Regulation of Kinase Activity of 3-Phosphoinositide-dependent Protein Kinase-1 by Binding to 14-3-3
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m205141200
ISSN1083-351X
AutoresSaori Sato, Naoya Fujita, Takashi Tsuruo,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
Resumo3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the protein kinase A, G, and C subfamily. In particular, PDK1 plays an important role in regulating the Akt survival pathway by phosphorylating Akt on Thr-308. PDK1 kinase activity was thought to be constitutively active; however, recent reports suggested that its activity is regulated by binding to other proteins, such as protein kinase C-related kinase-2 (PRK2), p90 ribosomal protein S6 kinase-2 (RSK2), and heat-shock protein 90 (Hsp90). Here we report that PDK1 binds to 14-3-3 proteins in vivo and in vitrothrough the sequence surrounding Ser-241, a residue that is phosphorylated by itself and is critical for its kinase activity. Mutation of PDK1 to increase its binding to 14-3-3 decreased its kinase activity in vivo. By contrast, mutation of PDK1 to decrease its interaction with 14-3-3 resulted in increased PDK1 kinase activity. Moreover, incubation of wild-type PDK1 with recombinant 14-3-3in vitro decreased its kinase activity. These data indicate that PDK1 kinase activity is negatively regulated by binding to 14-3-3 through the PDK1 autophosphorylation site Ser-241. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the protein kinase A, G, and C subfamily. In particular, PDK1 plays an important role in regulating the Akt survival pathway by phosphorylating Akt on Thr-308. PDK1 kinase activity was thought to be constitutively active; however, recent reports suggested that its activity is regulated by binding to other proteins, such as protein kinase C-related kinase-2 (PRK2), p90 ribosomal protein S6 kinase-2 (RSK2), and heat-shock protein 90 (Hsp90). Here we report that PDK1 binds to 14-3-3 proteins in vivo and in vitrothrough the sequence surrounding Ser-241, a residue that is phosphorylated by itself and is critical for its kinase activity. Mutation of PDK1 to increase its binding to 14-3-3 decreased its kinase activity in vivo. By contrast, mutation of PDK1 to decrease its interaction with 14-3-3 resulted in increased PDK1 kinase activity. Moreover, incubation of wild-type PDK1 with recombinant 14-3-3in vitro decreased its kinase activity. These data indicate that PDK1 kinase activity is negatively regulated by binding to 14-3-3 through the PDK1 autophosphorylation site Ser-241. phosphatidylinositide 3-OH kinase protein kinase A, G, and C glutathione S-transferase p70 ribosomal protein S6 kinase 3-phosphoinositide-dependent protein kinase-1 cAMP-dependent protein kinase protein kinase C PKC-related kinase p90 ribosomal protein S6 kinase serum and glucocorticoid-inducible kinase wild type hemagglutinin Many growth factors and cytokines have been reported to promote cell survival. Stimulation of cells with these factors activates phosphatidylinositide 3-OH kinase (PI3K),1 and activated PI3K generates the phospholipid second messenger molecules phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (1Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (827) Google Scholar, 2Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar, 3Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). These lipids then induce activation of several members of the AGC family of protein kinases including Akt, p70 ribosomal protein S6 kinase (p70S6K), serum, and glucocorticoid–inducible kinases (SGKs), protein kinase A (PKA), and protein kinase C (PKC) isoforms. Activated kinases, then, mediate survival-signal transduction by phosphorylating downstream key regulatory proteins.3-Phosphoinositide-dependent protein kinase-1 (PDK1) was originally identified as a kinase that could phosphorylate Akt on its activation loop (residue Thr-308) (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney R.P.J. Reeese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 5Stephens L. Anderson K. Stokoe D. Erdjument-Bromage 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, 6Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (969) Google Scholar). Later studies, however, have showed that PDK1 is not just an Akt kinase but also a kinase phosphorylating p70S6K, SGKs, PKC isoforms, and p90 ribosomal protein S6 kinases (RSKs) at the equivalent residues of Thr-308 of Akt (reviewed in Ref. 7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar). Therefore, PDK1 plays a central role in activating the AGC family of protein kinases. In the case of Akt, the interaction of phosphatidylinositol 3,4,5-trisphosphate with the pleckstrin homology domain recruits Akt to the plasma membrane and promotes conformational change, which results in phosphorylation of Akt at Thr-308 by PDK1 and at Ser-473 by an as yet unidentified kinase (so called PDK2) (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar, 8Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1514) Google Scholar). In the case of p70S6K, SGKs, and PKC isoforms, however, the precise mechanisms for PDK1- dependent phosphorylation and activation are not well understood.PDK1 itself is also a member of the AGC subfamily of protein kinases and is phosphorylated on the Ser-241 activation loop (equivalent to Thr-308 of Akt) (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar). As PDK1 expressed in bacteria is active and is phosphorylated at Ser-241 (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar), it is thought to phosphorylate itself at this same site. Further, mutation of Ser-241 to Ala was reported