Protein Kinase Cζ Is a Negative Regulator of Protein Kinase B Activity
1999; Elsevier BV; Volume: 274; Issue: 13 Linguagem: Inglês
10.1074/jbc.274.13.8589
ISSN1083-351X
AutoresRobert P. Doornbos, Marga Theelen, P.C.J. van der Hoeven, Wim J. van Blitterswijk, Arie J. Verkleij, Paul M.P. van Bergen en Henegouwen,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoProtein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKCζ but not PKCα and PKCδ can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKCζ was also found in COS-1 cells transiently expressing PKB and PKCζ, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with platelet-derived growth factor (PDGF) resulted in a decrease in the PKB-PKCζ interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGF-induced activation of PKCζ was not affected by co-expression of PKB. However, both PDGF- and p110-CAAX-induced activation of PKB were significantly abolished in cells co-expressing PKCζ. In contrast, co-expression of a kinase-dead PKCζ mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by co-expression of PKCζ. A clear inhibitory effect of PKCζ was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKCζ in vivoand that PKCζ acts as a negative regulator of PKB. Protein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKCζ but not PKCα and PKCδ can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKCζ was also found in COS-1 cells transiently expressing PKB and PKCζ, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with platelet-derived growth factor (PDGF) resulted in a decrease in the PKB-PKCζ interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGF-induced activation of PKCζ was not affected by co-expression of PKB. However, both PDGF- and p110-CAAX-induced activation of PKB were significantly abolished in cells co-expressing PKCζ. In contrast, co-expression of a kinase-dead PKCζ mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by co-expression of PKCζ. A clear inhibitory effect of PKCζ was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKCζ in vivoand that PKCζ acts as a negative regulator of PKB. Protein kinase B (PKB), 1The abbreviations used are:PKB, protein kinase B; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PI, phosphatidylinositol; GSK-3, glycogen synthase kinase-3; GS, glycogen synthase; PH, pleckstrin homology; AH, Akt homology; PDK, phosphatidylinositol-3,4,5-triphosphate-dependent protein kinase; PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-triphosphate; PDGF, platelet-derived growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; Btk, Bruton tyrosine kinase.also referred to as c-Akt or RAC-PK is a 60-kDa serine/threonine kinase which is the cellular homologue of the viral oncogene v-Akt (1Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (391) Google Scholar, 2Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemming B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (447) Google Scholar, 3Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 244-247Crossref Scopus (802) Google Scholar). So far, three isoforms of PKB have been isolated: PKBα, PKBβ, and PKBγ (1Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (391) Google Scholar, 2Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemming B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (447) Google Scholar, 4Jones P.F. Jakubowicz T. Hemming B.A. Cell Regul. 1991; 2: 1001-1009Crossref PubMed Scopus (141) Google Scholar, 5Konishi H. Kuroda S. Tanaka M. Matsuzaki H. Ono Y. Kameyama K. Haga T. Kikkawa U. Biochem. Biophys. Res. Commun. 1995; 216: 526-534Crossref PubMed Scopus (162) Google Scholar). Overexpression of PKB family members has been correlated with different cancers such as breast cancer and some pancreatic and ovarian cancers (2Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemming B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (447) Google Scholar, 6Cheng J.Q. Godwin A.K. Bellacosa A. Taguchi T. Franke T.F. Hamilton T.C. Tsichlis P.N. Testa J.