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• W2042121553 abstract "Protein phosphatase 4 (PP4; also named PPX or PPP4) is a PP2A-related protein serine/threonine phosphatase with important roles in a variety of cellular processes such as microtubule growth/organization, apoptosis, tumor necrosis factor (TNF)-α signaling, and activation of c-Jun N-terminal kinase and NF-κB. To further investigate the cellular functions of PP4, we isolated and identified PP4-interacting proteins using a proteomic approach. We found that insulin receptor substrate 4 (IRS-4) interacted with PP4 and that this interaction was enhanced following TNF-α stimulation. We also found that PP4, but not PP2A, down-regulated IRS-4 in a phosphatase activity-dependent manner. Pulse-chase analysis revealed that PP4 decreased the half-life of IRS-4 from 4 to 1 h. Moreover, we found that TNF-α stimulated a PP4-dependent degradation of IRS-4, as indicated by the blockage of the degradation by a potent PP4 inhibitor (okadaic acid) and a phosphatase-dead PP4 mutant (PP4-RL). Taken together, our studies indicate that IRS-4 is subject to regulation by TNF-α and that PP4 mediates TNF-α-induced degradation of IRS-4. Protein phosphatase 4 (PP4; also named PPX or PPP4) is a PP2A-related protein serine/threonine phosphatase with important roles in a variety of cellular processes such as microtubule growth/organization, apoptosis, tumor necrosis factor (TNF)-α signaling, and activation of c-Jun N-terminal kinase and NF-κB. To further investigate the cellular functions of PP4, we isolated and identified PP4-interacting proteins using a proteomic approach. We found that insulin receptor substrate 4 (IRS-4) interacted with PP4 and that this interaction was enhanced following TNF-α stimulation. We also found that PP4, but not PP2A, down-regulated IRS-4 in a phosphatase activity-dependent manner. Pulse-chase analysis revealed that PP4 decreased the half-life of IRS-4 from 4 to 1 h. Moreover, we found that TNF-α stimulated a PP4-dependent degradation of IRS-4, as indicated by the blockage of the degradation by a potent PP4 inhibitor (okadaic acid) and a phosphatase-dead PP4 mutant (PP4-RL). Taken together, our studies indicate that IRS-4 is subject to regulation by TNF-α and that PP4 mediates TNF-α-induced degradation of IRS-4. Insulin receptor substrate (IRS) 1The abbreviations used are: IRS, insulin receptor substrate; PP4, protein phosphatase 4; PP2A, protein phosphatase 2A; TNF-α, tumor necrosis factor-α; IGF-1, insulin-like growth factor-1; HA, hemagglutinin; HEK293, human embryonic kidney 293; Ab, antibody; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. proteins play a central role in signal transduction by the receptors for insulin, insulin-like growth factor 1 (IGF-1), and a growing number of cytokines and integrins (1White M.F. Am. J. Physiol. 2002; 283: E413-E422Crossref PubMed Scopus (44) Google Scholar). Four IRS proteins have been identified and characterized, including IRS-1, IRS-2, IRS-3 and IRS-4 (2Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1291) Google Scholar, 3Sun X.J. Wang L.M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (767) Google Scholar, 4Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 5Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Recently, two more signaling proteins, IRS5/DOK4 and IRS6/DOK5, have been identified as potentially new members of the IRS family (6Cai D. Dhe-Paganon S. Melendez P.A. Lee J. Shoelson S.E. J. Biol. Chem. 2003; 278: 25323-25330Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). IRSs are tyrosine-phosphorylated following insulin or IGF-1 stimulation and form signaling complexes at the receptor with Src homology 2 domain-containing proteins, including the p85 regulatory subunit of phosphatidylinositol 3-kinase, Grb2, Nck, and Shc (7White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (625) Google