Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Identification of Tyrosine Phosphorylation Sites on 3-Phosphoinositide-dependent Protein Kinase-1 and Their Role in Regulating Kinase Activity

2001; Elsevier BV; Volume: 276; Issue: 40 Linguagem: Inglês

10.1074/jbc.m105916200

ISSN

1083-351X

Autores

Jongsun Park, Michelle M. Hill, Daniel Heß, Derek P. Brazil, Jan Hofsteenge, Brian A. Hemmings,

Tópico(s)

Metabolism, Diabetes, and Cancer

Resumo

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287–292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased ∼6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinasein vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287–292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased ∼6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinasein vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family. 3-phosphoinositide-dependent protein kinase-1 protein kinase B p70 ribosomal protein S6 kinase cyclic AMP-dependent protein kinase protein kinase C serum and glucocorticoid-inducible kinase p90 ribosomal protein S6 kinase embryonic stem cells phosphoinositide 3-kinase phosphatidylinositol pleckstrin homology human embryonic kidney insulin-like growth factor-1 T-cell protein-tyrosine phosphatase insulin receptor phenylmethylsulfonyl fluoride dithiothreitol RRKDGATMKTFCGTPE protein phosphatase 2A catalytic subunit polyacrylamide gel electrophoresis 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine phosphoserine phosphothreonine phosphotyrosine high performance liquid chromatography mass spectrometry tandem mass spectrometry electrospray ionization collision-induced dissociation PDK1-interacting fragment Src homology-2 Lambda protein phosphatase polymerase chain reaction amino acid(s) second messenger regulated subfamily of protein kinases 3-Phosphoinositide-dependent protein kinase-1 (PDK1)1 appears to play a central regulatory role in many cell-signaling pathways (1–12). Several substrates of PDK1 have so far been identified, including protein kinase B (PKB), p70 ribosomal protein S6 kinase (p70S6K), cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC), serum and glucocorticoid-inducible kinase (SGK), p90 ribosomal protein S6 kinase (RSK), and p21-activated kinase-1 (1Alessi 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, 2Stokoe 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 (1043) Google Scholar, 6Galetic I. Andjelkovic M. Meier R. Brodbeck D. Park J. Hemmings B.A. Pharmacol. Ther. 1999; 82: 409-425Crossref PubMed Scopus (95) Google Scholar, 7Peterson R.T. Schreiber S.L. Curr. Biol. 1999; 9: R521-R524Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 8Flynn 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, 9Dempsey E.C. Newton A.C. Mochly-Rosen D. Fields A.P. Reyland M.E. Insel P.A. Messing R.O. Am. J. Physiol. Lung. Cell Mol. Physiol. 2000; 279: L429-L438Crossref PubMed Google Scholar, 13Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 14Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (718) Google Scholar, 15Romanelli A. Martin K.A. Toker A. Blenis J. Mol. Cell. Biol. 1999; 19: 2921-2928Crossref PubMed Google Scholar, 16Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (117) Google Scholar, 17Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9849-9854Crossref PubMed Scopus (190) Google Scholar, 18Chou M.M. Hou W. Johnson J. Graham L.K. Lee M.H. Chen C.S. Newton A.C. Schaffhausen B.S. Toker A. Curr. Biol. 1998; 8: 1069-1077Abstract Full Text Full Text PDF PubMed Google Scholar, 19Dutil E.M. Toker A. Newton A.C. Curr. Biol. 1998; 8: 1366-1375Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 20Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (967) Google Scholar, 21Dong L.Q. Zhang R.B. Langlais P. He H. Clark M. Zhu L. Liu F. J. Biol. Chem. 1999; 274: 8117-8122Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 22Inagaki M. Schmelzle T. Yamaguchi K. Irie K. Hall M.N. Matsumoto K. Mol. Cell. Biol. 1999; 19: 8344-8352Crossref PubMed Scopus (116) Google Scholar, 23Standaert M.L. Bandyopadhyay G. Perez L. Price D. Galloway L. Poklepovic A. Sajan M.P. Cenni V. Sirri A. Moscat J. Toker A. Farese R.V. J. Biol. Chem. 1999; 274: 25308-25316Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 24Sajan M.P. Standaert M.L. Bandyopadhyay G. Quon M.J. Burke Jr., T.R. Farese R.V. J. Biol. Chem. 1999; 274: 30495-30500Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 25Bandyopadhyay G. Standaert M.L. Sajan M.P. Karnitz L.M. Cong L. Quon M.J. Farese R.V. Mol. Endocrinol. 