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9267-9271Crossref PubMed Scopus (650) Google Scholar, 7Cheng J.Q. Ruggeri B. Klein W.M. Sonoda G. Altomare D.A. Watson D.K. Testa J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3636-3641Crossref PubMed Scopus (702) Google Scholar). Recently, PKB has been found to yield an anti-apoptotic signal, which is crucial for cell survival in both fibroblasts and neuronal cells (8Kauffmann-Zeh A. Rodriquez-Vicania P. Ulrich E. Gilbert C. Coffer P. Evans G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1077) Google Scholar, 9Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2230) Google Scholar). Other reports have indicated a role for PKB in the regulation of glycogen synthesis by inhibition of glycogen synthase kinase-3 (GSK-3) (10Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4426) Google Scholar, 11van Weeren P.C. de Bruyn K.M.T. de Vries-Smits A.M.M. van Lint J. Burgering B.M.T. J. Biol. Chem. 1998; 273: 13150-13156Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). In addition, glucose uptake and metabolism in 3T3-L1 adipocytes have been shown to be regulated by PKB by mediating the translocation of the glucose transporter GLUT4 to the plasma membrane (12Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar, 13Tanti J.F. Grillo S. Gremeaux T. Coffer P.J. Van Obberghen E. Le Marchand-Brustel Y. Endocrinology. 1997; 138: 2005-2010Crossref PubMed Google Scholar). Moreover, a role for PKB has been described in the regulation of protein synthesis through indirect activation of the p70 ribosomal S6 kinase (p70S6K) (14Burgering B.M.T. Cofer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1888) Google Scholar). PKB comprises a NH2-terminal Akt homology (AH) domain of 148 amino acids, a catalytic domain of 264 amino acids showing high homology with cyclic AMP-dependent protein kinase A (PKA) and protein kinase C (PKC) and a short COOH-terminal tail of 68 amino acids. A pleckstrin homology (PH) domain of 106 amino acids is present within the AH domain. Treatment of cells with different growth factors, insulin, or phosphatase inhibitors results in rapid activation of PKB (10Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4426) Google Scholar, 14Burgering B.M.T. Cofer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1888) Google Scholar, 15Andjelkovic M. Jakubowicz T. Cron P. Ming X.F. Han J.W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (431) Google Scholar). Also heat shock, hyperosmolarity stress, and intracellular cAMP elevation were shown to activate PKB in vivo (16Konishi H. Matsuzaki H. Tanaka M. Ono Y. Tokunaga C. Kuroda S. Kikkawa U. Proc. Natl. Acad. Sci. U. S. A. 1996; 91: 7639-7643Crossref Scopus (189) Google Scholar, 17Sable C.L. Filippa N. Hemmings B. Van Obberghen E. FEBS Lett. 1997; 409: 253-257Crossref PubMed Scopus (152) Google Scholar). Growth factor and insulin-induced activation is almost completely prevented by overexpression of a dominant negative form of phosphatidylinositol (PI) 3-kinase (Δp85) or by pretreatment of cells with the PI 3-kinase inhibitors wortmannin and LY294002 (14Burgering B.M.T. Cofer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1888) Google Scholar). Furthermore, a PDGF receptor mutant that is not able to stimulate PI 3-kinase activity also fails to activate PKB (14Burgering B.M.T. Cofer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1888) Google Scholar, 18Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1832) Google Scholar). These data demonstrate that insulin and growth factor-induced signals leading to PKB activation are transduced via the PI 3-kinase pathway. In contrast, stress, okadaic acid, and cAMP induced activation of PKB is PI 3-kinase independent since wortmannin is unable to block this pathway of PKB activation (15Andjelkovic M. Jakubowicz T. Cron P. Ming X.F. Han J.W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (431) Google