Cooperative Phosphorylation of the Tumor Suppressor Phosphatase and Tensin Homologue (PTEN) by Casein Kinases and Glycogen Synthase Kinase 3β

The phosphatase and tensin homologue (PTEN) tumor suppressor is a phosphatidylinositol D3-phosphatase that counteracts the effects of phosphatidylinositol 3-kinase and negatively regulates cell growth and survival. PTEN is itself regulated by phosphorylation on multiple serine and threonine residues in its C terminus. Previous work has implicated casein kinase 2 (CK2) as the kinase responsible for this phosphorylation. Here we showed that CK2 does not phosphorylate all sites in PTEN and that glycogen synthase kinase 3β (GSK3β) also participates in PTEN phosphorylation. Although CK2 mainly phosphorylated PTEN at Ser-370 and Ser-385, GSK3β phosphorylated Ser-362 and Thr-366. More importantly, prior phosphorylation of PTEN at Ser-370 by CK2 strongly increased its phosphorylation at Thr-366 by GSK3β, suggesting that the two may synergize. Using RNA interference, we showed that GSK3 phosphorylates PTEN in intact cells. Finally, PTEN phosphorylation was affected by insulin-like growth factor in intact cells. We concluded that multiple kinases, including CK2 and GSK3β, participate in PTEN phosphorylation and that GSK3β may provide feedback regulation of PTEN. The phosphatase and tensin homologue (PTEN) tumor suppressor is a phosphatidylinositol D3-phosphatase that counteracts the effects of phosphatidylinositol 3-kinase and negatively regulates cell growth and survival. PTEN is itself regulated by phosphorylation on multiple serine and threonine residues in its C terminus. Previous work has implicated casein kinase 2 (CK2) as the kinase responsible for this phosphorylation. Here we showed that CK2 does not phosphorylate all sites in PTEN and that glycogen synthase kinase 3β (GSK3β) also participates in PTEN phosphorylation. Although CK2 mainly phosphorylated PTEN at Ser-370 and Ser-385, GSK3β phosphorylated Ser-362 and Thr-366. More importantly, prior phosphorylation of PTEN at Ser-370 by CK2 strongly increased its phosphorylation at Thr-366 by GSK3β, suggesting that the two may synergize. Using RNA interference, we showed that GSK3 phosphorylates PTEN in intact cells. Finally, PTEN phosphorylation was affected by insulin-like growth factor in intact cells. We concluded that multiple kinases, including CK2 and GSK3β, participate in PTEN phosphorylation and that GSK3β may provide feedback regulation of PTEN. Phosphatase and tensin homologue (PTEN) 2The abbreviations used are: PTENphosphatase and tensin homologueCKcasein kinaseGSK3βglycogen synthase kinase 3βPI3kphosphatidylinositol 3-kinaseGSTglutathione S-transferaseLC-MS/MSliquid chromatography-tandem mass spectrometrysiRNAsmall interfering RNA. (1Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. 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Posttranslationally, PTEN is regulated through phosphorylation of a cluster of serine and threonine residues in its C terminus (15Georgescu M.-M. Kirsch K.H. Akagi T. Shishido T. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10182-10187Crossref PubMed Scopus (278) Google Scholar, 16Vazquez F. Ramaswamy S. Nakamura N. Sellers W.R. Mol. Cell. Biol. 2000; 20: 5010-5018Crossref PubMed Scopus (656) Google Scholar, 17Torres J. Pulido R. J. Biol. Chem. 2001; 276: 993-998Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar, 18Tolkacheva T. Boddapati M. Sanfiz A. Tsuchida K. Kimmelman A.C. Chan A.M. Cancer Res. 2001; 61: 4985-4989PubMed Google Scholar, 19Vazquez F. Grossman S.R. Takahashi Y. Rokas M.V. Nakamura N. Sellers W.R. J. Biol. Chem. 2001; 276: 48627-48630Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 20Birle D. Bottini N. Williams S. Huynh H. deBelle I. Adamson E. Mustelin T. J. Immunol. 