Phosphorylation of the Transcription Factor Forkhead Family Member FKHR by Protein Kinase B

Abstract

Protein kinase B lies “downstream” of phosphatidylinositide (PtdIns) 3-kinase and is thought to mediate many of the intracellular actions of insulin and other growth factors. Here we show that FKHR, a human homologue of the DAF16 transcription factor in Caenorhabditis elegans, is rapidly phosphorylated by human protein kinase Bα (PKBα) at Thr-24, Ser-256, and Ser-319in vitro and at a much faster rate than BAD, which is thought to be a physiological substrate for PKB. The same three sites, which all lie in the canonical PKB consensus sequences (Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr)), became phosphorylated when FKHR was cotransfected with either PKB or PDK1 (an upstream activator of PKB). All three residues became phosphorylated when 293 cells were stimulated with insulin-like growth factor 1 (IGF-1). The IGF-1-induced phosphorylation was abolished by the PtdIns 3-kinase inhibitor wortmannin but not by PD 98059 (an inhibitor of the mitogen-activated protein kinase cascade) or by rapamycin. These results indicate that FKHR is a physiological substrate of PKB and that it may mediate some of the physiological effects of PKB on gene expression. DAF16 is known to be a component of a signaling pathway that has been partially dissected genetically and includes homologues of the insulin/IGF-1 receptor, PtdIns 3-kinase and PKB. The conservation of Thr-24, Ser-256, and Ser-319 and the sequences surrounding them in DAF16 therefore suggests that DAF16 is also a direct substrate for PKB in C. elegans. Protein kinase B lies “downstream” of phosphatidylinositide (PtdIns) 3-kinase and is thought to mediate many of the intracellular actions of insulin and other growth factors. Here we show that FKHR, a human homologue of the DAF16 transcription factor in Caenorhabditis elegans, is rapidly phosphorylated by human protein kinase Bα (PKBα) at Thr-24, Ser-256, and Ser-319in vitro and at a much faster rate than BAD, which is thought to be a physiological substrate for PKB. The same three sites, which all lie in the canonical PKB consensus sequences (Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr)), became phosphorylated when FKHR was cotransfected with either PKB or PDK1 (an upstream activator of PKB). All three residues became phosphorylated when 293 cells were stimulated with insulin-like growth factor 1 (IGF-1). The IGF-1-induced phosphorylation was abolished by the PtdIns 3-kinase inhibitor wortmannin but not by PD 98059 (an inhibitor of the mitogen-activated protein kinase cascade) or by rapamycin. These results indicate that FKHR is a physiological substrate of PKB and that it may mediate some of the physiological effects of PKB on gene expression. DAF16 is known to be a component of a signaling pathway that has been partially dissected genetically and includes homologues of the insulin/IGF-1 receptor, PtdIns 3-kinase and PKB. The conservation of Thr-24, Ser-256, and Ser-319 and the sequences surrounding them in DAF16 therefore suggests that DAF16 is also a direct substrate for PKB in C. elegans. In recent years evidence has accumulated that many of the metabolic actions of insulin may be mediated by a protein kinase cascade that lies “downstream” of phosphatidylinositide (PtdIns) 1The abbreviations used are: PtdIns, phosphatidylinositide; PtdIns(3,4,5)P3, phosphatidylinositide 3,4,5-trisphosphate; PtdIns(3,4)P2, phosphatidylinositide 3,4-diphosphate; PKB, protein kinase B; PDK, 3-phosphoinositide-dependent protein kinase; IGF-1, insulin-like growth factor 1; IGFBP-1, IGF-binding protein 1; MAP, mitogen-activated protein; MAPKAP-K1, MAP kinase-activated protein kinase 1; PFK2, 6-phosphofructo-2-kinase; GSK3, glycogen synthase kinase 3; mTOR, mammalian target of rapamycin; PEPCK, phosphoenolpyruvate carboxykinase; PKI, specific peptide inhibitor of cAMP-dependent protein kinase; IRS, insulin response sequence; DAF, dauer arrest phenotype; GST, glutathioneS-transferase; EST, expressed sequence tag 3-kinase and the second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2 (reviewed in Refs. 1Cohen P. Alessi D.R. Cross D.A.E. FEBS Lett. 1997; 410: 3-10Crossref PubMed Scopus (236) Google Scholar and 2Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (677) Google Scholar). A central player in this cascade is protein kinase B (PKB, also called c-Akt). This enzyme is activated when it becomes phosphorylated at Thr-308 and Ser-473 (3Alessi D.R. Andjelkovic M. Caudwell F.B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar) by 3-phosphoinositide-dependent protein kinases 1 and 2 (PDK1, PDK2), respectively (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R.J. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 5Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 6Stokoe D. Stephens L.R. Copeland T. Gaffney P.R.J. Reese C.B. Painter G.F. Science. 1997; 277: 567-570Crossref PubMed Scopus (1054) Google Scholar, 7Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R.J. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (916) Google Scholar). The activation of PKB by PDK1 in vitro has an absolute requirement for PtdIns(3,4,5)P3 or PtdIns(3,4)P2 (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R.J. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar), and these mediators facilitate activation by binding to the pleckstrin homology domains of both PKB (5Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 7Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R.J. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (916) Google Scholar) and PDK1 (8Currie R.A. Walker K.S. Gray A. Deak M. Casamayor A. Downes C.P. Cohen P. Alessi D.R. Lucocq J. Biochem. J. 1999; 337: 575-583Crossref PubMed Scopus (272) Google Scholar). Consistent with these observations, the phosphorylation of PKB at Thr-308, induced by either insulin or insulin-like growth factor 1 (IGF-1) is prevented by inhibitors of PtdIns 3-kinase (3Alessi D.R. Andjelkovic M. Caudwell F.B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar). PDK2 has not yet been characterized although, like the phosphorylation of Thr-308, the insulin or IGF-1-induced phosphorylation of Ser-473 is prevented by inhibitors of PtdIns 3-kinase (3Alessi D.R. Andjelkovic M. Caudwell F.B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar). PKB mediates the metabolic actions of insulin by phosphorylating regulatory proteins at serine or threonine residues that lie in Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs (9Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (552) Google Scholar), of which the best characterized are the cardiac isoform of 6-phosphofructo-2-kinase (PFK2) (2Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (677) Google Scholar, 10Deprez J. Vertommen D. Alessi D.R. Hue L. Rider M.H. J. Biol. Chem. 1997; 272: 17269-17275Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar), the protein kinase glycogen synthase kinase 3 (GSK3) (11Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4397) Google Scholar, 12van Weeren P.C. de Bruyn K.M.T. de Vries-Smits A.M.M. van Lint J. Burgering B.M. Th. J. Biol. Chem. 1998; 273: 13150-13156Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), and the mammalian target of rapamycin (mTOR) (13Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (414) Google Scholar), as well as the proapoptotic protein BAD (reviewed in Ref. 2Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (677) Google Scholar). Phosphorylation by PKB activates cardiac PFK2, and this is thought to underlie the insulin-induced stimulation of glycolysis in the heart. Phosphorylation inhibits GSK3 and is thought to contribute to the stimulation of glycogen synthesis and global protein synthesis by insulin (1Cohen P. Alessi D.R. Cross D.A.E. FEBS Lett. 1997; 410: 3-10Crossref PubMed Scopus (236) Google Scholar, 2Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (677) Google Scholar). Phosphorylation by PKB activates mTOR, allowing it to catalyze several phosphorylation events that enhance the translation of specific proteins. The overexpression of PKB has also been shown to mimic other metabolic actions of insulin, such as the stimulation of glucose (14Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar) and amino acid transport (15Hajduch E. Alessi D.R. Hemmings B.A. Hundal H. Diabetes. 1998; 47: 1006-1013Crossref PubMed Scopus (296) Google Scholar). When cells are stimulated with IGF-1, PKB is initially translocated to the plasma membrane where it becomes activated by PDK1 and PDK2, but it subsequently accumulates in the nucleus (16Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J.C. Frech M. Cron P. Cohen P. Lucocq J.M. Hemming B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). This raises the question of whether PKB mediates some of the effects of insulin on specific gene transcription, and several pieces of evidence would appear to support this contention. For example, the overexpression of constitutively active mutants of PKB mimics the effects of insulin in stimulating the transcription of the obesity gene product leptin (17Barthel A. Kohn A.D. Luo Y.N. Roth R.A. Endocrinology. 