Scholar, 8Giovannone B. Scaldaferri M.L. Federici M. Porzio O. Lauro D. Fusco A. Sbraccia P. Borboni P. Lauro R. Sesti G. Diabetes Metab. Res. Rev. 2000; 16: 434-441Crossref PubMed Scopus (119) Google Scholar). All IRS proteins share a number of structural and functional characteristics, including an N-terminal pleckstrin homology and a phosphotyrosine binding domain, followed by a large C-terminal region containing multiple sites of tyrosine phosphorylation, which serve as docking sites for Src homology 2 domain-containing signaling proteins. IRS proteins also carry a large number of potential sites for serine and threonine phosphorylation, which may regulate protein-protein interactions (9Virkamaki A. Ueki K. Kahn C.R. J. Clin. Invest. 1999; 103: 931-943Crossref PubMed Scopus (726) Google Scholar). Despite the overall similarity, the IRS proteins differ in their subcellular distribution and tissue/developmental expression pattern. Each IRS is phosphorylated on a set of specific tyrosine residues, leading to its binding to the Src homology 2 domains of distinct signaling proteins and therefore, activation of specific signaling cascades. Disruption of each individual IRS gene causes distinct phenotypes in mice (10Araki E. Lipes M.A. Patti M.E. Bruning J.C. Haag 3rd, B. Johnson R.S. Kahn C.R. Nature. 1994; 372: 186-190Crossref PubMed Scopus (1099) Google Scholar, 11Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Sekihara H. Yoshioka S. Horikoshi H. Furuta Y. Ikawa Y. Kasuga M. Yazaki Y. Aizawa S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (906) Google Scholar, 12Withers D.J. Gutierrez J.S. Towery H. Burks D.J. Ren J.M. Previs S. Zhang Y. Bernal D. Pons S. Shulman G.I. Bonner-Weir S. White M.F. Nature. 1998; 391: 900-904Crossref PubMed Scopus (1347) Google Scholar, 13Liu S.C. Wang Q. Lienhard G.E. Keller S.R. J. Biol. Chem. 1999; 274: 18093-18099Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 14Fantin V.R. Wang Q. Lienhard G.E. Keller S.R. Am. J. Physiol. Endocrinol. Metab. 2000; 278: E127-E133Crossref PubMed Google Scholar), further indicating that the four IRS proteins play different roles in the regulation of the pleiotropic effects of insulin, IGF-1, and other growth factors. IRS-4 was initially detected in human embryonic kidney 293 (HEK293) cells (5Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 15Fantin V.R. Sparling J.D. Slot J.W. Keller S.R. Lienhard G.E. Lavan B.E. J. Biol. Chem. 1998; 273: 10726-10732Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Like other IRSs, IRS-4 enhances insulin- and IGF-1-induced mitogenesis in a variety of cells such as HEK293 cells (16Koval A.P. Karas M. Zick Y. LeRoith D. J. Biol. Chem. 1998; 273: 14780-14787Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 17Karas M. Koval A.P. Zick Y. LeRoith D. Endocrinology. 2001; 142: 1835-1840Crossref PubMed Scopus (12) Google Scholar), NIH3T3 cells (18Qu B.H. Karas M. Koval A. LeRoith D. J. Biol. Chem. 1999; 274: 31179-31184Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), adipose cells (19Zhou L. Chen H. Xu P. Cong L.N. Sciacchitano S. Li Y. Graham D. Jacobs A.R. Taylor S.I. Quon M.J. Mol. Endocrinol. 1999; 13: 505-514Crossref PubMed Google Scholar), and hematopoietic cells (20Fantin V.R. Keller S.R. Lienhard G.E. Wang L.M. Biochem. Biophys. Res. Commun. 1999; 260: 718-723Crossref PubMed Scopus (22) Google Scholar). It has also been shown that IRS-4 mediates mitogenic signaling by interleukin-4 in hematopoietic cells (20Fantin V.R. Keller S.R. Lienhard G.E. Wang L.M. Biochem. Biophys. Res. Commun. 1999; 260: 718-723Crossref PubMed Scopus (22) Google Scholar), by growth hormone in LB cells, a murine T-cell lymphoma devoid of IGF-I receptor (21Urso B. Ilondo M.M. Holst P.A. Christoffersen C.T. Ouwens M. Giorgetti S. Van Obberghen E. Naor D. Tornqvist H. De Meyts P. Cell. Signal. 