to abolish PDK1 kinase activity, and IGF-I stimulation did not cause further activation of PDK1 (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar). According to these results, PDK1 was thought to be constitutively active. Several recent reports, however, suggested that PDK1 kinase activity is controlled by PDK1-associating proteins, such as PKC-related kinase-1 (PRK1)/PRK2 (10Wick M.J. Dong L.Q. Riojas R.A. Ramos F.J. Liu F. J. Biol. Chem. 2000; 275: 40400-40406Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), PDK1-interacting fragment of PRK2 (11Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar), RSK2 (12Frodin M. Jensen C.J. Merienne K. Gammeltoft S. EMBO J. 2000; 19: 2924-2934Crossref PubMed Scopus (254) Google Scholar), and Hsp90 (13Fujita N. Sato S. Ishida A. Tsuruo T. J. Biol. Chem. 2002; 277: 10346-10353Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The interaction of PDK1 with the PDK1-interacting fragment of PRK2 converts PDK1 from an enzyme that phosphorylates Akt only at Thr-308 into a kinase that phosphorylates both Thr-308 and Ser-473 (11Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Further, the association of PDK1 with RSK2 stimulates PDK1 activation and autophosphorylation (12Frodin M. Jensen C.J. Merienne K. Gammeltoft S. EMBO J. 2000; 19: 2924-2934Crossref PubMed Scopus (254) Google Scholar). We recently reported that Hsp90 participates in stability and signaling of PDK1 (13Fujita N. Sato S. Ishida A. Tsuruo T. J. Biol. Chem. 2002; 277: 10346-10353Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Therefore, PDK1 is not a constitutively active kinase but a kinase regulated by other interacting proteins.Members of the 14-3-3 protein family are highly conserved and widely expressed 28–31 kDa proteins that naturally assemble as homodimers or heterodimers. They consists of at least seven isoforms in mammals (β, γ, ε, η, ς, θ, τ, and ζ). The 14-3-3 proteins have been shown to interact with and to regulate proteins controlling a wide array of signaling pathways, including Raf-1, Bad, FKHRL1, and Cdc25c (reviewed in Ref. 14Tzivion G. Avruch J. J. Biol. Chem. 2002; 277: 3061-3064Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Binding of 14-3-3 to its partners depends on phosphorylation of the Ser or Thr residue in the recognition domains. Using peptides derived from Raf-1, Muslin et al. (15Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar) identified that the motif optimal for association with 14-3-3 proteins was RSXpSXP, where pS represents phosphorylated Ser and X represents any amino acid. Moreover, using phosphopeptide libraries, Yaffe et al. (16Yaffe 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) and Rittingeret al. (17Rittinger K. Budman J., Xu, J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar) revealed that there are two preferred 14-3-3 binding motifs, RSXpSXP and RXXXpSXP, and most of the 14-3-3 partners identified to date contain one of these motifs. It is important to note that several proteins that associate with 14-3-3 proteins do not contain either of these motifs, indicating that 14-3-3 can bind to some variation (e.g. RXXpS in PTPH1 and TH, RX(X)pSXX(X)S in Cbl and PKCμ). Furthermore, 14-3-3 bound to some proteins and peptides in a phosphorylation-independent manner (14Tzivion G. Avruch J. J. Biol. Chem. 2002; 277: 3061-3064Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 18Yaffe M.B. FEBS Lett. 2002; 513: 53-57Crossref PubMed Scopus (549) Google Scholar).In the course of searching protein-protein interacting motifs in PDK1, we found four 14-3-3 binding motifs (RXXpS). Thus, we started to examine the interaction between PDK1 and 14-3-3 proteins, and of the five 14-3-3 isoforms we tested, 14-3-3θ and η were found to form a complex with PDK1. Using site-directed mutants, we identified the 14-3-3 recognition site in PDK1. Because the association of PDK1 with 14-3-3 reduced PDK1 kinase activity in vitro andin vivo, we concluded that 14-3-3 negatively regulates PDK1 by complex formation.DISCUSSIONIt is clear that PDK1 plays a central role in activating the AGC subfamily of protein kinases (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar, 24Williams M.R. Arthur J.S. Balendran A. van der Kaay J. Poli V. Cohen P. Alessi D.R. Curr. Biol. 2000; 10: 439-448Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). These kinases then mediate intracellular signaling such as cell survival, cell growth, protein synthesis, and gene expression. PDK1 phosphorylates AGC kinase members at the residues equivalent to Thr-308 of Akt (known as activation loop or T-loop) (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar). PDK1 is, itself, a member of the AGC subfamily of protein kinases. Thus, PDK1 phosphorylates itself at its activation loop (Ser-241), thereby activating itself (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar). Although PDK1 kinase activity has been thought to be constitutively active and not further activated by growth factor stimulation, recent findings suggest that its activity and its character are controlled by interaction with other proteins. For example, when PDK1 interacts with the PDK1-interacting fragment of PRK2, it is converted to exhibit PDK2-like activity (11Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). The association of PDK1 with RSK2 up-regulates PDK1 kinase activity and autophosphorylation (12Frodin M. Jensen C.J. Merienne K. Gammeltoft S. EMBO J. 2000; 19: 2924-2934Crossref PubMed Scopus (254) Google Scholar). We recently reported that PDK1 binds to Hsp90, and its binding prevents PDK1 from proteasome-dependent degradation and keeps it in a soluble and active conformational state (13Fujita N. Sato S. Ishida A. Tsuruo T. J. Biol. Chem. 2002; 277: 10346-10353Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Moreover, PDK1 kinase activity is promoted by phosphorylation at tyrosine residues, presumably by a member of the Src kinase family (25Grillo S. Gremeaux T. Casamayor A. Alessi D.R., Le Marchand-Brustel Y. Tanti J.F. Eur. J. Biochem. 