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Insulin-stimulated Protein Kinase B Phosphorylation on Ser-473 Is Independent of Its Activity and Occurs through a Staurosporine-insensitive Kinase

2001; Elsevier BV; Volume: 276; Issue: 28 Linguagem: Inglês

10.1074/jbc.c100174200

1083-351X

Michelle M. Hill, Mirjana Andjelković, Derek P. Brazil, Stefano Ferrari, Doriano Fabbro, Brian A. Hemmings,

Chronic Myeloid Leukemia Treatments

Full activation of protein kinase B (PKB, also called Akt) requires phosphorylation on two regulatory sites, Thr-308 in the activation loop and Ser-473 in the hydrophobic C-terminal regulatory domain (numbering for PKBα/Akt-1). Although 3′-phosphoinositide-dependent protein kinase 1 (PDK1) has now been identified as the Thr-308 kinase, the mechanism of the Ser-473 phosphorylation remains controversial. As a step to further characterize the Ser-473 kinase, we examined the effects of a range of protein kinase inhibitors on the activation and phosphorylation of PKB. We found that staurosporine, a broad-specificity kinase inhibitor and inducer of cell apoptosis, attenuated PKB activation exclusively through the inhibition of Thr-308 phosphorylation, with Ser-473 phosphorylation unaffected. The increase in Thr-308 phosphorylation because of overexpression of PDK1 was also inhibited by staurosporine. We further show that staurosporine (CGP 39360) potently inhibited PDK1 activity in vitro with an IC50 of ∼0.22 µm. These data indicate that agonist-induced phosphorylation of Ser-473 of PKB is independent of PDK1 or PKB activity and occurs through a distinct Ser-473 kinase that is not inhibited by staurosporine. Moreover, our results suggest that inhibition of PKB signaling is involved in the proapoptotic action of staurosporine. Full activation of protein kinase B (PKB, also called Akt) requires phosphorylation on two regulatory sites, Thr-308 in the activation loop and Ser-473 in the hydrophobic C-terminal regulatory domain (numbering for PKBα/Akt-1). Although 3′-phosphoinositide-dependent protein kinase 1 (PDK1) has now been identified as the Thr-308 kinase, the mechanism of the Ser-473 phosphorylation remains controversial. As a step to further characterize the Ser-473 kinase, we examined the effects of a range of protein kinase inhibitors on the activation and phosphorylation of PKB. We found that staurosporine, a broad-specificity kinase inhibitor and inducer of cell apoptosis, attenuated PKB activation exclusively through the inhibition of Thr-308 phosphorylation, with Ser-473 phosphorylation unaffected. The increase in Thr-308 phosphorylation because of overexpression of PDK1 was also inhibited by staurosporine. We further show that staurosporine (CGP 39360) potently inhibited PDK1 activity in vitro with an IC50 of ∼0.22 µm. These data indicate that agonist-induced phosphorylation of Ser-473 of PKB is independent of PDK1 or PKB activity and occurs through a distinct Ser-473 kinase that is not inhibited by staurosporine. Moreover, our results suggest that inhibition of PKB signaling is involved in the proapoptotic action of staurosporine. protein kinase B phosphoinositide 3-kinase 3′-phosphoinositide-dependent protein kinase 1 integrin-linked kinase insulin-like growth factor-1 human embryonic kidney protein kinase C glutathioneS-transferase pleckstrin homology 12-O-tetradecanoylphorbol-13-acetate hemagglutinin myristoylated/palmitylated Protein kinase B (PKB)1is activated by growth factors in a phosphoinositide 3-kinase (PI3K)-dependent manner, through translocation to the plasma membrane and phosphorylation on two regulatory sites, Thr-308 in the activation loop in the kinase domain and Ser-473 in the hydrophobic C-terminal regulatory domain (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2511) Google Scholar). Phosphorylation on both sites are required for full activation of PKB; however, the contribution of each site toward PKB activation is not equal. Thus, whereas phosphorylation on Thr-308 alone is able to increase PKB activity, phosphorylation on Ser-473 alone does not significantly stimulate the kinase (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2511) Google Scholar, 2Bellacosa A. Chan T.O. Ahmed N.N. Datta K. Malstrom S. Stokoe D. McCormick F. Feng J. Tsichlis P. Oncogene. 