2002; 169: 286-291Crossref PubMed Scopus (64) Google Scholar, 21Miller S.J. Lou D.Y. Seldin D.C. Lane W.S. Neel B.G. FEBS Lett. 2002; 528: 145-153Crossref PubMed Scopus (174) Google Scholar). Although not required for the activity of the catalytic domain, phosphorylation of the C-terminal region plays an important role in stabilizing the PTEN protein. In its phosphorylated form, the tail is thought to wrap unto the C2 and catalytic domains of PTEN and thereby block the translocation of PTEN to the cytoplasmic face of the plasma membrane (16Vazquez F. Ramaswamy S. Nakamura N. Sellers W.R. Mol. Cell. Biol. 2000; 20: 5010-5018Crossref PubMed Scopus (656) Google Scholar, 22Das S. Dixon J.E. Cho W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7491-7496Crossref PubMed Scopus (283) Google Scholar), thus effectively inhibiting the dephosphorylation of the substrates of PTEN. Tail mutants of PTEN tend to have increased catalytic activity but are rapidly degraded in cells. Using a mutagenesis approach, Torres and Pulido (17Torres J. Pulido R. J. Biol. Chem. 2001; 276: 993-998Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar) showed that the C-terminal region of PTEN is constitutively phosphorylated in vivo between residues 369 and 386, mostly on Ser-370 and Ser-385. They also found that the Ser/Thr protein kinase casein kinase 2 (CK2) can phosphorylate these residues in vitro as well as, to a lower extent, Ser-380, Thr-382, and Thr-383 (17Torres J. Pulido R. J. Biol. Chem. 2001; 276: 993-998Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). Vazquez et al. (16Vazquez F. Ramaswamy S. Nakamura N. Sellers W.R. Mol. Cell. Biol. 2000; 20: 5010-5018Crossref PubMed Scopus (656) Google Scholar) also found that all phosphorylation of PTEN occurred in the C-terminal region (residues 354–403) and identified Ser-370 plus at least one other residue (Ser-380, Thr-382, Thr-383, or Ser-385) as the sites in vivo. They also reported that mutation of Ser-380, Thr-382, or Thr-383 to alanine reduced the half-life and increased the catalytic activity of PTEN (16Vazquez F. Ramaswamy S. Nakamura N. Sellers W.R. Mol. Cell. Biol. 2000; 20: 5010-5018Crossref PubMed Scopus (656) Google Scholar). Finally, Miller et al. (21Miller S.J. Lou D.Y. Seldin D.C. Lane W.S. Neel B.G. FEBS Lett. 2002; 528: 145-153Crossref PubMed Scopus (174) Google Scholar) identified Ser-370 and Ser-385 as the major phosphorylation sites in vivo and also detected phosphate on Thr-366. They also found that CK2 readily phosphorylated Ser-370 and Ser-385 in vitro. Our own group (20Birle D. Bottini N. Williams S. Huynh H. deBelle I. Adamson E. Mustelin T. J. Immunol. 2002; 169: 286-291Crossref PubMed Scopus (64) Google Scholar) studied PTEN in a different cell type, the T lymphocyte, and we found that PTEN is heavily phosphorylated at Ser-380 and Ser-385 in these cells and that both residues can affect the half-life of PTEN. Other residues were not examined. We have now refined this study using mass spectrometry, phosphospecific antibodies, tryptic peptide mapping, phosphoamino acid analysis, site-directed mutagenesis, and RNA interference of kinases. These studies revealed a more complex regulation of PTEN by several kinases, which may act in concert or in response to different conditions or in different cell types. The participation of glycogen synthase kinase 3β (GSK3β) in PTEN phosphorylation in vivo suggested the possibility of PTEN regulation in a negative feedback loop or by stimuli that activate the PI3K/Akt pathway. Antibodies—Antibodies to PTEN were from Santa Cruz Biotechnology (Santa Cruz, CA) and Upstate Biotechnology (Lake Placid, NY), anti-CK2α′ and anti-GSK3α were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-PTEN-phospho-Ser-380, anti-phospho-Thr-Pro, anti-Akt, anti-Akt-phospho-S473, and anti-GSK3α/β-phospho-21/9 were from Cell Signaling Technology (Beverly, MA), anti-actin was from Sigma, and anti-CK2α was from StressGen Biotechnologies Corp. (Victoria, BC, Canada), anti-PTEN-phospho-Ser-370 was from Novus Biologicals (Littleton, CO), anti-PTEN-phospho-Ser-385 was from Ana-Spec, Inc. (San Jose, CA), and anti-GSK3β was from BD Transduction Laboratories and from Cell Signaling Technology (Beverly, MA). Recombinant insulin-like growth factor-1 was from PeproTech Inc. (Rocky Hill, NJ). Plasmids and Proteins—Constructs encoding