2000; 267: 6642-6649Crossref PubMed Scopus (45) Google Scholar, 26Park J. Hill M.M. Hess D. Brazil D.P. Hofsteenge J. Hemmings B.A. J. Biol. Chem. 2001; 276: 37459-37471Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).Here we provide evidence that PDK1 binds to 14-3-3 in vivoand in vitro through the residues surrounding the autophosphorylation site Ser-241 and that the association is achieved only when Ser-241 has been phosphorylated (Fig. 3). Although PDK1 contains other 14-3-3 binding motifs in its amino acid sequence (3RTTS6, 407RSGS410,546RYQS549), site-directed mutagenesis of the target Ser revealed that these sites were not involved in the PDK1–14-3-3 complex formation (Fig. 3 B). It should be noted that 14-3-3 also bound to the S410A-PDK1 mutant in which Ser-410 was converted to Ala although the Ser-410 residue has been reported to be phosphorylated in vivo (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar). There may be some conformational state essential for the 14-3-3 binding in addition to Ser phosphorylation in 14-3-3 binding motifs.Among the 14-3-3-binding partners, some proteins, like Raf-1, bind to almost all 14-3-3 isoforms with nearly equivalent affinity (18Yaffe M.B. FEBS Lett. 2002; 513: 53-57Crossref PubMed Scopus (549) Google Scholar). However, others, like A20 and Cdc25B, bind to different 14-3-3 isoforms with significantly different affinities (27Vincenz C. Dixit V.M. J. Biol. Chem. 1996; 271: 20029-20034Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 28Mils V. Baldin V. Goubin F. Pinta I. Papin C. Waye M. Eychene A. Ducommun B. Oncogene. 2000; 19: 1257-1265Crossref PubMed Scopus (91) Google Scholar). We recently reported that p27Kip1 binds to 14-3-3 after Akt-mediated phosphorylation at the COOH-terminal Thr-198 (29Fujita N. Sato S. Katayama K. Tsuruo T. J. Biol. Chem. 2002; 277: 28706-28713Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). The phosphorylated p27Kip1 could form complexes with 14-3-3θ, η, and ε but not with 14-3-3β and ζ (29Fujita N. Sato S. Katayama K. Tsuruo T. J. Biol. Chem. 2002; 277: 28706-28713Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), suggesting that 14-3-3 isoforms have different affinities to their partners. Among the five isoforms (β, ε, η, θ, and ζ), only 14-3-3θ and η interacted with PDK1, and 14-3-3θ bound more tightly than 14-3-3η (Fig.1 B). The difference among 14-3-3θ, η and other isoforms that accounts for the different affinities for PDK1 is still unclear, since many of the residues that form the ligand-binding groove are conserved in the seven isoforms. However, comparing the residues in 14-3-3θ necessary for the interaction with Raf-1 and PDK1 did reveal some differences. To form a complex with Raf-1, Arg-56, Arg-60, and Arg-127 were necessary (Fig. 2 B, lanes 3 and4), a finding consistent with a previous report (23Thorson J.A., Yu, L.W. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar). In the case of PDK1, Arg-56 and Arg-60 were not essential but Arg-127 was (Fig. 2 A). By contrast, both Arg-56 and Arg-60 are necessary but Arg-127 is not for p27Kip1 binding to 14-3-3 (Ref.29Fujita N. Sato S. Katayama K. Tsuruo T. J. Biol. Chem. 2002; 277: 28706-28713Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). 2N. Fujita, S. Sato, and T. Tsuruo, unpublished observations. These results indicate that the residues necessary for ligand-14-3-3 binding vary with the ligand. Moreover, there may be some residues essential for the association with ligands that are not conserved in the 14-3-3 isoforms. However, we could not exclude the possibility that the differences we observed above were a result of the differences between the ligand that possessed at least two 14-3-3 binding motifs (Raf-1) and the one that had only one (PDK1). The distinct mechanism of isoform-specific interaction is a problem to be solved in future studies.The general mechanisms by which 14-3-3 regulates partner protein functions are 4-fold: 1) regulation of subcellular localization of the target protein; 2) direct regulation of catalytic activity of the binding partner; 3) protection of the ligands from proteolysis or dephosphorylation; and 4) regulation of interaction between bound protein and other molecules. The identified 14-3-3 binding residue of PDK1 (Ser-241) is thought to be phosphorylated by itself and it be essential for its kinase activity (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar). Consistent with previous reporting (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar), we confirmed that S241A-PDK1 exhibited weak Akt and SGK phosphorylation activity when compared with WT-PDK1 (Fig. 4). We thus generated PDK1 point mutants in which residues other than Ser-241 are mutated (Fig. 3 C) and examined their activity to phosphorylate Akt (Fig. 4 A) or SGK (Fig. 4 C). Mutation of PDK1 to promote 14-3-3 (V243P-PDK1) association exhibited reduced PDK1 kinase activity to phosphorylate Akt and SGK in cells, while mutation of PDK1 to inhibit the interaction (R238E-PDK1) slightly increased its activity (Fig. 4). Moreover, incubation with recombinant 14-3-3θ decreased PDK1 autophosphorylation activity (Fig.5 A) and kinase activity to phosphorylate SGK (Fig.5 B) in a dose-dependent manner in