2003; 15: 385-394Crossref PubMed Scopus (9) Google Scholar), and by hepatocyte growth factor in pancreatic β-cells (22Gahr S. Merger M. Bollheimer L.C. Hammerschmied C.G. Scholmerich J. Hugl S.R. J. Mol. Endocrinol. 2002; 28: 99-110Crossref PubMed Scopus (39) Google Scholar). IRS-4 has been implicated in liver regeneration as indicated by the substantial induction after partial hepatectomy (23Escribano O. Fernandez-Moreno M.D. Zueco J.A. Menor C. Fueyo J. Ropero R.M. Diaz-Laviada I. Roman I.D. Guijarro L.G. Hepatology. 2003; 37: 1461-1469Crossref PubMed Scopus (34) Google Scholar). Recently, it was found that IRS-4 expression level is decreased in polycystic ovary syndrome (PCOS) theca cells (24Yen H.W. Jakimiuk A.J. Munir I. Magoffin D.A. Mol. Hum. Reprod. 2004; 10: 473-479Crossref PubMed Scopus (35) Google Scholar). IRS-4 has a compensatory role for IRS-1 in adipocyte differentiation (25Tseng Y.H. Kriauciunas K.M. Kokkotou E. Kahn C.R. Mol. Cell. Biol. 2004; 24: 1918-1929Crossref PubMed Scopus (149) Google Scholar) and for IRS-2 in pancreatic β-cells (26Lingohr M.K. Dickson L.M. Wrede C.E. Briaud I. McCuaig J.F. Myers M.G. Rhodes C.J. Mol. Cell. Endocrinol. 2003; 209: 17-31Crossref PubMed Scopus (64) Google Scholar). However, the negative regulation of IRS-1 and IRS-2 by IRS-4 in IGF-1 signaling has also been suggested (27Tsuruzoe K. Emkey R. Kriauciunas K.M. Ueki K. Kahn C.R. Mol. Cell. Biol. 2001; 21: 26-38Crossref PubMed Scopus (85) Google Scholar). IRS-4 lacks both putative SHP-2 binding motifs present in IRS-1, IRS-2, and IRS-3 (15Fantin V.R. Sparling J.D. Slot J.W. Keller S.R. Lienhard G.E. Lavan B.E. J. Biol. Chem. 1998; 273: 10726-10732Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In comparison with other IRSs, IRS-4 exhibits a more limited tissue expression. Besides HEK293 cells, the IRS-4 protein has only been detected in heart and skeletal muscle cells (28Schreyer S. Ledwig D. Rakatzi I. Kloting I. Eckel J. Endocrinology. 2003; 144: 1211-1218Crossref PubMed Scopus (25) Google Scholar), although IRS-4 mRNA is expressed in a variety of human and rodent tissues including pituitary, thyroid, ovary, prostate, hypothalamus, liver, heart, and skeletal muscle (29Sesti G. Federici M. Hribal M.L. Lauro D. Sbraccia P. Lauro R. FASEB J. 2001; 15: 2099-2111Crossref PubMed Scopus (285) Google Scholar). Tissue-specific expression of IRS-4 suggests the potential involvement of IRS-4 in the regulation of insulin/IGF-1 signaling in these tissues. Five amino acid polymorphisms have been identified in IRS-4, although their functional relevance remains to be determined (30Almind K. Frederiksen S.K. Ahlgren M.G. Urhammer S. Hansen T. Clausen J.O. Pedersen O. Diabetologia. 1998; 41: 969-974Crossref PubMed Scopus (18) Google Scholar). To date, only a few IRS-4-interacting proteins have been identified, including the regulatory and catalytic subunits of phosphatidylinositol 3-kinase (23Escribano O. Fernandez-Moreno M.D. Zueco J.A. Menor C. Fueyo J. Ropero R.M. Diaz-Laviada I. Roman I.D. Guijarro L.G. Hepatology. 2003; 37: 1461-1469Crossref PubMed Scopus (34) Google Scholar, 31Sano H. Liu S.C. Lane W.S. Piletz J.E. Lienhard G.E. J. Biol. Chem. 2002; 277: 19439-19447Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), the imidazoline receptor antisera selected (31Sano H. Liu S.C. Lane W.S. Piletz J.E. Lienhard G.E. J. Biol. Chem. 2002; 277: 19439-19447Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), the suppressor of cytokine signaling-6 (32Krebs D.L. Uren R.T. Metcalf D. Rakar S. Zhang J.G. Starr R. De Souza D.P. Hanzinikolas K. Eyles J. Connolly L.M. Simpson R.J. Nicola N.A. Nicholson S.E. Baca M. Hilton D.J. Alexander W.S. Mol. Cell. Biol. 2002; 22: 4567-4578Crossref PubMed Scopus (126) Google Scholar), Src homology phosphatase (23Escribano O. Fernandez-Moreno M.D. Zueco J.A. Menor C. Fueyo J. Ropero R.M. Diaz-Laviada I. Roman I.D. Guijarro L.G. Hepatology. 