METHOD FOR SCREENING FOR SUBSTANCES WHICH ARE ACTIVATORS, INHIBITORS OR BINDERS OF PKB BY THE USE OF A SUBSTRATE PEPTIDE AND THE USE OF THE SUBSTRATE PEPTIDE

Abstract: The invention relates to a method for screening for substances which are activators or inhibitors of Protein kinase B (PKB) and can be used as a kinase substrate for PKB. The substrate is a peptide comprising the two sequences R-R-R-A-A/V-S-M and R-P/S-R-S/T-S/N-S-N and preferably comprising the three sequences R-P-R-S-C-T-W, R-R-R-A-A/V-S-M and R-P/S-R-S/T-S/N-S-N. These sequences as well as the human Afx, amino acids 12-505 or amino acids 1-505, can be used in assays measuring the activity of PKB, and in screening for substances which are activators or inhibitors of gene transcriptional regulation of forkhead proteins through the catalytic activities of PKB. These sequences can also be used for discrimination between the effects of compounds which mediate insulin action through transcription from those which modulate activity of enzymes involved in metabolism by phosphorylation.

Description: Method for screening for substances which are activators, inhibitors or binders of PKB by the use of a substrate peptide and the use of the substrate peptide Summary The present invention relates to a method for screening for substances which are activators or inhibitors of Protein kinase B (PKB) and can be used as a kinase substrate for PKB. The substrate is a peptide comprising the two sequences R-R-R-A-A/N-S-M and R-P/S-R-S/T-S/Ν-S-Ν and preferably comprising the three sequences R-P-R-S-C- T-W, R-R-R-A-A/V-S-M and R-P/S-R-S/T-S/Ν-S-Ν. These sequences as well as the human Afx , amino acids 12-505 or amino acids 1-505, can be used in assays measuring the activity of PKB, and in screening for substances which are activators or inhibitors of gene transcriptional regulation of forkhead proteins through the catalytic activities of PKB. These sequences can also be used for discrimination between the effects of compounds which mediate insulin action through transcription from those which modulate activity of enzymes involved in metabolism by phosphorylation. Background The mechanistic basis by which insulin regulates the disposition of glucose by animals has been elucidated in close detail in recent years. A cascade of interacting proteins has been described which, when functioning in the normal situation, serves to transduce the signals emanating from insulin, causing cells of different origin to take up glucose from the bloodstream and store it (White 1997). In the current understanding, activation of the insulin receptor by insulin causes the phosphorylation and activation of insulin receptor substrate (IRS) proteins. These serve to act as docking proteins for a variety of downstream proteins leading to their activation. A key downstream protein in insulin signalling is phosphoinositide 3-kinase (PI3K) which catalyses the production of the second messenger phosphatidylinositol 3,4,5-trisphosphate. This is a lipid and is central to the activation of PKB ((Franke et al. 1995); (James et al. 1996); (Franke et al. 1997); (Klippel et al. 1997); (Alessi et al. 1997); (Stokoe et al. 1997)). It is bound by the pleckstrin homology (PH) domains of PKB and of an upstream kinase called 3 -phosphoinositi de-dependent kinase 1 (PDK1) which is involved in the activation of PKB. PKB appears to be a key intermediary in the regulation of glucose utilisation and control of protein synthesis by insulin (Cross et al. 1995); (Cohen et al. 1997); (Peak et al. 1998); (Gingras et al. 1998)). Thus, it has been demonstrated to phosphorylate and inactivate glycogen synthase kinase 3 (GSK3; (Cross et al. 1995)), permitting the synthesis of glycogen from glucose. Furthermore, in cardiac myocytes, PKB has been shown to phosphorylate and activate phosphofructo kinase-2 (Deprez et al. 1997) whose product, fructose 2,6-bisphosphate, acts as an allosteric activator of glycolysis. A third likely substrate for PKB is the type 3B cyclic AMP phosphodiesterase (Wijkander et al. 1998), which in insulin-responsive tissues is activated by phosphorylation, leading to the inactivation of adrenergic-stimulated processes. Although the range of substrates phosphorylated by PKB is diverse, all of them described share a common short primary sequence which serves as the target region for PKB. The first identified consensus sequence of amino acids was in GSK-3, described as gly-arg-pro-arg-thr-ser-ser-phe-ala-glu-gly (GRPRTSSFAEG ) (Cross et al. 1995). See also WO 97/22360. Comparison with other substrates allows a consensus sequence to be derived from this which is likely to contain the essential features for phosphorylation by PKB. This consensus is: RXRXXS/TF (Alessi et al. 1996) where