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• W2009240129 abstract "3-Phosphoinositide-dependent protein kinase-1 (PDK-1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase Cζ. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser399and Thr516 as the major mPDK-1 autophosphorylation sitesin vitro. Furthermore, we have found that these two residues, as well as Ser244 in the activation loop, are phosphorylated in cells and demonstrated that Ser244 is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser244, but not at Ser399 or Thr516, led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser399 or Thr516 is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1T516E, but not of mPDK-1S244E or mPDK-1S399D, in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr308 to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo. 3-Phosphoinositide-dependent protein kinase-1 (PDK-1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase Cζ. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser399and Thr516 as the major mPDK-1 autophosphorylation sitesin vitro. Furthermore, we have found that these two residues, as well as Ser244 in the activation loop, are phosphorylated in cells and demonstrated that Ser244 is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser244, but not at Ser399 or Thr516, led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser399 or Thr516 is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1T516E, but not of mPDK-1S244E or mPDK-1S399D, in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr308 to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo. 3-Phosphoinositide-dependent protein kinase-1 (PDK-1) 1The abbreviations used are: PDK-13-phosphoinositide-dependent protein kinase-1mPDK-1mouse PDK-1hPDK-1human PDK-1CHOChinese hamster ovaryIRinsulin receptorPHpleckstrin homology, PI, phosphatidylinositol is a recently identified protein kinase that functions downstream from PI 3-kinase and upstream of protein kinase B (Akt) in receptor tyrosine kinase signal transduction pathways (1.Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-567Crossref PubMed Scopus (1400) Google Scholar). PDK-1 phosphorylates Thr308 in the activation loop of Akt and contributes to the activation of the enzyme (2.Alessi 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, 3.Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (916) Google Scholar). In addition to Akt, PDK-1 also phosphorylates and activates several other downstream effectors of PI 3-kinase including p70 S6 kinase (4.Alessi 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 (519) Google Scholar, 5.Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (731) Google Scholar), protein kinase C isoforms (6.Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar, 7.Chou 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, 8.Dong L.Q. Zhang L.-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, 9.Dutil E.M. Toker A. Newton A.C. Curr. Biol. 1998; 8: 1366-1375Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), serum and glucocorticoid-inducible kinase (10.Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (530) Google Scholar, 11.Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (335) Google Scholar, 12.Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (482) Google Scholar), and protein kinase C-related kinases 1 and 2 (13.Flynn P. Mellor H. Casamassima A. Parker P.J. J. Biol. Chem. 2000; 275: 11064-11070Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 14.Dong L.Q. Landa L.R. Wick M.J. Zhu L. Mukai H. Ono Y. Liu F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5089-5094Crossref PubMed Scopus (87) Google Scholar). These kinases play versatile roles in numerous cellular events; thus, the activation state of PDK-1 toward these substrates may play an important physiological role in the regulation of a variety of biological activities including cell proliferation, differentiation, and apoptosis. 3-phosphoinositide-dependent protein kinase-1 mouse PDK-1 human PDK-1 Chinese hamster ovary insulin receptor pleckstrin homology, PI, phosphatidylinositol Although the activation of several PDK-1 substrates has been characterized, the mechanism by which PDK-1 activity is regulated in cells is largely unknown. Phosphorylation of protein kinase Cζ and protein kinase Cδ by PDK-1 in vivo has been shown to be inhibited by the PI 3-kinase inhibitor LY294002 (6.Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar), suggesting that PDK-1 activity is mediated by a PI 3-kinase-dependent mechanism. Consistent with this finding, it has recently been found that insulin stimulates the activity of PDK-1 toward Akt and that this activation is blocked by wortmannin, another inhibitor of PI 3-kinase (15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar). However, others have found PDK-1 to be constitutively activated (4.Alessi 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 (519) Google Scholar, 8.Dong L.Q. Zhang L.