1997; 183: 3559-3562Crossref Scopus (31) Google Scholar) and in inhibiting the transcription of IGF-binding protein 1 (IGFBP-1) (18Cichy S.B. Uddin S. Danilkovich A. Guo S. Klippel A. Unterman T.G. J. Biol. Chem. 1998; 273: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The insulin-induced suppression of phosphoenolpyruvate carboxykinase (19Sutherland C. Waltner-Law M. Gnudi L. Kahn B. Granner D.K. J. Biol. Chem. 1998; 273: 3198-3204Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and IGFBP-1 (18Cichy S.B. Uddin S. Danilkovich A. Guo S. Klippel A. Unterman T.G. J. Biol. Chem. 1998; 273: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) is prevented by inhibitors of PtdIns 3-kinase (wortmannin, LY 294002) and unaffected by the drugs that inhibit mTOR (rapamycin) or the classical mitogen-activated protein (MAP) kinase cascade (18Cichy S.B. Uddin S. Danilkovich A. Guo S. Klippel A. Unterman T.G. J. Biol. Chem. 1998; 273: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 19Sutherland C. Waltner-Law M. Gnudi L. Kahn B. Granner D.K. J. Biol. Chem. 1998; 273: 3198-3204Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Studies with constitutively active and dominant negative forms of PKB have shown that PKB may mediate transcriptional effects of insulin through a conserved insulin response sequence present in a number of genes known to be inhibited by insulin in the liver, such as IGFBP-1 and PEPCK (18Cichy S.B. Uddin S. Danilkovich A. Guo S. Klippel A. Unterman T.G. J. Biol. Chem. 1998; 273: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). This suggests that PKB may indeed play an important role in mediating the effects of insulin on hepatic gene expression The insulin/IGF-1-stimulated PKB cascade has also been identified inCaenorhabditis elegans, where it is known to stimulate metabolism, to inhibit dauer arrest and to shorten the life span of this nematode (reviewed in Ref. 20Wood W.B. Cell. 1998; 95: 147-150Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In this pathway, which has been partially dissected by genetic techniques, the DAF2 gene encodes a homologue of the IGF-1 receptor and lies “upstream” of the AGE1 gene that encodes a PtdIns 3-kinase homologue and the AKT1 and AKT2 genes that encode homologues of PKB. Downstream of PKB is the transcription factor DAF16; mutations inDAF16 return life span to normal that has been lengthened by inactivating mutations in AGE1 or AKT1/2 (21Lin K. Dorman J.B. Rodan A. Kenyon C. Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1214) Google Scholar,22Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1551) Google Scholar). Whether DAF16 is phosphorylated directly by AKT1/AKT2 is unknown, but we noticed that it possesses three consensus sequences for phosphorylation by PKB, all of which are highly conserved in several mammalian DAF16 homologues, namely the “Forkhead” family members FKHR, FKHRL1, and AFX (23Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (558) Google Scholar). Two of the three sites are conserved in a further DAF16 homologue encoded by the AF6q21 gene (24Hillion J. LeConiat M. Jonveaux P. Berger R. Bernard O.A. Blood. 1997; 90: 3714-3719Crossref PubMed Google Scholar). Here we establish that FKHR is phosphorylated at these three sites by PKB in vitro and in cotransfection experiments and that the same sites become phosphorylated in response to IGF-1 in 293 cells via a PtdIns 3-kinase-dependent pathway that is independent of mTOR or the classical MAP kinase cascade. The accompanying paper (25Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar) demonstrates that FKHR-stimulated reporter gene expression is dependent on an intact insulin response sequence (IRS) and that by FKHR is inhibited by insulin via the phosphorylation of (25Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). The conservation of Thr-24, Ser-256, and Ser-319 and the sequences surrounding them in DAF16 suggests that DAF16 is to be a direct substrate for PKB in C. elegans. these results indicate that PKB the of FKHR to stimulate and IGF-1 were and protein PKI, the specific peptide inhibitor of cAMP-dependent protein kinase was by F. B. Caudwell in the Protein Phosphorylation and other by G. of was PD 98059 and rapamycin and the and were and expressed in was by of this PDK1 (5Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar) and (3Alessi