vitro. These results indicate that 14-3-3 is not only a PDK1-associating protein but also a protein that negatively regulates PDK1 kinase activity.Several reports indicate that PDK1 binds to phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate through its pleckstrin homology domain (19Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 30Filippa N. Sable C.L. Hemmings B.A. Van Obberghen E. Mol. Cell. Biol. 2000; 20: 5712-5721Crossref PubMed Scopus (112) Google Scholar). Because mutants of PDK1 deleted in its pleckstrin homology domain have been reported to prevent translocation of Akt, the relocalization of PDK1 from cytosol to the plasma membrane is suggested to play an important role in the recruitment of Akt to the plasma membrane and the subsequent Akt activation in stimulated cells (30Filippa N. Sable C.L. Hemmings B.A. Van Obberghen E. Mol. Cell. Biol. 2000; 20: 5712-5721Crossref PubMed Scopus (112) Google Scholar). PDK1 catalytic activity is not required for the PDK1 translocation to the plasma membrane (30Filippa N. Sable C.L. Hemmings B.A. Van Obberghen E. Mol. Cell. Biol. 2000; 20: 5712-5721Crossref PubMed Scopus (112) Google Scholar). Although mutating PDK1 to increase its binding to 14-3-3 (V243P) reduced kinase activity to phosphorylate Akt at Thr-308, the mutation did not affect the binding affinity of PDK1 to Akt (Fig. 4). Therefore, the decrease in kinase activity of V243P-PDK1 to phosphorylate Akt in cells may be caused by down-regulation of its kinase activity but not by suppressing translocation. This notion was supported by the fact that the amount of the phosphorylated form of Myr-Akt was reduced by 14-3-3 protein expression even though Myr-Akt constitutively targeted the plasma membrane (Fig. 4 D). In the case of SGK, translocation to the plasma membrane is not required for PDK1-dependent phosphorylation of SGK at Thr-256 (31Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (524) Google Scholar). SGK phosphorylation at Thr-256 was also suppressed by PDK1 mutation to increase its binding to 14-3-3 (V243P; Fig. 4 C). These results strongly suggest that inhibition of PDK1 activity but not PDK1 translocation is the main mechanism of 14-3-3-mediated inactivation of PDK1-dependent signal transduction. Many growth factors and cytokines have been reported to promote cell survival. Stimulation of cells with these factors activates phosphatidylinositide 3-OH kinase (PI3K),1 and activated PI3K generates the phospholipid second messenger molecules phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (1Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (827) Google Scholar, 2Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar, 3Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). These lipids then induce activation of several members of the AGC family of protein kinases including Akt, p70 ribosomal protein S6 kinase (p70S6K), serum, and glucocorticoid–inducible kinases (SGKs), protein kinase A (PKA), and protein kinase C (PKC) isoforms. Activated kinases, then, mediate survival-signal transduction by phosphorylating downstream key regulatory proteins. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) was originally identified as a kinase that could phosphorylate Akt on its activation loop (residue Thr-308) (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney R.P.J. Reeese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 5Stephens L. Anderson K. Stokoe D. Erdjument-Bromage 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, 6Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (969) Google Scholar). Later studies, however, have showed that PDK1 is not just an Akt kinase but also a kinase phosphorylating p70S6K, SGKs, PKC isoforms, and p90 ribosomal protein S6 kinases (RSKs) at the equivalent residues of Thr-308 of Akt (reviewed in Ref. 7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar). Therefore, PDK1 plays a central role in activating the AGC family of protein kinases. In the case of Akt, the interaction of phosphatidylinositol 3,4,5-trisphosphate with the pleckstrin homology domain recruits Akt to the plasma membrane and promotes conformational change, which results in phosphorylation of Akt at Thr-308 by PDK1 and at Ser-473 by an as yet unidentified kinase (so called PDK2) (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar, 8Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1514) Google Scholar). In the case of p70S6K, SGKs, and PKC isoforms, however, the precise mechanisms for PDK1- dependent phosphorylation and activation are not well understood. PDK1 itself is also a member of the AGC subfamily of protein kinases and is phosphorylated on the Ser-241 activation loop (equivalent to Thr-308 of Akt) (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar). As PDK1 expressed in bacteria is active and is phosphorylated at Ser-241 (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar), it is thought to phosphorylate itself at this same site. Further, mutation of Ser-241 to Ala was reported to abolish PDK1 kinase activity, and IGF-I stimulation did not cause further activation of PDK1 (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar). According to these results, PDK1 was thought to be constitutively active. Several recent reports, however, suggested that PDK1 kinase activity is controlled by PDK1-associating proteins, such as PKC-related kinase-1 (PRK1)/PRK2 (10Wick M.J. Dong L.Q. Riojas R.A. Ramos F.J. Liu F. J. Biol. Chem. 2000; 275: 40400-40406Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), PDK1-interacting fragment of PRK2 (11Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar), RSK2 (12Frodin M. Jensen C.J. Merienne K. Gammeltoft S. EMBO J. 2000; 19: 2924-2934Crossref PubMed Scopus (254) Google Scholar), and Hsp90 (13Fujita N. Sato S. Ishida A. Tsuruo T. J. Biol. Chem. 2002; 277: 10346-10353Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The interaction of PDK1 with the PDK1-interacting fragment of PRK2 converts PDK1 from an enzyme that phosphorylates Akt only at Thr-308 into a kinase that phosphorylates both Thr-308 and Ser-473 (11Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Further, the association of PDK1 with RSK2 stimulates PDK1 activation and autophosphorylation (12Frodin M. Jensen C.J. Merienne K. Gammeltoft S. EMBO J. 2000; 19: 2924-2934Crossref PubMed Scopus (254) Google Scholar). We recently reported that Hsp90 participates in stability and signaling of PDK1 (13Fujita N. Sato S. Ishida A. Tsuruo T. J. Biol. Chem. 2002; 277: 10346-10353Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Therefore, PDK1 is not a constitutively active kinase but a kinase regulated by other interacting proteins. Members of the 14-3-3 protein family are highly conserved and widely expressed 28–31 kDa proteins that naturally assemble as homodimers or heterodimers. They consists of at least seven isoforms in mammals (β, γ, ε, η, ς, θ, τ, and ζ). The 14-3-3 proteins have been shown to interact with and to regulate proteins controlling a wide array of signaling pathways, including Raf-1, Bad, FKHRL1, and Cdc25c (reviewed in Ref. 14Tzivion G. Avruch J. J. Biol. Chem. 2002; 277: 3061-3064Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Binding of 14-3-3 to its partners depends on phosphorylation of the Ser or Thr residue in the recognition domains. Using peptides derived from Raf-1, Muslin et al. (15Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar) identified that the motif optimal for association with 14-3-3 proteins was RSXpSXP, where pS represents phosphorylated Ser and X represents any amino acid. Moreover, using phosphopeptide libraries, Yaffe et al. (16Yaffe 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) and Rittingeret al. (17Rittinger K. Budman J., Xu, J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar) revealed that there are two preferred 14-3-3 binding motifs, RSXpSXP and RXXXpSXP, and most of the 14-3-3 partners identified to date contain one of these motifs. It is important to note that several proteins that associate with 14-3-3 proteins do not contain either of these motifs, indicating that 14-3-3 can bind to some variation (e.g. RXXpS in PTPH1 and TH, RX(X)pSXX(X)S in Cbl and PKCμ). Furthermore, 14-3-3 bound to some proteins and peptides in a phosphorylation-independent manner (14Tzivion G. Avruch J. J. Biol. Chem. 2002; 277: 3061-3064Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 18Yaffe M.B. FEBS Lett. 2002; 513: 53-57Crossref PubMed Scopus (549) Google Scholar). In the course of searching protein-protein interacting motifs in PDK1, we found four 14-3-3 binding motifs (RXXpS). Thus, we started to examine the interaction between PDK1 and 14-3-3 proteins, and of the five 14-3-3 isoforms we tested, 14-3-3θ and η were found to form a complex with PDK1. Using site-directed mutants, we identified the 14-3-3 recognition site in PDK1. Because the association of PDK1 with 14-3-3 reduced PDK1 kinase activity in vitro andin vivo, we concluded that 14-3-3 negatively regulates PDK1 by complex formation. DISCUSSIONIt is clear that PDK1 plays a central role in activating the AGC subfamily of protein kinases (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar, 24Williams M.R. Arthur J.S. Balendran A. van der Kaay J. Poli V. Cohen P. Alessi D.R. Curr. Biol. 2000; 10: 439-448Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). These kinases then mediate intracellular signaling such as cell survival, cell growth, protein synthesis, and gene expression. PDK1 phosphorylates AGC kinase members at the residues equivalent to Thr-308 of Akt (known as activation loop or T-loop) (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar). PDK1 is, itself, a member of the AGC subfamily of protein kinases. Thus, PDK1 phosphorylates itself at its activation loop (Ser-241), thereby activating itself (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar). Although PDK1 kinase activity has been thought to be constitutively active and not further activated by growth factor stimulation, recent findings suggest that its activity and its character are controlled by interaction with other proteins. For example, when PDK1 interacts with the PDK1-interacting fragment of PRK2, it is converted to exhibit PDK2-like activity (11Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). The association of PDK1 with RSK2 up-regulates PDK1 kinase activity and autophosphorylation (12Frodin M. Jensen C.J. Merienne K. Gammeltoft S. EMBO J. 2000; 19: 2924-2934Crossref PubMed Scopus (254) Google Scholar). We recently reported that PDK1 binds to Hsp90, and its binding prevents PDK1 from proteasome-dependent degradation and keeps it in a soluble and active conformational state (13Fujita N. Sato S. Ishida A. Tsuruo T. J. Biol. Chem. 2002; 277: 10346-10353Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Moreover, PDK1 kinase activity is promoted by phosphorylation at tyrosine residues, presumably by a member of the Src kinase family (25Grillo S. Gremeaux T. Casamayor A. Alessi D.R., Le Marchand-Brustel Y. Tanti J.F. Eur. J. Biochem. 