-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), and PDK-1-mediated phosphorylation of p70 S6 kinase in cells has been shown to be unaffected by wortmannin treatment (5.Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (731) Google Scholar). Although activation of PI 3-kinase may be important for the activity of PDK-1 toward some of its downstream substrates, differential phosphorylation of PDK-1 itself may also play an important role in regulating the kinase activity of the enzyme. A recent study identified Ser25, Ser241, Ser393, Ser396, and Ser410 on human PDK-1 (hPDK-1) as phosphorylated sites in cells (17.Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (292) Google Scholar). Of these sites, phosphorylation at Ser241 was found to be essential for hPDK-1 activityin vitro (17.Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (292) Google Scholar). However, the functional role(s) of PDK-1 phosphorylation at the other sites is unknown. Recently, we found that purified mPDK-1 underwent significant autophosphorylation in vitro (8.Dong L.Q. Zhang L.-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, 18.Wick M.J. Dong L.Q. Riojas R.A. Ramos F. Liu F. J. Biol. Chem. 2000; 275: 40400-40406Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). However, the identity of these sites and the extent to which phosphorylation of these residues played a role in PDK-1 function remained unclear. In the present study, we report the identification of two major in vitro autophosphorylation sites on mPDK-1: Thr516 and Ser399. Interestingly, we found that Ser244 in the activation loop of mPDK-1 is only marginally phosphorylatedin vitro. On the other hand, we demonstrate that Ser244, Ser399, and Thr516 of mPDK-1 are phosphorylated in cells and that Ser244 is a major in vivo phosphorylation site. Overexpression of mPDK-1T516E, but not mPDK-1S244E, mPDK-1S399D, or mPDK-1T516A, resulted in Akt Thr308 phosphorylation to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation is independent of the PH domain of Akt. Taken together, our results suggest that phosphorylation of PDK-1 at multiple sites plays distinct and important roles in the regulation of PDK-1 function in vivo under various cellular conditions. Buffer A consisted of 50 mm Hepes, pH 7.6, 150 mm NaCl, and 0.1% Triton X-100. Buffer B consisted of 50 mm Hepes, pH 7.6, 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, 1 mm NaF, 1 mm sodium pyrophosphate, 1 mmNa3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride. Buffer C consisted of 50 mm Tris-HCl, pH 7.5, 5 mmMgCl2, 1 mm Na3VO4, 1 mm sodium pyrophosphate, 1 mm NaF, and 1 mm phenylmethylsulfonyl fluoride. cDNAs encoding Myc-tagged full-length mPDK-1, mPDK-1ΔPH, mPDK-1A280V, Myc-tagged Akt-1, and FLAG-tagged full-length and Akt-1ΔPH were described previously (18.Wick M.J. Dong L.Q. Riojas R.A. Ramos F. Liu F. J. Biol. Chem. 2000; 275: 40400-40406Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Polyclonal anti-Akt and anti-phospho-Akt (Thr308) antibodies were obtained from New England Biolabs. Monoclonal anti-Myc was obtained from Santa Cruz Biotechnology, Inc. The polyclonal antibody to PDK-1 was described previously (8.Dong L.Q. Zhang L.-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). All mPDK-1 mutants were generated using single-stranded site-directed mutagenesis according to the protocol described by Kunkel et al. (19.Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-383Crossref PubMed Scopus (4560) Google Scholar) using customized primers. All constructs were verified by restriction mapping and DNA sequencing. Chinese hamster ovary cells overexpressing the insulin receptor (CHO/IR), and human embryonic kidney (HEK293) cells were used in all experiments. Transfections were performed using LipofectAMINE (Invitrogen) according to the manufacturer's protocol. The cells were lysed in Buffer B. The cell lysates were centrifuged at 12,000 ×g for 10 min at 4 °C, and the clarified supernatant was used for either immunoprecipitation or Western blot analysis. The proteins in cell lysates were immunoprecipitated by incubation with the primary antibody conjugated to protein G-Sepharose beads for 6–18 h at 4 °C. The immunoprecipitates were washed three times with ice-cold Buffer A. For immunoblot analysis, the cell lysates or immunoprecipitates were separated by SDS-PAGE using 10 or 15% polyacrylamide gels. Following electrophoresis, the proteins were transferred onto nitrocellulose membranes, and the bound proteins were detected by blotting with primary antibody, followed by horseradish peroxidase- or alkaline phosphatase-conjugated secondary antibodies (Promega, Madison, WI). CHO/IR cells transiently expressing Myc-tagged wild type or mutant PDK-1 were washed once with phosphate-buffered saline followed by serum starvation for 1 h at 37 °C. The cells were lysed in Buffer B, and the proteins were immunoprecipitated using antibody to the tag as described above. The bound protein was washed twice with ice-cold Buffer A and once with Buffer C. Phosphorylation was initiated with the addition of 30 μl of