F is phenylalanine but could be replaced by another bulky hydrophobic residue. Data in the literature (not only Alessi et al. 1996 but also Walker et al. 1998) strongly suggest that the amino acid sequence for phophorylation by PKB must include a large hydrophobic residue directly C- terminal to the phosphorylation site. Such a residue has thus been described as crucial for phosphorylation by PKB. Studies in C. elegans have recently suggested that an important effect of insulin may be to suppress the transcriptional activity in the family of transcription factors (Ogg et al. 1997). By inactivating a specific transcription factor in C. elegans (daf-16), the authors have shown that development in C. elegans can bypass a modulated insulin receptor signalling pathway. We have previously shown and claimed (see patent application SE9801530-8) that PKB phosphorylates a peptide sequence derived from the transcription factor forkhead FKHR. This is a member of a subfamily of the wider forkhead superfamily, which is related to the C. elegans nematode worm forkhead protein daf-16. However, in SE9801530-8 we disclosed peptides which do not include a large hydrophobic residue directly C-terminal to the phosphorylation site and we demonstrated that these peptides are similarly good substrates for PKB as the earlier known peptides with the large hydrophobic residue directly C-terminal to the phosphorylation site. Figure 1 shows an alignment of the daf-16-related fork head proteins taken from the literature and we refer here to Parry, P et al, Genes Chromosomes Cancer 11 (2), 79- 84 (1994) and Borkhardt, A et al., Oncogene 14 (2), 195-202 (1997) when relating to Afx and to Anderson, M. J et al., Genomics 47 (2), 187- 199 (1998) and Galili,N et al, Na Genet. 5 (3), 230-235 (1993), when relating to FKHR, SEQ ID No 8 The sequence and its close homologues disclosed in SE9801530-8 is underlined. Afx, SEQ ID No 7 is a human homologue of daf-16. Paradis and Ruvkun in Genes & Development, 12:2488-98, 1998 show that two C. elegans Akt/PKB homologues, akt-1 and akt-2, transduce insulin receptor-like signalling in C. elegans and discuss the target of Akt/PKB signalling as a transcription factor. The questions whether PKB phosphorylates the intact forkhead protein, whether it does this in an insulin-sensitive manner and what effect this has on the activity if the forkhead protein itself, i.e. does insulin regulate forkhead transcriptional activity via PKB, are not answered by the earlier work on Afx, daf-16, parts of FKHR or PKB. If Afx could be inhibited, such an inhibitor may be of great use in the metabolic therapeutic area, such as in the treatment of metabolic syndrome, insulin resistance and insulin resistance related diseases such as diabetes type 2. Figures Figure 1. Alignment of forkhead proteins Figure 2. Phosphorylation of human recombinant Afx by recombinant PKB Figure 3a to 3d. The effect of insulin on phosphorylation of Afxl2-505 by PKB Figure 4A and 4 B. Expression constructs Figure 4C. Relative alkaline phosphatase activities, Example 3 Figure 5. Relative alkaline phosphatase activities, Example 4 The invention We have earlier (see SE9801530-8) found that PKBα activity is stimulated to phosphorylate a small peptide. Our earlier data (patent applied for) also suggest that we know where in the protein PKB acts to add phosphate, namely within the consensus sequences for PKB action. The small peptides can be useful in screening methods. But for a eukaryote cell based screening, the function of the whole protein is of importance. We have now found that we can also use a substrate peptide which comprises the phosphorylation sites of FKHR, FKHRL 1 and Afx, (residues 19-26, 251 -257 and 314- 321 (FKHR), 27-33, 248-254 and 310-316 (FKHRL1), 27-33, 192-198 and 257-263 (Afx, our numbering from our genomic clone). Afx has accession number X939996 . The sequences, included in the family of forkhead protein FKHR, SEQ ID No 8, FKHRL 1, SEQ ID No 9 and preferably Afx, SEQ ID No 7, have the potential to be a target for regulation by PKB and this has been supported by in vitro experiments that show that the recombinant protein can be phosphorylated by PKB. Our data show that phosphorylation of Afx protein by PKB is greater when the PKB activity is isolated from cells which have been stimulated with insulin or IGF-1 than in cells which are not stimulated. These data suggest that insulin and IGF-1 primary messengers stimulate PKB activity against a variety of intracellular targets, including the forkhead proteins. This is the first time that this has been shown in a mammalian cellular system. Afx has thus been shown as good substrate for PKB and can therefore be used in screening for substances which are activators, inhibitors