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J. Biol. Chem., Vol. 276, Issue 28, 25643-25646, July 13, 2001

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

Michelle M. Hill, Mirjana Andjelkovic, Derek P. Brazil, Stefano Ferrari^§, Doriano Fabbro^§, and Brian A. Hemmings^¶

From the Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel and the ^§ Department of Oncology, Novartis Pharma AG, CH-4057 Basel, Switzerland

Received for publication, April 6, 2001, and in revised form, May 9, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 PKB/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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein kinase B (PKB)¹ is 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 (1). 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 (1, 2). 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 (3, 4).

Mitogen-activated protein kinase-activated protein kinase-2 was the first kinase shown to phosphorylate PKB on Ser-473 in vitro (1). 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 (1). Integrin-linked kinase (ILK) was also shown to phosphorylate Ser-473 on PKB in vitro, and overexpression of a kinase-inactive ILK inhibited Ser-473 phosphorylation (5). 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 (6). Two further candidate Ser-473 kinases have been proposed recently, PDK1 (7) and PKB itself (8). 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 PKB (7). 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 (9). Autophosphorylation was originally ruled out, because kinase-inactive PKB 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 (1). In contrast to these observations, Toker and Newton (8) recently demonstrated that IGF-1 stimulated phosphorylation of kinase-inactive PKB on Thr-308 but not on Ser-473 when overexpressed in the same cells and that PKB is able to autophosphorylate on Ser-473 in vitro (8). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression Constructs and Transfection of Cells-- Culture and transfection of HEK293 cells and the expression constructs used in this study have been described previously (1, 10-12).

Recombinant Proteins-- Expression and infection of insect Sf9 cells have been described previously for PKB and PKC (13, 14). Human PDK1-glutathione S-transferase fusion protein (GST-PDK1) was cloned in a modified pFastBac vector (Life Technologies, Inc.) and prepared as described previously (13).

Immunoprecipitation, Immunoblotting, and in Vitro Kinase Assays-- Cell lysis, immunoprecipitation, immunoblotting, and PKB assay using Crosstide peptide (GRPRTSSAEG) were performed as described previously (11). 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 (15). Assays for recombinant PKC and PKC have been described previously (14). Recombinant GST-PDK1 was similarly assayed, using 0.1 mg/ml casein (Sigma) as substrate.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have previously reported the characterization of a PKB construct in which the pleckstrin homology (PH) domain was replaced by the C1 domain of PKC (C1-PKB-PH) (11). C1-PKB-PH 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 (11). TPA-stimulated activation of C1-PKB-PH was inhibited by PI3K inhibitors, as well as the broad range protein kinase inhibitor, staurosporine (11). Interestingly, whereas the former inhibited phosphorylation on both sites, staurosporine treatment specifically attenuated phosphorylation on Thr-308 without affecting Ser-473 phosphorylation (11).

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-PKB-PH, without affecting Ser-473 phosphorylation (Fig. 1B). 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. 1B). 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-PKB-PH but was much less potent than CGP 39360 (Fig. 1B). The inactive analog of CGP 41251, CGP 42700, had no significant effect (Fig. 1B). 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. 1C). Notably, an effect of CGP 57148B (STI571 or Glivec) was only observed at 40 µM (Fig. 1C). CGP 57148B is a potent inhibitor of Abl and PDGF receptor tyrosine kinases that selectively inhibits the growth of Bcr/Abl-transformed cells (16) 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. 1C).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effect of staurosporine and its derivatives on TPA-stimulated activation of C1-PKB-PH. HEK293 cells transiently transfected with HA-C1-PKB-PH were treated with the indicated concentration of staurosporine (Stauro) (Alexis), CGP 39360, CGP 41251, CGP 42700 (B), CGP 25956, CGP 45910, or CGP 57148B/STI571 (C) for 30 min prior to stimulation with TPA (200 ng/ml; Life Technologies, Inc.) for 15 min. HA-C1-PKB-PH was immunoprecipitated and assayed for kinase activity or analyzed by immunoblotting with phospho-specific antibodies. Structures of the compounds used are shown in A.