2003; 37: 1461-1469Crossref PubMed Scopus (34) Google Scholar), and protein kinase C-ζ (23Escribano O. Fernandez-Moreno M.D. Zueco J.A. Menor C. Fueyo J. Ropero R.M. Diaz-Laviada I. Roman I.D. Guijarro L.G. Hepatology. 2003; 37: 1461-1469Crossref PubMed Scopus (34) Google Scholar). Identification and characterization of novel IRS-4-interacting proteins will help in understanding the cellular functions of IRS-4. Protein phosphatase 4 (PP4; previously called PPX) (33Brewis N.D. Street A.J. Prescott A.R. Cohen P.T. EMBO J. 1993; 12: 987-996Crossref PubMed Scopus (200) Google Scholar) is a member of the PP2A subfamily of protein serine/threonine phosphatases, along with PP2A and PP6 (34Bastians H. Ponstingl H. J. Cell Sci. 1996; 109: 2865-2874Crossref PubMed Google Scholar). PP4, PP6, and PP2A are highly homologous; human PP4 shares 65% identity with PP2A (33Brewis N.D. Street A.J. Prescott A.R. Cohen P.T. EMBO J. 1993; 12: 987-996Crossref PubMed Scopus (200) Google Scholar, 35Brewis N.D. Cohen P.T. Biochim. Biophys. Acta. 1992; 1171: 231-233Crossref PubMed Scopus (38) Google Scholar). PP4, like PP2A, is a holoenzyme composed of catalytic (C), structural (A), and regulatory (B) subunits. To date, three subunits have been identified for PP4: α4, PP4-R1, and PP4-R2 (36Chen J. Peterson R.T. Schreiber S.L. Biochem. Biophys. Res. Commun. 1998; 247: 827-832Crossref PubMed Scopus (159) Google Scholar, 37Nanahoshi M. Tsujishita Y. Tokunaga C. Inui S. Sakaguchi N. Hara K. Yonezawa K. FEBS Lett. 1999; 446: 108-112Crossref PubMed Scopus (59) Google Scholar, 38Kloeker S. Wadzinski B.E. J. Biol. Chem. 1999; 274: 5339-5347Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 39Hastie C.J. Carnegie G.K. Morrice N. Cohen P.T. Biochem. J. 2000; 347: 845-855Crossref PubMed Scopus (48) Google Scholar, 40Wada T. Miyata T. Inagi R. Nangaku M. Wagatsuma M. Suzuki D. Wadzinski B.E. Okubo K. Kurokawa K. J. Am. Soc. Nephrol. 2001; 12: 2601-2608Crossref PubMed Google Scholar). Like PP2A, PP4 contains a putative binding domain for okadaic acid, a potent tumor promoter toxin (41Dounay A.B. Forsyth C.J. Curr. Med. Chem. 2002; 9: 1939-1980Crossref PubMed Scopus (109) Google Scholar). Okadaic acid inhibits PP4 with a similar range of concentration (IC50 = 0.1 nmin vitro) as PP2A (42Hastie C.J. Cohen P.T. FEBS Lett. 1998; 431: 357-361Crossref PubMed Scopus (99) Google Scholar). PP4 is involved in the regulation of microtubule growth or organization at the centrosomes (43Helps N.R. Brewis N.D. Lineruth K. Davis T. Kaiser K. Cohen P.T. J. Cell Sci. 1998; 111: 1331-1340Crossref PubMed Google Scholar) and the centrosome maturation in mitosis and meiosis (44Sumiyoshi E. Sugimoto A. Yamamoto M. J. Cell Sci. 2002; 115: 1403-1410PubMed Google Scholar). It has been recently shown that PP4 plays an important proapoptotic role in T lymphocytes (45Mourtada-Maarabouni M. Kirkham L. Jenkins B. Rayner J. Gonda T.J. Starr R. Trayner I. Farzaneh F. Williams G.T. Cell Death Differ. 2003; 10: 1016-1024Crossref PubMed Scopus (40) Google Scholar). Our previous studies found that PP4 interacts with members of the NF-κB family of transcription factors c-Rel and RelA and activates NF-κB-mediated transcription (46Hu M.C.-T. Tang-Oxley Q. Qui W.R. Wang Y.-P. Mihindukulasuriya K.A. Afshar R. Tan T-H. J. Biol. Chem. 1998; 273: 33561-33565Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Recent studies have demonstrated that PP4 dephosphorylates RelA (also called NF-κB p65), primarily on Thr435, and that this dephosphorylation is required for NF-κB activation induced by cisplatin and extracellular signal-regulated kinase suppression (47Yeh P.Y. Yeh K.H. Chuang S.E. Song Y.C. Cheng A.L. J. Biol. Chem. 2004; 279: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). We have also found that PP4 plays a role as a positive regulator of the c-Jun N-terminal kinase pathway in TNF-α signaling (48Zhou G. