Peptide and Protein Library Screening Defines Optimal Substrate Motifs for AKT/PKB
2000; Elsevier BV; Volume: 275; Issue: 46 Linguagem: Inglês
10.1074/jbc.m005497200
ISSN1083-351X
AutoresToshiyuki Obata, Michael B. Yaffe, Germán Leparc, Elizabeth T. Piro, Hiroshi Maegawa, Atsunori Kashiwagi, Ryuichi Kikkawa, Lewis C. Cantley,
Tópico(s)Protein Degradation and Inhibitors
ResumoAKT was originally identified as a proto-oncogene with a pleckstrin homology and Ser/Thr protein kinase domains. Recent studies revealed that AKT regulates a variety of cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism. To clarify the substrate specificity of AKT, we have used an oriented peptide library approach to determine optimal amino acids at positions N-terminal and C-terminal to the site of phosphorylation. The predicted optimal peptide substrate (Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-Phe-Gly where Ser* is the phosphorylation site) has similarities to but is distinct from optimal substrates that we previously defined for related basophilic protein kinases such as protein kinase A, Ser/Arg-rich kinases, and protein kinase C family members. The positions most important for highV max/K m ratio were Arg-3>Arg-5>Arg-7. The substrate specificity of AKT was further investigated by screening a λGEX phage HeLa cell cDNA expression library. All of the substrates identified by this procedure contained Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs and were in close agreement with the motif identified by peptide library screening. The results of this study should help in prediction of likely AKT substrates from primary sequences. AKT was originally identified as a proto-oncogene with a pleckstrin homology and Ser/Thr protein kinase domains. Recent studies revealed that AKT regulates a variety of cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism. To clarify the substrate specificity of AKT, we have used an oriented peptide library approach to determine optimal amino acids at positions N-terminal and C-terminal to the site of phosphorylation. The predicted optimal peptide substrate (Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-Phe-Gly where Ser* is the phosphorylation site) has similarities to but is distinct from optimal substrates that we previously defined for related basophilic protein kinases such as protein kinase A, Ser/Arg-rich kinases, and protein kinase C family members. The positions most important for highV max/K m ratio were Arg-3>Arg-5>Arg-7. The substrate specificity of AKT was further investigated by screening a λGEX phage HeLa cell cDNA expression library. All of the substrates identified by this procedure contained Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs and were in close agreement with the motif identified by peptide library screening. The results of this study should help in prediction of likely AKT substrates from primary sequences. protein kinase C cAMP-dependent protein kinase A phosphatidylinositol 3′-kinase 3-phosphoinositide-dependent protein kinase 1 glycogen synthase kinase 3 aminoethylsulfonyl fluoride isopropyl-thiogalactopyranoside glutathioneS-transferase iminodiacetic acid hemagglutinin polymerase chain reaction granulocyte colony-stimulating factor receptor The AKT protein kinase (also referred to as protein kinase B or Rac-protein kinase) was initially identified as an acute transforming component of the AKT8 virus isolated from a murine T cell lymphoma (1Staal S.P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5034-5037Crossref PubMed Scopus (635) Google Scholar,2Staal S.P. Hartley J.W. J. Exp. Med. 1988; 167: 1259-1264Crossref PubMed Scopus (82) Google Scholar). The catalytic domain of AKT is closely related to those of protein kinase C (PKC)1 and protein kinase A (PKA) family members (3Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (786) Google Scholar, 4Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (439) Google Scholar, 5Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (387) Google Scholar). The kinase activity of AKT is stimulated by a variety of growth factors (6Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar), cytokines, chemokines, heat shock, hyperosmolarity, hypoxia, integrin engagement, and T cell receptor (7Andjelkovic M. Suidan H.S. Meier R. Frech M. Alessi D.R. Hemmings B.A. Eur. J. Biochem. 1998; 251: 195-200Crossref PubMed Scopus (57) Google Scholar, 8Ahmed N.N. Grimes H.L. Bellacosa A. Chan T.O. Tsichlis P.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3627-3632Crossref PubMed Scopus (486) Google Scholar, 9King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (383) Google Scholar, 10Konishi H. Matsuzaki H. Tanaka M. Takemura Y. Kuroda S. Ono Y. Kikkawa U. FEBS Lett. 1997; 410: 493-498Crossref PubMed Scopus (234) Google Scholar, 11Mazure N.M. Chen E.Y. Laderoute K.R. Giaccia A.J. Blood. 1997; 90: 3322-3331Crossref PubMed Google Scholar, 12Reif K. Lucas S. Cantrell D. Curr. Biol. 1997; 7: 285-293Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 13Songyang Z. Baltimore D. Cantley L.C. Kaplan D.R. Franke T.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11345-11350Crossref PubMed Scopus (322) Google Scholar, 14Tilton B. Andjelkovic M. Didichenko S.A. Hemmings B.A. Thelen M. J. Biol. Chem. 1997; 272: 28096-28101Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 15Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3707) Google Scholar). The pleckstrin homology domain of AKT binds to the lipid products of phosphoinositide 3′-kinase (PI3K) and thereby mediates recruitment to the membrane in response to PI3K (6Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar, 16Sable C.L. Filippa N. Filloux C. Hemmings B.A. Van O.E. J. Biol. Chem. 1998; 273: 29600-29606Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 17Datta K. Franke T.F. Chan T.O. Makris A. Yang S.I. Kaplan D.R. Morrison D.K. Golemis E.A. Tsichlis P.N. Mol. Cell. Biol. 1995; 15: 2304-2310Crossref PubMed Scopus (156) Google Scholar, 18Alberti S. Proteins. 1998; 31: 1-9Crossref PubMed Scopus (23) Google Scholar). The translocation of AKT allows phosphorylation at Thr-308 by another Ser/Thr kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK-1) (19Stephens L. Anderson K. Stokoe D. Erdjument B.H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (910) Google Scholar, 20Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 21Vanhaesebroeck, B., and Alessi, D. R. (2000) Biochem. J. 561–576.Google Scholar). Full activation of AKT requires phosphorylation at Ser-473. Although there may be multiple mechanisms for phosphorylation at Ser-473, recent studies indicate that phosphorylation is enhanced by PDK-1-dependent phosphorylation of Thr-308 and is dependent on a kinase-active AKT supporting an autophosphorylation model (22Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 23Biondi R.M. Cheung P.C. Casamayor A. Deak M. Currie R.A. Alessi D.R. EMBO J. 2000; 19: 979-988Crossref PubMed Scopus (248) Google Scholar).Given the importance of AKT in a variety of cellular functions, numerous laboratories have sought in vivo substrates of AKT that could explain its various roles in signaling. However, identification of in vivo substrates of protein kinases is complicated by the existence of protein kinase cascades. Thus, even ifin vivo phosphorylation of a specific site on a protein is blocked by disrupting the function of a single protein kinase, one cannot conclude that the kinase of interest directly phosphorylates the site. A downstream kinase could be responsible. Support for direct phosphorylation can be obtained by demonstrating that the site is preferentially phosphorylated by the kinase of interest in a purein vitro assay. Knowledge of the optimal motif of a protein kinase can also accelerate discovery of targets by allowing prediction of sites from a global search of genome sequences or by a restricted search of candidate proteins. Ultimately, such predictions must be tested by both in vivo and in vitroexperiments.Recently, a number of in vivo and/or in vitrostudies have suggested substrates for AKT, including BAD (24Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4915) Google Scholar, 25del Peso L. Gonzalez G.M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar), glycogen synthase kinase 3 (GSK3) (26Cross D.A. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4326) Google Scholar), 6-phosphofructo-2-kinase (27Deprez J. Vertommen D. Alessi D.R. Hue L. Rider M.H. J. Biol. 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Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell. Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (307) Google Scholar), rac1 (34Kwon T. Kwon D.Y. Chun J. Kim J.H. Kang S.S. J. Biol. Chem. 2000; 275: 423-428Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), raf-1 protein kinase (35Zimmermann S. Moelling K. Science. 1999; 286: 1741-1744Crossref PubMed Scopus (901) Google Scholar, 36Rommel C. Clarke B.A. Zimmermann S. Nunez L. Rossman R. Reid K. Moelling K. Yancopoulos G.D. Glass D.J. Science. 