2000; 267: 6642-6649Crossref PubMed Scopus (45) Google Scholar, 26Park J. Hill M.M. Hess D. Brazil D.P. Hofsteenge J. Hemmings B.A. J. Biol. Chem. 2001; 276: 37459-37471Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).Here we provide evidence that PDK1 binds to 14-3-3 in vivoand in vitro through the residues surrounding the autophosphorylation site Ser-241 and that the association is achieved only when Ser-241 has been phosphorylated (Fig. 3). Although PDK1 contains other 14-3-3 binding motifs in its amino acid sequence (3RTTS6, 407RSGS410,546RYQS549), site-directed mutagenesis of the target Ser revealed that these sites were not involved in the PDK1–14-3-3 complex formation (Fig. 3 B). It should be noted that 14-3-3 also bound to the S410A-PDK1 mutant in which Ser-410 was converted to Ala although the Ser-410 residue has been reported to be phosphorylated in vivo (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar). There may be some conformational state essential for the 14-3-3 binding in addition to Ser phosphorylation in 14-3-3 binding motifs.Among the 14-3-3-binding partners, some proteins, like Raf-1, bind to almost all 14-3-3 isoforms with nearly equivalent affinity (18Yaffe M.B. FEBS Lett. 2002; 513: 53-57Crossref PubMed Scopus (549) Google Scholar). However, others, like A20 and Cdc25B, bind to different 14-3-3 isoforms with significantly different affinities (27Vincenz C. Dixit V.M. J. Biol. Chem. 1996; 271: 20029-20034Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 28Mils V. Baldin V. Goubin F. Pinta I. Papin C. Waye M. Eychene A. Ducommun B. Oncogene. 2000; 19: 1257-1265Crossref PubMed Scopus (91) Google Scholar). We recently reported that p27Kip1 binds to 14-3-3 after Akt-mediated phosphorylation at the COOH-terminal Thr-198 (29Fujita N. Sato S. Katayama K. Tsuruo T. J. Biol. Chem. 2002; 277: 28706-28713Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). The phosphorylated p27Kip1 could form complexes with 14-3-3θ, η, and ε but not with 14-3-3β and ζ (29Fujita N. Sato S. Katayama K. Tsuruo T. J. Biol. Chem. 2002; 277: 28706-28713Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), suggesting that 14-3-3 isoforms have different affinities to their partners. Among the five isoforms (β, ε, η, θ, and ζ), only 14-3-3θ and η interacted with PDK1, and 14-3-3θ bound more tightly than 14-3-3η (Fig.1 B). The difference among 14-3-3θ, η and other isoforms that accounts for the different affinities for PDK1 is still unclear, since many of the residues that form the ligand-binding groove are conserved in the seven isoforms. However, comparing the residues in 14-3-3θ necessary for the interaction with Raf-1 and PDK1 did reveal some differences. To form a complex with Raf-1, Arg-56, Arg-60, and Arg-127 were necessary (Fig. 2 B, lanes 3 and4), a finding consistent with a previous report (23Thorson J.A., Yu, L.W. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar). In the case of PDK1, Arg-56 and Arg-60 were not essential but Arg-127 was (Fig. 2 A). By contrast, both Arg-56 and Arg-60 are necessary but Arg-127 is not for p27Kip1 binding to 14-3-3 (Ref.29Fujita N. Sato S. Katayama K. Tsuruo T. J. Biol. Chem. 2002; 277: 28706-28713Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). 2N. Fujita, S. Sato, and T. Tsuruo, unpublished observations. These results indicate that the residues necessary for ligand-14-3-3 binding vary with the ligand. Moreover, there may be some residues essential for the association with ligands that are not conserved in the 14-3-3 isoforms. However, we could not exclude the possibility that the differences we observed above were a result of the differences between the ligand that possessed at least two 14-3-3 binding motifs (Raf-1) and the one that had only one (PDK1). The distinct mechanism of isoform-specific interaction is a problem to be solved in future studies.The general mechanisms by which 14-3-3 regulates partner protein functions are 4-fold: 1) regulation of subcellular localization of the target protein; 2) direct regulation of catalytic activity of the binding partner; 3) protection of the ligands from proteolysis or dephosphorylation; and 4) regulation of interaction between bound protein and other molecules. The identified 14-3-3 binding residue of PDK1 (Ser-241) is thought to be phosphorylated by itself and it be essential for its kinase activity (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar). Consistent with previous reporting (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar), we confirmed that S241A-PDK1 exhibited weak Akt and SGK phosphorylation activity when compared with WT-PDK1 (Fig. 4). We thus generated PDK1 point mutants in which residues other than Ser-241 are mutated (Fig. 3 C) and examined their activity to phosphorylate Akt (Fig. 4 A) or SGK (Fig. 4 C). Mutation of PDK1 to promote 14-3-3 (V243P-PDK1) association exhibited reduced PDK1 kinase activity to phosphorylate Akt and SGK in cells, while mutation of PDK1 to inhibit the interaction (R238E-PDK1) slightly increased its activity (Fig. 4). Moreover, incubation with recombinant 14-3-3θ decreased PDK1 autophosphorylation activity (Fig.5 A) and kinase activity to phosphorylate SGK (Fig.5 B) in a dose-dependent manner in vitro. These results indicate that 14-3-3 is not only a PDK1-associating protein but also a protein that negatively regulates PDK1 kinase activity.Several reports indicate that PDK1 binds to phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate through its pleckstrin homology domain (19Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 30Filippa N. Sable C.L. Hemmings B.A. Van Obberghen E. Mol. Cell. Biol. 2000; 20: 5712-5721Crossref PubMed Scopus (112) Google Scholar). Because mutants of PDK1 deleted in its pleckstrin homology domain have been reported to prevent translocation of Akt, the relocalization of PDK1 