than that of unstimulated wild type HAPKB ⁇ protein, and similar to that of wild-type HA-PKB ⁇ phosphorylated at Ser-473.
• HA-473D PKB ⁇ was also not activated further by incubation with MAPKAP kinase-2 and MgATP.
• the activity of HA-308A PKB ⁇ was about 40% that of the unstimulated wild type enzyme, and after phosphorylation with MAPKAP kinase-2 is activity increased to a level similar to that of wild type HA-PKB ⁇ phosphorylated at Ser-473.
• HA-308D PKB ⁇ which (like HA-473D PK) was five-fold more active than dephosphorylated wild type HA-PKB ⁇ , was activated dramatically by phosphorylation of Ser-473.
• MAPKAP kinase-2 and MgATP the activity of HA-308D PKB ⁇ was nearly five-fold higher than that of wild type HA-PKB ⁇ phosphorylated at Ser-473 (Fig 12B) .
• Example 5 Phosphorylation of both Thr-308 and Ser-473 is required for a high level of activation of PKB ⁇ in vivo.
• 293 cells were transiently transfected with DNA constructs expressing wild type PKBa, HA-D473- PKB ⁇ , and HA-308D/473D-PKB ⁇ . After treatment for 10 min with or without 100 nM wortmannin, cells were stimulated for 10 min with or without 100 nM insulin in the continued presence of wortmannin.
• PKB ⁇ was immunoprecipitated from the lysates and assayed, and activities corrected for the relative levels of HA-PKB ⁇ expression as described in the methods. The results are expressed relative to the specific activity of wild type HA-PKB ⁇ obtained from unstimulated 293 cells.
• HA-308A PKB ⁇ The basal activity of HA-308A PKB ⁇ in unstimulated cells was also similar to that of wild type HA-PKB ⁇ derived from unstimulated cells, but virtually no activation of this mutant occurred following stimulation of the cells with insulin or IGF-1.
• These data are consistent with in vitro experiments and indicate that maximal activation of PKB ⁇ requires phosphorylation of both Ser-473 and Thr-308 and that phosphorylation of both residues results in a synergistic activation of the enzyme. Consistent with these results, HA-473D PKB ⁇ displayed five-fold higher activity and the HA-308D/HA473D double mutant 40-fold higher activity than wild type HA-PKB ⁇ when expressed in unstimulated cells.
• HA-473D PKB ⁇ was activated to a level similar to that observed with the wildtype enzyme, while the HA-308D/HA-473D double mutant could not be activated further (Fig 13) .
• activation of HA-473D PKB ⁇ by insulin was prevented by wortmannin, and the activity of the HA-308D/ HA-473D double mutant was resistant to wortmannin (Fig 13) .
• Example 6 Phosphorylation of Thr-308 is not dependent on phosphorylation of Ser-473 or vice versa (in 293 cells) .
• Fig 10 A 10 cm dish of 293 cells were transfected with either HA-308A PKB ⁇ or HA-473A PKB ⁇ , 2 P-labelled, then stimulated for 10 min with either IGF-1 (50 ng/ml) or buffer.
• the 32 P-labelled PKB ⁇ mutants were immunoprecipitated from the lysates, treated with 4-vinylpyridine, electrophoresed on a 10% polyacrylamide gel, excised from the gel and digested with trypsin, then chromatographed on a C18 column.
• the tryptic peptides containing the phosphorylated residues Ser-124, Thr-308, Thr-450, Ser-473 are marked and their assignments were confirmed by phosphoamino acid analysis and sequencing to identify the sites of phosphorylation (data not shown) .
• the phosphopeptides containing Thr-308 and Ser-473 were absent if stimulation with IGF-1 was omitted, while the phosphopeptides containing Ser-124 and Thr-450 were present at similar levels as observed with wild-type PKB ⁇ (see Fig 9A) . Similar results were obtained in 3 separate experiments.
• Example 7 IGF-1 or insulin induces phosphorylation of Thr-308 and Ser-473 in a catalytically inactive mutant of PKB ⁇ .
• Fig 15. The catalytically inactive PKB ⁇ mutant (HA-KD-PKB ⁇ ) expressed in 293 cells is phosphorylated at Thr-308 and Ser-473 after stimulation with IGF-1.