or binders of PKB. When screening for substances which are activators or inhibitors of PKB itself, it is important to know if the substance stimulates or inhibits PKB in a cell and especially in an insulin-stimulated cell. This can only be measured when a substrate is used which includes most of the primary sequence of the fork head protein. Assays which measure the functional activity of Afx can be used for discrimination between the effects of compounds which mediate insulin action through transcriptional regulation, from those compounds which modulate activity of key metabolic enzymes by phosphorylation. Gene transcriptional regulation involves activation or repression of the transcription of the enzymes and other components involved in metabolism, for example, repression of phosphoenolpyruvate carboxykinase (PEPCK) in liver. Afx can be used in the search for new substrates for PKB as templates for sequence searches and/or for primers in techniques of molecular biology such as PCR. By PKB any isoform thereof is included. The invention is defined in the attached claims. Example 1. Phosphorylation of human recombinant Afx by recombinant PKB. GST-PKB pre-coupled to sepharose beads was activated by incubation with lysate (approx. 200μg/ml) from IGF-1 -stimulated H9C2 cardiac myocytes in the presence of unlabelled ATP and phosphatase inhibitors. After thorough washing of the beads to ensure that contamination with cell lysate was minimal, beads were incubated with approximately 5μg recombinant human Afx (comprising amino acids 12-505) termed Afxl2-505 or truncated Afx comprising amino acids 86-236, termed Afx86-236, which we hypothesise still contains a PKB phosphorylation site, in the presence of radioactive ATP. Pre-activated PKB was incubated with Afx proteins (, lanes 1-4, Afx 12-505: lanes 5-6 Afx86-236 in Figure 2) in the presence of radioactive ATP and proteins were subsequently resolved by SDS-PAGE. In lanes 3-6, kinase-permissive conditions were used and each Afx protein was phosphorylated. Control reactions included a buffer which does not support kinase activity (lanes 1 and 2 236 in Figure 2) and omission of PKB (lanes 7-10 236 in Figure 2). Incubations were stopped after 30 min, proteins were separated by SDS-PAGE, using a 4-20% gradient gel, and after drying, the gel was exposed by autoradiography. Results. Figure 2 shows that in reactions containing both PKB and Afx (12-505 or 86- 236) performed in a buffer which supports kinase reactions (50mM Tris/HCl pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, lOmM MgCl 2 plus various protease and phosphatase inhibitors), both the full length (lanes 3 and 4) and truncated forms (lanes 5 and 6) of the transcription factor become phosphorylated. When PKB was omitted from the reactions, neither protein became labelled with radioactive phosphate (lanes 7-10) indicating that phosphorylation was not due to an endogenous contaminating kinase activity. Furthermore, reactions between PKB and Afx were precluded in a buffer which does not support a kinase reaction (lanes 1 and 2, performed in a buffer containing Tris/HCl pH 7.5, high NaCl and 1M imidazole). Thus, recombinant forkhead transcription factor does appear to be a substrate for PKB in vitro. Example 2. The effect of insulin on phosphorylation of Afxl2-505 by PKB To address whether insulin stimulation of cells leads to increased phosphorylation of Afxl2-505 by PKB, quiescent L6 muscle cells were pre-treated with rapamycin and PD-98059 to inhibit activation of p70S6kinase and MAP kinase, respectively, and stimulated with lOOnM insulin for 5 min. Cells were lysed and lysates from resting and insulin-stimulated cells were resolved using MonoQ anion exchange chromatography, as has been previously described by Walker et al (Walker, K.S. (1998) Biochem. J 331, 299-308). Three discrete peaks of PKB activity were observed, identified by measuring phosphorylation of the PKB substrate peptide (RPRTSSF), which is a known sequence exactly as described by Walker et al (1998) (data not shown). The first peak was PKBγ, whilst peaks 2 and 3 contained PKBα (identified by Western blotting, data not shown). Each Mono Q fraction was analysed for the ability to phosphorylate Afx 12-505. lμg recombinant Afx 12-505 was incubated with lOμl each fraction in the presence of radioactive ATP and lOμM unlabelled ATP for 1 hour at 37°C. Reactions were terminated by addition of SDS reducing buffer, and after boiling, samples were resolved by 10% SDS-PAGE. Gels were stained with coomassie stain (Figure 3a), which revealed a single band at the expected molecular weight for Afxl2-505, dried and exposed on the phosphorimager for 24 hours. The gel in Figure 3a is representative of four gels where reactions