1999; 13: 1766-1772PubMed Google Scholar, 26Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (521) Google Scholar, 27Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (479) Google Scholar, 28Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (332) Google Scholar, 29Jensen 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 (212) Google Scholar, 30Richards S.A. Fu J. Romanelli A. Shimamura A. Blenis J. Curr. Biol. 1999; 9: 810-820Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 31King C.C. Gardiner E.M. Zenke F.T. Bohl B.P. Newton A.C. Hemmings B.A. Bokoch G.M. J. Biol. Chem. 2000; 275: 41201-41209Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). However, most of these putative PDK1 substrates were identified usingin vitro phosphorylation experiments, and not all the substrate proteins appear to be physiological targets for PDK1 in vivo. Experiments with embryonic stem (ES) cells ablated for both alleles of PDK1 revealed that PKB, p70S6K, and RSK are no longer phosphorylated or activated by agonists that are potent stimuli in normal ES cells, providing in vivo evidence that these three enzymes are dependent on PDK1 for activation (32Williams 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 (392) Google Scholar). The regulation of the other potential substrates of PDK1 is apparently unaffected in the knock-out cell line (32Williams 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 (392) Google Scholar). PKB, p70S6K, RSK, and SGK, members of the AGC family of protein kinases, are apparently maintained in the cytoplasm in an inactive state. Upon stimulation of receptor tyrosine kinases, phosphoinositide 3-kinase (PI3K) becomes activated, generating the phospholipid second messengers, PtdIns(3,4,5)P3 and PtdIns(3,4)P2. These lipids then mediate the phosphorylation and activation of PDK1 targets through diverse mechanisms (1Alessi 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, 2Stokoe 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 (1043) Google Scholar, 13Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 14Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (718) Google Scholar, 27Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (479) Google Scholar, 28Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (332) Google Scholar, 29Jensen 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 (212) Google Scholar, 30Richards S.A. Fu J. Romanelli A. Shimamura A. Blenis J. Curr. Biol. 1999; 9: 810-820Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 33Walker K.S. Deak M. Paterson A. Hudson K. Cohen P. Alessi D.R. Biochem. J. 1998; 331: 299-308Crossref PubMed Scopus (238) Google Scholar). In the case of PKB, this activation appears to be facilitated by lipid binding to pleckstrin homology (PH) domains that mediate the recruitment of both PKB and PDK1 to the plasma membrane, thereby promoting phosphorylation of PKB by PDK1 and the as-yet-unidentified Ser-473 kinase (34Hemmings B.A. Science. 1997; 275: 628-630Crossref PubMed Scopus (434) Google Scholar, 35Frech M. Andjelkovic 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 (355) Google Scholar, 36Hemmings B.A. Science. 1997; 277: 534Crossref PubMed Scopus (77) Google Scholar, 37Hemmings B.A. Science. 1997; 275: 1899Crossref PubMed Scopus (67) Google Scholar, 38Meier R. Alessi D.R. Cron P. Andjelkovic M. Hemmings B.A. J. Biol. Chem. 1997; 272: 30491-30497Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 39Andjelkovic 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 (892) Google Scholar, 40Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1178) Google Scholar, 41Haas-Kogan D. Shalev N. Wong M. Mills G. Yount G. Stokoe D. Curr. Biol. 1998; 8: 1195-1198Abstract Full Text Full Text PDF PubMed Google Scholar, 42Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 43Aoki M. Batista O. Bellacosa A. Tsichlis P. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14950-14955Crossref PubMed Scopus (258) Google Scholar, 44Frech M. Hemmings B.A. Methods Mol. Biol. 1998; 88: 197-210PubMed Google Scholar, 45Andjelkovic M. Suidan H.S. Meier R. Frech M. Alessi D.R. Hemmings B.A. Eur. J. Biochem. 1998; 251: 195-200Crossref PubMed Scopus (57) Google Scholar, 45Andjelkovic M. Suidan H.S. Meier R. Frech M. Alessi D.R. Hemmings B.A. Eur. J. Biochem. 1998; 251: 195-200Crossref PubMed Scopus (57) Google Scholar). In the case of SGK/RSK/p70S6K, the precise mechanism for stimulatory phosphorylation of the activation loop by PDK1 is less well understood. It is possible that there is a mechanism for inducing transient association of PDK1 and SGK/RSK/p70S6K. Indeed, PDK1 and SGK are able to form a stable complex (27Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (479) Google Scholar), and phosphorylation of a hydrophobic motif on RSK serves as a docking site for PDK1 (47Frodin M. Jensen C.J. Merienne K. Gammeltoft S. EMBO J. 2000; 19: 2924-2934Crossref PubMed Scopus (251) Google Scholar). Although a role for PDK1 in insulin signaling is well established, the mechanism of regulation of PDK1 activity remains controversial. Previously, it was thought that PDK1 is constitutively active in resting cells and not further activated by growth factor stimulation (48Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (284) Google Scholar). In addition, the subcellular localization of PDK1 also appears to be growth factor-insensitive (49Currie R.A. Walker K.S. Gray A. Deak M. Casamayor A. Downes C.P. Cohen P. Alessi D.R. Lucocq J. Biochem. J. 1999; 337: 575-583Crossref PubMed Scopus (272) Google Scholar), although others have reported growth factor-dependent translocation of PDK1 to the plasma membrane (42Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 50Grillo S. Gremeaux T. Casamayor A. Alessi D.R. Le Marchand-Brustel Y. Tanti J.F. Eur. J. Biochem. 2000; 267: 6642-6649Crossref PubMed Scopus (45) Google Scholar). Several serine sites (Ser-25, Ser-241, Ser-393/396, Ser-410) are phosphorylated on PDK1 in unstimulated human embryonic kidney (HEK) 293 cells, as well as insulin-like growth factor-1 (IGF-1)-stimulated cells (48Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (284) Google Scholar). However, only phosphorylation on the activation loop Ser-241 (equivalent to Thr-308 of PKB) is necessary for PDK1 activity (48Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (284) Google Scholar). Furthermore, this study examined the effect of IGF-1 stimulation on PDK1 activity and was unable to find any activation (48Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (284) Google Scholar). Although physiologically relevant, signal transduction events elicited by IGF-1 may be transient, and/or too small in magnitude for detection in this experimental paradigm. To further probe the regulation of PDK1, we have utilized pervanadate, an inhibitor of protein-tyrosine phosphatases that apparently mimics insulin action, to stimulate cells (51Posner B.I. Faure R. Burgess J.W. Bevan A.P. Lachance D. Zhang-Sun G. Fantus I.G. Ng J.B. Hall D.A. Lum B.S. et al.J. Biol. Chem. 1994; 269: 4596-4604Abstract Full Text PDF PubMed Google Scholar). Pervanadate stimulates PKB activity to a much higher extent than insulin or IGF-1 (52Andjelkovic 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 (425) Google Scholar), and through this amplification of signal intensity, we hoped to observe changes in PDK1 activity and/or subcellular localization previously not detectable with insulin stimulation. Indeed, stimulation of cells with pervanadate resulted in a significant increase in PDK1 activity and translocation to the plasma membrane, together with an induction of tyrosine phosphorylation. During the course of our experiments, work from two independent groups (50Grillo S. Gremeaux T. Casamayor A. Alessi D.R. Le Marchand-Brustel Y. Tanti J.F. Eur. J. Biochem. 2000; 267: 6642-6649Crossref PubMed Scopus (45) Google Scholar, 53Prasad N. Topping R.S. Zhou D. Decker S.J. Biochemistry. 2000; 39: 6929-6935Crossref PubMed Scopus (74) Google Scholar) also reported that PDK1 becomes tyrosine-phosphorylated following stimulation with pervanadate or hydrogen peroxide in adipocytes, HEK 293 cells, and A20 lymphoma cells. In this report, we identify three tyrosine phosphorylation sites on PDK1 using in vivo labeling and mass spectrometry: Tyr-9 and Tyr-373/376. Using site-directed mutants, we show that although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both sites can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data show that PDK1 activity is regulated by reversible phosphorylation, possibly by a Src family kinase. Purified recombinant T-cell protein-tyrosine phosphatase (TC-PTP (54Tiganis T. Flint A.J. Adam S.A. Tonks N.K. J. Biol. Chem. 1997; 272: 21548-21557Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar)) and v-Src were kindly provided by Dr. Nick Tonks (Cold Spring Harbor, NY) and Dr. Doriano Fabbro (Novartis, Switzerland), respectively. Monoclonal anti-Src and anti-insulin receptor (IR) antibody were gifts from Dr. Kurt Ballmer-Hofer (Paul Scherrer Institute, Switzerland) and Dr. Kenneth Siddle (University of Cambridge, UK), respectively. Anti-Myc 9E10 and anti-phosphotyrosine 4G10 monoclonal antibody were from commercial sources. The Myc-tagged full-length PDK1 (Myc-PDK1) was generated by a two-step PCR using Myc-PDK1-Δ50 lacking the first 50 amino acids (14Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (718) Google Scholar) as a template with the following primers (1st round PCR, 5′-CCC GGT ACC ACC ATG GCT TAC CCA TAC GAT GTT CCA GAT TAC GCT TCG ACC GTC AAA ACC GAG GCT GCT CGA; 2nd round PCR, 5′-CCC GGT ACC ACC ATG GCT TAC CCA TAC GAT GTT CCA GAT TAC GCT TCG ACC and 3′-CCC GGT ACC ACC ATG GCT TAC CCA TAC GAT GTT CCA GAT) and subcloned into pCMV5 vector. The PH domain deletion mutant of PDK1 was generated by a standard PCR-cloning strategy (Myc-ΔPH-PDK1). The mutants at Tyr-9 (Myc-PDK1 Y9F), Tyr-373/376 (Myc-PDK1 Y373/376F), and both Tyr-9 and Tyr-373/376 (Myc-PDK1 FFF) were created by using the QuikChange site-directed mutagenesis kit (Stratagene) as described by the manufacturer with pCMV5 Myc-PDK1 as template. v-Src and kinase-dead mutants of v-Src constructs in pcDNA3.1 (v-Src and v-Src-KD) were a gift from Dr. Monilola Olayioye (FMI, Switzerland). Wild type Fyn (Fyn-WT) and insulin-receptor tyrosine kinase (IR-WT) expression constructs have been described previously (55Dunant N.M. Messerschmitt A.S. Ballmer-Hofer K. J. Virol. 1997; 71: 199-206Crossref PubMed Google Scholar, 56Baltensperger K. Kozma L.M. Cherniack A.D. Klarlund J.K. Chawla A. Banerjee U. Czech M.P. Science. 1993; 260: 1950-1952Crossref PubMed Scopus (230) Google Scholar). All constructs were confirmed by automated DNA sequencing. Sequences of the mutagenic oligonucleotides are available upon request. HEK 293 cells were maintained and transfected using a modified calcium phosphate method as previously described (27Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (479) Google Scholar). The transfection mixture was removed after 16-h incubation, and cells were serum-starved for 24 h before stimulation for 15 min with 100 nm insulin (Roche Molecular Biochemicals), 50 ng/ml IGF-1 (Life Technologies), 100 nmcalyculin-A (Alexis), or 100 μm pervanadate prepared with 0.2 mm H2O2 (51Posner B.I. Faure R. Burgess J.W. Bevan A.P. Lachance D. Zhang-Sun G. Fantus I.G. Ng J.B. Hall D.A. Lum B.S. et al.J. Biol. Chem. 1994; 269: 4596-4604Abstract Full Text PDF PubMed Google Scholar), or for 1 h with 1 μm okadaic acid (Alexis). Pretreatment with 50 μm LY294002 (Alexis) or 10 μm4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1, Alexis) was for 15 or 30 min before cell stimulation, respectively. HEK 293 cells were placed on ice and extracted with lysis buffer containing 50 mm Tris-HCl, pH 7.5, 1% v/v Nonidet P-40, 120 mm NaCl, 25 mm sodium fluoride, 40 mm β-glycerol phosphate, 0.1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm benzamidine, and 2 μm microcystin-LR. Lysates were centrifuged for 15 min at 12,000 × g and the Myc-PDK1 protein was immunoprecipitated from 200–400 μg of cell-free extracts with the anti-Myc 9E10 monoclonal antibody immobilized on protein G-Sepharose (Amersham Pharmacia Biotech). The immune complexes were washed once with lysis buffer containing 0.5m NaCl, followed by lysis buffer and finally with kinase assay buffer (50 mm Tris-HCl, pH 7.5, 0.1% v/v 2-mercaptoethanol). In vitro kinase assays were performed for 60 min at 30 °C in a 50-μl reaction volume containing 30 μl of immunoprecipitates in kinase buffer, 100 μm Suntide (RRKDGATMKTFCGTPE) as substrate, 10 mmMgCl2, 1 μm protein kinase A inhibitor peptide (Bachem), and 100 μm [γ-32P]ATP (Amersham Pharmacia Biotech; 1000–2000 cpm/pmol). Reactions were stopped by adding EDTA to a final concentration of 50 mmand processed as described previously (52Andjelkovic 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 (425) Google Scholar). Protein concentrations were determined by the method of Bradford (Bio-Rad) using bovine serum albumin as a standard. Polyclonal antisera that recognize specific phosphorylation sites were raised against the following peptides: RTTSQLYDAVPIQS (Tyr-9; 3–16 aa), VLCSCPSPSMVRTQ (Ser-25; 19–32 aa), KQARANSFVGTAQY (Ser-241; 235–248 aa), EDDEDCYGNYDNLLSQF (Tyr-373/376; 367–383 aa), VSSSSSSHSLSASDTG (Ser-393/396; 388–403 aa), LPQRSGSNIEQYIH (Ser-410; 404–417 aa), where the phosphorylated amino acids are underlined. An antisera against the C-terminal region of PDK1 was generated by injecting with the following peptide: KIQEVWRQRYQSHPDAAVQ (538–556 aa). In addition, antisera were prepared by simultaneously immunizing with the four phosphoserine peptides and the C-terminal peptide. All peptides were coupled with Keyhole-Limpet hemocyanin and injected into rabbits. After purification by Protein A-Sepharose (Amersham Pharmacia Biotech) chromatography, some antibodies were affinity-purified using antigenic peptides coupled to Affi-Gel 10 or 15 (Bio-Rad). All procedures were performed at 4 °C. For protein phosphatase treatment, immunoprecipitates of Myc-PDK1 from the pervanadate-treated HEK 293 cells were incubated with 25 ng of purified recombinant TC-PTP (54Tiganis T. Flint A.J. Adam S.A. Tonks N.K. J. Biol. Chem. 1997; 272: 21548-21557Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) in 45 μl of buffer containing 50 mm Tris-HCl (pH 7.5), 1 mmdithiothreitol (DTT), 0.1 mm EDTA, and 0.1% bovine serum albumin, or 25 ng of purified recombinant protein phosphatase 2A catalytic subunit (PP2Ac) purified from the baculovirus-infected Sf21 cells (57Myles T. Schmidt K. Evans D.R.H. Cron P. Hemmings B.A. Biochem. J. 2001; 356: 225-232Crossref Google Scholar) in 45 μl of buffer containing 50 mm Tris-HCl (pH 7.5), 1% 2-mercaptoethanol, 1 mm MnCl2, 1 mm benzamidine, and 0.5 mm PMSF, or 400 units of recombinant Lambda protein phosphatase (Lambda-PP, New England BioLabs) in 45 μl of buffer containing 50 mm Tris-HCl (pH 7.5), 0.1 mmNa2EDTA, 5 mm DTT, 0.01% Brij 35, and 2 mm MnCl2 for 30 min at 30 °C. The reactions were stopped by addition of 0.1 mm sodium orthovanadate for TC-PTP and Lambda-PP or 1 μm okadaic acid for PP2Ac. The immune complexes were washed three times with 50 mmTris-HCl (pH 7.5), containing 1 mm benzamidine, 0.5 mm PMSF, 0.1 mm sodium orthovanadate, and 1 μm okadaic acid, and then analyzed for PDK1 activity, or by immunoblot analysis. For protein-tyrosine kinase treatment, immunoprecipitated Myc-PDK1 from untreated HEK 293 cells was prepared as above. In vitrophosphorylation of PDK1 were performed with 250 ng of purified v-Src in 100 μl of Src kinase buffer containing 25 mm Tris-HCl (pH 7.5), 30 mm MgCl2, 0.5 mm EGTA, 60 μm sodium orthovanadate, 0.5 mm DTT, 10 mm MnCl2, 1 μm protein kinase A inhibitor peptide (Bachem), and 100 μm[γ-32P]ATP (1000–2000 cpm/pmol; Amersham Pharmacia Biotech) for 60 min at 30 °C. Reactions were stopped by addition of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and PDK1 was purified