from cytosol to the plasma membrane is suggested to play an important role in the recruitment of Akt to the plasma membrane and the subsequent Akt activation in stimulated cells (30Filippa N. Sable C.L. Hemmings B.A. Van Obberghen E. Mol. Cell. Biol. 2000; 20: 5712-5721Crossref PubMed Scopus (112) Google Scholar). PDK1 catalytic activity is not required for the PDK1 translocation to the plasma membrane (30Filippa N. Sable C.L. Hemmings B.A. Van Obberghen E. Mol. Cell. Biol. 2000; 20: 5712-5721Crossref PubMed Scopus (112) Google Scholar). Although mutating PDK1 to increase its binding to 14-3-3 (V243P) reduced kinase activity to phosphorylate Akt at Thr-308, the mutation did not affect the binding affinity of PDK1 to Akt (Fig. 4). Therefore, the decrease in kinase activity of V243P-PDK1 to phosphorylate Akt in cells may be caused by down-regulation of its kinase activity but not by suppressing translocation. This notion was supported by the fact that the amount of the phosphorylated form of Myr-Akt was reduced by 14-3-3 protein expression even though Myr-Akt constitutively targeted the plasma membrane (Fig. 4 D). In the case of SGK, translocation to the plasma membrane is not required for PDK1-dependent phosphorylation of SGK at Thr-256 (31Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (524) Google Scholar). SGK phosphorylation at Thr-256 was also suppressed by PDK1 mutation to increase its binding to 14-3-3 (V243P; Fig. 4 C). These results strongly suggest that inhibition of PDK1 activity but not PDK1 translocation is the main mechanism of 14-3-3-mediated inactivation of PDK1-dependent signal transduction. It is clear that PDK1 plays a central role in activating the AGC subfamily of protein kinases (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar, 24Williams M.R. Arthur J.S. Balendran A. van der Kaay J. Poli V. Cohen P. Alessi D.R. Curr. Biol. 2000; 10: 439-448Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). These kinases then mediate intracellular signaling such as cell survival, cell growth, protein synthesis, and gene expression. PDK1 phosphorylates AGC kinase members at the residues equivalent to Thr-308 of Akt (known as activation loop or T-loop) (7Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar). PDK1 is, itself, a member of the AGC subfamily of protein kinases. Thus, PDK1 phosphorylates itself at its activation loop (Ser-241), thereby activating itself (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar). Although PDK1 kinase activity has been thought to be constitutively active and not further activated by growth factor stimulation, recent findings suggest that its activity and its character are controlled by interaction with other proteins. For example, when PDK1 interacts with the PDK1-interacting fragment of PRK2, it is converted to exhibit PDK2-like activity (11Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). The association of PDK1 with RSK2 up-regulates PDK1 kinase activity and autophosphorylation (12Frodin M. Jensen C.J. Merienne K. Gammeltoft S. EMBO J. 2000; 19: 2924-2934Crossref PubMed Scopus (254) Google Scholar). We recently reported that PDK1 binds to Hsp90, and its binding prevents PDK1 from proteasome-dependent degradation and keeps it in a soluble and active conformational state (13Fujita N. Sato S. Ishida A. Tsuruo T. J. Biol. Chem. 2002; 277: 10346-10353Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Moreover, PDK1 kinase activity is promoted by phosphorylation at tyrosine residues, presumably by a member of the Src kinase family (25Grillo S. Gremeaux T. Casamayor A. Alessi D.R., Le Marchand-Brustel Y. Tanti J.F. Eur. J. Biochem. 2000; 267: 6642-6649Crossref PubMed Scopus (45) Google Scholar, 26Park J. Hill M.M. Hess D. Brazil D.P. Hofsteenge J. Hemmings B.A. J. Biol. Chem. 2001; 276: 37459-37471Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Here we provide evidence that PDK1 binds to 14-3-3 in vivoand in vitro through the residues surrounding the autophosphorylation site Ser-241 and that the association is achieved only when Ser-241 has been phosphorylated (Fig. 3). Although PDK1 contains other 14-3-3 binding motifs in its amino acid sequence (3RTTS6, 407RSGS410,546RYQS549), site-directed mutagenesis of the target Ser revealed that these sites were not involved in the PDK1–14-3-3 complex formation (Fig. 3 B). It should be noted that 14-3-3 also bound to the S410A-PDK1 mutant in which Ser-410 was converted to Ala although the Ser-410 residue has been reported to be phosphorylated in vivo (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar). There may be some conformational state essential for the 14-3-3 binding in addition to Ser phosphorylation in 14-3-3 binding motifs. Among the 14-3-3-binding partners, some proteins, like Raf-1, bind to almost all 14-3-3 isoforms with nearly equivalent affinity (18Yaffe M.B. FEBS Lett. 2002; 513: 53-57Crossref PubMed Scopus (549) Google Scholar). However, others, like A20 and Cdc25B, bind to different 14-3-3 isoforms with significantly different affinities (27Vincenz C. Dixit V.M. J. Biol. Chem. 1996; 271: 20029-20034Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 28Mils V. Baldin V. Goubin F. Pinta I. Papin C. Waye M. Eychene A. Ducommun B. Oncogene. 