from each reaction were separated. Images from the phosphorimager showed that after incubation with samples from individual Mono Q fractions which contained PKB activities in the presence of radioactive ATP as described above, the major Afxl 2-505 band was radioactively labelled, along with two minor contaminants. Fraction numbers are shown at the top of respective lanes and the position of Afxl2-505 is indicated. Figure 3bl : fractions from insulin- stimulated cells; Figure 3b2: fractions from control resting cells. Result: Labelling was greater when Afx 12-505 was incubated with column fractions from insulin-stimulated cell lysates. It is clear from figure 3b 1 and 3b2 that cells which were stimulated with insulin have higher activity than cells without insulin. Bands corresponding to Afxl2-505 from each lane in Figure 3b were quantitated using the phosphorimager and the extent of phosphorylation is shown using the arbitrary pixel units of the software. Open symbols in Figure 3 c shows insulin-stimulated cells; closed symbols in Figure 3 c shows resting cells. Figure 3c shows that radioactive labelling of Afxl2-505 by column fractions from non-stimulated L6 cells remained fairly constant across the column, with minor peaks observed. However, for insulin-stimulated lysates, phosphorylation of Afxl 2-505 fell into three distinct peaks, corresponding with the peaks of PKB activity. Identical experiments as in Example 2, Figure 3c, but for a separate experiment in which PKB from lysates of resting L6 cells was compared with IGF-1 -stimulated cells, show that the peaks of PKBα, (peaks 2 and 3 on Mono Q column) activity are stimulated to phosphorylate Afx compared with activities in unstimulated cells, whereas the activity of the γ-isoform of PKB is similar in both cell lysates (Figure 3d). These data show both insulin and IGF-1 lead to the activation of PKB isoforms whose activity against Afx in vitro is markedly enhanced. Thus, stimulation of cells with these agonists may lead to phosphorylation of Afx by PKB, demonstrating a novel regulation of transcription by PKB. Example 3, Afx enhances transcription of an IRE driven reporter gene The ability of Afxl2-505 to activate an IRE (insulin responsive enhancer) DNA sequence was investigated by transient transfection assays in HepG2 cells. HepG2 cells were transfected with 1.5 μg expression plasmid, 0.5 μg reporter plasmid and 1 μg RSV LUC (Control for transfection efficiency) using CaP0 4 method. Cells were grown in MEM with 10% FBS. Differences in transfection efficiency are corrected for by measured Luciferase activity. Transfections have been performed in triplicate and one representative experiment is shown. The expression and reporter plasmids are shown in Figure 4A. The sequence of the IRE, which is derived from the IGFBP-1 gene, is shown in Figure 4B. Relative alkaline phosphatase activities 48 hr. after transfection are shown in Figure 4C. This shows that transfection of Afxl2-505 specifically induces the alkaline phosphatase reporter gene when an IRE DNA sequence is inserted upstream (Compare columns 4 and 3, respectively). Some IRE specific activity is seen when the cells are transfected with the pEGFP-C3 plasmid, lacking Afx sequences (columns 1 and 2, respectively). This is presumably due to endogenous transcription factors activating via this DNA sequence in HepG2 cells. Furthermore, expression of Axf86-282 induces no additional alkaline phosphatase activity above that seen with pEGFP-C3. Example 4. The ability of Afx to activate transcription is suppressed by insulin The ability of insulin to suppress the Afx mediated stimulation of the IRE driven reporter gene was investigated by culturing HepG2 cells transfected with an expression plasmid which expresses Afxl 2-505 and a reporter plasmid with an IRE driven alkaline phosphatase reporter gene in serum free medium in the presence and absence of insulin. HepG2 cells were transfected with 1.5 μg expression plasmid expressing Afxl 2-505 and 0.5 μg reporter plasmid pSEAP2-IRE using CaPO4. Cells were grown in MEM with 10% FBS. 24 hr. after transfection the medium was changed to serum free medium with (see Figure 5, left column) and without (see Figure 5, right column) lOOnM Insulin. Relative alkaline phosphatase activities 48 hr. after the change to serum free medium are shown. Figure 5 shows that the ability of Afxl2-505 to activate the IRE driven reporter gene is markedly suppressed in the presence of insulin (left column). The mechanism by which insulin suppress the Afx mediated stimulation of the IRE driven reporter gene was investigated by culturing HepG2 cells transfected with an expression plasmid which expresses Afxl2-505, a reporter plasmid with an IRE driven alkaline phosphatase