• the 2 P-labelled HA-KD-PKB ⁇ was immunoprecipitated from the lysates, treated with 4 vinylpyridine, electrophoresed on a 10% polyacrylamide gel, excised from the gel and digested with trypsin, then chromatographed on a C18 column.
• the tryptic peptides containing the phosphorylated residues Ser-124, Thr-308, Thr-450 and Ser-473 are marked. Similar results were obtained in 3 separate experiments for (A) and (B) , and in two experiments for (C) .
• HA-KD-PKB ⁇ This kinase dead mutant of PKB ⁇ , termed HA-KD-PKB ⁇ , in which Lys-179 was changed to Ala (see above) was transiently expressed in 293 cells and its level of expression found to be several-fold lower than that of wild type HA-PKB ⁇ expressed under identical conditions (Fig 8B) .
• Fig 8B As expected, no PKB ⁇ activity was detected when 293 cells expressing HA-KD-PKB ⁇ , were stimulated with insulin or IGF-1 (Fig 7A) .
• HA-KD-PKB ⁇ 293 cells that had been transiently transfected with HA-KD-PKB ⁇ were 32 P-labelled, then stimulated with buffer, insulin or IGF-1. and sites on PKB ⁇ phosphorylated under these conditions were mapped.
• HA-KD PKB ⁇ was phosphorylated to a much lower level at Ser-124, but phosphorylated similarly at Thr-450 (Fig 15A) .
• HA-KD-PKB ⁇ Following stimulation with IGF-1 (Fig 15B) or insulin (Fig 14C) HA-KD-PKB ⁇ became phosphorylated at the peptides containing Thr-308 and Ser-473, the extent of phosphorylation of these sites being at least as high as wild type PKB ⁇ . Amino acid sequencing of these peptides established that they were phosphorylated at Thr-308 and Ser-473, respectively.
• Thr-308 is conserved in rat PKB/3 and PKB7 but, interestingly, Ser-473 (and the sequence surrounding it) is only conserved in PKB ⁇ . In rat PKB7, Ser-473 is missing because the C-terminal 23 residues are deleted. This suggests that the regulation of PKB7 may differ significantly from that of PKB ⁇ and PKBjS in the rat.
• Thr-308 is located in subdomain VIII of the kinase catalytic domain, nine residues upstream of the conserved Ala-Pro-Glu motif, the same position as activating phosphorylation sites found in many other protein kinases.
• Ser-473 is located C-terminal to the catalytic domain in the consensus sequence Phe-Xaa-Xaa-Phe/Tyr-Ser/Thr-Phe/Tyr which is present in several protein kinases that participate in growth factor-stimulated kinase cascades, such as p70 S6 kinase, PKC and p90rsk (Pearson et al, 1995) .
• MAPKAP kinase-2 is a component of a protein kinase cascade which becomes activated when cells are stimulated with interleukin-1 or tumour necrosis factor or exposed cellular stresses (Rouse et al, 1994; Cuenda et al, 1995) .
• MAPKAP kinase-2 phosphorylates PKB ⁇ stoichiometrically at Ser-473 (Fig 11) and this finding was useful in establishing the role of Ser473 phosphorylation in regulating PKB ⁇ activity.
• MAPKAP kinase-2 activity is stimulated to a small extent by insulin in L6 cells, no activation could be detected in 293 cells in response to insulin or IGF-1.
• MAPKAP kinase-2 (D. Alessi, unpublished work) as found in other cells (Rouse et al, 1994; Cuenda et al, 1995), but did not activate PKB ⁇ at all.
• the drug SB 203580 which is a specific inhibitor of the protein kinase positioned immediately upstream of MAPKAP kinase-2 (Cuenda et al, 1995) , prevented the activation of MAPKAP kinase-2 by arsenite but had no effect on the activation of PKB ⁇ by insulin or IGF-1.
• the Km for phosphorylation of the synthetic peptide comprising residues 465-478 is nearly 100-fold higher than the Km for the standard MAPKAP kinase-2 substrate peptide (data not shown) . It is therefore unlikely that MAPKAP kinase-2 mediates the phosphorylation of Ser-473 in vivo.
• the enzyme(s) which phosphorylates Thr-308 and Ser-473 in vivo does not appear to be PKB ⁇ itself.