Scholar, 16Konishi H. Matsuzaki H. Tanaka M. Ono Y. Tokunaga C. Kuroda S. Kikkawa U. Proc. Natl. Acad. Sci. U. S. A. 1996; 91: 7639-7643Crossref Scopus (189) Google Scholar, 17Sable C.L. Filippa N. Hemmings B. Van Obberghen E. FEBS Lett. 1997; 409: 253-257Crossref PubMed Scopus (152) Google Scholar). Thus, in vivo, PKB can be activated via at least two pathways: a PI 3-kinase dependent and a PI 3-kinase independent pathway. The mechanism of PKB activation through the PI 3-kinase signaling pathway is not completely understood. In vitro, phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), one of the lipid products generated by PI 3-kinase, stimulates PKB activity by binding to the PH domain (19Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (448) Google Scholar, 20Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1313) Google Scholar). Furthermore, PKB activation was shown to be dependent on its phosphorylation of Thr308 and Ser473 (21Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2537) Google Scholar). Phosphorylation of Thr308 is mediated by an upstream kinase, called phosphatidylinositol 3,4,5-triphosphate-dependent protein kinase-1 (PDK-1), while the kinase responsible for phosphorylation of Ser473, already designated as PDK-2, remains to be identified (22Stokoe D. Stephens L.R. Copeland T. Gaffney P.R.J. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1057) Google Scholar, 23Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R.J. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar). The proposed mechanism for PKB activation is that PI(3,4)P2 and phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) generated by PI 3-kinase recruit PKB to the plasma membrane where Thr308 is phosphorylated by PDK-1 and Ser473 by PDK-2 (24Downward J. Science. 1998; 279: 673-674Crossref PubMed Scopus (181) Google Scholar). The activation of PKB by both PI(3,4)P2 and PDK-1 and -2 makes the activation of PKB a multistep process. Initial studies by Konishi and co-workers (25Konishi H. Shinomura T. Kuroda S. Ono Y. Kikkawa U. Biochem . Biophys. Res. Commun. 1994; 205: 817-825Crossref PubMed Scopus (70) Google Scholar, 26Konishi H. Kuroda S. Kikkawa U. Biochem. Biophys. Res. Commun. 1994; 205: 1770-1775Crossref PubMed Scopus (90) Google Scholar) showed that the α, δ, and ζ isoforms of PKC are able to interact with PKB in vitro. In this paper we show that PKB can only be co-immunoprecipiatated with PKCζ and in addition we found that this interaction is under control of PKB activity. To understand the possible function of the PKB-PKCζ association, we investigated whether the interacting kinases regulate the activity of the respective kinases. Although no effect was found of PKB on PKCζ activity, both PDGF- and p110-CAAX-induced activation of PKB is abolished by co-expression of PKCζ. The activity of GSK-3, a downstream target of PKB is also affected by PKCζ co-expression. Finally, we found that the constitutive active PKB mutant (T308D/S473D) is inhibited by PKCζ in a PDGF-independent fashion. The results obtained establish PKCζ as a negative regulator of PKB activity. The pSG5 (Stratagene, La Jolla, CA) constructs containing HA-tagged wild-type bovine PKBα, PKBα "kinase dead" (K179A), PKBDD and PKBAA were a gift from Dr. Paul Coffer (Department of Pulmonary Diseases, University Hospital Utrecht, The Netherlands). The DNA fragments encoding the AH domain of PKB (PKBAH) and PKB lacking the AH domain (PKBΔAH) were amplified by polymerase chain reaction and subcloned as a BamHI/KpnI fragment into the eukaryotic expression vector pBK-CMV (Stratagene, La Jolla, CA) containing a HA epitope tag (pBK-HA). p110-CAAX, p110-R916P-CAAX (PLAP-CAAX), and the pMT2SM constructs containing Myc-tagged wild-type mouse PKCζ and Myc-tagged kinase-dead PKCζ have been described earlier (27Van Dijk M.C.M. Muriana F.J.G. Van der Hoeven P.C.J. De Wit J. Schaap D. Moolenaar W.H. Van Blitterswijk W.J. Biochem. J. 1997; 323: 693-699Crossref