To extend our observations to wild type PKB, we examined the effect of staurosporine on insulin-stimulated activation of HA-PKB expressed in HEK293 cells. Staurosporine treatment inhibited insulin-stimulated HA-PKB activation in a dose-dependent manner, with complete inhibition observed at 1 µM (Fig. 2A). Similar to C1-PKB-PH, this inhibitory effect of staurosporine on HA-PKB activity correlated with an inhibition of Thr-308 phosphorylation (Fig. 2A). In contrast, phosphorylation on Ser-473 was slightly enhanced with increasing concentrations of staurosporine (Fig. 2A). A similar inhibitory effect of staurosporine was observed for insulin-stimulated Thr-308 phosphorylation of endogenous PKB in HEK293 cells (Fig. 2B). Two other modes of PKB activation were also examined, coexpression of PDK1 and constitutive membrane targeting. In agreement with previous results (3), coexpression of PDK1 with HA-PKB resulted in a 3-fold increase in basal PKB activity, together with constitutive phosphorylation of Thr-308 (Fig. 2C). Treatment with staurosporine reduced Thr-308 phosphorylation and kinase activity (Fig. 2C). 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. 2C). Targeting of PKB to the plasma membrane using the Lck myristoylation/palmitylation signal (m/p-PKB) results in the constitutive activation and phosphorylation of PKB (10). In contrast to Thr-308 phosphorylation induced by insulin or coexpression of PDK1, staurosporine did not reduce Thr-308 phosphorylation of m/p-PKB (Fig. 2D). 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.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Differential effects of staurosporine on PKB phosphorylation induced by insulin, coexpression of PDK1, or membrane targeting. HEK293 cells were transiently transfected with wild type HA-PKB (A, C, and D), myristoylated/palmitylated HA-PKB (D), and myc-PDK1 (C). Cells were treated with the indicated concentration of staurosporine (Stauro) for 30 min prior to stimulation with 0.1 µM insulin (Life Technologies, Inc.) for 15 min. HA-PKB was immunoprecipitated and assayed for kinase activity or analyzed by immunoblotting with phospho-specific antibodies. The phosphorylation status of endogenous PKB was determined by immunoblotting cell lysates (20 µg) with phospho-specific antibodies (B).

Staurosporine is a competitive inhibitor that is thought to bind in the ATP pocket of target protein kinases (17). 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-PKB, PKC, and PKC. CGP 39360 (staurosporine) was most potent against PKC (IC₅₀ < 3 nM) but also inhibited PDK1 (IC₅₀ = 0.22 ± 0.09 µM), PKB (IC₅₀ = 0.83 ± 0.19 µM), and PKC (IC₅₀ = 1.03 ± 0.37 µM) at higher concentrations. CGP 41251 selectively inhibited PKC (IC₅₀ = 0.04 ± 0.02 µM) and also inhibited PDK1 (IC₅₀ = 1.72 ± 0.21 µM) but did not have significant effects on PKB or PKC (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 PKB was examined (Fig. 3). In agreement with previously reported results (1), 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 PKB (Fig. 3), but its effect on Ser-473 phosphorylation of the different PKB constructs was somewhat varied. Staurosporine at 0.1 and 1 µM did not inhibit insulin-stimulated Ser-473 phosphorylation of wild type and T308A-PKB 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 PKB 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.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   Effect of staurosporine on insulin-stimulated phosphorylation and activation of PKB mutants. HEK293 cells transiently transfected with wild type or mutant HA-PKB were treated with 0.1 or 1 µM staurosporine (Stauro) for 30 min prior to stimulation with 0.1 µM insulin for 15 min. HA-PKB expression was determined by blotting with an anti-HA antibody. PKB activity and phosphorylation was analyzed as for Fig. 1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phosphorylation at Thr-308 and Ser-473 is required for full activation of PKB. 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 (8). Our previous results with m/p-PKB (10) and C1-PKB-PH (11) 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 µM staurosporine 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 (1) that kinase-inactive PKB 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 PKB on Ser-473 in the presence of an exogenous peptide that resembles phosphorylated Ser-473 (7). 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 (9), 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. 2C). In this case, however, Ser-473 phosphorylation is dependent on PDK1 activity, as it is attenuated by staurosporine treatment (Fig. 2C), 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 (5). 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 (6). It was recently shown that the hydrophobic phosphorylation site in p90 ribosomal S6 kinase acts as a docking site for the recruitment of PDK1 (18). 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 (19-22) 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 (23). Apart from PKB, PDK1 also phosphorylates and activates other kinases that are involved in cell survival, including p70 ribosomal S6 kinase (12), p90 ribosomal S6 kinase (24), and serum- and glucocorticoid-inducible protein kinase (25). 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.