• incubation of the partially active Asp-308 mutant with MgATP did not result in the phosphorylation of Ser-473, phosphorylation of the latter residue only occurring when MAPKAP kinase-2 was added (Fig 11A, Fig 12) .
• Thr-308 did not become phosphorylated when either the partially active D473 mutant or the partially active Ser-473 phosphorylated form of PKB ⁇ were incubated with MgATP.
• Ser-124 is located in the linker region between the PH domain and the catalytic domain of the mammalian PKB ⁇ isoforms but, unlike Thr-450, is not conserved in the Drosophila homologue (Andjelkovic et al, 1995) .
• PIP3 or PI3,4-bisP may activate one or more protein kinases which then phosphorylate PKB ⁇ at Thr-308 and Ser-473.
• the formation of PIP3 may lead to the recruitment of PKB ⁇ to the plasma membrane where it is activated by a membrane-associated protein kinase(s) .
• Thr-308 and Ser-473 kinases might themselves be activated by PIP3 and the possibility that Thr-308 and/or Ser-473 are phosphorylated directly by PI 3-kinase has also not been excluded, because this enzyme is known to phosphorylate itself (Dhand et al, 1994) and other proteins (Lam et al, 1994) on serine residues.
• Example 8 Molecular basis for substrate specificity of PKB.
• PKB ⁇ has been shown to influence GSK3 activity.
• GSK3 ⁇ and GSK3/3 are phosphorylated at Ser-21 and Ser-9, respectively, by two other insulin-stimulated protein kinases, namely p70 S6 kinase and MAP kinase-activated protein kinase-1 (MAPKAP-K1, also known as p90 S6 kinase) .
• MAPKAP-K1 MAP kinase-activated protein kinase-1
• PI 3-kinase The activation of PI 3-kinase is essential for many of the effects of insulin and growth factors, including the stimulation of glucose transport, fatty acid synthesis and DNA synthesis, protection of cells against apoptosis and actin cytoskeletal rearrangements (reviewed in Carpenter et al., 1996) . These observations raise the question of whether PKB ⁇ mediates any of these events by phosphorylating other proteins. To address this issue we characterised the substrate specificity requirements of PKB ⁇ .
• IGF-1 stimulation resulted in a 38-fold activation of PKB ⁇ (Fig 16) and analysis of the immunoprecipitates by SDS-polyacrylamide gel electrophoresis revealed that the 60 kDa PKB ⁇ was the major protein staining with Coomassie Blue apart from the heavy and light chains of the haemagglutonin monoclonal antibody (Fig 16, Lanes 2 and 3) .
• the minor contaminants were present in control immunoprecipitates derived from 293 cells transfected with an empty pCMV5 vector but lacked HA-PKB activity (Fig 16, Lane 4) .
• HA- PKB ⁇ _immunoprecipitated from the lysates of IGF-1 stimulated 293 cells had no Crosstide kinase activity (Alessi et al., 1996) .
• all the Crosstide activity in HA-PKB immunoprecipitates is catalysed by PKB ⁇ .
• Ser-36 of histone H2B lies in an Arg-Xaa-Arg-Xaa-Xaa-Ser-Hyd motif (where Hyd is a bulky hydrophobic residue -Phe in Crosstide, Tyr in H2B) .
• MAPKAP-K1 and p70 S6 kinase phosphorylate the same residue in GSK3 phosphorylated by PKB ⁇
• studies with synthetic peptides have established that MAPKAP-K1 and p70 S6 kinase also preferentially phosphorylate peptides in which basic residues are present at positions n-3 and n-5 (Leighton et al., 1995)
• MAPKAP kinase-1 and p70 S6 kinase phosphorylate the peptides KKKNRTLSVA and KKRNRTLSVA with extremely low Km values of 0.2- 3.3 ⁇ M, respectively (Table 7.2) .
• these peptides were phosphorylated by PKB ⁇ with 50-900 fold higher Km values (Table 7.2A, peptides 1 and 2) .
• the peptide KKRNRTLTV which is a relatively specific substrate for p70 S6 kinase (Leighton et al., 1995) was also phosphorylated very poorly by PKB ⁇ (Table 7.2A, peptide 4) .