1998; 17: 313-325Crossref PubMed Scopus (456) Google Scholar). Although the upstream kinase responsible for phosphorylation of Thr-308 has been identified as 3′-phosphoinositide-dependent kinase-1 (PDK1), the identity of the Ser-473 kinase has yet to be determined (3Alessi 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, 4Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1046) Google Scholar). Mitogen-activated protein kinase-activated protein kinase-2 was the first kinase shown to phosphorylate PKBα on Ser-473 in vitro (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2511) Google Scholar). However, mitogen-activated protein kinase-activated protein kinase-2 is unlikely to be the physiological Ser-473 kinase as it is activated downstream of p38 mitogen-activated protein kinase in response to stress, in a PI3K-independent manner, and inhibition of p38 mitogen-activated protein kinase by SB 203580 did not interfere with activation of PKB (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2511) Google Scholar). Integrin-linked kinase (ILK) was also shown to phosphorylate Ser-473 on PKBα in vitro, and overexpression of a kinase-inactive ILK inhibited Ser-473 phosphorylation (5Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (945) Google Scholar). However, certain kinase-inactive ILK mutants also induced Ser-473 phosphorylation, suggesting that ILK is unlikely to be the direct Ser-473 kinase in vivo (6Lynch D.K. Ellis C.A. Edwards P.A. Hiles I.D. Oncogene. 1999; 18: 8024-8032Crossref PubMed Scopus (183) Google Scholar). Two further candidate Ser-473 kinases have been proposed recently, PDK1 (7Balendran 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 (383) Google Scholar) and PKB itself (8Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). PDK1, in the presence of a peptide resembling the phosphorylated Ser-473 region of PKB, is able to phosphorylate Ser-473, in addition to Thr-308 of PKBα (7Balendran 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 (383) Google Scholar). However, PDK1 is clearly not the in vivo Ser-473 kinase, as PDK1-null embryonic stem cells are impaired in Thr-308 but not Ser-473 phosphorylation (9Williams 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 (395) Google Scholar). Autophosphorylation was originally ruled out, because kinase-inactive PKBα undergoes insulin-like growth factor-1 (IGF-1)-induced phosphorylation at both Thr-308 and Ser-473 when overexpressed in human embryonic kidney (HEK) 293 cells (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2511) Google Scholar). In contrast to these observations, Toker and Newton (8Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar) recently demonstrated that IGF-1 stimulated phosphorylation of kinase-inactive PKBα on Thr-308 but not on Ser-473 when overexpressed in the same cells and that PKBα is able to autophosphorylate on Ser-473 in vitro (8Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Thus, it seems possible that agonist-induced Ser-473 phosphorylation may be mediated by PKB itself. To further characterize the upstream kinase(s) involved in the activation of PKB, we have adopted a pharmacological approach by screening for protein kinase inhibitors that differentially inhibit either Thr-308 or Ser-473 phosphorylation. We found that staurosporine, a broad-specificity protein kinase inhibitor, attenuated PKB activation specifically through inhibition of PDK1, with an IC50 of ∼0.22 µm in vitro. Staurosporine has been widely used as an inducer of apoptosis; however, the cellular target(s) of its proapoptotic action are not known. Our data suggest that at least part of the apoptotic effects of staurosporine is due to inhibition of PKB signaling. In contrast to Thr-308 phosphorylation, insulin-stimulated phosphorylation of the Ser-473 site was not reduced by staurosporine treatment (up to 1 µm). Taken together, our results suggest that phosphorylation of PKB on Ser-473 does not occur by autophosphorylation but rather through the action of an