Buffer C plus 2 μCi of [γ-32P]ATP (PerkinElmer Life Sciences) and incubated for 20 min at 30 °C. The bound protein was eluted by heating at 95 °C for 3 min in SDS-PAGE sample buffer. The proteins were separated by SDS-PAGE and blotted to nitrocellulose or polyvinylidene difluoride membrane. Phosphorylation of PDK-1 was visualized by autoradiography, and the bands corresponding to PDK-1 were excised and used in various assays. CHO/IR cells overexpressing wild type or mutant mPDK-1 were washed with phosphate-buffered saline and serum starved for 1 h. The cells were lysed in Buffer B, and Myc-tagged PDK-1 was immunoprecipitated using antibody to the tag. Immunoprecipitates were washed twice with ice-cold Buffer A and once with Buffer C. The activity of mPDK-1 was determined by incubation of the immunoprecipitates with 40 μl of Buffer C containing 2 μCi of [γ-32P]ATP and PDK1 substrate peptide corresponding to the sequence surrounding Thr308 of Akt-1 (KTFCGTPEYLAPEVRR) for 30 min at 30 °C. Phosphorylated peptide substrate was precipitated with trichloroacetic acid, spotted on p81 Whatman paper, and washed three times in 1% H3PO4. Peptide phosphorylation by PDK-1 was determined by scintillation counting. CHO/IR cells overexpressing Myc-tagged wild type or mutant PDK-1 were washed once and incubated with warmed Krebs-Ringer bicarbonate buffer for 30 min at 37 °C. The cells were labeled with 400–800 μCi of [32P]orthophosphate (PerkinElmer Life Sciences) for 4 h and then left untreated or treated with 10 nminsulin for 5 min at 37 °C. The cells were lysed in Buffer B and immunoprecipitated as described above. The bound protein was washed two times with high and low salt wash buffers (20 mmNa2HPO4, pH 8.6, 0.5% Triton X-100, 0.1% SDS, 0.02% NaN3, and either 1 or 0.15 m NaCl) and eluted by heating at 95 °C for 3 min in SDS-PAGE sample buffer. The proteins were separated by SDS-PAGE, blotted to nitrocellulose or polyvinylidene difluoride membrane, and utilized as described above. In vitro or metabolically 32P-labeled proteins were separated by SDS-PAGE, transferred to nitrocellulose (phosphopeptide mapping) or polyvinylidene difluoride (phosphoamino acid analysis) membranes, and excised as described above. The bound protein was blocked with 0.5% polyvinylpyrrolidone-360 (Sigma) in 100 mm acetic acid for 30 min at 37 °C. The protein was washed twice with double distilled H2O, washed once with 50 mm ammonium bicarbonate, and subjected to trypsin digest for 18 h at 37 °C. The digests were collected and lyophilized, and the samples were desalted using C18 ZipTips (Millipore, Bedford, MA) according to a modified protocol by the manufacturer. The samples were loaded on TLC cellulose plates and subjected to electrophoresis and liquid chromatography as described previously (20.Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). Phosphopeptides were visualized by autoradiography using x-ray film. Phosphoamino acid analysis was carried out using a protocol described previously (21.Dong L.Q. Du H.-Y. Porter S. Kolakowski J.L.F. Lee A.V. Mandarino L.J. Fan J.B. Yee D. Liu F. J. Biol. Chem. 1997; 272: 29104-29112Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). We (8.Dong L.Q. Zhang L.-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, 18.Wick M.J. Dong L.Q. Riojas R.A. Ramos F. Liu F. J. Biol. Chem. 2000; 275: 40400-40406Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) and others (22.Currie 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) have previously demonstrated that PDK-1 undergoes significant autophosphorylationin vitro. In an attempt to identify PDK-1 autophosphorylation sites, we carried out phosphoamino acid analysis and two-dimensional phosphopeptide mapping studies on in vitro phosphorylated wild type mPDK-1. Our results showed that mPDK-1 autophosphorylated in vitro on both serine and threonine residues at approximately a 1:1 ratio (Fig. 1 A, left panel). Noin vitro phosphorylation was observed for a kinase-inactive mutant of mPDK-1 (mPDK-1K114G) (data not shown). Phosphopeptide analysis of wild type mPDK-1 revealed three major (Fig. 1 A, right panel, 1–3) and nine minor (Fig. 1 A, right panel, a–i) phosphopeptides, suggesting that PDK-1 autophosphorylates multiple sites in vitro. Phosphoamino acid analysis of phosphopeptides 1–3 revealed that phosphopeptides 1 and 2 contained exclusively phosphothreonine, whereas phosphopeptide 3 contained only phosphoserine (data not shown). We have recently shown that Ser244 of mPDK-1 is important for PDK-1 function in cells (15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar). To determine whether Ser244 is an in vitro autophosphorylation site, we examined the phosphorylation of wild type and Ser244mutants of mPDK-1 (Fig. 1 B). We found that wild type mPDK-1 underwent significant autophosphorylation in vitro.Replacing Ser244 of mPDK-1 with alanine resulted in a dramatic decrease in the in vitro phosphorylation of the protein. This decrease in phosphorylation was partially recovered by replacing Ser244 with