1999; 286: 1738-1741Crossref PubMed Scopus (661) Google Scholar), mammalian target of rapamycin (37Nave B.T. Ouwens M. Withers D.J. Alessi D.R. Shepherd P.R. Biochem. J. 1999; 2: 427-431Crossref Google Scholar), breast cancer susceptibility gene 1 (BRCA1) (38Altiok S. Batt D. Altiok N. Papautsky A. Downward J. Roberts T.M. Avraham H. J. Biol. Chem. 1999; 274: 32274-32278Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), insulin receptor substrate 1 (39Paz K. Liu Y.F. Shorer H. Hemi R. LeRoith D. Quan M. Kanety H. Seger R. Zick Y. J. Biol. Chem. 1999; 274: 28816-28822Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), and fork head transcription factors (40Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (551) Google Scholar, 41Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Google Scholar). The sites identified in these substrates define a minimal motif, Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr), that is similar to motifs identified for some of the PKC family members (42Nishikawa K. Toker A. Johannes F.J. Songyang Z. Cantley L.C. J. Biol. Chem. 1997; 272: 952-960Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar).In this study, to better understand the substrate specificity of AKT and compare it to other basophilic protein kinases, we have used a degenerate peptide library approach that provides an unbiased evaluation of the importance of the residues surrounding the site of phosphorylation for an optimal substrate. To further test the specificity in a protein context and to identify candidate substrates, we performed phosphorylation screening of a λ phage cDNA expression library for AKT using purified protein kinase and [γ-32P]ATP, and searched for evidence of AKT phosphorylation motifs within these newly identified substrates.DISCUSSIONIn this study, we have determined the optimal phosphorylation motif for Akt by oriented peptide library screening and corroborated the result by identifying similar motifs in AKT protein substrates isolated by screening of a HeLa cell cDNA expression library. The optimal motif was confirmed by a detailed kinetic analysis of individual peptides containing Ala substitutions at positions within the motif. We constructed a non-phosphorylatable substrate analog, and showed that this peptide could function as a competitive inhibitor of AKT phosphorylation in vitro.As shown in Table II, most known physiological substrates of AKT have Arg at both the −3 and −5 positions and hydrophobic amino acids (e.g. Phe) in the +1 position. However several other basophilic protein kinases have similar requirements for optimal substrates. For example, PKCα, PKCγ, and PKCδ also select substrates with Arg at −3 and −5 and Phe at +1 (TableV). A distinguishing feature of optimal AKT substrate is selection for residues at S+2 that form tight turns (e.g. Gly), in contrast to optimal PKC substrates which select for basic or aromatic residues in the S+2 position. In addition, selection for Thr at S−2 also distinguishes AKT from all other basophilic kinases investigated to date (Table I and Table V). Thus, the information provided by peptide library screening can help to distinguish AKT phosphorylation sites from sites likely to be phosphorylated by other related basophilic protein kinases. Our results are in good agreement with those of Alessi et al. (50Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (550) Google Scholar) who showed that substitution of Ala for Arg in the S−5 or S−3 positions or Ala for Phe in the S+1 position of a peptide derived from the AKT phosphorylation site in GSK3 resulted in reduced phosphorylation. Our results also agree with those of Kobayashi and Cohen (51Kobayashi, T., and Cohen, P. (1999) Biochem. J. 319–328.Google Scholar) who found a critical dependence on Arg in the S−3 position. Mutation of this residue, even to Lys, another basic amino acid, severely compromised the ability of AKT to phosphorylate a peptide substrate. Similarly, Kobayashi and Cohen (51Kobayashi, T., and Cohen, P. (1999) Biochem. J. 319–328.Google Scholar) reported that mutation of Phe in the S+1 position to the charged amino acids Lys or Glu decreased the efficiency of phosphorylation by 30 and 13%, respectively. In agreement with these data, Phe was the optimal residue in the S+1 position by peptide library screening, and as shown in Table I, most known substrates of Akt have hydrophobic amino acids (often Phe) in this position.Table VComparison of the substrate specificities of basophilic protein kinases determined by soluble oriented peptide library screening−7−6−5−4−3−2−10+1+2+3+4+5PKARRRRSIIFIPKCαRRRRRKGSFRRKAPKCβ I, IIRKLKRKGSFRRKAPKCγRRRRRKGSFKKFAPKCδAARKRKGSFFYGGPKCɛYYXKRKMSFFEFDPKCηARLRRRRSFRRXRPKCζRRFKRQGSFFYFFPKCμAALVRQMSVAFFFCaM kinase IIKRQQSFDLFPhosphorylase kinaseFRMMSFFLFSLK1RRFGSLRRFSRPK2RRRHSRRRRAKT/PKBRKRXRTYSFGA list of motifs obtained by soluble peptide library screening for a variety of basophilic Ser/Thr protein kinase is