PubMed Scopus (69) Google Scholar, 30Didichenko S.A. Tilton B. Hemmings B.A. Balmer H.K. Thelen M. Curr. Biol. 1996; 6: 1271-1278Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). COS-1 and CHO cells were grown in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal calf serum (Life Technologies, Inc.) at 37 °C in a humidified atmosphere with 7% CO2. Transient transfections in COS-1 cells were performed at 40% confluency by a DEAE-dextran method. In short, DNA was diluted in 500 μg/ml DEAE-dextran (Sigma) in phosphate-buffered saline and added to the cells. Following a 30-min incubation at 37 °C, medium containing 80 μm chloroquine (Sigma) was added and the cells were incubated for 2.5–3 h at 37 °C and subsequently shocked with 10% dimethyl sulfoxide (Sigma) for 2.5 min. Twenty-four hours after transfection cells were serum starved for 16 h. Stimulated and unstimulated cells were washed once with ice-cold phosphate-buffered saline and lysed in lysis buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 5 mm EDTA, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 40 mmβ-glycerophosphate, 1 mm sodium vanadate, 50 mm sodium fluoride, and 10 μg/ml aprotinin) and incubated on ice for 5 min. Lysates were centrifuged and supernatants were precleared with protein A-Sepharose beads (Pharmacia, Uppsala, Sweden) for 1 h at 4 °C. HA-PKB was immunoprecipitated from aliquots (200 μg of protein) of the precleared extracts using 6 μg of the monoclonal anti-HA antibody (12CA5) coupled to protein G-Sepharose beads (Sigma), whereas Myc-PKCζ was immunoprecipitated from precleared lysates by 1 μg of the monoclonal anti-Myc antibody (9E10) (Boehringer, Mannheim, Germany) coupled to protein A-Sepharose beads. Endogenous PKCζ was immunoprecipitated from CHO cells using a polyclonal PKCζ antibody (27Van Dijk M.C.M. Muriana F.J.G. Van der Hoeven P.C.J. De Wit J. Schaap D. Moolenaar W.H. Van Blitterswijk W.J. Biochem. J. 1997; 323: 693-699Crossref PubMed Scopus (69) Google Scholar) coupled to protein G-Sepharose beads, whereas PKCα and PKCδ were immunoprecipitated by monoclonal PKCα and PKCδ antibodies (Transduction Laboratories, Lexington, KY), respectively. Normal rabbit serum was used as control antiserum for the co-immunoprecipitation studies. Immunoprecipitations were washed twice with lysis buffer and twice with low salt buffer (50 mmTris-HCl, pH 7.5, 10 mm MgCl2) prior to Western blot analysis or twice with high salt buffer (50 mmTris-HCl, pH 7.5, 10 mm MgCl2, and 0.5m LiCl) and twice with low salt buffer prior to activity measurements. Cell extracts and immunoprecipitations were separated on an 8% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Boehringer, Mannheim, Germany). Membranes were blocked in 5% Protifar (Nutricia, Zoetermeer, The Netherlands) in TBST buffer (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, and 0.1% Tween 20) for 1 h at room temperature. For detection of the Myc-tagged or HA-tagged proteins the membranes were incubated with the monoclonal 9E10 or 12CA5 antibody in 1% protifar in TBST buffer subsequently followed by incubation with peroxidase-conjugated rabbit anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA). Detection of endogenous PKB was performed using the monoclonal PKB/Akt antibody (Transduction Laboratories, Lexington, KY) or the polyclonal Akt-C20 (Santa Cruz Biochemical Corp., Santa Cruz, CA) subsequently followed by incubation with peroxidase-conjugated rabbit anti-mouse or donkey anti-goat (Jackson ImmunoResearch) secondary antibody, respectively. Endogenous PKCα, PKCδ, and PKCζ were detected using the monoclonal PKCα, PKCδ (Transduction Laboratories), or polyclonal PKCζ antibody followed by incubation with peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibody (Jackson ImmunoResearch). For PKB detection with the phospho-specific Akt (Ser473) antibody (New England Biolabs, Beverly, MA) polyvinylidene difluoride membranes were incubated with the polyclonal