a negatively charged glutamate. Interestingly, substitution of Ser244 with a phosphorylatable threonine residue resulted in an overall increase in autophosphorylation compared with wild type. Replacing Ser244 of mPDK-1 with alanine also led to a significant decrease of the in vitro kinase activity of the enzyme, which was partially recovered by substitution of this residue with either glutamate or threonine (Fig. 1 C). These findings further support Ser244 as an autophosphorylation site and demonstrate that phosphorylation at this site is important for mPDK-1 autokinase activity. To further characterize the role of Ser244 in mPDK-1in vitro autophosphorylation, we performed phosphoamino acid analysis and two-dimensional phosphopeptide mapping studies on in vitro autophosphorylated mPDK-1S244A. Our results showed that although overall autophosphorylation of the mutant PDK-1 was significantly decreased compared with wild type (Fig. 1 B), there was no significant change in the overall serine:threonine ratio (Fig. 1, D, left panel versus A, left panel), suggesting that Ser244 is not a major autophosphorylation site in vitro. Consistent with this, two-dimensional phosphopeptide mapping studies revealed that despite the significant decrease in overall phosphorylation of this mutant (Fig. 1 B), mutating Ser244 to alanine did not abolish the in vitroautophosphorylation of the three major peptides (peptides1–3) (Fig. 1, D, right panel versus A, right panel). On the other hand, replacing Ser244 of mPDK-1 with threonine led to a notable increase in threonine:serine autophosphorylation compared with the wild type enzyme (Fig. 1, E, left panel versus A, left panel). Two-dimensional phosphopeptide mapping studies of the mPDK-1S244T mutant showed a significant increase in autophosphorylation of phosphopeptidee compared with wild type protein (Fig. 1, E,right panel versus A, right panel). Phosphoamino acid analysis of phosphopeptide e revealed that this peptide contained only phosphoserine for the wild type and exclusively phosphothreonine for the S244T mutant of mPDK-1 (data not shown). Taken together, these results identified phosphopeptidee of wild type mPDK-1 as a Ser244-containing peptide. Because Ser244 of mPDK-1 was found to be only marginally phosphorylated in vitro, we attempted to identify the major mPDK-1 in vitroautophosphorylation sites. At least five serine residues in hPDK-1, including Ser25, Ser241, Ser393, Ser396, and Ser410 were found phosphorylated in cells (17.Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (292) Google Scholar). To test whether the corresponding sites in mPDK-1 are phosphorylated in vitro, we generated mutants of mPDK-1 in which the corresponding sites, Ser25, Ser244, Ser396, Ser 399, and Ser413, were replaced with either alanine or glycine. Mutant mPDK-1 proteins were expressed in CHO/IR cells, purified by immunoprecipitation, and autophosphorylated in vitro in the presence of [γ-32P]ATP. Phosphoamino acid analysis of the in vitro phosphorylated mPDK-1 mutants revealed that mutations at Ser25, Ser244, Ser396, and Ser413 had no significant effect on mPDK-1 in vitro autophosphorylation (data not shown). On the other hand, replacing Ser399 with glycine (mPDK-1S399G) significantly decreased phosphoserine content compared with that of wild type (Fig. 2, left panel versus Fig. 1 A, left panel). Phosphopeptide mapping studies revealed that mutation at this site resulted in the loss of phosphopeptide 3 (Fig. 2, right panel), implicating Ser399 of mPDK-1 as a major in vitroautophosphorylation site. In an attempt to localize potential threonine autophosphorylation site(s), we examined autophosphorylation of a mPDK-1 mutant lacking the carboxyl-terminal PH domain (mPDK-1ΔPH, residues 1–459). Phosphoamino acid analysis revealed that deletion of the PH domain resulted in a complete abolishment of threonine autophosphorylation (Fig. 3 A, left panel). Phosphopeptide mapping studies of mPDK-1ΔPH revealed a loss of both phosphopeptides 1 and 2 (Fig. 3 A, right panel), suggesting that the major mPDK-1 threonine autophosphorylation site(s) might be localized in the PH domain. Sequence analysis of mPDK-1 revealed the presence of four threonine residues in the PH domain: Thr482, Thr516, Thr521, and Thr525 (Fig. 3 B). To identify potential mPDK-1 threonine autophosphorylation sites, we mutated these threonine residues individually to alanine. The mPDK-1 threonine mutants were then expressed in CHO/IR cells, purified by immunoprecipitation, and autophosphorylated in vitro in the presence of [γ-32P]ATP. We found that mutations at Thr482, Thr521, and Thr525 had no significant effect on mPDK-1 autophosphorylation in vitro(data not shown). On the other hand, replacing Thr516 with alanine significantly decreased phosphothreonine content compared with that of the wild type enzyme (Fig. 3 C, left panel versus Fig. 1 A, left panel). Interestingly, mutating Thr516 to alanine resulted in the loss of both phosphothreonine-containing peptides 1 and 2 in the two-dimensional