2000; 19: 1257-1265Crossref PubMed Scopus (91) Google Scholar). We recently reported that p27Kip1 binds to 14-3-3 after Akt-mediated phosphorylation at the COOH-terminal Thr-198 (29Fujita N. Sato S. Katayama K. Tsuruo T. J. Biol. Chem. 2002; 277: 28706-28713Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). The phosphorylated p27Kip1 could form complexes with 14-3-3θ, η, and ε but not with 14-3-3β and ζ (29Fujita N. Sato S. Katayama K. Tsuruo T. J. Biol. Chem. 2002; 277: 28706-28713Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), suggesting that 14-3-3 isoforms have different affinities to their partners. Among the five isoforms (β, ε, η, θ, and ζ), only 14-3-3θ and η interacted with PDK1, and 14-3-3θ bound more tightly than 14-3-3η (Fig.1 B). The difference among 14-3-3θ, η and other isoforms that accounts for the different affinities for PDK1 is still unclear, since many of the residues that form the ligand-binding groove are conserved in the seven isoforms. However, comparing the residues in 14-3-3θ necessary for the interaction with Raf-1 and PDK1 did reveal some differences. To form a complex with Raf-1, Arg-56, Arg-60, and Arg-127 were necessary (Fig. 2 B, lanes 3 and4), a finding consistent with a previous report (23Thorson J.A., Yu, L.W. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar). In the case of PDK1, Arg-56 and Arg-60 were not essential but Arg-127 was (Fig. 2 A). By contrast, both Arg-56 and Arg-60 are necessary but Arg-127 is not for p27Kip1 binding to 14-3-3 (Ref.29Fujita N. Sato S. Katayama K. Tsuruo T. J. Biol. Chem. 2002; 277: 28706-28713Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). 2N. Fujita, S. Sato, and T. Tsuruo, unpublished observations. These results indicate that the residues necessary for ligand-14-3-3 binding vary with the ligand. Moreover, there may be some residues essential for the association with ligands that are not conserved in the 14-3-3 isoforms. However, we could not exclude the possibility that the differences we observed above were a result of the differences between the ligand that possessed at least two 14-3-3 binding motifs (Raf-1) and the one that had only one (PDK1). The distinct mechanism of isoform-specific interaction is a problem to be solved in future studies. The general mechanisms by which 14-3-3 regulates partner protein functions are 4-fold: 1) regulation of subcellular localization of the target protein; 2) direct regulation of catalytic activity of the binding partner; 3) protection of the ligands from proteolysis or dephosphorylation; and 4) regulation of interaction between bound protein and other molecules. The identified 14-3-3 binding residue of PDK1 (Ser-241) is thought to be phosphorylated by itself and it be essential for its kinase activity (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar). Consistent with previous reporting (9Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (288) Google Scholar), we confirmed that S241A-PDK1 exhibited weak Akt and SGK phosphorylation activity when compared with WT-PDK1 (Fig. 4). We thus generated PDK1 point mutants in which residues other than Ser-241 are mutated (Fig. 3 C) and examined their activity to phosphorylate Akt (Fig. 4 A) or SGK (Fig. 4 C). Mutation of PDK1 to promote 14-3-3 (V243P-PDK1) association exhibited reduced PDK1 kinase activity to phosphorylate Akt and SGK in cells, while mutation of PDK1 to inhibit the interaction (R238E-PDK1) slightly increased its activity (Fig. 4). Moreover, incubation with recombinant 14-3-3θ decreased PDK1 autophosphorylation activity (Fig.5 A) and kinase activity to phosphorylate SGK (Fig.5 B) in a dose-dependent manner in vitro. These results indicate that 14-3-3 is not only a PDK1-associating protein but also a protein that negatively regulates PDK1 kinase activity. Several reports indicate that PDK1 binds to phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate through its pleckstrin homology domain (19Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 30Filippa N. Sable C.L. Hemmings B.A. Van Obberghen E. Mol. Cell. Biol. 2000; 20: 5712-5721Crossref PubMed Scopus (112) Google Scholar). Because mutants of PDK1 deleted in its pleckstrin homology domain have been reported to prevent translocation of Akt, the relocalization of PDK1 from cytosol to the plasma membrane is suggested to play an important role in the recruitment of Akt to the plasma membrane and the subsequent Akt activation in stimulated cells (30Filippa N. Sable C.L. Hemmings B.A. Van Obberghen E. Mol. Cell. Biol. 2000; 20: 5712-5721Crossref PubMed Scopus (112) Google Scholar). PDK1 catalytic activity is not required for the PDK1 translocation to the plasma membrane (30Filippa N. Sable C.L. Hemmings B.A. Van Obberghen E. Mol. Cell. Biol. 2000; 20: 5712-5721Crossref PubMed Scopus (112) Google Scholar). Although mutating PDK1 to increase its binding to 14-3-3 (V243P) reduced kinase activity to phosphorylate Akt at Thr-308, the mutation did not affect the binding affinity of PDK1 to Akt (Fig. 4). Therefore, the decrease in kinase activity of V243P-PDK1 to phosphorylate Akt in cells may be caused by down-regulation of its kinase activity but not by suppressing translocation. This notion was supported by the fact that the amount of the phosphorylated form of Myr-Akt was reduced by 14-3-3 protein expression even though Myr-Akt constitutively targeted the plasma membrane (Fig. 4 D). In the case of SGK, translocation to the plasma membrane is not required for PDK1-dependent phosphorylation of SGK at Thr-256 (31Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (524) Google Scholar). SGK phosphorylation at Thr-256 was also suppressed by PDK1 mutation to increase its binding to 14-3-3 (V243P; Fig. 4 C). These results strongly suggest that inhibition of PDK1 activity but not PDK1 translocation is the main mechanism of 14-3-3-mediated inactivation of PDK1-dependent signal transduction. We thank Drs. Philip Hawkins and Karen Anderson for providing the pCMV3-PDK1.
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