antibody followed by incubation with peroxidase-conjugated goat anti-rabbit secondary antibody. Proteins were visualized by Enhanced Chemiluminescence (Renaissance, NEN Life Science Products Inc., Boston, MA). For quantification of protein amounts a densitometer (Molecular Dynamics) and ImageQuant software were used. PKCζ activity was measured with the ε-peptide (ERMRPRKRQGSVRRRV) as substrate as described previously (27Van Dijk M.C.M. Muriana F.J.G. Van der Hoeven P.C.J. De Wit J. Schaap D. Moolenaar W.H. Van Blitterswijk W.J. Biochem. J. 1997; 323: 693-699Crossref PubMed Scopus (69) Google Scholar, 28Ways D.K. Cook P.P. Webster C. Parker P.J. J. Biol. Chem. 1992; 267: 4799-4805Abstract Full Text PDF PubMed Google Scholar). Immunoprecipitations were incubated with 45 μl of kinase buffer (50 mm Tris-HCl, pH 7.5, 10 mm MgCl2) containing 50 μm ε-peptide, 0.2 mm EGTA, 50 μm unlabeled ATP, and 3 μCi of [γ-32P]ATP (Amersham International, United Kingdom). PKB activity was assayed with the Crosstide peptide (GRPRTSSFAEG) as a substrate (10Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4426) Google Scholar). Immunoprecipitations were incubated with 45 μl of kinase assay mixture (50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol, 30 μm Crosstide peptide, 1 μm of the specific peptide inhibitor of cyclic AMP-dependent protein kinase (PKI) (Bachem, Bubendorf, Switzerland), 50 μm unlabeled ATP, and 3 μCi of [γ-32P]ATP). GSK-3 activity was measured using the GS peptide (YRRAAVPPSPSLSRHSSPHQSEDEEE) (29Welsh G.I. Patel J.C. Proud C.G. Anal. Biochem. 1997; 244: 16-21Crossref PubMed Scopus (56) Google Scholar). Cell lysates were incubated with 60 μm GS peptide, 2 mm MgCl2, 100 μm ATP, and 2 μCi of [γ-32P]ATP. After incubation for 20 min at 30 °C under continuous shaking, reactions were stopped by addition of 200 mm EDTA. Proteins were precipitated by the addition of 25% trichloroacetic acid and centrifuged for 1 min at 14,000 rpm. Supernatants containing the phosphorylated peptide were spotted onto p81 phosphocellulose filters (Whatman), washed three times with 1% (v/v) orthophosphoric acid, and analyzed by Cerenkov counting. Control experiments revealed that phosphorylation of the GS peptide is highly specific for GSK-3β and that neither PKB nor PKCζ is able to phosphorylate the peptide. 2R. P. Doornbos, unpublished observations.Under the conditions used the kinase assays are linear for at least 60 min. In vitrobinding studies have recently shown that PKB associates with the α, δ, and ζ isoforms of PKC (5Konishi H. Kuroda S. Tanaka M. Matsuzaki H. Ono Y. Kameyama K. Haga T. Kikkawa U. Biochem. Biophys. Res. Commun. 1995; 216: 526-534Crossref PubMed Scopus (162) Google Scholar). In order to investigate the possible interaction of these PKC isoforms with PKB in vivo, we performed co-immunoprecipitation studies using CHO cells. Endogenous PKCα, PKCδ, and PKCζ were immunoprecipitated from cell lysates and Western blot analysis shows that similar amounts of the three PKC isoforms were precipitated (Fig.1 B). The presence of PKB was analyzed using Western blot detection and, as shown in Fig. 1A, PKB is present in the PKCζ but not in the PKCα and PKCδ immunoprecipitates. As a control, normal rabbit serum was incubated with lysates of CHO cells and only a faint band is visible possibly reflecting aspecific binding to the non-immune control (Fig. 1A). The binding of PKB with PKCζ was subsequently investigated in more detail by transient expression of HA-tagged PKB and Myc-tagged PKCζ (Myc-PKCζ) in COS-1 cells. HA-PKB was immunoprecipitated using a monoclonal antibody against the HA-tag (12CA5) and co-immunoprecipitation of Myc-PKCζ was observed on Western blot using a monoclonal antibody against the Myc-tag (9E10) (Fig. 1 C). To identify the domain of PKB that is necessary for the association with PKCζ in vivo, we generated HA-tagged PKB constructs lacking the AH domain (HA-PKBΔAH) or comprising the AH domain (HA-PKBAH). Co-expression of these constructs with PKCζ revealed that the interaction of PKB with PKCζ depends entirely on the presence of the AH domain. This observation is in agreement with the in vitro data obtained by Konishi and co-workers (26Konishi H. Kuroda S. Kikkawa U. Biochem. Biophys. Res. Commun. 