Role of Translocation in the Activation and Function of Protein Kinase B
1997; Elsevier BV; Volume: 272; Issue: 50 Linguagem: Inglês
10.1074/jbc.272.50.31515
ISSN1083-351X
AutoresMirjana Andjelković, Dario R. Alessi, Roger Meier, Anne Fernandez, Ned Lamb, Matthias Frech, Peter Cron, Philip Cohen, John M. Lucocq, Brian A. Hemmings,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoWe have investigated the role of subcellular localization in the regulation of protein kinase B (PKB) activation. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKBα was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBα was 60-fold higher compared with the unstimulated wild-type enzyme, and could not be stimulated further by growth factors or phosphatase inhibitors.In vivo 32P labeling and mutagenesis demonstrated that m/p-PKBα activity was due to phosphorylation on Thr308 and Ser473, that are normally induced on PKB following stimulation of the cells with insulin or insulin-like growth factor-1 (IGF-1). A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBα activity. The pleckstrin homology (PH) domain of m/p-PKBα was not required for its activation or phosphorylation on Thr308 and Ser473, suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKBα was translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation required the PH domain and was sensitive to wortmannin. Our results indicate that PI3-K activity is required for translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of the same sites that are induced by insulin or IGF-1. Following activation the kinase detached from the membrane and translocated to the nucleus. We have investigated the role of subcellular localization in the regulation of protein kinase B (PKB) activation. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKBα was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBα was 60-fold higher compared with the unstimulated wild-type enzyme, and could not be stimulated further by growth factors or phosphatase inhibitors.In vivo 32P labeling and mutagenesis demonstrated that m/p-PKBα activity was due to phosphorylation on Thr308 and Ser473, that are normally induced on PKB following stimulation of the cells with insulin or insulin-like growth factor-1 (IGF-1). A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBα activity. The pleckstrin homology (PH) domain of m/p-PKBα was not required for its activation or phosphorylation on Thr308 and Ser473, suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKBα was translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation required the PH domain and was sensitive to wortmannin. Our results indicate that PI3-K activity is required for translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of the same sites that are induced by insulin or IGF-1. Following activation the kinase detached from the membrane and translocated to the nucleus. Many growth factors elicit cellular responses by activating phosphoinositide 3-kinase (PI3-K 1The abbreviations used are: PI3-K, phosphoinositide 3-kinase; PKB, protein kinase B; PH, pleckstrin homology; IGF-1, insulin-like growth factor 1; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4,-bisphosphate; PDK1, 3-phosphoinositide-dependent protein kinase-1; HA, hemagglutinin; m/p, myristoylated/palmitylated; FCS, fetal calf serum; REF-52, rat embryo fibroblasts; Abα469/480 and Abα1/131, antibodies raised against the C terminus and the PH domain of PKBα, respectively; MAPKAP, mitogen-activated protein kinase-activated protein kinase; Pipes, 1,4-piperazinediethanesulfonic acid; HPLC, high performance liquid chromatography; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline.1The abbreviations used are: PI3-K, phosphoinositide 3-kinase; PKB, protein kinase B; PH, pleckstrin homology; IGF-1, insulin-like growth factor 1; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4,-bisphosphate; PDK1, 3-phosphoinositide-dependent protein kinase-1; HA, hemagglutinin; m/p, myristoylated/palmitylated; FCS, fetal calf serum; REF-52, rat embryo fibroblasts; Abα469/480 and Abα1/131, antibodies raised against the C terminus and the PH domain of PKBα, respectively; MAPKAP, mitogen-activated protein kinase-activated protein kinase; Pipes, 1,4-piperazinediethanesulfonic acid; HPLC, high performance liquid chromatography; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline.; reviewed in Ref. 1Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 253-258Crossref Scopus (575) Google Scholar). Recently, protein kinase B (PKB), also known as RAC protein kinase or c-Akt (2Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (787) Google Scholar, 3Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (387) Google Scholar, 4Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (440) Google Scholar) was recognized as a downstream target of PI3-K (5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1875) Google Scholar, 6Franke 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 (1821) Google Scholar). Three mammalian isoforms of PKB have been identified so far, termed PKBα, -β, and -γ (7Jones P.F. Jakubowicz T. 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PKBα was found to mediate insulin- and insulin-like growth factor (IGF-1)-induced cellular responses, such as the inhibition of glycogen synthase kinase-3 (11Cross D.A.E. Alessi D.R. Cohen P. Andjelković M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4339) Google Scholar), the stimulation of glucose uptake (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 (1087) Google Scholar), and the promotion of cell survival by inhibiting apoptosis (Ref. 13Dudek 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 (2215) Google Scholar; reviewed in Refs. 14Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1520) Google Scholar and 15Hemmings B.A. Science. 1997; 275: 628-630Crossref PubMed Scopus (435) Google Scholar). PKBα is the cellular homologue of the oncogene product v-Akt encoded by the AKT8 retrovirus, which induces thymic lymphomas in mice (16Staal S.P. Hartley J.W. J. Exp. Med. 1988; 167: 1259-1264Crossref PubMed Scopus (82) Google Scholar). Cloning of v-akt revealed that it was created by fusion of viral Gag sequences to the N terminus of mouse PKBα, which adds an N-terminal myristoylation signal to the oncoprotein and could account for its transforming ability (2Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (787) Google Scholar, 17Ahmed N.N. Franke T.F. Bellacosa A. Datta K. Gonzales-Portal M.-E. Taguchi T. Testa J.R. Tsichlis P.N. Oncogene. 1993; 8: 1957-1963PubMed Google Scholar). Overexpression of PKBα or -β is associated with some human ovarian, pancreatic, and breast carcinomas (8Cheng 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 (646) Google Scholar, 18Staal S.P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5034-5037Crossref PubMed Scopus (640) Google Scholar, 19Bellacosa A. de Feo D. Godwin A.K. Bell D.W. Cheng J.Q. Altomare D.A. Wan M. Dubeau L. Scambia G. Masciullo V. Ferrandina G. Benedetti Oanici P. Mancuso S. Neri G. Testa J.R. Int. J. Cancer. 1995; 64: 280-285Crossref PubMed Scopus (735) Google Scholar, 20Cheng 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 (699) Google Scholar). PKBα is activated by a variety of growth factors and phosphatase inhibitors (5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1875) Google Scholar, 6Franke 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 (1821) Google Scholar, 21Andjelković M. Jakubowicz T. Cron P. Ming X.-F. Han J.H. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (428) Google Scholar) through a phosphorylation mechanism (21Andjelković M. Jakubowicz T. Cron P. Ming X.-F. Han J.H. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (428) Google Scholar, 22Alessi D.R. Andjelković M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2500) Google Scholar, 23Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). The activation of PKBα by insulin or IGF-1 is mediated by phosphorylation of Thr308 in the catalytic domain and Ser473 at the C terminus (22Alessi D.R. Andjelković M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2500) Google Scholar). The phosphorylation of both sites is blocked by pretreatment of the cells with the PI3-K inhibitor wortmannin. Substitution of both regulatory sites by aspartic acid residues to mimic phosphorylation by the introduction of a negative charge, produces a constitutively active enzyme (22Alessi D.R. Andjelković M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2500) Google Scholar). This work predicted the existence of an upstream kinase(s) that phosphorylate(s) these sites, and recently a protein kinase activity was identified and purified capable of phosphorylating Thr308 in the presence of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) or phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) (Refs. 24Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R.J. Reese C. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar and 25Stokoe 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 (1045) Google Scholar; reviewed in Ref. 26Hemmings B.A. Science. 1997; 277: 534Crossref PubMed Scopus (77) Google Scholar). The enzyme has therefore been termed 3-phosphoinositide-dependent protein kinase-1 (PDK1). The PH domain of PKB has been reported to play a role in the activation process (6Franke 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 (1821) Google Scholar), but PKB activation can also occur in its absence, depending on the agonist and the type of deletion mutants used (21Andjelković M. Jakubowicz T. Cron P. Ming X.-F. Han J.H. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (428) Google Scholar, 23Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar,27Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar). The PH domain of PKB binds PtdIns(3,4,5)P3 and PtdIns(3,4)P2 at low micromolar concentrations (28James S.R. Downes C.P. Gigg R. Grove S.J.A. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar, 29Frech M. Andjelković M. Ingley E. Reddy K.K. Falck J.R. Hemmings B.A. J. Biol. Chem. 1997; 272: 8474-8481Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar), but the precise role of inositol phospholipid binding to PKB is not fully understood (28James S.R. Downes C.P. Gigg R. Grove S.J.A. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar, 29Frech M. Andjelković M. Ingley E. Reddy K.K. Falck J.R. Hemmings B.A. J. Biol. Chem. 1997; 272: 8474-8481Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 30Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1298) Google Scholar, 31Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (446) Google Scholar). Since PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are located in the plasma membrane, the interaction of PKB with one or both of these phosphoinositides may play a role in recruiting PKB to the membrane. To address this question, we have added a membrane targeting signal to the N terminus of PKBα and find that this is sufficient for maximal phosphorylation of Thr308 and Ser473, and activation of PKBα in the presence or absence of the PH domain. Furthermore, we provide evidence that the IGF-1-induced activation of PKB is accompanied by its translocation to the membrane, followed by the translocation to the nucleus. The pECE and pCMV constructs encoding hemagglutinin (HA) epitope-tagged PKBα have been described (21Andjelković M. Jakubowicz T. Cron P. Ming X.-F. Han J.H. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (428) Google Scholar, 22Alessi D.R. Andjelković M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2500) Google Scholar). Myristoylated/palmitylated (m/p)-HA-PKBα was created by polymerase chain reaction with HA-PKBα as template, using a 5′ oligonucleotide encoding the 12-amino acid N-terminal sequence of Lck, which carries the myristoylation/palmitylation signal, followed by two Ala residues and the HA epitope, and a 3′ oligonucleotide encoding amino acids 468–480 of PKBα. The resulting product was subcloned as a SalI/NotI fragment into pECE.HA-PKBα. The pECE expression constructs encoding PKBα phosphorylation site mutants at Ser473 and Thr308, or both have been described (22Alessi D.R. Andjelković M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2500) Google Scholar). To create the membrane targeted versions of these mutants, the CelII/EcoRII fragment of pECE.HA-PKBα-S473A and NotI/EcoRI fragment of pECE.HA-PKBα-T308A were subcloned into the respective restriction sites of pECE.m/p-HA-PKBα. The pECE construct encoding m/p-HA-PKBα-ΔPH was made by polymerase chain reaction using a 5′ oligonucleotide encoding the 12-amino acid N terminus of Lck, followed by two Ala residues, the HA epitope and amino acids 119–125 of PKBα, and a 3′ oligonucleotide encoding amino acids 468–480. The product was subcloned as a SalI/EcoRI fragment into the same restriction sites of the pECE vector (32Ellis L. Clauser E. Morgan D.O. Edery M. Roth R.A. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (696) Google Scholar). TheBglII/XbaI fragments from the above described pECE constructs were transferred into the same restriction sites of the pCMV5 vector (33Andersson S. Davis D.N. Dahlbäck H. Jörnvall H. Russell D.W J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar). m/p-HA-PKBα-T308D/S473D was created by subcloningNotI/XbaI fragments from pECE-HA-PKBα-T308D/S473D and pECE.HA-PKBα-ΔC14 into pCMV5.m/p-HA-PKBα. The constructs were confirmed by restriction analysis and sequencing. The construct SRα-p85αΔ478–513 has been described (34Dhand R. Hara K. Hiles I. Bax B. Gout I. Panayotou G. Fry M.J. Yonezawa K. Kasuga M. Waterfield M.D. EMBO J. 1994; 13: 511-521Crossref PubMed Scopus (295) Google Scholar). Human embryonic kidney 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS; Life Technologies, Inc.) at 37 °C, in an atmosphere containing 5% CO2. Cells seeded at 106/10-cm dish and 0.5 × 106/6-cm dish, respectively, were transfected the following day by a modified calcium phosphate method (35Chen C. Okayama H. Biotechniques. 1988; 6: 632Crossref PubMed Scopus (28) Google Scholar), with 1–2 μg/ml plasmid DNA. The transfection mixture was removed after a 16-h incubation and cells were serum-starved for 24 h before stimulation for 15 min with 100 nm insulin (Novo-Nordisk, Bagsvaerd, Denmark), 50 or 100 ng/ml IGF-1 (Life Technologies, Inc.) or 0.1 mm pervanadate prepared as described previously (21Andjelković M. Jakubowicz T. Cron P. Ming X.-F. Han J.H. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (428) Google Scholar). Pretreatment with 100 μm LY 294002 (Calbiochem) was performed during the last 12 h of serum-starvation. Rat embryo fibroblast (REF-52) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS as described previously (36Lamb N.J.C. Fernandez A. Watrin A. Labbé J.C. Cavadore J.C. Cell. 1990; 60: 151-165Abstract Full Text PDF PubMed Scopus (107) Google Scholar). Transfected 293 cells were collected in ice-cold hypotonic buffer containing 5 mm Tris, pH 7.4, 25 mm NaF, 5 mm MgCl2, 1 mm EGTA, and 0.1 mm sodium orthovanadate, and lysed by 10 strokes in a Dounce homogenizer. Nuclei were removed by centrifugation for 10 min at 1,000 × g at 4 °C. The P100 and S100 fraction were obtained by additional centrifugation at 100,000 × g for 30 min at 4 °C. P100 was resuspended in lysis buffer. Cells were extracted on plates in lysis buffer containing 50 mmTris-HCl, pH 7.5, 1% w/v Nonidet P-40, 120 mm NaCl, 25 mm NaF, 40 mm β-glycerol phosphate, 0.1 mm sodium orthovanadate, 100 nm okadaic acid (LC Laboratories), 0.1% v/v 2-mercaptoethanol, 1 mmphenylmethylsulfonyl fluoride, and 1 mm benzamidine. Lysates were centrifuged for 15 min at 12,000 × g. The HA-PKBα protein was immunoprecipitated from 100–200 μg of cell-free extracts, or 50 μg of the S100 and P100 fractions with the anti-HA epitope 12CA5 monoclonal antibody coupled to protein A-Sepharose. The immune complexes on beads were washed once with lysis buffer containing 0.5 m NaCl, followed by lysis buffer and finally with kinase buffer (50 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 1 mm benzamidine). In vitro kinase assays were performed for 30 min at 30 °C in 50 μl of reaction volume containing 30 μl of immunoprecipitate in kinase buffer, 30 μm peptide GRPRTSSFAEG as substrate (11Cross D.A.E. Alessi D.R. Cohen P. Andjelković M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4339) Google Scholar), 10 mm MgCl2, 1 μm protein kinase A inhibitor peptide (Bachem), and 50 μm[γ-32P]ATP (Amersham; 1,000–2,000 cpm/pmol) and the activity determined as described previously (22Alessi D.R. Andjelković M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2500) Google Scholar). Cell extracts and immunoprecipitates were resolved by 7.5% SDS-polyacrylamide gel electrophoresis, and transferred to Immobilon P membranes (Millipore). The filters were blocked for 30 min with 5% skimmed milk in 1 × Tris-buffered saline, 1% Triton X-100, 0.5% Tween 20, followed by a 2-h incubation with 50-fold diluted rabbit polyclonal anti-PKBα antisera specific for the C terminus (Abα469/480; Ref. 4Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (440) Google Scholar), or recombinant PH domain containing N-terminal 131 amino acids (Abα1/131) or with the anti-HA epitope 12CA5 monoclonal antibody that was 1,000-fold diluted in the same blocking solution. The secondary antibodies were 1,000-fold diluted alkaline-phosphatase conjugated anti-rabbit IgG (Sigma) and anti-mouse Ig (Southern Biotechnology Associated), or 5,000-fold diluted horseradish peroxidase-linked Ig (Amersham). Detection was performed using the AP color development reagents from Bio-Rad or by enhanced chemiluminescence (Amersham). To normalize expression levels of PKB, FITC-labeled secondary antibodies were employed at a 200-fold dilution, the signal detected by chemifluorescence using a Storm 840/860 PhosphorImager and quantified with ImageQuant Software (Molecular Dynamics). 293 cells were plated and transfected on sterile coverslips. Fixation of cells with formaldehyde and permeabilization with 0.2% Triton X-100 were performed according to Ref. 37Harlow E. Lane D. Antibodies: A Laboratory Manuel. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar. The 12CA5 monoclonal antibody diluted 50-fold in phosphate-buffered saline (PBS) was applied for one hour at 37 °C. The cells were subsequently washed twice with PBS and incubated with FITC-conjugated anti-mouse IgG (Sigma) at a 50-fold dilution, or with 100-fold diluted biotinylated anti-mouse IgG (Sigma), followed by 200-fold diluted streptavidin coupled to Texas Red (Amersham). DNA was stained with 4,6-diamidino-2-phenylindole. The coverslips were washed twice with PBS, once with H2O, mounted on glass slides using Gelvatol, and photographed with a LEITZ DMRD Leica camera. Confocal images were collected on a Leica TCS 4D microscope. REF-52 cells were subcultured on either 25-mm glass coverslips (Schutt Labortechnik, Göttingen, Germany) or acid-washed coverslips. Microinjection was performed with a normal Leitz micromanipulator, as described previously (38Turowski P. Fernandez A. Favre B. Lamb N.J.C. hemmingsi B.A. J. Cell. Biol. 1995; 129: 397-410Crossref PubMed Scopus (136) Google Scholar). Cells were injected with 0.5 mg/ml PKB construct in the presence of 1 mg/ml biotinylated rabbit IgG (Sigma). Cells were serum-starved for 36–48 h before stimulation with 1 μm okadaic acid/10% FCS. Cells were fixed with 3.7% formaldehyde and further treated as described previously (36Lamb N.J.C. Fernandez A. Watrin A. Labbé J.C. Cavadore J.C. Cell. 1990; 60: 151-165Abstract Full Text PDF PubMed Scopus (107) Google Scholar). PKB was detected using Abα469/480 followed by a FITC-conjugated anti-rabbit antibody, and HA-PKBα was detected with the 12CA5 monoclonal antibody followed by a FITC-conjugated anti-mouse antibody. Microinjected cells were identified by costaining the cells with streptavidin coupled to Texas Red (Amersham). DNA was stained using Hoechst stain 33358 (1 μg/ml bisbenzemidine). The cells were fixed in 4% formaldehyde in 0.2 m Pipes, pH 7.2, for at least 20 min, washed in PBS, scraped from the dish using a rubber policeman, and embedded in 10% pig skin gelatin before cryoprotection in 2.3 m sucrose in PBS. Ultrathin sections were cut at −110 °C in a Reichert Ultracut E cryomicrotome, mounted on carbon/Formvar-coated grids, and labeled using the 12CA5 monoclonal antibody followed by a rabbit anti-mouse antibody and finally protein A gold (7 or 5 nm particle size prepared as described by Lucocq; Ref.39Lucocq J.M. Griffiths G. Fine Structure Immunocytochemistry. Springer-Verlag, Berlin1993: 279-302Crossref Google Scholar). Sections were embedded in methylcellulose uranyl acetate as described in Ref. 40Griffiths G. McDowall A. Back R. Dubochet J. J. Ultrastr. Res. 1984; 89: 65-78Crossref PubMed Scopus (341) Google Scholar. To quantitate immunolabeling, sections were scanned systematically and gold label identified. All visible parts of the plasma membrane and adjacent cytoplasm of labeled cells were photographed at magnification ×15,000. Cytoplasm areas and membrane profile length were estimated using a square lattice grid with 1-cm line spacing as described in Ref. 41Lucocq J.M. J. Anat. 1994; 184: 1-13PubMed Google Scholar. Gold particles over the nucleus were not included in the analysis and gold particles were only assigned to the plasma membrane if they lay within 2 particle widths of the plasma membrane profile. 293 cells transfected with HA-PKBα DNA constructs were serum-starved overnight, washed twice with phosphate-free DMEM, and incubated for 4 h (Fig. 5) or 12 h (Fig. 10) with carrier-free [32P]orthophosphate (1 mCi/ml) in either the presence or absence of 100 μm LY294002 (Fig. 10). The cells were then stimulated for 10 min at 37 °C in the presence or absence of 100 ng/ml IGF-1 and placed on ice. The medium was aspirated, the cells washed twice with ice-cold DMEM buffer and then lysed. HA-PKBα was immunoprecipitated from the lysates, alkylated with 4-vinylpyridine, digested with trypsin, and analyzed by C18 chromatography exactly as described previously (22Alessi D.R. Andjelković M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2500) Google Scholar).Figure 10Morphological demonstration of IGF-1-induced translocation of HA-PKBα to the plasma membrane, which can be prevented by wortmannin treatment. 293 cells expressing HA-PKBα were fixed and processed as described for Fig. 9. A, significant labeling of HA-PKBα in the cytoplasm of unstimulated cells with low labeling over the plasma membrane. B, the plasma membrane of cells stimulated with 100 ng/ml IGF-1 for 5 min is intensely labeled with gold particles and, in this case, there is little cytoplasmic label. C, lack of appreciable labeling at the plasma membranes in cells pretreated with 100 nmwortmannin for 10 min before IGF-1 stimulation. A and B, 7 nm protein A gold; C, 5 nm protein A gold. Bars, 100 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We previously proposed that PKB activation occurs by phosphorylation, following recruitment of the kinase to the membrane via its PH domain (21Andjelković M. Jakubowicz T. Cron P. Ming X.-F. Han J.H. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (428) Google Scholar, 22Alessi D.R. Andjelković M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2500) Google Scholar). To investigate the role of membrane targeting in the activation of PKB, the N-terminal membrane localization sequence from Lck was attached to the N terminus of HA-PKBα (see Fig.1). This signal was chosen because it contains the consensus sequence for both myristoylation and palmitylation (42Resh M. Cell. 1994; 76: 411-413Abstract Full Text PDF PubMed Scopus (590) Google Scholar) and has been shown to be sufficient to localize a number of cytosolic proteins to the plasma membrane (43Zlatkine P. Mehul B. Magee A.I. J. Cell Sci. 1997; 110: 673-679PubMed Google Scholar). Several mutants of m/p-HA-PKBα were also prepared in which the ATP-binding site (Lys179) or the phosphorylation sites (Thr308, Ser473) were mutated to Ala, or in which the N-terminal 118 amino acids containing the PH domain, or the C-terminal 14 amino acids encompassing the Ser473phosphorylation site were deleted (Fig. 1). To confirm that the Lck myristoylation/palmitylation signal provides membrane attachment of m/p-HA-PKBα, we determined the subcellular localization of the proteins expressed in 293 cells by immunofluorescence using the anti-HA epitope antibody. HA-PKBα was found in the cytosol of serum-starved, unstimulated 293 cells (Fig.2, A and B). However, all forms of m/p-HA-PKBα (Fig. 2, C–G) were highly concentrated at the plasma membrane. No immunostaining occurred if the 293 cells were transfected with vector alone (Fig.2 H). Overexpression of m/p-HA-PKBα, m/p-HA-PKBα-S473A, or m/p-HA-PKBα-ΔPH in 293 cells resulted in rounding of the cells, which was not observed when either wild-type HA-PKB or other m/p-HA-PKBα mutants were overexpressed. The activity of m/p-HA-PKBα in unstimulated cells was over 60-fold higher than that of HA-PKBα. This is higher than the activity of PKBα obtained after stimulation of the wild-type kinase with insulin, IGF-1, or vanadate (Fig. 3 A). Consistent with this finding, the activity of m/p-HA-PKBα could not be increased further by stimulation of the cells with insulin, IGF-1, or vanadate (Fig. 3 A). m/p-HA-PKBα from unstimulated cells, like HA-PKBα from IGF-1-stimulated cells, could be inactivated by treatment with protein phosphatase 2A in vitro (data not shown). No PKB activity was detected when kinase-inactive m/p-HA-PKBα-K179A was expressed in 293 cells (Fig.3 A). The intracellular localization of wild-type HA-PKBα and m/p-HA-PKBα were confirmed by biochemical studies. About 80% of HA-PKBα activity and 75% of HA-PKBα protein were detected in the 100,000 ×g supernatant (S100) of unstimulated 293 cells, wherea
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