upstream kinase that is resistant to staurosporine and distinct from PDK1. Culture and transfection of HEK293 cells and the expression constructs used in this study have been described previously (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2511) Google Scholar, 10Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar, 11Andjelkovic M. Maira S.M. Cron P. Parker P.J. Hemmings B.A. Mol. Cell. Biol. 1999; 19: 5061-5072Crossref PubMed Scopus (101) Google Scholar, 12Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (724) Google Scholar). Expression and infection of insect Sf9 cells have been described previously for PKB and PKC (13Fabbro D. Batt D. Rose P. Schacher B. Roberts T.M. Ferrari S. Protein Expression Purif. 1999; 17: 83-88Crossref PubMed Scopus (17) Google Scholar,14Geiges D. Meyer T. Marte B. Vanek M. Weissgerber G. Stabel S. Pfeilschifter J. Fabbro D. Huwiler A. Biochem. Pharmacol. 1997; 53: 865-875Crossref PubMed Scopus (89) Google Scholar). Human PDK1-glutathione S-transferase fusion protein (GST-PDK1) was cloned in a modified pFastBac vector (Life Technologies, Inc.) and prepared as described previously (13Fabbro D. Batt D. Rose P. Schacher B. Roberts T.M. Ferrari S. Protein Expression Purif. 1999; 17: 83-88Crossref PubMed Scopus (17) Google Scholar). Cell lysis, immunoprecipitation, immunoblotting, and PKB assay using Crosstide peptide (GRPRTSSAEG) were performed as described previously (11Andjelkovic M. Maira S.M. Cron P. Parker P.J. Hemmings B.A. Mol. Cell. Biol. 1999; 19: 5061-5072Crossref PubMed Scopus (101) Google Scholar). Phospho-specific PKB antibodies were purchased from Cell Signaling Technologies. In addition, we also produced and purified an anti-phospho-Ser-473 PKB antibody using the peptide Arg-Pro-His-Phe-Pro-Gln-Phe-Ser(PO3H2)-Tyr-Ser-Ala-Ser (15Hill, M. M., and Hemmings, B. A. (2001) Methods Enzymol. 345, in press.Google Scholar). Assays for recombinant PKCα and PKCζ have been described previously (14Geiges D. Meyer T. Marte B. Vanek M. Weissgerber G. Stabel S. Pfeilschifter J. Fabbro D. Huwiler A. Biochem. Pharmacol. 1997; 53: 865-875Crossref PubMed Scopus (89) Google Scholar). Recombinant GST-PDK1 was similarly assayed, using 0.1 mg/ml casein (Sigma) as substrate. We have previously reported the characterization of a PKBα construct in which the pleckstrin homology (PH) domain was replaced by the C1 domain of PKC (C1-PKBα-ΔPH) (11Andjelkovic M. Maira S.M. Cron P. Parker P.J. Hemmings B.A. Mol. Cell. Biol. 1999; 19: 5061-5072Crossref PubMed Scopus (101) Google Scholar). C1-PKBα-ΔPH translocated to the membrane upon stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and was activated and phosphorylated on both Thr-308 and Ser-473 (11Andjelkovic M. Maira S.M. Cron P. Parker P.J. Hemmings B.A. Mol. Cell. Biol. 1999; 19: 5061-5072Crossref PubMed Scopus (101) Google Scholar). TPA-stimulated activation of C1-PKBα-ΔPH was inhibited by PI3K inhibitors, as well as the broad range protein kinase inhibitor, staurosporine (11Andjelkovic M. Maira S.M. Cron P. Parker P.J. Hemmings B.A. Mol. Cell. Biol. 1999; 19: 5061-5072Crossref PubMed Scopus (101) Google Scholar). Interestingly, whereas the former inhibited phosphorylation on both sites, staurosporine treatment specifically attenuated phosphorylation on Thr-308 without affecting Ser-473 phosphorylation (11Andjelkovic M. Maira S.M. Cron P. Parker P.J. Hemmings B.A. Mol. Cell. Biol. 1999; 19: 5061-5072Crossref PubMed Scopus (101) Google Scholar). To extend our observations, the effect of a panel of protein kinase inhibitors was compared with staurosporine (Fig.1). Staurosporine obtained from Alexis or Novartis (CGP 39360) inhibited TPA-stimulated Thr-308 phosphorylation and activation of C1-PKBα-ΔPH, without affecting Ser-473 phosphorylation (Fig. 1 B). CGP 39360 was more potent than staurosporine (Alexis), requiring 0.1 and 1 µm to reduce kinase activity and Thr-308 phosphorylation to basal levels, respectively (Fig. 1 B). This difference may be because of improved purity of the chemical produced by Novartis. The staurosporine derivative CGP 41251 also inhibited Thr-308 phosphorylation and activation of C1-PKBα-ΔPH