1994; 205: 1770-1775Crossref PubMed Scopus (90) Google Scholar). PKCζ could not be observed on a Western blot when PKBΔAH was immunoprecipitated from cells expressing PKBΔAH and PKCζ (Fig. 1 C). As a control, the expression levels of the transiently expressed proteins were analyzed in total cell lysates and similar expression levels were found for all constructs (Fig. 1 C). In conclusion, our observations clearly demonstrate that PKCζ associates with PKBin vivo. Furthermore, the in vivo association of PKB and PKCζ is mediated via the AH domain of PKB. To investigate the effect of PDGF on the PKB-PKCζ complex, COS-1 cells were transiently co-transfected with both HA-PKB and Myc-PKCζ. The cells were serum-starved overnight and either left untreated or stimulated with 25 ng/ml PDGF for 10 min. PKB was immunoprecipitated and co-immunoprecipitation of PKCζ was determined by Western blot analysis (Fig. 2 A). Upon PDGF treatment the interaction decreased with approximately 75% indicating that PDGF induces the dissociation of the complex. As previously reported, PDGF induces the activation of PKB and, albeit to a lesser extent, also of PKCζ (27Van Dijk M.C.M. Muriana F.J.G. Van der Hoeven P.C.J. De Wit J. Schaap D. Moolenaar W.H. Van Blitterswijk W.J. Biochem. J. 1997; 323: 693-699Crossref PubMed Scopus (69) Google Scholar). In order to establish whether the activity of these kinases is involved in PKB·PKCζ complex formation, we analyzed the effect of kinase-dead mutants of both PKB and PKCζ. In repeated experiments expression of kinase-dead PKCζ resulted in a reduction of complex formation which, however, can be explained by the reduction in PKCζkd expression (Fig.2 B). In contrast, expression of kinase-dead PKB resulted in a dramatic increase in the PKB-PKCζ interaction (Fig. 2 A). This demonstrates that PKB activity induces the dissociation of the complex, whereas PKCζ activity seems not to be required for the regulation of the complex. As a control experiment, we incubated the same blot with anti-HA antibodies showing that similar amounts of HA-PKB were precipitated (Fig. 2 B). In order to establish the physiological role for the PKB-PKCζ interaction we investigated the effect on the activity of both kinases. To test a possible role for PKB on PKCζ activity, COS-1 cells were transiently transfected with PKCζ alone or co-transfected with PKB. After stimulation of the cells with PDGF, PKCζ was immunoprecipitated and its activity was measured by an in vitro kinase assay using the ε-peptide as a substrate (28Ways D.K. Cook P.P. Webster C. Parker P.J. J. Biol. Chem. 1992; 267: 4799-4805Abstract Full Text PDF PubMed Google Scholar). PDGF stimulation resulted in an increase of PKCζ activity (Fig. 3) which is in agreement with previous studies (27Van Dijk M.C.M. Muriana F.J.G. Van der Hoeven P.C.J. De Wit J. Schaap D. Moolenaar W.H. Van Blitterswijk W.J. Biochem. J. 1997; 323: 693-699Crossref PubMed Scopus (69) Google Scholar). Co-expression of PKB did not affect activation of PKCζ upon PDGF treatment, demonstrating that PKB has no effect on the PDGF-induced activity of PKCζ (Fig. 3). To demonstrate that PKCζ and not PKB activity accounts for the observed change in ε-peptide phosphorylation we co-transfected PKCζ with kinase-dead PKB. Similar results were obtained as with wild-type PKB showing that PKB activity does not influence the observed change in ε-peptide phosphorylation (Fig. 3). Using the same approach, we investigated whether PKCζ has an effect on PKB activity. For these experiments, COS-1 cells were transiently transfected with wild-type PKB or co-transfected with wild-type PKB and either wild-type PKCζ or kinase-dead PKCζ. After