reporter gene and a plasmid which expresses full length human PKBa. Control transfections included the vector alone, into which the PKBα gene had been inserted. HepG2 cells were transfected with 1 μg expression plasmid expressing Afxl2-505, 0.5 μg reporter plasmid pSEAP2-IRE, 1 μg PKBa expressing or control plasmid and lμg RSV LUC (Control for transfection efficiency) using a CaPO4 method. Cells were grown in MEM with 10% FBS. 24 hours after transfection the medium was changed to serum free medium with and without 100 ng/ml insulin. Differences in transfection efficiency are corrected for by measured Luciferase activity. Relative alkaline phosphatase activities (%) 48 hours after the change to serum free medium are shown (Figure 6). These data show that the ability of Afx 12- 505 to activate an IRE driven reporter gene is markedly suppressed by overexpressed PKB and that the remaining activity is further suppressed by insulin. This is genetic evidence for the direct regulation of Afx by PKB, which we infer from our biochemical data (where PKB phosphorylates Afx protein directly). We have demonstrated (data not shown) that Afx alone allowed yeast cells to grow, after transfection with Afx constructs, under non-permissive conditions. Thus, fusion constructs, consisting of a heterologous DNA-binding domain, e.g. the DNA-binding domain from the yeast transcription factor Gal4, fused to Afxl2-505, Afx233-505 or Afx283-505 act as strong transcriptional activators of genes including appropriate regulatory sequences, e.g. Gal4 response elements. This can be exploited in screens to search for inhibitors of Afx transactivation function. These data show that Afx is a strong transcriptional activator in yeast cells and allows yeast cells to grow in hostile conditions. Therefore, we could use Afx-transfected yeast cells as a screen for Afx inhibitors. Successful hits would be detected because no cells would be able to grow. A screening method for compounds which are inhibitors of Afx could thus be by determining cell number in the presence of a test compound and a reference compound. The compound which reduces growth would be an inhibitor of Afx. One function of Afx is thus to activate genes which are normally repressed by insulin and on these genes insulin acts to suppress the transcriptional activation of Afx. The experiments shown in Figures 4C and 5 support this hypothesis. Discussion. Our initial prediction that the family of forkhead proteins related to FKHR may be a target for regulation by PKB has been supported by in vitro experiments that show recombinant protein can be phosphorylated by PKB. Our earlier data (patent applied for) also suggest that we know where in the protein PKB acts to add phosphate, namely within the consensus sequences for PKB action, shown in Figure 1. Our data also show that phosphorylation of Afx proteins by PKB is greater when the PKB activity is isolated from cells which have been stimulated with insulin or IGF-1, than in cells which are not stimulated. These data suggest that insulin and IGF-1 primary messengers stimulate PKB activity against a variety of intracellular targets, including the forkhead proteins. This is the first time that this has been shown in a mammalian cellular system. Thus we can construct a signal transduction cascade from the insulin or IGF-1 receptor all the way via a family of transcription factors to a DNA response element regulating genes involved in the metabolic response. Our prediction is that PKB-mediated phosphorylation of the forkhead proteins may act to inhibit the activity of the transcription factor against certain genes (e.g. PEPCK), whilst perhaps increasing the transcription of other genes involved in the metabolic response. Afx peptide can be used for finding compounds useful for treating patients having a dysfunction of essential components in metabolism such as transducers of the insulin signalling pathway and enzymes involved in metabolism. The compounds found from the claimed screening are anticipated also to be used against long term complications resulting from insulin resistance, such as vascular dysfunction, loss of neuronal cells, retinopathy and beta-cells in pancreas. These compounds are not possible to find by using the modulators as described in WO 97/22360. References Alessi, D.et al. (1996).FEBS Letters 399: 333-338. Alessi, D. R. et al. (1997). Current Biology 7: 261-269. Cohen, P et al. (1997). FEBS Letters 410: 3-10. Cross, D., et al. (1995). Nature 378: 785-789. Deprez, D et al. (1997). Journal of Biological Chemistry 272: 17269-17275. Franke, T. F. et al. (1997).Science 275: 665-668. Franke, T. F. et al. (1995). Cell 81 : 727-736. Gingras, A.