• Crosstide is phosphorylated by p70 S6 kinase and MAPKAP kinase-1 with similar efficiency to PKB ⁇ ((Leighton et al. , 1995); Table 7.2B-peptide 1 and Fig 18) .
• truncation of Crosstide to generate the peptide RPRTSSF was deleterious for phosphorylation by MAPKAP-K1 and even worse for p70 S6 kinase (Table 7.2B-peptides 1 and 2 and Fig 18) .
• This peptide was phosphorylated by PKB ⁇ with a Km of 25 ⁇ M and similar Vmax to RPRTSSF, but was not phosphorylated to a significant extent by either MAPKAP-Kl or p70 S6 kinase (Table 7.2B-peptide 5, Fig 18) .
• the protein kinase concentration in the assays towards Crosstide were 0.2 U/ml, and each peptide substrate was assayed at a concentration of 30 ⁇ M.
• Filled bars denote PKB ⁇ activity, hatched bars MAPKAP kinase-1 activity, and grey bars p70 S6 kinase activity.
• the activities of each protein kinase are given relative to their activity towards Crosstide (100) . The results are shown ⁇ SEM for two experiments each carried out in triplicate.
• Vmax for this peptide (250 nmoles min-1 mg-1) may be an underestimate because the PKB ⁇ was obtained by immunoprecipitation from extracts of IGF-1 stimulated 293 cells over-expressing this protein kinase, and a significant proportion of the PKB ⁇ may not have been activated by IGF-1 treatment.
• the peptide RPRAATF which was phosphorylated with a Km of 25 ⁇ M and similar Vmax to RPRTSSF, may therefore be a better substrate for assaying PKB ⁇ in partially purified preparations, because unlike Crosstide, it contains only one phosphorylatable residue and is not phosphorylated significantly by MAPKAP-Kl or p70 S6 kinase (Table 7.2, Fig 18 and see below).
• histone H2B the residue located four amino acids N-terminal to the PKB ⁇ phosphorylation site is serine (Fig 17) .
• the presence of Glu and Lys at positions n-l and n-2 may explain why histone H2B is phosphorylated by PKB ⁇ with a four-fold lower Vmax than the peptide RPRTSSF.
• MAPKAP- Kl plays a role in the inhibition of GSK3 by EGF because, unlike inhibition by insulin which is prevented by inhibitors of PI 3-kinase, the inhibition of GSK3 by EGF is only suppressed partially by inhibitors of PI 3-kinase. Moreover, in NIH 3T3 cells, the inhibition of GSK3 ⁇ and GSK3 ⁇ by EGF is largely prevented by expression of a dominant negative mutant of MAP kinase kinase-1 (Eldar et al., 1995).
• p70 S6 kinase is not rate limiting for the inhibition of GSK3 in the cells that have been examined so far because rapamycin, which prevents the activation of p70 S6 kinase by EGF or insulin, has no effect on the inhibition of GSK3 by these agonists (Cross et al., 1995 and Saito et al., 1994) .
• MAPKAP-Kl and (to a lesser extent) p70 S6 kinase can tolerate substitution of the Arg at position n-5 by lysine whereas PKB ⁇ cannot (see Table 7.1A, Table 7.2A and (Leighton et al., 1995)) .
• MAPKAP-Kl and p70 S6 kinase can also tolerate, to some extent, substitution of Arg at position n-3 by Lys.
• the peptide KKRNKTLSVA is phosphorylated by MAPKAP-Kl and p70 S6 kinase with Km values of 17 and 34 ⁇ M, respectively, as compared to Km values of 0.7 and 1.5 ⁇ M for the peptide KKRNRTLSVA (Table 7.2A) .
• PKB ⁇ does not phosphorylate the peptide KKRNKTLSVA (Table 7.2A) or any other peptide that lacks Arg at position n-3.
• Example 9 This example demonstrates that coexpression of GSK3 in 293 cells with either the wild type or a constitutively activated PKB results in GSK3 becoming phosphorylated and inactivated. However coexpression of a mutant of GSK3 in which Ser-9 is mutated to an Ala residue is not inactivated under these conditions. These experiments provide further evidence that PKBa activation can mediate the phosphorylation and inactivation of GSK3 in a cellular environment, and could be used as an assay system to search for specific PKB inhibitors.