phosphopeptide map of this mutant (Fig. 3 C, right panel). One possible explanation for this observation may be that two phosphopeptides were generated because of incomplete digestion of the Thr516-containing tryptic peptide. Consistent with this idea, Thr516 is immediately flanked on the amino terminus by a lysine residue (Lys515). Phosphorylation at Thr516 could potentially impair the ability of trypsin to cleave the peptide bond between Lys515 and Thr516, resulting in two Thr516-containing phosphopeptides. To confirm this, we mutated Thr516 of mPDK-1 to serine. mPDK-1T516S was expressed in CHO/IR cells, immunoprecipitated, autophosphorylated in vitro, and subjected to two-dimensional phosphopeptide mapping analysis. Phosphopeptide mapping showed a significant decrease in phosphorylation at phosphopeptides 1 and 2. Subsequent phosphoamino acid analysis revealed that both phosphopeptides 1 and 2 contained exclusively phosphoserine (data not shown). These results provide further evidence that phosphopeptides 1 and 2 are due to incomplete tryptic digestion of the Thr516-containing peptide. To determine whether Ser244 , Ser399, and Thr516 are phosphorylated in cells, phosphopeptide mapping studies were performed on wild type and point-mutated mPDK-1 overexpressed and metabolically labeled in CHO/IR cells. Two-dimensional phosphopeptide mapping of the metabolically labeled wild type mPDK-1 revealed the presence of phosphopeptides 1–3 (Fig. 4, panel a), indicating that both Ser399 and Thr516 are phosphorylated in intact cells. Consistent with the results from in vitroautophosphorylation and two-dimensional phosphopeptide mapping studies, mutation of Thr516 to alanine resulted in a loss of both phosphopeptides 1 and 2 for the metabolically labeled mPDK-1 (Fig. 4,panel b). Similarly, mutating Ser399 to glycine resulted in the loss of phosphopeptide 3 (Fig. 4, panel c). Our results also showed phosphopeptide e to be heavily phosphorylated in vivo, indicating that Ser244is one of the major phosphorylation sites in cells (Fig. 4). Phosphoamino acid analysis of phosphopeptide e recovered from the maps of metabolically labeled wild type or the S244T mutant of mPDK-1 indicated that this peptide contained solely phosphoserine or phosphothreonine, respectively (data not shown). Interestingly, phosphoamino acid analysis of phosphopeptides f andi recovered from the maps of metabolically labeled wild type or mPDK-1S244T indicated that these two peptides also contained only phosphoserine or phosphothreonine, respectively (data not shown). These findings implicate Ser244 as a major phosphorylation site of mPDK-1 in cells and suggest that phosphopeptides e, f, and i are most likely due to incomplete digestion of Ser244-containing peptide. Interestingly, we found that the in vivophosphorylation pattern of kinase-inactive PDK-1K114G is similar to that of the wild type enzyme, suggesting that PDK-1 may undergo transphosphorylation in vivo. 2Wick, M. J., and Liu, F., manuscript in preparation. We have recently found that insulin stimulates mPDK-1 phosphorylation in NIH3T3 cells and rat adipocytes (15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar). To test whether a mutation at Ser399 or Thr516 affects the overall phosphorylation of mPDK-1 in vivo, we examined basal and insulin-stimulated phosphorylation of wild type and mPDK-1 phosphorylation site mutants in CHO/IR cells. Consistent with our recent findings (15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar), insulin treatment resulted in a significant increase in mPDK-1 phosphorylation (Fig. 5 A, lane 2 versus lane 1). Interestingly, we found that despite an overall decrease in phosphorylation, the mPDK-1S244A mutant was still phosphorylated in cells, and the phosphorylation was enhanced in response to insulin stimulation (Fig. 5 A, lanes 3and 4 versus lanes 1 and2). These findings suggest that insulin stimulates the phosphorylation at a site(s) in addition to or other than Ser244 in CHO/IR cells. Although mutating Ser399 to glycine or aspartate had no significant effect on mPDK-1 phosphorylation in vivo (data not shown), replacing Thr516 of mPDK-1 with glutamate (Fig. 5 A,lane 7 versus lane 2), but not alanine (Fig. 5 A, lanes 5 versus lane 2), resulted in an increase in the basal phosphorylation of mPDK-1 to the same level as that of the insulin-stimulated wild type protein. Furthermore, this phosphorylation was not further increased following insulin treatment (Fig. 5 A, lane 8 versus lane 7). Interestingly, the recently identified constitutively active mutant of mPDK-1, mPDK-1A280V (18.Wick M.J. Dong L.Q. Riojas R.A. Ramos F. Liu F. J. Biol. Chem. 2000; 275: 40400-40406Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), also showed an enhanced and insulin-independent increase in basal phosphorylation (Fig. 5 A, lanes 9 and 10 versus lanes 1 and 2). These findings suggest that phosphorylation of Thr516 may result in a similar conformational change induced by the alanine to valine mutation at position 280, which subsequently leads to enhanced phosphorylation of mPDK-1. To determine whether autophosphorylation plays a role in the function of mPDK-1, we examined the kinase activity of mPDK-1, both in vitro and in cells.In vitro, we found that mutation of Ser399 of mPDK-1 to glycine or aspartate, or Thr516 to alanine or glutamate, had no significant effect of mPDK-1 kinase activity (data not shown). To examine the effect of PDK-1 autophosphorylation on its kinase activity in cells, phosphorylation of the PDK-1 substrate Akt by wild type and autophosphorylation mutants was examined. As expected, insulin treatment resulted in a significant increase in Akt phosphorylation at Thr308 (Fig. 5 B, lane 7 versus lane 1). Although co-expression of mPDK-1S244Eor mPDK-1S399D mutants had no effect on Akt phosphorylation at Thr308 under basal conditions (Fig. 5 B,lanes 2 and 3 versus lane 1), co-expression of mPDK-1T516E resulted in a significant increase in Akt phosphorylation to a level similar to that induced by insulin stimulation (Fig. 5 B, lane 5 versus lane 7). Under this condition, only a small increase in Akt phosphorylation at Thr308 was observed in cells co-expressing mPDK-1T516A (Fig. 5 B, lane 4 versus lane 1), suggesting that a negatively charged residue at this site plays a critical role in promoting PDK-1 to phosphorylate Akt in cells. Furthermore, insulin stimulation did not increase the elevated Akt Thr308phosphorylation in cells co-expressing mPDK-1T516E (Fig. 5 B, lane 11 versus lane 5). Similar results were obtained for mPDK-1T516E co-expressed with Akt in CHO and HEK-293 cells (data not shown). In agreement with our recent study (18.Wick M.J. Dong L.Q. Riojas R.A. Ramos F. Liu F. J. Biol. Chem. 2000; 275: 40400-40406Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), co-expression of mPDK-1A280V with Akt also resulted in a similar increase in basal Akt phosphorylation at Thr308 (Fig. 5 B, lane 6). Previous studies have suggested that translocation of Akt to the plasma membrane is required for full phosphorylation and activation of the enzyme (23.Stokoe 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 (1054) Google Scholar, 24.Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar). Studies have also shown that this translocation requires an intact PH domain (25.Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J.C. 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 (904) Google Scholar). To better understand the mechanism by which autophosphorylation of PDK-1 regulates its activation of downstream substrates, we co-expressed FLAG-tagged wild type Akt-1 or the PH domain deletion mutant (Akt-1ΔPH) with either wild type or mutant mPDK-1 in CHO/IR cells. Insulin stimulation (Fig. 5 C, lane 2 versus lane 1) or co-expression of either mPDK-1T516E (Fig. 5C, lane 3 versus lane 1) or mPDK-1A280V (Fig. 5 C, lane 4 versus lane 1) resulted in significant Akt phosphorylation at Thr308. The PH domain-deleted mutant of Akt was not phosphorylated on Thr308 in response to insulin stimulation (Fig. 5 C, lane 6). However, co-expression of PDK-1T516E was sufficient to restore Akt-1ΔPHphosphorylation at Thr308 to the same level as that of insulin-stimulated, full-length Akt (Fig. 5 C, lane 7 versus lane 2). It is of interest to note that the insulin-independent and Akt PH domain-independent phosphorylation of Akt at Thr308 can be mediated by both PDK-1T516E and PDK-1A280V mutants (Fig. 5,B, lanes 5 and 6 and C,lanes 7 and 8). These data suggest that substituting Val280 with alanine or Thr516 with glutamate may lead to a similar conformational change of mPDK-1, which is essential for interaction and phosphorylation of downstream substrates such as Akt in cells. The mechanism(s) by which PDK-1 activity is regulated has remained, for a large part, unclear. Subcellular localization and substrate conformation have been hypothesized as the primary means for regulating PDK-1 activity enzyme (22.Currie 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, 26.Toker A. Newton A.C. Cell. 2000; 103: 185-188Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 27.Wick K.L.R. Liu F. Current Drug Targets Immune, Endocrine Metabolic Disorders. 2001; 1: 209-221Crossref PubMed Scopus (29) Google Scholar); however, some studies have indicated that phosphorylation of PDK-1 may also play a role (8.Dong L.Q. Zhang L.-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,15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar). PDK-1 is phosphorylated both in vitro and in cells (2.Alessi 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,17.Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (292) Google Scholar), and treatment of PDK-1-expressing cells with insulin or hydrogen peroxide has been shown to increase phosphorylation of PDK-1 on specific residues (15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar, 28.Park J. Hill M.M. Hess D. Brazil D.P. Hofsteenge J. Hemmings B.A. J. Biol. Chem. 2001; 31: 37459-37471Abstract Full Text Full Text PDF Scopus (103) Google Scholar). Several studies have focused on Ser244 (mPDK-1) or Ser241 (hPDK-1) in the activation loop as a critical phosphorylation site, because phosphorylation upon this residue is essential for PDK-1 kinase activity (15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar, 17.Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (292) Google Scholar). Some studies have indicated that phosphorylation at Ser241 is not regulated by growth factor stimulation (17.Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (292) Google Scholar). On the other hand, we have found that in certain cell types, mPDK-1 phosphorylation and activity increase following insulin stimulation in a Ser244-dependent manner (15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar). However, in CHO/IR cells, we found that the mPDK-1S244A mutant still underwent insulin-stimulated phosphorylation (Fig. 5 A). These findings suggest that insulin-stimulated phosphorylation of mPDK-1 occurs at sites other than or in addition to Ser244, and this phosphorylation may function in conjunction with Ser244 to regulate mPDK-1 activity in cells. In the present study, we identify Ser399 and Thr516 on mPDK-1 as two major in vitroautophosphorylation sites, which can also be phosphorylated in cells. This finding is in agreement with a recent study by Park et al. (28.Park J. Hill M.M. Hess D. Brazil D.P. Hofsteenge J. Hemmings B.A. J. Biol. Chem. 2001; 31: 37459-37471Abstract Full Text Full Text PDF Scopus (103) Google Scholar), who showed the Thr516-corresponding site in hPDK-1 to be phosphorylated in vitro. However, our results also show that phosphorylation at these sites may play different roles in PDK-1 function in cells. Indeed, substitution of Thr516with glutamate increases basal PDK-1 phosphorylation in cells to that seen with insulin stimulation, whereas replacing Ser399with aspartate does not significantly affect either basal or insulin-stimulated phosphorylation of the enzyme. In addition, overexpression of mPDK-1T516E, but not mPDK-1S399D, led to Akt phosphorylation at Thr308 to the same level as that induced by insulin treatment. Taken together, these findings suggest that autophosphorylation at Thr516, but not Ser399, regulates PDK-1 phosphorylation upon additional residues in cells and subsequently functions to regulate the activity of PDK-1 toward its substrates. Previous studies have shown that hPDK-1 isolated from cells is constitutively active in vitro, presumably as a result of autophosphorylation upon Ser241 (4.Alessi 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 (519) Google Scholar, 8.Dong L.Q. Zhang L.-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, 22.Currie 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). In agreement with this, we have found that a mutation of the corresponding Ser244 on mPDK-1 renders the kinase inactive in vitro (Fig. 1 C) and in cells (Ref. 15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar and data not shown). Interestingly, although we found the kinase activity of mPDK-1 to be significantly reduced by a mutation at Ser244, we found the ratio of serine:threonine autophosphorylation unchanged in its absence. This suggests that Ser244 is not a major site for in vitro autophosphorylation. Low autophosphorylation at Ser244 may result from phosphorylation at this site in cells that remains stable in vitro. In support of this, phosphorylation at Ser244 is less stable if the residue is replaced with threonine, resulting in an increase in autophosphorylation at this site in vitro (Fig. 1 B). Although Ser244 is a minor phosphorylation site, phosphopeptide maps of metabolically labeled wild type and S244T mutant of mPDK-1 indicated that Ser244 is highly phosphorylated in CHO/IR cells, which is in agreement with our recent studies showing that Ser244 is a major phosphorylation site in NIH-3T3 cells (15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar). In contrast, autophosphorylation upon the majorin vitro sites, Ser399 and Thr516, is less stable in cells, which indicates that phosphorylation upon these residues is reversible and may be tightly regulated in cells. Insulin stimulation has been shown to increase PDK-1 phosphorylation in both NIH-3T3 and rat adipose cells (15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar). Consistent with this, we observed insulin-stimulated phosphorylation of mPDK-1 when overexpressed in CHO/IR cells (Fig. 5 A); however, we found that this insulin-stimulated increase could occur in the absence of Ser244 phosphorylation. Mutations at Ser399 and Thr516 did not significantly affect insulin-stimulated mPDK-1 phosphorylation (Fig. 5 A and data not shown), suggesting the presence of other insulin-stimulated phosphorylation site(s). On the other hand, replacement of Thr516 with a negatively charged glutamate residue resulted in a significant increase in basal phosphorylation. The increase in basal mPDK-1T516Ephosphorylation also correlates with an increase in mPDK-1T516E-mediated phosphorylation of Akt at Thr308 (Fig. 5 B), which is in agreement with our recent finding that insulin-stimulated phosphorylation of mPDK-1 correlates with an increase in activity (15.Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar). It has been previously shown that growth factor-mediated phosphorylation and activation of Akt requires a translocation event via an intact PH domain (23.Stokoe 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 (1054) Google Scholar, 24.Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar, 25.Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J.C. 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 (904) Google Scholar, 29.Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (677) Google Scholar). Recently, we (18.Wick M.J. Dong L.Q. Riojas R.A. Ramos F. Liu F. J. Biol. Chem. 2000; 275: 40400-40406Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) and others (22.Currie 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,30.Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar) have shown that phosphorylation at Thr308 of Akt by PDK-1 is facilitated by an intact PDK-1-PH domain, whereas others have suggested that translocation of PDK-1 to the membrane may play a role in the phosphorylation and activation of the enzyme (31.Filippa N. Sable C.L. Hemmings B.A. Van Obberghen E. Mol. Cell. Biol. 2000; 20: 5712-5722Crossref PubMed Scopus (113) Google Scholar). In the present study, we have found that mutating Thr516 to glutamate allows PDK-1 to phosphorylate Thr308 not only in a growth factor-independent fashion but also without the requirement for the PH domain of Akt (Fig. 5 C). These data suggest that the T516E mutation of mPDK-1 bypasses the normal requirement for translocation and allows it to interact with and subsequently phosphorylate Akt at Thr308. One possible explanation for this observation may be that the introduction of a negatively charged residue at Thr516 results in a conformational change in mPDK-1, which normally occurs after growth factor-mediated translocation of the enzyme to the plasma membrane. This resulting conformation may result in an increase in autophosphorylation at Ser244 and a subsequent increase in mPDK-1 activity. Consistent with this, a mPDK-1S244A/T516E mutant was unable to increase phosphorylation of mPDK-1 or Thr308 of Akt in cells (data not shown), suggesting that phosphorylation at Thr516 modifies activity of mPDK-1 through a Ser244 phosphorylation-dependent mechanism. In Caenorhabditis elegans, a natural point mutation (A277V) allows the mammalian PDK-1 homolog to bypass the need for PI 3-kinase for its activation (16.Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (558) Google Scholar). We recently reported that a corresponding mutation in mPDK1 (A280V) resulted in an increase in the in vitro autophosphorylation of mPDK-1 and constitutive activation of the kinase toward Akt in cells (18.Wick M.J. Dong L.Q. Riojas R.A. Ramos F. Liu F. J. Biol. Chem. 2000; 275: 40400-40406Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Whereas this mutation has not been found to occur naturally in higher species, it is interesting to note the similarity between mPDK-1A280V and mPDK-1T516E. We found that although neither had any significant effect on mPDK-1 activity in vitro (data not shown), both elevated mPDK-1 basal phosphorylation in cells (Fig. 5 A). Furthermore, overexpression of either mPDK-1A280V or mPDK-1T516E was sufficient to elevate Akt phosphorylation at Thr308 in a growth factor- and Akt PH domain-independent manner (Fig. 5, B and Fig.C). These findings suggest that each mutation may result in a conformational change and/or cellular relocalization of mPDK1, which allows the enzyme to interact with and phosphorylate its downstream substrates such as Akt. In conclusion, our results suggest that phosphorylation at Thr516 plays an important role in the regulation of mPDK-1in vivo activity toward its substrates. Although it remains to be established whether phosphorylation upon Thr516 is directly regulated by insulin stimulation, autophosphorylation at this site may specifically direct the extent of phosphorylation at other site(s) and subsequently the activity of mPDK-1 in response to insulin or other growth factors. Autophosphorylation at Thr516 may be specifically regulated in cells by various cellular events, such as conformational changes of the kinase following growth factor stimulation. Alternatively, Thr516 may be constitutively autophosphorylated in cells, and this phosphorylation may be tightly controlled by a serine/threonine phosphatase, allowing dynamic regulation of this pivotal kinase in response to growth factor stimulation. Further studies will be needed to test these possibilities." @default.
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• W2009240129 title "Substitution of the Autophosphorylation Site Thr516with a Negatively Charged Residue Confers Constitutive Activity to Mouse 3-Phosphoinositide-dependent Protein Kinase-1 in Cells" @default.
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