-Cet al. (1998). Genes and Development 12: 502-513. James, S. R. et al. (1996). Biochemical Journal 315: 709-713. Klippel, A. et al. (1997). Molecular and Cellular Biology 17: 338-344. Ogg, S. et al. (1997). Nature 389: 994-999. Peak, M. J. et al. (1998). Diabetologia 41 : 16-25. Stokoe, D. et al. (1997). Science 277: 567-570. Walker, K. S. et al. (1998). Biochemical Journal 331: 299-308. White, M. F. (1997). Diabetologia 40: S2-S17. Wijkander, J. et al. (1998). Endocrinology 139: 219-227. SEQUENCE LISTING <110> Pharmacia & Upjohn AB <120> Method for screening <130> Afx and PKB <140> <141> <160>9 <170>PatentInVer.2.0 <210> 1 <211>533 <212>PRT <213> Homo sapiens <400> 1 Arg Arg Arg Ala Val Ser Met 1 5 <210>2 <211>7 <212> PRT <213> Homo sapiens <400> 2 Arg Ser Arg Thr Asn Ser Asn 1 5 <210> 3 <211>7 <212> PRT <213> Homo sapiens <400> 3 Arg Arg Arg Ala Ala Ser Met 1 5 <210>4 <211>7 <212>PRT <213> Homo sapiens <400> 4 Arg Pro Arg Thr Ser Ser Asn 1 5 <210> 5 <21 1> 7 <212> PRT <213> Homo sapiens <400> 5 Arg Pro Arg Ser Ser Ser Asn 1 5 <210> 6 <21 1> 7 <212> PRT <213> Homo sapiens <400> 6 Arg Pro Arg Ser Cys Thr Trp 1 5 <210> 7 <211> 501 <212> PRT <213> Homo sapiens <400> 7 Met Arg He Gin Pro Gin Lys Ala Ala Ala He He Asp Leu Asp Pro 1 5 10 15 Asp Phe Glu Pro Gin Ser Arg Pro Arg Ser Cys Thr Trp Pro Leu Pro 20 25 30 Arg Pro Glu He Ala Asn Gin Pro Ser Glu Pro Pro Glu Val Glu Pro 35 40 45 Asp Leu Gly Glu Lys Val His Thr Glu Gly Arg Ser Glu Pro He Leu 50 55 60 Leu Pro Ser Arg Leu Ser Glu Pro Ala Gly Gly Pro Gin Pro Gly He 65 70 75 80 Leu ( ily Ala Val Thr Gly Pro Λrg Lys Gly Gly Ser Λrg Λrg Λsn Λla 85 90 95 Trp Gly Asn Gin Ser Tyr Ala Glu Phe He Ser Gin Λla l ie Glu Ser 100 105 1 10 Ala Pro Glu Lys Arg Leu Thr Leu Ala Gin He Tyr Glu Trp Met Val 1 1 5 120 125 Arg Thr Val Pro Tyr Phe Lys Asp Lys Gly Asp Ser Asn Ser Ser Ala 130 135 140 Gly Trp Lys Asn Ser He Arg His Asn Leu Ser Leu His Ser Lys Phe 145 150 155 160 He Lys Val His Asn Glu Ala Thr Gly Lys Ser Ser Trp Trp Met Leu 165 170 175 Asn Pro Glu Gly Gly Lys Ser Gly Lys Ala Pro Arg Arg Arg Ala Ala 180 185 190 Ser Met Asp Ser Ser Ser Lys Leu Leu Arg Gly Arg Ser Lys Ala Pro 195 200 205 Lys Lys Lys Pro Ser Val Leu Pro Ala Pro Pro Glu Gly Ala Thr Pro 210 215 220 Thr Ser Pro Val Gly His Phe Ala Lys Trp Ser Gly Ser Pro Cys Ser 225 230 235 240 Arg Asn Arg Glu Glu Ala Asp Met Tφ Thr Thr Phe Arg Pro Arg Ser 245 250 255 Ser Ser Asn Ala Ser Ser Val Ser Thr Arg Leu Ser Pro Leu Arg Pro 260 265 270 Glu Ser Glu Val Leu Ala Glu Glu He Pro Ala Ser Val Ser Ser Tyr 275 280 285 Ala Gly Gly Val Pro Pro Thr Leu Asn Glu Gly Leu Glu Leu Leu Asp 290 295 300 Gly Leu Asn Leu Thr Ser Ser His Ser Leu Leu Ser Arg Ser Gly Leu 305 310 315 320 Ser Gly Phe Ser Leu Gin His Pro Gly Val Thr Gly Pro Leu His Thr 325 330 335 Tyr Ser Ser Ser Leu Phe Ser Pro Ala Glu Gly Pro Leu Ser Ala Gly 340 345 350 Glu Gly Cys Phe Ser Ser Ser Gin Ala Leu Glu Ala Leu Leu Thr Ser 355 360 365 Asp Thr Pro Pro Pro Pro Ala Asp Val Leu Met Thr Gin Val Asp Pro 370 375 380 He Leu Ser Gin Ala Pro Thr Leu Leu Leu Leu Gly Gly Leu Pro Ser 385 390 395 400 Ser Ser Lys Leu Ala Thr Gly Val Gly Leu Cys Pro Lys Pro Leu Glu 405 410 415 Ala Arg Gly Pro Ser Ser Leu Val Pro Thr Leu Ser Met He Ala Pro 420 425 430 Pro Pro Val Met Ala Ser Ala Pro He Pro Lys Ala Leu Gly Thr Pro 435 440 445 Val Leu Thr Pro Pro Thr Glu Ala Ala Ser Gin Asp Arg Met Pro Gin 450 455 460 Asp Leu Asp Leu Asp Met Tyr Met Glu Asn Leu Glu Cys Asp Met Asp 465 470 475 480 Asn He He Ser Asp Leu Met Asp Glu Gly Glu Gly Leu Asp Phe Asn 485 490 495 Phe Glu Pro Asp Pro 500 <210> 8 <211> 655 <212> PRT <213> Homo sapiens <400> 8 Met Ala Glu Ala Pro Gin Val Val Glu He Asp Pro Asp Phe Glu Pro 1 5 10 15 Leu Pro Arg Pro Arg Ser Cys Thr Tφ Pro Leu Pro Arg Pro Glu Phe 20 25 30 Ser Gin Ser Asn Ser Ala Thr Ser Ser Pro Ala Pro Ser Gly Ser Ala 35 40 45 Ala Ala Asn Pro Asp Ala Ala Ala Gly Leu Pro Ser Ala Ser Ala Ala 50 55 60 Ala Val Ser Ala Asp Phe Met Ser Asn Leu Ser Leu Leu Glu Glu Ser 65 70 75 80 Glu Asp Phe Pro Gin Ala Pro Gly Ser Val Ala Ala Ala Val Ala Ala 85 90 95 Ala Ala Ala Ala Ala Ala Thr Gly Gly Leu Cys Gly Asp Phe Gin Gly 100 105 1 10 Pro Glu Ala Gly Cys Leu His Pro Ala Pro Pro Gin Pro Pro Pro Pro 1 15 ' 120 125 Gly Pro Val Ser Gin His Pro Pro Val Pro Pro Ala Ala Ala Gly Pro 130 135 140 Leu Ala Gly Gin Pro Arg Lys Ser Ser Ser Ser Arg Arg Asn Ala Tφ 145 150 155 160 Gly Asn Leu Ser Tyr Ala Asp Leu He Thr Lys Ala He Glu Ser Ser 165 170 175 Ala Glu Lys Arg Leu Thr Leu Ser Gin He Tyr Glu Tφ Met Val Lys 180 185 190 Ser Val Pro Tyr Phe Lys Asp Lys Gly Asp Ser Asn Ser Ser Ala Gly 195 200 205 Tφ Lys Asn Ser He Arg His Asn Leu Ser Leu His Ser Lys Phe lie 210 215 220 Arg Val Gin Asn Glu Gly Thr Gly Lys Ser Ser Tφ Tφ Met Leu Asn 225 230 235 240 Pro Glu Gly Gly Lys Ser Gly Lys Ser Pro Arg Arg Arg Ala Ala Ser 245 250 255 Met Asp Asn Asn Ser Lys