• Monoclonal antibodies recognising the sequence EFMPME (EE) antibodies and the (EQKLISEEDL) c-Myc purchased from Boehringer (Lewis, UK) .
• HA-PKBa Construction of expression vectors and transfections into 293 cells.
• a DNA construct expressing human GSK3B with the EFMPME (EE) epitope tag at the N-terminus was prepared as follows: A standard PCR reaction was carried out using as a template the human GSK33 cDNA clone in the pBluescript SK+ vector and the oligonucleotides
• the c-Myc GSK3, BA9 construct encodes GSK3/3 in which Ser-9 is mutated to Ala and possesses a c-myc epitope tag at the C-terminus and was prepared as described in Sperber et al., 1995.
• the c-Myc GSK33 A9 gene was then subcloned into xba I/ECOR I sites of the pCMV5 eukaryotic expression vector. cotransfection of GSK3/3 with PKBa and its assay.
• the cells were grown in the absence of serum for 16 h prior to lysis, and then lysed in 1.0 ml of ice-cold Buffer A (50 mM Tris/HCl pH 7.5,1 mM EDTA 1 mM EGTA, 1% (by vol) Triton X100, 1 mM sodium orthopervanadate, 10 mM sodium glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, luM Microcystin-LR, 0.27 M sucrose, 1 mM benzamidine, 0.2 mM phenylmethylsulphonyl fluoride, 10 ug/ml leupeptin, and 0.1% (by vol) 2-mercaptoethanol ) .
• Buffer A 50 mM Tris/HCl pH 7.5,1 mM EDTA 1 mM EGTA, 1% (by vol) Triton X100, 1 mM sodium orthopervanadate,
• the lysate was centrifuged at 4°C for 10 min at 13, 000 x g and an aliquot of the supernatant (100 ug protein) was incubated for 30 min on a shaking platform with 5 ul of protein G-Sepharose coupled to lug of EE monoclonal antibody.
• the suspension was centrifuged for lmin at 13,000 x g, the Protein G-Sepharose-antibody-EE-GSK3
• Buffer B 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.01% (by vol) Brij-35 and 0.1% (by vol) 2-mercaptoethanol
• Example 10 basic assay for identifying agents which affect the activity of PKB.
• a 40 ⁇ l assay mix was prepared containing protein kinase (0.2U/ml) in 50 mM Tris/HCI pH 7.5, 0.1 mM EGTA, 0.1% (by vol) 2-mercaptoethanol, 2.5 ⁇ M PKI, protein kinase substrate (30 ⁇ M) , and the indicated concentration of Ro-318220 or GC 109203X (test inhibitors) . After incubation on ice for 10 min the reaction was started by the addition of 10 ⁇ l of 50mM magnesium acetate and 0.5 mM [7 3 P]ATP (100-200 cpm/pmol) .
• Protein kinase B an enzyme that is activated in response to insulin and growth factors was inhibited by Ro 318220 (IC S0 of 1 ⁇ M, Fig 2OB) similar to the IC 50 for PKA.
• Ro 318220 IC S0 of 1 ⁇ M, Fig 2OB
• Tissue culture reagents myelin basic protein (MBP), microcystin-LR, and IGF-1 were obtained from Life Technologies Inc. (Paisley, UK), insulin from Novo-Nordisk (Bagsvaerd, Denmark), phosphate free Dulbecco's minimal essential medium (DMEM) from (ICN, Oxon, UK), Protein G-Sepharose and CH-Sepharose from Pharmacia (Milton Keynes, UK), alkylated trvpsin from Promega (Southampton, UK), 4-vinylpyridine, wortmannin and fluroisothiocyanante-labelled antimou ⁇ e IgG from goat from Sigma-Aldrich (Poole, Dorset, UK).
• DMEM Dulbecco's minimal essential medium
• Polyclonal antibodies were raised in sheep against the peptides RPHFPQFSYSASGTA (corresponding to the last 15 residues of rodent PKB ⁇ ) and MTSALATMRVDYEQIK (corresponding to residues 352 to 367 of human MAPKAPkinase-2) and affinity purified on peptide-CH-Sepharose.
• Monoclonal HA antibodies were purified from the tissue culture medium of 12CA5 hybridoma and purified by chromatography on Protein GSepharose.