but was much less potent than CGP 39360 (Fig. 1 B). The inactive analog of CGP 41251, CGP 42700, had no significant effect (Fig. 1 B). Three other protein kinase inhibitor compounds examined (CGP 25956, CGP 45910, and CGP 57148B) had an inhibitory effect only at concentrations above 10 µm, where they reduced phosphorylation at both sites to below basal levels (Fig. 1 C). Notably, an effect of CGP 57148B (STI571 or Glivec) was only observed at 40 µm (Fig. 1 C). CGP 57148B is a potent inhibitor of Abl and PDGF receptor tyrosine kinases that selectively inhibits the growth of Bcr/Abl-transformed cells (16Druker B.J. Tamura S. Buchdunger E. Ohno S. Segal G.M. Fanning S. Zimmermann J. Lydon N.B. Nat. Med. 1996; 2: 561-566Crossref PubMed Scopus (3150) Google Scholar) and is now in clinical trials for treatment of chronic myeloid leukemia. The effects of CGP 57148B were observed concentrations <10 µm, and our results show that it does not significantly affect the PDK1/PKB pathway at this concentration (Fig. 1 C). To extend our observations to wild type PKB, we examined the effect of staurosporine on insulin-stimulated activation of HA-PKBα expressed in HEK293 cells. Staurosporine treatment inhibited insulin-stimulated HA-PKBα activation in a dose-dependent manner, with complete inhibition observed at 1 µm (Fig.2 A). Similar to C1-PKBα-ΔPH, this inhibitory effect of staurosporine on HA-PKBα activity correlated with an inhibition of Thr-308 phosphorylation (Fig.2 A). In contrast, phosphorylation on Ser-473 was slightly enhanced with increasing concentrations of staurosporine (Fig.2 A). A similar inhibitory effect of staurosporine was observed for insulin-stimulated Thr-308 phosphorylation of endogenous PKB in HEK293 cells (Fig. 2 B). Two other modes of PKB activation were also examined, coexpression of PDK1 and constitutive membrane targeting. In agreement with previous results (3Alessi 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), coexpression of PDK1 with HA-PKBα resulted in a 3-fold increase in basal PKBα activity, together with constitutive phosphorylation of Thr-308 (Fig. 2 C). Treatment with staurosporine reduced Thr-308 phosphorylation and kinase activity (Fig. 2 C). Interestingly, overexpression of PDK1 also induced an increase in Ser-473 phosphorylation, reaching ∼10% of the insulin-stimulated levels, that was reduced with staurosporine treatment, suggesting that it occurs through a mechanism different from insulin-stimulated Ser-473 phosphorylation (Fig. 2 C). Targeting of PKB to the plasma membrane using the Lck myristoylation/palmitylation signal (m/p-PKBα) results in the constitutive activation and phosphorylation of PKB (10Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar). In contrast to Thr-308 phosphorylation induced by insulin or coexpression of PDK1, staurosporine did not reduce Thr-308 phosphorylation of m/p-PKBα (Fig. 2 D). This observation indicates that dephosphorylation of PKB does not occur readily at the plasma membrane and that the phosphorylation step is the target of staurosporine. Staurosporine is a competitive inhibitor that is thought to bind in the ATP pocket of target protein kinases (17Toledo L.M. Lydon N.B. Elbaum D. Curr. Med. Chem. 1999; 6: 775-805PubMed Google Scholar). The observed effect on Thr-308 may occur via direct inhibition of PDK1, or staurosporine could bind to PKB, thus blocking the access to the phosphorylation site in the catalytic domain. To distinguish between these possibilities, we determined the inhibitory profiles of the CGP inhibitor compounds using recombinant GST-PDK1, GST-PKBα, PKCα, and PKCζ. CGP 39360 (staurosporine) was most potent against PKCα (IC50 < 3 nm) but also inhibited PDK1 (IC50 = 0.22 ± 0.09 µm), PKBα (IC50 = 0.83 ± 0.19 µm), and PKCζ (IC50 = 1.03 ± 0.37 µm) at higher concentrations. CGP 41251 selectively inhibited PKCα (IC50 = 0.04 ± 0.02 µm) and also inhibited PDK1 (IC50 = 1.72 ± 0.21 µm) but did not have significant effects on PKBα or PKCζ (up to 10 µm). These data suggest that the target of staurosporine and its derivative CGP 41251 is PDK1 rather than PKB. CGP 42700, CGP 25956, CGP 45910, and CGP 57148B did not