PDGF treatment, PKB was immunoprecipitated and its activity was measured by an in vitro kinase assay using Crosstide as substrate (10Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4426) Google Scholar). Activity measurements showed that PKB activity was increased more than 3-fold upon stimulation of the cells with PDGF (Fig.4A). However, PDGF-induced PKB activation was almost completely abolished when PKB was co-expressed with wild-type PKCζ (Fig. 4 A). This indicates that PKCζ is able to inhibit PDGF induced activity of PKB. In contrast, PKB activity was increased more than 7-fold by PDGF when the cells were co-transfected with the kinase-dead mutant of PKCζ (Fig.4 A). These data clearly show that PKB activity is negatively regulated by PKCζ. An important step in the full activation of PKB is the phosphorylation of residues Thr308 and Ser473 by PDK1 and -2 (21Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2537) Google Scholar). To establish whether the inhibition of PKB activation by PKCζ is due to a reduced increase in the phosphorylation of PKB we used a polyclonal antibody against PKB when phosphorylated on Ser473. As shown in Fig. 5,A and C, PDGF treatment induced an significant increase (p < 0.05) in Ser473phosphorylation when PKB was the only transfected protein in COS-1 cells. In contrast, no significant increase in Ser473phosphorylation was observed upon PDGF treatment when PKCζ was co-expressed with PKB (Fig. 5, A and C). As a control, expression levels of total PKB protein were determined and as shown in Fig. 5 B the amount of PKB in all lanes was equal indicating that the observed differences in phosphorylated PKB on Ser473 was not due to differences in PKB expression levels. Taken together, from these experiments it can be concluded that PKCζ is a negative regulator of PDGF-induced PKB activity. As already mentioned, both PKB and PKCζ are activated by PDGF most probably through the PI 3-kinase signaling pathway. In order to find out whether the negative regulation of PKB by PKCζ is mediated by the PI 3-kinase/PKB signal transduction pathway we expressed a catalytically active membrane-targeted PI 3-kinase (p110-CAAX) together with PKB in COS-1 cells. p110-CAAX caused a significant, ligand-independent increase in PKB activity (Fig.6 A). In contrast, a catalytically inactive membrane-targeted PI 3-kinase (PLAP-CAAX) was unable to do so (Fig. 6 A), which is in agreement with the work of Didichenko and co-workers (30Didichenko S.A. Tilton B. Hemmings B.A. Balmer H.K. Thelen M. Curr. Biol. 1996; 6: 1271-1278Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Co-expression of PKCζ with p110-CAAX and PKB reduced the p110-CAAX-induced PKB activity with almost 80% (Fig. 6 A). Interestingly co-expression of PKCζ with PLAP-CAAX and PKB also resulted in a decrease in basal PKB activity (Fig. 6 A). These observations demonstrate that basal activity of PKCζ is already sufficient to inhibit PKB. It has previously been shown that GSK-3 is phosphorylated and inactivated by PKB in vitro and in vivo (10Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4426) Google Scholar,11van Weeren P.C. de Bruyn K.M.T. de Vries-Smits A.M.M. van Lint J. Burgering B.M.T. J. Biol. Chem. 1998; 273: 13150-13156Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). In order to test whether this downstream effector of PKB is also affected by co-expression of PKCζ, we measured GSK-3 activities in cells expressing PKB alone, PKB and PKCζ, and PKB and kinase-dead PKCζ using a peptide phosphorylation assay (29Welsh G.I. Patel J.C. Proud C.G. Anal. Biochem. 1997; 244: 16-21Crossref PubMed Scopus (56) Google Scholar). As expected, upon treatment of cells with PDGF the GSK-3 activity is decreased (Fig.4 B). However, when PKB is co-expressed with PKCζ the PDGF-induced reduction in GSK-3 activity is completely overcome (Fig.4 B). In contrast, cells co-expressing a kinase inactive PKCζ mutant exhibits a normal reduction in
Referência(s)