• the peptide RPRHFPQFSYSAS corresponding to residues 465-478 of PKB ⁇ , was synthesized on an Applied Biosystems 430A peptide synthesizer.
• cDNA encoding residues 46-400 of human MAPKAP kinase-2 was expressed in E.coli as a glutathione S-transferase fusion protein and activated with p38/RK MAP KINASE by Mr A.Clifton (University of Dundee) as described previously (Ben-Levy et al., 1995).
• Monoclonal antibodies recognising the haemagglutonin (HA) epitope sequence YPYDVPDYA, Protein G-Sepharose and histone H2B were obtained from Boehringer (Lewes, UK).
• MAPKAP kinase-1 (Sutherland et al., 1993) and p70 S6 kinases (Leighton et al., 1995) were purified from rabbit skeletal muscle and rat liver respectively.
• the pECE constructs encoding the human HAPKB ⁇ and kinase-dead (K179A) HA-KD-PKB ⁇ have already been described (Andjelkovic at al., 1996).
• the mutants at Ser-473 (HA-473A PKB ⁇ and HA-473D PKB ⁇ were created by PCR using a 5' oligonucleotide encoding amino acids 406 - 414 and mutating 3' oligonucleotide encoding amino acids 468 - 480, and the resulting PCR products subcloned as Cel ⁇ I-EcoRI fragment into pECE.HA-PKB ⁇ .
• Thr-308 mutants (HA-308A PKB ⁇ and HA308D PKB ⁇ ) were created by the two-stage PCR technique (No et al., 1989) and subcloned as Notl-EcoRI fragments into pECE.HA-PKB.
• the double mutant HA-308D/473D PKB was made by subcloning the Celll-EcoRI fragment encoding 473D into pECE.HA-308D PKB ⁇ .
• Bglll-Xbal fragments from the appropriate pECE constructs were subcloned into the same restriction sites of the pCMV5 vector (Andersson et al., 1989). All constructs were confirmed by restriction analysis and sequencing and purified using Quiagen Plasmid Maxi Kit according to the manufacturer's protocol. All oligonucleotide sequences are available upon request.
• L6 myotubes 2 P-labelling of L6 myotubes and iuiwunoprecipitation of PKB ⁇ .
• L6 cells were differentiated into myotubes on 10 cm diameter dishes (Hundal et al., 1992). The myotubes were deprived of serum overnight in DMEM, washed three times in phosphate free DMEM and incubated for a further 1 h with 5 ml of this medium. The myotubes were then washed twice with phosphate free DMEM and incubated for 4 h with carrier-free [ 32 P]orthophosphate (1 mCi/ml).
• the myotubes were stimulated for 5 min at 37°c in the presence or absence of 100 nM insulin and placed on ice, The medium was aspirated,the myotubes washed twice with ice-cold DMEM buffer and then lysed with 1.0 ml of ice-cold Buffer A (50 mM Tris/HCI pH 7.5,1 mM EDTA 1 mM EGTA, 1% (by vol) Triton X100, 1 mM sodium orthopervanadate, 10 mM sodium glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 1 ⁇ M Microcystin-LR, 0.27 M sucrose, 1 mM benzamidine, 0.2 mM phenylmethylsulphonyl fluoride, 10 ⁇ g/ml leupeptin, and 0.1% (by vol) 2-mercaptoethanol
• the lysates were centrifuged at 4°C for 10 min at 13,000 x g and the supernatants incubated for 30 min on a shaking platform with 50 ⁇ l of Protein G-Sepharose coupled to 50 ⁇ g of preimmune sheep IgG.
• the suspensions were centrifuged for 2 min at 13,000 x g and the supernatants incubated for 60 min with 30 ⁇ l of Protein G— Sepharose covalently coupled to 60 ⁇ g of PKB ⁇ antibody (Harlow and Lane, 1988).
• the Protein G-Sepharose-antibody-PKB ⁇ complex was washed eight times with 1.0 ml of Buffer A containing 0.5 M NaCl, and twice with 50 mM Tris/HCI pH 7.5, 0.1 mM EGTA and 0.1% (by vol) 2-mercaptoethanol (Buffer B).
• the 60 kDa band corresponding to rat PKB ⁇ was excised and the gel piece homogenized in five vols of 25 mM N-ethylmorpholine HCl, pH 7.7, containing 0.1% (by mass) SDS and 5 % (by vol) 2-mercaptoethanol.
• the suspension was incubated for 1 h at 37°C on a shaking platform, then centrifuged for 1 min at 13,000 x g and the supernatant collected.
• the pellet was incubated for a further lh with five vols of the same buffer and centrifuged for lmin at 13,000 xg.
• the two supernatants (containing 80-90% of the 32 P-radioactivity) were combined, 0.2 vols of 100% (by mass) trichloroacetic acid added, and the sample incubated for 1 h on ice.
• the suspension was centrifuged for 10 min at 13,000 x g, the supernatant discarded and the pellet washed five times with 0.2 ml of water.
• the pellet was then incubated at 30°C with 0.3 ml of 50 mM Tris/HCI pH 8.0, 0.1% (by vol) Triton X100 containing l ⁇ g of alkylated trypsin. After 3 h another l ⁇ g of trypsin was added and the suspension left for a further 12 h.
• Guanidinium hydrochloride (8 M) was added to bring the final concentration to 1.0 M in order to precipitate any residual SDS and, after standing on ice for 10 min, the suspension was centrifuged for 5 min at 13, 000 x g. The supernatant containing 90 % of the 32 P-radioactivity was chromatographed on a Vydac C18 column as described in the legend to Fig 2.
• the cells were placed in an atmosphere of 3% C02, for 16 h at 37°C, then the medium was aspirated, and replaced with fresh DMEM containing 10% foetal calf serum.
• the cells were incubated for 12 h at 37°C in an atmosphere of 5% C02,, and then for 12 h in DMEM in the absence of serum.
• Cells were preincubated for 10 min in the presence of 0.1% DMSO or 100 nM wortmannin in 0.1% DMSO and then stimulated for 10 min with either 100 nM insulin or 50 ng/ml IGF-1 in the continued presence of wortmannin.
• the cells were lysed in 1.0 ml of icecold Buffer A, the lysate was centrifuged at 4°C for 10 min at 13,000 x g and an aliquot of the supernatant (10 ⁇ g protein) was incubated for 60 min on a shaking platform with 5 ⁇ l of protein G-Sepharo ⁇ e coupled to 2 ⁇ g of HA monoclonal antibody.
• the suspension was centrifuged for 1 min at 13,000 x g, the Protein G-Sepharose-antibody-HA-PKB ⁇ complex washed twice with 1.0 ml of Buffer A containing 0.5 M NaCl, and twice with Buffer B, and the i munoprecipitate assayed for PKB ⁇ activity as described above.
• COS-1 cells were maintained in DMEM supplemented with 10% FCS at 370C in an atmosphere of 5% C02,.
• Cells at 70 - 80% confluency were transfected by a DEAE-dextran method (Seed & Aruffo, 1987), and 48 hours later serum-starved for 24 hours.
• Cells were lysed in a buffer containing 50 mM Tris-HCl, pH 7.5,120 M NaCl, 1% Nonidet P-40, 25 mM NaF, 40 mM sodium-, ⁇ -glycerophosphate, 0.1 mM sodium orthopervanadate, 1 mM EDTA, ImM benzamidine, 1 mM phenylmethylsulphonyl fluoride, and lysates centrifuged for 15 min at 13,000 x g at 4°C.
• TTYADFIASGRTGRRNAIHD the specific peptide inhibitor of cyclic AMP dependent protein kinase - PKI
• cytomegalovirus vectors pCMV5 of the human haemagglutonin epitope-tagged wild type _(HA-PKB ⁇ ) was described previously (Alessi et al., 1996). 293 cells grown on 10 cm dishes were transfected with a DNA construct expressing HA-PKB ⁇ using a modified calcium phosphate procedure (Alessi et al., 1996). The cells were deprived of serum for 16h prior to lysis and, where indicated, were stimulated for 10 min in the presence of 50 ng/ml IGF-1 to activate PKB ⁇ .
• the cells were lysed in 1.0 ml ice-cold Buffer A (50 mM Tris/HCI pH 7.5, 1 mM EDTA 1 mM EGTA, 1% (by vol) Triton X-100, 1 mM sodium orthovanadate, 10 mM sodium ⁇ -glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 1 ⁇ M Microcystin-LR, 0.27 M sucrose, 1 mM benzamidine, 0.2 mM phenylmethylsulphonyl fluoride, 10 ⁇ g/ml leupeptin, and 0.1 % (by vol) 2-mercaptoethanol) the lysate centrifuged at 4°C for 10 min at 13, 000 x g and the supernatant obtained from one 10 cm dish of cells (2-3 mg protein) was incubated for 60 min on a shaking platform with 20 ⁇ l of protein G-Sepharose coupled to 10 ⁇ g of
• the suspension was centrifuged for 1 min at 13, 000 x g, the Protein G-Sepharose-antibody-HA-PKB ⁇ complex washed twice with 1.0 ml of Buffer A containing 0.5 M NaCl, and twice with Buffer B (50 mM Tris/HCI pH 7.5, 0.1 mM EGTA, 0.01% (by vol) Brij-35 and 0.1% (by vol) 2-mercaptoethanol) .
• Buffer B 50 mM Tris/HCI pH 7.5, 0.1 mM EGTA, 0.01% (by vol) Brij-35 and 0.1% (by vol) 2-mercaptoethanol
• the PKB ⁇ immunoprecipitates were diluted in Buffer B to an activity of 2.0 U/ml towards the Crosstide peptide GRPRTSSFAEG and 0.1 ml aliquots snap frozen in liquid nitrogen and stored at -80 oC.
• the standard PKB ⁇ _assay contained: 50 mM Tris/HCI pH 7.5, 0.1 mM EGTA, 0.1% (by vol) 2-mercaptoethanol, 2.5 ⁇ M PKI, 0.2 U/ml PKB ⁇ , Crosstide (30 ⁇ M), 10 mM magnesium acetate and 0.1 mM ( ⁇ 32 P]ATP (100-200 cpm/p ol).
• the assays were carried out for 15 min at 30oC, the assay tubes being agitated continuously to keep the immunoprecipitate in suspension, then terminated and analysed as described (Alessi et al., 1995).
• One unit of activity was that amount of enzyme which catalysed the phosphorylation of 1 nmol of Crosstide in 1 min.
• the phosphorylation of other peptides, histone H2B and MBP were carried out in an identical manner.
• Histone H2B (30 ⁇ M) was phosphorylated with 0.2 U/ml HA-PKB ⁇ . After 60 min 0.2 vol of 100% (by mass) trichloroacetic acid was added, and the sample incubated for 1 h on ice. The suspension was centrifuged for 10 min at 13, 000 x g, the supernatant discarded and the pellet washed five times with 0.2 ml of ice cold acetone.
• the pellet was resuspended in 0.3 ml of 50 mM Tris/HCI pH 8.0, 0.1% (by vol) reduced Triton-XlOO containing 2 ⁇ g of alkylated trypsin and, after incubation for 16 h at 30oC, the digest was centrifuged for 5 min at 13, 000 x g. The supernatant, containing 95% of the 32 P-radioactivity, was chromatographed on a Vydac C18 column equilibrated with 0.1% (by vol) trifluoroacetic acid (TFA) in water.
• TFA trifluoroacetic acid
• the phosphorylated residue is shown in boldface type, the altered residue is underiined.V(100 ⁇ M) is the relative rate of phosphorylation at 0.1 mM peptide relative to peptide 1. ND , no t determined. *An aianine residue was added to the C-terminal of he peptide RPRTSSP , since we have experienced difficulty in synthesing peptides terminating in proline.
• RPRAGSF 49 114 70 Table J. Comparison of the substrate specificities of PKBn, MAPKAP kinase-1 , and p70S6 kinase.
• Peptides 1 and 2 are very good substrates for MAPKAP kinase- 1 and p70 S6 kinase, and peptide 3 is a relatively specific substrate for p70 S6 kinase [16]. * Data reported previously [16]; ND, not determined.
• KKRNRTLSVA 80 300 0.7* 1800* 1.5* 1520*
• Hyvonen M. , Macias, M.J., Nilges, M., Oschkinat, H. , Saraste, M. and Wilmanns, M. (1995) EMBO J.14, 4676-4685.

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