inhibit any of the four kinases tested. To further examine the regulation of PKB activation by upstream kinases, the effect of staurosporine on insulin-stimulated phosphorylation and activity of kinase-inactive (K179A) or phosphorylation-site mutants (T308A and S473A) of PKBα was examined (Fig. 3). In agreement with previously reported results (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2511) Google Scholar), Thr-308 and Ser-473 phosphorylation occurred independently of each other upon insulin stimulation, as observed in the phosphorylation-site mutants (Fig. 3). In addition, the kinase-inactive mutant (K179A) was phosphorylated on both Thr-308 and Ser-473 upon insulin stimulation (Fig. 3). Staurosporine treatment inhibited insulin-stimulated Thr-308 phosphorylation on wild type, K179A, and S473A PKBα (Fig. 3), but its effect on Ser-473 phosphorylation of the different PKBα constructs was somewhat varied. Staurosporine at 0.1 and 1 µm did not inhibit insulin-stimulated Ser-473 phosphorylation of wild type and T308A-PKBα but even enhanced their phosphorylation, which was more readily observed in the T308A mutant (Fig. 3). Interestingly, insulin-stimulated Ser-473 phosphorylation of kinase-inactive PKBα was inhibited by staurosporine at 1 µm but not at 0.1 µm (Fig. 3). As staurosporine is a broad specificity kinase inhibitor, it is possible that complex effects are observed because of inhibition of numerous kinases/pathways at higher doses. Phosphorylation at Thr-308 and Ser-473 is required for full activation of PKBα. Although the Thr-308 kinase has been identified (PDK1), the Ser-473 kinase remains elusive. Most recently, it has been suggested that autophosphorylation may be the mechanism by which PKB is phosphorylated on Ser-473 and that the previously reported phosphorylation of kinase-deficient PKB at this site is due to the activity of endogenous PKB (8Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Our previous results with m/p-PKBα (10Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar) and C1-PKBα-ΔPH (11Andjelkovic M. Maira S.M. Cron P. Parker P.J. Hemmings B.A. Mol. Cell. Biol. 1999; 19: 5061-5072Crossref PubMed Scopus (101) Google Scholar) suggest that both upstream kinases are present in a constitutively active form at the plasma membrane. As PKB is present largely in the cytosol prior to stimulation, it seems unlikely that PKB itself is the physiological Ser-473 kinase. More directly, here we show that pretreatment with 1 µmstaurosporine abolished insulin-stimulated PKB activation of both transiently expressed and endogenous PKB, without affecting Ser-473 phosphorylation (Fig. 2). In addition, using phospho-specific antibodies, we have confirmed our previous results (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2511) Google Scholar) that kinase-inactive PKBα can be phosphorylated on both Thr-308 and Ser-473 in response to insulin (Fig. 3). Taken together, these data do not support the hypothesis that phosphorylation on Ser-473 occurs via autophosphorylation or trans-phosphorylation. Rather, it confirms the existence of a distinct Ser-473 kinase that is constitutively active at the plasma membrane of quiescent cells. One kinase that fulfills the above criteria is PDK1. Indeed, PDK1 has the ability to phosphorylate PKBα on Ser-473 in the presence of an exogenous peptide that resembles phosphorylated Ser-473 (7Balendran 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 (383) Google Scholar). However, PDK1 is not the physiological Ser-473 kinase, because PDK1-null embryonic stem cells are not impaired in Ser-473 phosphorylation in response to IGF-1 (9Williams 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 (395) Google Scholar), and staurosporine inhibits PDK1 activity without affecting insulin-stimulated Ser-473 phosphorylation (Fig. 2). Interestingly, overexpression of PDK1 in HEK293 cells not only induced constitutive phosphorylation of PKB at Thr-308 but also caused a slight elevation of Ser-473 phosphorylation (Fig. 2 C). In this case, however, Ser-473 phosphorylation is dependent on PDK1 activity, as it is attenuated by staurosporine treatment (Fig. 2 C), and overexpression of a kinase-inactive PDK1 mutant did not increase basal Ser-473 phosphorylation in HEK293 cells (data not shown). Thus, although PDK1 is not the physiological Ser-473 kinase, it likely plays a role in Ser-473 phosphorylation, with the nature of this interaction yet to be defined. ILK has recently come to attention as a prime candidate kinase for Ser-473 phosphorylation (5Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (945) Google Scholar). According to our observations, the Ser-473 kinase should be staurosporine-resistant. Unfortunately, we were unable to determine the effect of staurosporine on ILK as we have not been able to detect any significant kinase activity of overexpressed or endogenous ILK by autophosphorylation or on myelin basic protein (data not shown). Intriguingly, ILK possesses a hydrophobic motif similar to the Ser-473 site, and when this serine was mutated to an acidic residue to mimic phosphorylation, the ability of a kinase-inactive ILK to induce Ser-473 phosphorylation was rescued (6Lynch D.K. Ellis C.A. Edwards P.A. Hiles I.D. Oncogene. 1999; 18: 8024-8032Crossref PubMed Scopus (183) Google Scholar). It was recently shown that the hydrophobic phosphorylation site in p90 ribosomal S6 kinase acts as a docking site for the recruitment of PDK1 (18Frodin M. Jensen C.J. Merienne K. Gammeltoft S. EMBO J. 2000; 19: 2924-2934Crossref PubMed Scopus (256) Google Scholar). Thus, it is possible that ILK mediates the colocalization of PKB with PDK1 and the Ser-473 kinase. Staurosporine exhibits anti-proliferative properties on a wide range of mammalian cells, and its derivatives UCN-01, CGP 41251, Ro 31–8220, and PKC412 (19Shao R.G. Shimizu T. Pommier Y. Exp. Cell. Res. 1997; 234: 388-397Crossref PubMed Scopus (84) Google Scholar, 20Begemann M. Kashimawo S.A. Lunn R.M. Delohery T. Choi Y.J. Kim S. Heitjan D.F. Santella R.M. Schiff P.B. Bruce J.N. Weinstein I.B. Anticancer Res. 1998; 18: 3139-3152PubMed Google Scholar, 21Begemann M. Kashimawo S.A. Heitjan D.F. Schiff P.B. Bruce J.N. Weinstein I.B. Anticancer Res. 1998; 18: 2275-2282PubMed Google Scholar, 22Fabbro D. Ruetz S. Bodis S. Pruschy M. Csermak K. Man A. Campochiaro P. Wood J. O'Reilly T. Meyer T. Anticancer Drug Des. 2000; 15: 17-28PubMed Google Scholar) are being examined as potential therapeutic agents for the treatment of cancer. Despite the common use of staurosporine as an inducer of apoptosis, the direct cellular target of staurosporine is not known. Our finding that staurosporine inhibits PDK1 activity raises the possibility that staurosporine and its derivatives may induce apoptosis by interfering with survival signaling mediated by PDK1. Indeed, it was recently shown that reduction of PDK1 expression by antisense oligonucleotides induced apoptosis (23Flynn P. Wongdagger M. Zavar M. Dean N.M. Stokoe D. Curr. Biol. 2000; 10: 1439-1442Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Apart from PKB, PDK1 also phosphorylates and activates other kinases that are involved in cell survival, including p70 ribosomal S6 kinase (12Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (724) Google Scholar), p90 ribosomal S6 kinase (24Jensen C.J. Buch M.B. Krag T.O. Hemmings B.A. Gammeltoft S. Frodin M. J. Biol. Chem. 1999; 274: 27168-27176Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), and serum- and glucocorticoid-inducible protein kinase (25Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (482) Google Scholar). Thus the mechanisms of staurosporine-induced apoptosis needs to be readdressed in light of its effects on PDK1 activity. In summary, we have demonstrated that staurosporine attenuates PKB activation through direct inhibition of PDK1 activity, without affecting insulin-stimulation of Ser-473 phosphorylation. These results strongly suggest that insulin-stimulated phosphorylation on Ser-473 is not dependent on the activity of PDK1 or PKB. Our data is consistent with a model in which phosphorylation on Thr-308 and Ser-473 occurs via two distinct upstream kinases that are constitutively active at the plasma membrane, and of these, only the Ser-473 kinase is staurosporine-resistant.