glutathione S-transferase (GST)-fused PTEN and its phosphorylation site mutants were cloned by standard PCR and recombinant DNA methods. Briefly, fragments containing the entire open reading frame of PTEN were subcloned into the pGEX-2T vector (Amersham Biosciences), in-frame with the (GST) polypeptide using EcoRI and XhoI restriction sites flanking the 5′ and 3′ ends, respectively. The phosphorylation site mutants of PTEN (S362A/T366A, S370A, S380A, S385A, S380A/385A) were generated using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA). All constructs were verified by sequencing. Proteins were expressed and purified according to standard techniques. Cells, Transfection, and RNA Interference—Human embryonic kidney 293T cells were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, l-glutamine, and antibiotics. These cells were transfected with siRNAs using Lipofectamine 2000 (Invitrogen) following the recommendations of the manufacturer. The siRNAs used for transfections were: CK2α (Dharmacon, M-003480-00-05) and CK2α′ (Qiagen, 5′-AGC UGC GAC UGA UAG AUU G-3′), GSK3β (Dharmacon, M-003009-00), and GSK3α (Dharmacon, M-003010-00-05). Cells were lysed 48 h after transfections. In initial experiments, we co-transfected a fluorescein-labeled luciferase GL2 duplex (Dharmacon, D-001120-01-20) and then sorted out the fluorescent cells. However, this did not have any significant impact on the experimental results, and this step was therefore omitted in later experiments. Experiments were also performed with Jurkat cells, as before (12Wang X. Gjörloff-Wingren A. Saxena M. Pathan N. Reed J.C. Mustelin T. J. Immunol. 2000; 164: 1934-1939Crossref PubMed Scopus (59) Google Scholar, 20Birle D. Bottini N. Williams S. Huynh H. deBelle I. Adamson E. Mustelin T. J. Immunol. 2002; 169: 286-291Crossref PubMed Scopus (64) Google Scholar). Cell Lysis, Immunoprecipitation, and Immunoblotting—Cells were rinsed twice with ice-cold phosphate-buffered saline and lysed in 20 mm Tris/HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, and 1% Nonidet P-40, 1 mm Na3VO4, 2 mm NaF, 10 μg/ml aprotinin and leupeptin, 100 μg/ml soybean trypsin inhibitor, and 1 mm phenylmethylsulfonyl fluoride. Cell lysates were then clarified by centrifugation at 13,800 × g for 10 min at 4 °C. For PTEN immunoprecipitation, the clarified lysates were incubated with protein G-Sepharose beads and the PTEN antibody from Santa Cruz Biotechnology for 3 h. Immune complexes were washed in and in Proteins were in to and incubated with The were by the according to the In and in vitro kinase were incubated with GSK3β CK2α for min at in 20 mm pH 7.4, mm 10 mm 1 mm 2 mm and 5 of 1 mm The were by and to for 5 min by and peptide was performed as before M. Williams S. K. Mustelin T. Nat. Cell Biol. 1999; PubMed Scopus Google Scholar, A. S. Williams S. M. L. A. S. Mustelin T. Nat. Immunol. 2003; PubMed Scopus Google Scholar, H. Bottini N. Williams S. V. L. K. S. E. Wang X. J. M. E. P. W. Mustelin T. Nat. Cell Biol. PubMed Scopus Google Scholar) with the of et al. K. B.M. 5: Google Scholar). acid was performed by acid in 1 and in two in the of 293T cell lysates were incubated with or to The beads were then washed with in and by of phosphorylated residues by was performed on in vitro phosphorylated and with The was into a liquid which the on a from which into a mass with a of CK2 on of PTEN at Ser-380 in a we showed that PTEN is phosphorylated at Ser-380 in Jurkat T cells and in human T (20Birle D. Bottini N. Williams S. Huynh H. deBelle I. Adamson E. Mustelin T. J. Immunol. 