Phe Ala Lys Ser Arg Ser Arg Ala Ala Lys 260 265 270 Lys Lys Ala Ser Leu Gin Ser Gly Gin Glu Gly Ala Gly Asp Ser Pro 275 280 285 Gly Ser Gin Phe Ser Lys Trp Pro Ala Ser Pro Gly Ser His Ser Asn 290 295 300 Asp Asp Phe Asp Asn Trp Ser Thr Phe Arg Pro Arg Thr Ser Ser Asn 305 310 315 320 Ala Ser Thr He Ser Gly Arg Leu Ser Pro He Met Thr Glu Gin Asp 325 330 335 Asp Leu Gly Glu Gly Asp Val His Ser Met Val Tyr Pro Pro Ser Ala 340 345 350 Ala Lys Met Ala Ser Thr Leu Pro Ser Leu Ser Glu He Ser Asn Pro 355 360 365 Glu Asn Met Glu Asn Leu Leu Asp Asn Leu Asn Leu Leu Ser Ser Pro 370 375 380 Thr Ser Leu Thr Val Ser Thr Gin Ser Ser Pro Gly Thr Met Met Gin 385 390 395 400 Gin Thr Pro Cys Tyr Ser Phe Ala Pro Pro Asn Thr Ser Leu Asn Ser 405 410 415 Pro Ser Pro Asn Tyr Gin Lys Tyr Thr Tyr Gly Gin Ser Ser Met Ser 420 425 430 Pro Leu Pro Gin Met Pro He Gin Thr Leu Gin Asp Asn Lys Ser Ser 435 440 445 Tyr Gly Gly Met Ser Gin Tyr Asn Cys Ala Pro Gly Leu Leu Lys Glu 450 455 460 Leu Leu Thr Ser Asp Ser Pro Pro His Asn Asp He Met Thr Pro Val 465 470 475 480 Asp Pro Gly Val Ala Gin Pro Asn Ser Arg Val Leu Gly Gin Asn Val 485 490 495 Met Met Gly Pro Asn Ser Val Met Ser Thr Tyr Gly Ser Gin Ala Ser 500 505 510 His Asn Lys Met Met Asn Pro Ser Ser His Thr His Pro Gly His Ala 515 520 525 Gin Gin Thr Ser Ala Val Asn Gly Arg Pro Leu Pro His Thr Val Ser 530 535 540 Thr Met Pro His Thr Ser Gly Met Asn Arg Leu Thr Gin Val Lys Thr 545 550 555 560 Pro Val Gin Val Pro Leu Pro His Pro Met Gin Met Ser Ala Leu Gly 565 570 575 Gly Tyr Ser Ser Val Ser Ser Cys Asn Gly Tyr Gly Arg Met Gly Leu 580 585 590 Leu His Gin Glu Lys Leu Pro Ser Asp Leu Asp Gly Met Phe He Glu 595 600 605 Arg Leu Asp Cys Asp Met Glu Ser He He Arg Asn Asp Leu Met Asp 610 615 620 Gly Asp Thr Leu Asp Phe Asn Phe Asp Asn Val Leu Pro Asn Gin Ser 625 630 635 640 Phe Pro His Ser Val Lys Thr Thr Thr His Ser Trp Val Ser Gly 645 650 655 <210> 9 <21 1> 673 <212> PRT <213> Homo sapiens <400> 9 Met Ala Glu Ala Pro Ala Ser Pro Ala Pro Leu Ser Pro Leu Glu Val 1 5 10 15 Glu Leu Asp Pro Glu Phe Glu Pro Gin Ser Arg Pro Arg Ser Cys Thr 20 25 30 Tφ Pro Leu Gin Arg Pro Glu Leu Gin Ala Ser Pro Ala Lys Pro Ser 35 40 45 Gly Glu Thr Ala Ala Asp Ser Met He Pro Glu Glu Glu Asp Asp Glu 50 55 60 Asp Asp Glu Asp Gly Gly Gly Arg Ala Gly Ser Ala Met Ala He Gly 65 70 75 80 Gly Gly Gly Gly Ser Gly Thr Leu Gly Ser Gly Leu Leu Leu Glu Asp 85 90 95 Ser Ala Arg Val Leu Ala Pro Gly Gly Gin Asp Pro Gly Ser Gly Pro 100 105 1 10 Ala Thr Ala Ala Gly Gly Leu Ser Gly Gly Thr Gin Ala Leu Leu Gin 1 15 120 125 Pro Gin Gin Pro Leu Pro Pro Pro Gin Pro Gly Λla Ala Gly Gly Ser 130 135 140 Gly Gin Pro Arg Lys Cys Ser Ser Arg Arg Asn Ala Trp Gly Asn Leu 145 150 155 160 Ser Tyr Ala Asp Leu He Thr Arg Ala He Glu Ser Ser Pro Asp Lys 165 170 175 Arg Leu Thr Leu Ser Gin He Tyr Glu Tφ Met Val Arg Cys Val Pro 180 185 190 Tyr Phe Lys Asp Lys Gly Asp Ser Asn Ser Ser Ala Gly Trp Lys Asn 195 200 205 Ser He Arg His Asn Leu Ser Leu His Ser Arg Phe Met Arg Val Gin 210 215 220 Asn Glu Gly Thr Gly Lys Ser Ser Tφ Tφ He He Asn Pro Asp Gly 225 230 235 240 Gly Lys Ser Gly Lys Ala Pro Arg Arg Arg Ala Val Ser Met Asp Asn 245 250 255 Ser Asn Lys Tyr Thr Lys Ser Arg Gly Arg Ala Ala Lys Lys Lys Ala 260 265 270 Ala Leu Gin Thr Ala Pro Glu Ser Ala Asp Asp Ser Pro Ser Gin Leu 275 280 285 Ser Lys Tφ Pro Gly Ser Pro Thr Ser Arg Ser Ser Asp Glu Leu Asp 290 295 300 Ala Tφ Thr Asp Phe Arg Ser Arg Thr Asn Ser Asn Ala Ser Thr Val 305 310 315 320 Ser Gly Arg Leu Ser Pro He Met Ala Ser Thr Glu Leu Asp Glu Val 325 330 335 Gin Asp Asp Asp Ala Pro Leu Ser Pro Met Leu Tyr Ser Ser Ser Ala 340 345 350 Ser Leu Ser Pro Ser Val Ser Lys Pro Cys Thr Val Glu Leu Pro Arg 355 360 365 Leu Thr Asp Met Ala Gly Thr Met Asn Leu Asn Asp Gly Leu Thr Glu 370 375 380 Λsn Leu Met Asp Asp Leu Leu Asp Λsn He Thr Leu Pro Pro Ser Gin 385 390 395 400 Pro Ser Pro Thr Gly Gly Leu Met Gin Arg Ser Ser Ser Phe Pro Tyr 405 410 415 Thr Thr Lys Gly Ser Gly Leu Gly Ser Pro Thr Ser Ser Phe Asn Ser 420 425 430 Thr Val Phe Gly Pro Ser Ser Leu Asn Ser Leu Arg Gin Ser Pro Met 435 440 445 Gin Thr He Gin Glu Asn Lys Pro Ala Thr Phe Ser Ser Met Ser His 450 455 460 Tyr Gly Asn Gin Thr Leu Gin Asp Leu Leu Thr Ser Asp Ser Leu Ser 465 470 475 480 His Ser Asp Val Met Met Thr Gin Ser Asp Pro Leu Met Ser Gin Ala 485 490 495 Ser Thr Ala Val Ser Ala Gin Asn Ser Arg Arg Asn Val Met Leu Arg 500 505 510 Asn Asp Pro Met Met Ser Phe Ala Ala Gin Pro Asn Gin Gly Ser Leu 515 520 525 Val Asn Gin Asn Leu Leu His His Gin His Gin Thr Gin Gly Ala Leu 530 535 540 Gly Gly Ser Arg Ala Leu Ser Asn Ser Val Ser Asn Met Gly Leu Ser 545 550 555 560 Glu Ser Ser Ser Leu Gly Ser Ala Lys His Gin Gin Gin Ser Pro Val 565 570 575 Ser Gin Ser Met Gin Thr Leu Ser Asp Ser Leu Ser Gly Ser Ser Leu 580 585 590 Tyr Ser Thr Ser Ala Asn Leu Pro Val Met Gly His Glu Lys Phe Pro 595 600 605 Ser Asp Leu Asp Leu Asp Met Phe Asn Gly Ser Leu Glu Cys Asp Met 610 615 620 Glu Ser He He Arg Ser Glu Leu Met Asp Ala Asp Gly Leu Asp Phe 625 630 635 640 Asn Phe Λsp Ser Leu He Ser Thr Gin Λsn Val Val Gly Leu Asn Val 645 650 655 Gly Asn Phe Thr Gly Ala Lys Gin Ala Ser Ser Gin Ser Trp Val Pro 660 665 670 Gly