shown. The optimal amino acids at the critical residues are denoted by bold letters. SRPK, SR protein kinases; SLK, Ste20-like kinase. Data are from Songyanget al. (45Songyang Z. Cantley L.C. Methods Mol. Biol. 1998; 87: 87-98PubMed Google Scholar), Songyang et al. (47Songyang Z. Lu K.P. Kwon Y.T. Tsai L.H. Filhol O. Cochet C. Brickey D.A. Soderling T.R. Bartleson C. Graves D.J. DeMaggio A.J. Hoekstra M.F. Blenis J. Hunter T. Cantley L.C. Mol. Cell. Biol. 1996; 16: 6486-6493Crossref PubMed Scopus (486) Google Scholar), Nishikawaet al. (42Nishikawa K. Toker A. Johannes F.J. Songyang Z. Cantley L.C. J. Biol. Chem. 1997; 272: 952-960Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar) and Wang et al. (52Wang H.Y. Lin W. Dyck J.A. Yeakley J.M. Songyang Z. Cantley L.C. Fu X.D. J. Cell Biol. 1998; 140: 737-750Crossref PubMed Scopus (250) Google Scholar). Open table in a new tab A Structural Model for AKT Motif SelectionWe have previously proposed a structural basis for substrate selectivity of basophilic protein kinases based on the crystal structure of the PKA/PKI complex and residues conserved between PKA and other basophilic protein kinases (42Nishikawa K. Toker A. Johannes F.J. Songyang Z. Cantley L.C. J. Biol. Chem. 1997; 272: 952-960Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, 47Songyang Z. Lu K.P. Kwon Y.T. Tsai L.H. Filhol O. Cochet C. Brickey D.A. Soderling T.R. Bartleson C. Graves D.J. DeMaggio A.J. Hoekstra M.F. Blenis J. Hunter T. Cantley L.C. Mol. Cell. Biol. 1996; 16: 6486-6493Crossref PubMed Scopus (486) Google Scholar, 52Wang H.Y. Lin W. Dyck J.A. Yeakley J.M. Songyang Z. Cantley L.C. Fu X.D. J. Cell Biol. 1998; 140: 737-750Crossref PubMed Scopus (250) Google Scholar). To rationalize the substrate specificity we observed for AKT, we modeled the structure of the AKT kinase domain using the x-ray structure of the PKA·PKI·ATP ternary complex as a basis set. The resulting molecular surface of the modeled AKT protein, shaded by electrostatic potential, is shown in Fig.5. All of the basophilic kinases in TableV preferentially phosphorylate substrates with Arg in the S−3 position. These results can be rationalized by a weakly acidic pocket in PKA at the S−3 position (Fig. 5, red) with contributions from both a highly conserved acidic residue (Glu-127 in PKA; Glu-234 in AKT) that salt bridges to the guanidino nitrogens and the proximity of the β- and γ-phosphates of ATP to this residue. In addition, both PKC and AKT contain residues predicted to form an acidic patch at the S−5 position (e.g. Asp-467, Glu-530, and Asp-503 of PKCα and Glu-278, Glu-341, and Glu-314 in AKT that loosely map to the S−2 pocket in PKA (Glu-170, Glu-230, and Glu-202)). Similarly, nearly all basophilic kinases contain a hydrophobic pocket at S+1 which is particularly deep in the case of AKT (e.g. Phe-309, Pro-313, and Leu-316) rationalizing selection for Phe in the S+1 position. As discussed above, the selection for peptides with residues at S+2 that tend to form tight turns is unique to AKT. This may be explained if the substrate binding cleft terminates at this position, forcing a tight turn to exit the pocket. Interestingly, several of the in vivo substrates of AKT have Pro at the S+2 position and are known to bind to 14-3-3 proteins after phosphorylation (53Yaffe M.B. Cantley L.C. Nature. 1999; 402: 30-31Crossref PubMed Scopus (70) Google Scholar, 54Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar, 55Franke T.F. Cantley L.C. Nature. 1997; 390: 116-117Crossref PubMed Scopus (168) Google Scholar). The crystal structures of 14-3-3 protein·phosphopeptide complexes reveal the necessity for a tight turn residue at S+2 for exit from a deep binding cleft (53Yaffe M.B. Cantley L.C. Nature. 1999; 402: 30-31Crossref PubMed Scopus (70) Google Scholar, 54Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar). In addition, tight binding to 14-3-3 protein is favored by Arg at S−3 and Ser or Thr at S−2 and is further facilitated by Arg at S−5 (54Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar) (Table VI). Thus, AKT appears to have evolved a substrate selection that is skewed toward motifs recognized by 14-3-3 proteins. Consistent with this idea, the sites on Forkhead-related proteins that are phosphorylated by AKT and bind to 14-3-3 are highly conserved from Caenorhabditis elegans to mammals (Table VI).Table VIComparison of the AKT phosphorylation sites of Daf-16 family members with the predicted optimal motifs for AKT and for 14-3-3 bindingPosition−7−6−5−4−3−2−10+1+2Optimal 14-3-3 binding motifRRRSQ/Y/FpSL/E/A/MPOptimal AKT phosphorylation motifRKRXRTY/F/G/MSF/MG/T/SAKT phosphorylation sites in C. elegans Daf-16 and mammalian homologs Daf-16a (T54)IPRDRCNTWP Daf-16b (T19)EPRGRCYTWP FKHRL1 (T31)QSRPRSCTWP FKHR (T23)LPRPRSCTWP AFX (T27)QSRPRSCTWPThe AKT phosphorylation motif is from Table I. The 14-3-3 binding motif is from Yaffe et al. (54Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar). Residues in bold are strongly selected in the peptide library screens. See Paradis and Ruvkun (40Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (551) Google Scholar), Brunet et al. (62Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5367) Google Scholar), del Paso et al. (63del Peso L. Gonzalez V.M. Hernandez R. Barr F.G. Nunez G. Oncogene. 