D.R. Andjelkovic M. Caudwell F.B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar) were by Deak in the at 293 cells were at in an of with of cells was the of to cells were for were in 1 A 1 1 1 and The were at and the were in and at of the identified that encoded of of these sequences with the sequence of the second FKHR sequence to be number M.J. S. 1998; 47: PubMed Scopus Google human FKHR was as A was in which the the the of FKHR This the in a with a second to of the FKHR This product was the The of FKHR were by a the and sites G. 1998; PubMed Scopus Google Scholar). A a of FKHR was by the of FKHR in This was and to expression of FKHR in mammalian the FKHR was a for expression of a protein in E. was by and at in at a of 2 (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R.J. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar). and were to residues and of FKHR, where the of phosphorylation Ser-256, and The were to both and and at the were through a to which a been by on to which the been were with to with and at A further was residues of FKHR that the as well as the phosphorylated of the that been expressed in cells and activated by phosphorylation with PDK1 (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R.J. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar) was for at with FKHR or BAD in PKI, of PKB was that that the phosphorylation of 1 of the peptide in 1 (11Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4397) Google Scholar). the of phosphorylation of FKHR and BAD, of the were in and to with and the were and the of and were by a with of in on the same The of and were as and The that FKHR three at Thr-24, Ser-256, and that lie in consensus sequences for phosphorylation by PKB to the phosphorylation of this protein by PKB in FKHR was phosphorylated by PKB to a of of of protein The rate of phosphorylation was much faster than that of the protein BAD 1 which is thought to be an in for PKB S.R. H. X. S. H.A. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the sites of we that FKHR it is phosphorylated at Thr-24, Ser-256, or The of these is in The a to the sequence surrounding FKHR phosphorylation by PKB, and of was prevented the was with the used to it but not by with either of the other The of the to the sequences surrounding and Ser-319 were in an These results that PKB FKHR at Thr-24, Ser-256, and The phosphorylation of all three sites a when of PKB were present in the 3 when the of PKB was to to of was to be phosphorylated rapidly than the other sites 3 FKHR at three expressed was with and PKB the were in on to and with the in same as that the of PKB was to and the were by to the of phosphorylation of whether FKHR be phosphorylated by PKB in a we 293 cells FKHR with that a constitutively active PKB and FKHR phosphorylation by with the These experiments that Thr-24, Ser-256, and Ser-319 were phosphorylated to when FKHR was and that phosphorylation of when FKHR was cotransfected with PKB also that FKHR became phosphorylated at all three sites when cotransfected with PDK1 of the upstream of PKB (reviewed in Refs. 1Cohen P. Alessi D.R. Cross D.A.E. FEBS Lett. 1997; 410: 3-10Crossref PubMed Scopus (236) Google Scholar and 2Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (677) Google Scholar). In 293 cells PKB is activated stimulation with IGF-1 with a for activation of 1 (3Alessi D.R. Andjelkovic M. Caudwell F.B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar). IGF-1 also stimulated the phosphorylation of FKHR at Thr-24, Ser-256, and phosphorylation not Consistent with the IGF-1-induced phosphorylation of FKHR mediated by PKB, phosphorylation of Thr-24, Ser-256, and Ser-319 was prevented the cells were with the PtdIns 3-kinase inhibitor wortmannin to stimulation with IGF-1 The of phosphorylation was also abolished by and all lie in Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) sequences that are not consensus sequences for phosphorylation by PKB but also for phosphorylation by MAP kinase-activated protein kinase 1 also called and kinase (9Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (552) Google Scholar, Caudwell F.B. Cohen Cohen P. FEBS Lett. 1995; PubMed Scopus Google Scholar). lies of MAP and kinase lies of are both activated in response to insulin or IGF-1, and activation is also inhibited by wortmannin D.A.E. Alessi D.R. Hundal Cohen P. Biochem. J. PubMed Scopus Google Scholar, C. R. J. Biochem. 