	FOOTNOTES

^* This work was supported in part by the Swiss Cancer League (to M. M. H., M. A., and B. A. H.), and the Friedrich Miescher Institute is supported by the Novartis Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Vascular and Metabolic Diseases, F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland.

^¶ To whom correspondence should be addressed. Tel.: 41-61-697-40-46; Fax: 41-61-697-39-76.

Published, JBC Papers in Press, May 23, 2001, DOI 10.1074/jbc.C100174200

	ABBREVIATIONS

The abbreviations used are: PKB, protein kinase B; PI3K, phosphoinositide 3-kinase; PDK1, 3'-phosphoinositide-dependent protein kinase 1; ILK, integrin-linked kinase; IGF-1, insulin-like growth factor-1; HEK, human embryonic kidney; PKC, protein kinase C; GST, glutathione S-transferase; PH, pleckstrin homology; TPA, 12-O-tetradecanoylphorbol-13-acetate; HA, hemagglutinin; m/p, myristoylated/palmitylated.

	REFERENCES

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				K. Sakamoto, W. G. Aschenbach, M. F. Hirshman, and L. J. Goodyear Akt signaling in skeletal muscle: regulation by exercise and passive stretch Am J Physiol Endocrinol Metab, November 1, 2003; 285(5): E1081 - E1088. [Abstract] [Full Text] [PDF]

				L. Koivisto, K. Alavian, L. Hakkinen, S. Pelech, C. A. McCulloch, and H. Larjava Glycogen synthase kinase-3 regulates formation of long lamellipodia in human keratinocytes J. Cell Sci., September 15, 2003; 116(18): 3749 - 3760. [Abstract] [Full Text] [PDF]

				X. Chen, H. Al-Hasani, T. Olausson, A.-M. Wenthzel, U. Smith, and S. W. Cushman Activity, phosphorylation state and subcellular distribution of GLUT4-targeted Akt2 in rat adipose cells J. Cell Sci., September 1, 2003; 116(17): 3511 - 3518. [Abstract] [Full Text] [PDF]

				R. Tamaskovic, S. J. Bichsel, H. Rogniaux, M. R. Stegert, and B. A. Hemmings Mechanism of Ca2+-mediated Regulation of NDR Protein Kinase through Autophosphorylation and Phosphorylation by an Upstream Kinase J. Biol. Chem., February 21, 2003; 278(9): 6710 - 6718. [Abstract] [Full Text] [PDF]

				I. M. Bird, L. Zhang, and R. R. Magness Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2003; 284(2): R245 - R258. [Abstract] [Full Text] [PDF]

				M. P. Scheid, P. A. Marignani, and J. R. Woodgett Multiple Phosphoinositide 3-Kinase-Dependent Steps in Activation of Protein Kinase B Mol. Cell. Biol., September 1, 2002; 22(17): 6247 - 6260. [Abstract] [Full Text] [PDF]

				D. Decraene, P. Agostinis, R. Bouillon, H. Degreef, and M. Garmyn Insulin-like Growth Factor-1-mediated AKT Activation Postpones the Onset of Ultraviolet B-induced Apoptosis, Providing More Time for Cyclobutane Thymine Dimer Removal in Primary Human Keratinocytes J. Biol. Chem., August 30, 2002; 277(36): 32587 - 32595. [Abstract] [Full Text] [PDF]

C. B. Knobbe, A. Merlo, and G. Reifenberger Pten signaling in gliomas Neuro-oncol, July 1, 2002; 4(3): 196 - 211. [Abstract] [PDF]