by 12% preparative SDS-PAGE. HEK 293 cell extracts and immunoprecipitates were resolved by 7.8% SDS-PAGE, and transferred to Immobilon-P membranes (Millipore). The filters were blocked for 30 min in 1 × phosphate-buffered saline (140 mm NaCl, 2.7 mm KCl, 4.3 mmNa2HPO4·2H2O, 1.5 mmKH2PO4, pH 7.4), containing 5% skimmed milk, 0.5% Triton X-100, and 0.5% Tween 20, followed by a 2-h incubation with the anti-Myc 9E10 or anti-phosphotyrosine 4G10 monoclonal antibody diluted 1000-fold, or the anti-phospho-site-specific polyclonal antisera in the same blocking solution. The secondary antibody was alkaline phosphatase-conjugated anti-mouse IgG or anti-rabbit IgG (Sigma Chemical Co.), diluted 2500-fold in the blocking buffer. The detection and quantitation of PDK1 expression was carried out by using the alkaline phosphatase color development reagents from Bio-Rad. HEK 293 cells were treated as described, and then placed on ice. After washing once in ice-cold phosphate-buffered saline, cells were scraped in 500 μl of ice-cold fractionation buffer containing 20 mmHEPES-NaOH, pH 7.4, 250 mm sucrose, 25 mmsodium fluoride, 1 mm sodium pyrophosphate, 0.1 mm sodium orthovanadate, 2 μm microcystin LR, 1 mm PMSF, and 1 mm benzamidine and then homogenized by passing through a 26-gauge needle 10 times. Homogenates were centrifuged at 14,000 × g for 10 min to separate the cytosolic fraction (supernatant) from organelles (pellet). The resulting pellet was resuspended in 1 ml of fractionation buffer and layered onto 10 ml of sucrose cushion (20 mm HEPES-NaOH, pH 7.4, 1.15 m sucrose) and centrifuged at 77,000 ×g for 60 min in a Beckman SW41 rotor. The diffuse band at the interface of the sucrose solutions was collected (1 ml), mixed with 1 ml of fractionation buffer, and centrifuged at 100,000 ×g for 20 min to pellet crude plasma membrane fraction. The pellet was resuspended in fractionation buffer, and the protein concentration was determined. HEK 293 cells transiently transfected with wild type Myc-PDK1 were serum-starved for 24 h, and then incubated for 4 h with 32Pi (1 mCi/10-cm plate, Amersham Pharmacia Biotech) in phosphate-free Dulbecco's modified Eagle's medium. Cells were stimulated with buffer or 100 μm pervanadate for 15 min (4 × 10-cm plates per each condition). Lysates were prepared and Myc-PDK1-immunoprecipitated as described above, and then resolved by 12% preparative SDS-PAGE. After staining with Coomassie Blue and autoradiography, the32P-labeled band corresponding to Myc-PDK1 were excised from the gel, reduced with 10 mm DTT, alkylated with 100 mm iodoacetamide, and cleaved with 1 μg of trypsin (Promega, sequencing grade). After the trypsin cleavage the gel pieces were dried and 1 μg of Asp-N (Roche Molecular Biochemicals, sequencing grade) was added in 40 μl of 50 mm sodium phosphate buffer (pH 8.0) containing 10% acetonitrile and incubated for 6 h at 37 °C. The peptides were then extracted (58Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7735) Google Scholar) and processed as described below. Phosphoamino acids were identified following hydrolysis in 6 m HCl containing 0.1 mg/ml bovine serum albumin at 110 °C for 60 min. The hydrolysate was separated by thin-layer electrophoresis at pH 3.5 to resolve phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) (59Duclos B. Marcandier S. Cozzone A.J. Methods Enzymol. 1991; 201: 10-21Crossref PubMed Scopus (209) Google Scholar), and radioactivity was detected using a PhosphorImager (Molecular Dynamics). Peptides were analyzed by high performance liquid chromatography (HPLC) interfaced with electrospray ionization mass spectrometry (ESI-MS) using a Rheos 4000 chromatograph. This chromatograph was equipped with a 1- x 250-mm Vydac (Hesperia, CA) C18 column and interfaced with a Sciex API 300 mass spectrometer (PE Sciex, Toronto, Ontario, Canada) operated in the single quadrupole. The mass range from 300 to 2400 Da was scanned with a step size of 0.3 Da and a dwell time of 3.6 s per scan. The HPLC column was equilibrated in 95% solvent A (2% CH3CN, 0.05% trifluoroacetic acid in H2O), 5% solve

Referência(s)