Claims: CLAIMS 1. Method for screening for substances which are activators, inhibitors or binders of Protein kinase B (PKB) by the use of a substrate peptide comprising two sequences selected from SEQ ID No 1 and SEQ ID No 2; SEQ ID No 3 and SEQ ID No 4 and SEQ ID No 1 and SEQ ID No 5. 2. Method for screening according to claim 1 in which the substrate peptide comprises the three sequences selected from SEQ ID No 6, SEQ ID No 1 and SEQ ID No 2; SEQ ID No 6, SEQ ID No 3 and SEQ ID No 4 or SEQ ID No 6, SEQ ID No 1 and SEQ ID No 5. 3. Method for screening according to claim 2 in which the substrate peptide comprises the three sequences SEQ ID No 6, SEQ ID No 1 and SEQ ID No 5. 4. Method for screening according to any of claim 1 to 3 in which the substrate peptide is forkhead protein FKHRL 1, FKHR or Afx from amino acid number 12. 5. Method for screening according to any of claim 1 to 3 in which the substrate peptide is the forkhead protein Afx 1- 501, SEQ ID No 7. 6. Method for screening for substances which are activators, inhibitors and binders of gene transcriptional regulation by forkhead proteins through the catalytic activities of PKB characterised by the use of any of the substrate peptide according to any of claims 1 - 4. 7. Method for screening for substances which have the capacity to modulate PKB activity characterised by the use of any of the substrate peptide according to any of claims 1 - 4. 8. Method for identifying a compound that activates PKB in vitro, comprising: a) contacting a test compound with the substrate peptide according to any of claims 1 - 4 for a time sufficient to form a complex with the substrate peptide b) detecting the complex by detecting the phosphorylation in the complex, so that if a complex is detected a compound that activates PKB is identified. 9. Method for aiding in the identification of a compound for use in treating metabolic disease comprising the steps of a) incubating a cell in the presence and absence of a test compound, wherein said cell has been altered to contain a nucleic acid molecule comprising an insulin response element operable linked to a reporter gene and said cell expresses a wild-type Afx protein; b) determining the level of expression of said reporter gene in said cell incubated in the presence and absence of said test compound; and c) selecting a compound that induces a higher level of expression of said reporter gene in step b) than that expressed in the absence of said compound, for use in treating metabolic disease. 10. Use of a substrate peptide according to any of claims 1 - 4 in assays measuring the activity of PKB. 11. Use of a substrate peptide according to any of claims 1 - 4 in screening for substances which are activators or inhibitors of gene transcriptional regulation by forkhead proteins through the catalytic activities of PKB. 12. Use of substrate peptide according to any of claims 1 - 4 for discrimination between the effects of compounds which mediate insulin action through transcription from those which modulate activity of enzymes involved in metabolism by phosphorylation. 13. Use according to claim 9 for discrimination between the effects of compounds which mediate insulin action through transcription via forkhead transcription factor family from those which modulate activity of enzymes involved in metabolism by phosphorylation. 14. A method for making a pharmaceutical formulation for the treatment of disease, comprising a) contacting a test compound with the substrate peptide according to any of claims 1 to 4 for a time sufficient to form a complex with the substrate peptide b) detecting the complex by detecting the phosphorylation in the complex, so that if a complex is detected a compound that activates PKB is identified. c) manufacturing bulk quantities of the compound identified in step b) and d) formulating the compound manufactured in step c) in a pharmaceutically acceptable carrier.

Inventors: BUTCHER STEVE, CLIMENT ISABEL, DAHLMAN-WRIGHT KARIN, JAMES STEPHEN, LAKE STAFFAN, BUTCHER, STEVE, CLIMENT, ISABEL, DAHLMAN-WRIGHT, KARIN, JAMES, STEPHEN, LAKE, STAFFAN
Applicants: BUTCHER STEVE, CLIMENT ISABEL, DAHLMAN WRIGHT KARIN, JAMES STEPHEN, LAKE STAFFAN, PHARMACIA & UPJOHN AB, BUTCHER, STEVE, CLIMENT, ISABEL, DAHLMAN-WRIGHT, KARIN, JAMES, STEPHEN, LAKE, STAFFAN
Application year: 1999
Main classification: C12Q1
IPC codes: A61K45, A61P43, C07K7, C12Q1, G01N33