1995; PubMed Scopus Google Scholar). which of these protein kinases mediates the IGF-1-induced phosphorylation of FKHR in we therefore experiments in which 293 cells were with either rapamycin the activation of kinase by inhibiting mTOR G. Curr. Opin. Biol. 1997; PubMed Scopus Google or PD 98059 the activation of MAP kinase and the activation of D.R. A. Cohen P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). of these drugs the or IGF-1-induced phosphorylation of FKHR at Thr-24, Ser-256, and although, at the same prevented the and IGF-1-induced phosphorylation of kinase and not In this we have a phosphorylation of FKHR by PKB at Thr-24, Ser-256, and This both in vitro 2 and and in cotransfection experiments either PKB or PDK1 PDK1 is an upstream activator of PKB (reviewed in Refs. 1Cohen P. Alessi D.R. Cross D.A.E. FEBS Lett. 1997; 410: 3-10Crossref PubMed Scopus (236) Google Scholar and 2Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (677) Google Scholar), and the phosphorylation of FKHR when cotransfected with PDK1 is by the PKB that becomes activated by the were with GSK3, physiological substrate of PKB M. Cohen P. Alessi D.R. FEBS Lett. 1997; PubMed Scopus Google Scholar). have also shown that all three sites on FKHR become rapidly phosphorylated when 293 cells are stimulated with IGF-1 The IGF-1-induced phosphorylation is prevented by inhibitors of PtdIns 3-kinase but not by inhibitors of the activation of the MAP kinase cascade or These with the in vitro and cotransfection indicate that the IGF-1-induced phosphorylation of FKHR is mediated by PKB or a A pathway in C. has been partially dissected by genetic in which DAF16 (an FKHR is a component of a signaling cascade that includes homologues of the insulin/IGF-1 receptor, PtdIns and PKB PDK1 has not yet been identified as a component of this pathway, has other role in protein kinases that lie in protein kinase This may of the PDK1 homologues in is also A. T. J. Alessi D.R. Curr. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). have noticed as the of C. indeed encode a PDK1 homologue on the number The accompanying paper (25Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar) that FKHR the transcription of a reporter that this stimulation is dependent on an intact insulin response sequence and that transcription is by when cells are to insulin or with constitutively active PKB. In this phosphorylation of to be and to mediate the of insulin to the to acid mimics is the in FKHR that is phosphorylated rapidly by PKB in vitro 3 In of or Ser-319 to has on the of insulin or constitutively active PKB to activation by FKHR, and the of these other phosphorylation events to be the conservation of the sequences that and Ser-319 in other mammalian FKHR homologues (23Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (558) Google Scholar) as well as DAF16 of C. (21Lin K. Dorman J.B. Rodan A. Kenyon C. Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1214) Google Scholar, S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1551) Google Scholar) suggests that phosphorylation is to play a in In this with that in the accompanying paper (25Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar), the direct evidence that the kinase cascade to the of DAF16 is present in mammalian The that phosphorylation of FKHR inhibits to stimulate transcription is with genetic evidence in C. that has shown that mutations in gene have the to mutations in either the AGE1 or AKT1/2 genes (21Lin K. Dorman J.B. Rodan A. Kenyon C. Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1214) Google Scholar, S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1551) Google Scholar, S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (558) Google Scholar). These the that phosphorylation of FKHR the of at some of the genes transcription is inhibited by in this paper was results to and in the paper (25Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar) have been by three other In of family members namely A. A. M.J. P. Anderson M.J. J. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), AFX de Vries-Smits A.M.M. D.R. Burgering Nature. 1999; PubMed Scopus Google Scholar), and J. T. Proc. Natl. Acad. Sci. U. S. A. 1999; Google Scholar), phosphorylation was by A. A. M.J. P. Anderson M.J. J. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, de Vries-Smits A.M.M. D.R. Burgering Nature. 1999; PubMed Scopus Google Scholar) as a of of the phosphorylated transcription factor with proteins A. A. M.J. P. Anderson M.J. J. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). and activation of Deak of PDK1 and constitutively active and BAD for