2002; 169: 286-291Crossref PubMed Scopus (64) Google Scholar). CK2 is responsible for this phosphorylation in cells, we used RNA interference to the of the two catalytic of this kinase, CK2α and 293T cells can transfected to were transfected with small interfering for kinase to and to the required and then with both siRNAs after these cells showed an in CK2α and in were Cell and also with the antibody revealed that phosphorylation of PTEN at this site was not affected at all by this in CK2 CK2 PTEN at Ser-370 and Ser-385 but of effects of the of CK2 on PTEN phosphorylation at Ser-380 that of CK2 are to out a phosphorylation at this site or that kinase is the we phosphorylated PTEN in vitro with CK2 and the phosphorylation by tryptic peptide mapping, phosphoamino acid analysis, and mass These showed that CK2 readily phosphorylated PTEN recombinant Akt did not and that this phosphorylation occurred on two both of which The two a on the in the that are both and These are found in the two C-terminal tryptic to residues and of the containing the major phosphorylation sites in PTEN in T cells, Ser-380 and Ser-385 (20Birle D. Bottini N. Williams S. Huynh H. deBelle I. Adamson E. Mustelin T. J. Immunol. 2002; 169: 286-291Crossref PubMed Scopus (64) Google Scholar). Using PTEN with or both of these two residues mutated to alanine as substrates showed that CK2 readily phosphorylated all these with for PTEN with Ser-385 mutated Finally, mass spectrometry detected phosphorylation of two tryptic at residues Ser-370 and Ser-385, In peptide containing phosphate at Ser-380 was the peptide with phosphate at Ser-385 was readily that Ser-380 was not phosphorylated at all by all these that CK2 can phosphorylate PTEN well but that is not required for phosphorylation of PTEN at Ser-380 in intact cells. CK2 did not phosphorylate this residue in vitro but phosphorylates serine residues and We also found that CK2 phosphorylates serine any of phosphate on threonine We concluded that is that participate in the phosphorylation of PTEN at serine and threonine residues in intact cells. of PTEN by GSK3β and but by we a of other Ser/Thr for to phosphorylate PTEN. in recombinant Akt readily phosphorylated other in not was to phosphorylate protein kinase, protein kinase and the protein and were all to phosphorylate In GSK3β and of into PTEN peptide of PTEN phosphorylated by GSK3β showed a which was both and suggesting that to of the two C-terminal tryptic of PTEN. acid revealed that GSK3β phosphorylated PTEN on both threonine and serine mass spectrometry identified the phosphorylated residues as Ser-362 and Thr-366 of the several other serine or threonine residues any the of PTEN was not phosphorylated at all by GSK3β in vitro The phosphorylation of PTEN by or GSK3β in vitro was with and and which that CK2 readily phosphorylated both sites phosphorylated Ser-370 CK2 but Ser-385 a CK2 In GSK3β did not phosphorylate site and Ser-362 or Thr-366 are not but Thr-366 is by a we this site with an GSK3 PTEN readily with this antibody was by mutation of Thr-366 was in PTEN at CK2 was not to any phosphorylation at this of PTEN by GSK3β and phosphorylates or that are residues of an phosphorylated a that the that Ser-362 a Thr-366 has phosphorylation of Ser-370 by CK2 have the on phosphorylation of Thr-366 by this we with or kinase in the of and washed the and then with GSK3β in kinase with or with kinase and The also as a to that CK2 did not affect the in PTEN with CK2 was more phosphorylated by GSK3β PTEN with kinase and CK2 this was with the of CK2 was lower were with was to PTEN with the antibody phosphorylate PTEN with both CK2 and more strongly In did not at all with the antibody and the of CK2 was in the these showed that phosphorylation of PTEN by CK2 at Ser-370 phosphorylation at Thr-366 by GSK3β PTEN at Thr-366 in GSK3β participates in the phosphorylation of PTEN in intact cells, we reduced the of GSK3β, as well as the by RNA interference, and with the antibody In cells with reduced of both GSK3 phosphorylation of PTEN at Thr-366 was that GSK3 is for this phosphorylation in intact cells. In with the and was not and PTEN and were We concluded that GSK3 phosphorylates at least Thr-366 in intact cells. was by the of a small of GSK3β well as to incubated with cell lysates by and not of by 1 PTEN at Thr-366 in we to stimuli that affect the activity of GSK3α and the phosphorylation of PTEN by this kinase in intact cells. growth factor 1 is to a of in phosphorylates GSK3 and the of this growth factor to cells a in and the of phosphorylated at was a in the of PTEN with the The of Akt and PTEN in these experiments, the of GSK3 to at least in to translocation of this kinase J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). these that stimuli that activate the PTEN phosphorylation at a regulation of this phosphorylation of PTEN. of PTEN at Thr-366 the of PTEN in to phosphorylation at Thr-366 has any impact on we expressed PTEN or the in Jurkat T cells, which and effects on the phosphorylation of In these experiments, reduced Akt phosphorylation to a PTEN that phosphorylation of PTEN at Thr-366 by GSK3 its Although with the from studies that CK2 plays a role in PTEN phosphorylation (17Torres J. Pulido R. J. Biol. Chem. 2001; 276: 993-998Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar), we found that CK2 is not the kinase in this important several other (16Vazquez F. Ramaswamy S. Nakamura N. Sellers W.R. Mol. Cell. Biol. 2000; 20: 5010-5018Crossref PubMed Scopus (656) Google Scholar, 17Torres J. Pulido R. J. Biol. Chem. 2001; 276: 993-998Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar, 21Miller S.J. Lou D.Y. Seldin D.C. Lane W.S. Neel B.G. FEBS Lett. 2002; 528: 145-153Crossref PubMed Scopus (174) Google Scholar), we found that Ser-370 and Ser-385 are phosphorylated in vivo and by CK2 in vitro. However, we phosphorylation of other residues by and phosphorylation of threonine residues at We also found that CK2α and are not required in intact cells for phosphorylation of Ser-380, phosphorylation of PTEN was by CK2 by RNA interference not CK2 not the kinase in PTEN phosphorylation in cells. we found that GSK3β also phosphorylates PTEN at two Ser-362 and the detected by Miller et al. (21Miller S.J. Lou D.Y. Seldin D.C. Lane W.S. Neel B.G. FEBS Lett. 2002; 528: 145-153Crossref PubMed Scopus (174) Google Scholar) as a site in intact cells. We also detected phosphate at Thr-366 in and this phosphate of GSK3 or the of insulin-like growth factor 1 to the cells. GSK3β and CK2 phosphorylated and we found that phosphorylation of Ser-370 by CK2 strongly phosphorylation of Thr-366 by these two and in the phosphorylation of PTEN in cells. also that can phosphorylate at Ser-385, suggesting that this kinase may also a role in intact cells. a of the in PTEN tail phosphorylation. We reported that PTEN phosphorylation is by phosphorylated substrates for in to a negative feedback loop (20Birle D. Bottini N. Williams S. Huynh H. deBelle I. Adamson E. Mustelin T. J. Immunol. 2002; 169: 286-291Crossref PubMed Scopus (64) Google Scholar). in this was that CK2 is not to regulated by these or The of GSK3β this kinase is to by phosphorylation by suggesting that of in Jurkat T may the phosphorylation at Thr-366 expressed in Jurkat T cells was phosphorylated on serine but (20Birle D. Bottini N. Williams S. Huynh H. deBelle I. Adamson E. Mustelin T. J. Immunol. 2002; 169: 286-291Crossref PubMed Scopus (64) Google Scholar). et al. K. M. F.B. Cavenee W.K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) showed that the C terminus of PTEN with the protein. Thr-366 of PTEN and was by a suggesting that phosphorylation at Thr-366 was for the with PTEN in a that did not the phosphatase activity of PTEN K. M. F.B. Cavenee W.K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). we found that GSK3 is responsible for phosphorylation of PTEN at that GSK3 plays an role in the tumor suppressor of PTEN by of which kinase phosphorylates CK2 GSK3β phosphorylated this site in and with the antibody was not affected by of that a kinase is responsible for PTEN phosphorylation at possibility is which phosphorylated PTEN in in to a of other Ser/Thr kinases, including protein kinase, protein kinase and protein kinases. possibility is a group of Ser/Thr as the kinases, which a and to the C-terminal tail of PTEN M. A. B. Torres J. A. C. Pulido R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Finally, out that PTEN is also regulated both and and both these can to in PTEN protein and to of PTEN in PTEN is regulated provide into cell signaling and may to the of

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