1999; 18: 7328-7333Crossref PubMed Scopus (110) Google Scholar) and Kopset al. (61Kops G.J. de Ruiter N.D. De Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (948) Google Scholar) for mapping phosphorylation sites in Daf-16, FKHRL1, FKHR, and AFX. Open table in a new tab Identification of in Vitro AKT SubstratesSeveral newin vitro AKT substrates were identified using solid-phase phosphorylation screening. Many of the positive clones we identified were spliceosomal proteins. This selection may result, in part, from cDNA bias, because spliceosomal proteins tend to be expressed in relatively large amounts within cells (56Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar). Furthermore, many have Arg/Ser (RS)-rich domains that are the targets for SR kinase-mediated phosphorylation events. These Arg/Ser domains frequently contain multiple AKT phosphorylation motifs lying within narrow stretches of sequence, as described for Zis above. The splicing factor SC35, for example, has more than 20 possible AKT phosphorylation motifs in its protein sequence. When the entire GenPept data base was searched using a profile-based bioinformatics program and the optimal consensus motif determined for AKT, 2M. B. Yaffe, G. G. Leparc, J. Lai, T. Obata, S. Volinia, and L. C. Cantley, submitted for publication. 81 of the top 300 scoring sequences were spliceosomal proteins. Thus there is consistency between the peptide library screening/data base search approach and an independent cDNA expression library screening for AKT substrates. Intriguingly, this bioinformatics search also identified all known AKT substrates including BAD, and members of the Forkhead transcription factor family. An AKT phosphorylation site on mTOR, the mammalian target of rapamycin, at Ser-2448 was also identified, and this site has recently been reported by Nave et al. (37Nave B.T. Ouwens M. Withers D.J. Alessi D.R. Shepherd P.R. Biochem. J. 1999; 2: 427-431Crossref Google Scholar).The relevance of our finding that many proteins involved in RNA processing and previously identified as substrates of SR kinases are also good substrates for AKT is not yet clear. This may reflect similarities between optimal substrates for SR kinases and AKT (TableV). One major difference between SR kinases and AKT is that SR kinases prefer substrates with Arg at the S+1 position (52Wang H.Y. Lin W. Dyck J.A. Yeakley J.M. Songyang Z. Cantley L.C. Fu X.D. J. Cell Biol. 1998; 140: 737-750Crossref PubMed Scopus (250) Google Scholar), whereas AKT prefers hydrophobic residues. Thus, the substrates in Table IV with Arg at S+1 are more likely to be SR kinase substrates in vivowhereas those with hydrophobic residues at S+1 are good candidates forin vivo AKT substrates. Further work will be necessary to evaluate the in vivo relevance of these candidate substrates.AKT is known to participate in a wide range of normal cell functions and up-regulation of its activity has been noted in a variety of neoplasms. Identification of the optimal substrate motif using oriented peptide library screening and protein library screening should facilitate the identification of additional in vivo AKT targets relevant to tumorigenesis and cancer progression. The AKT protein kinase (also referred to as protein kinase B or Rac-protein kinase) was initially identified as an acute transforming component of the AKT8 virus isolated from a murine T cell lymphoma (1Staal S.P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5034-5037Crossref PubMed Scopus (635) Google Scholar,2Staal S.P. Hartley J.W. J. Exp. Med. 1988; 167: 1259-1264Crossref PubMed Scopus (82) Google Scholar). The catalytic domain of AKT is closely related to those of protein kinase C (PKC)1 and protein kinase A (PKA) family members (3Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (786) Google Scholar, 4Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (439) Google Scholar, 5Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (387) Google Scholar). 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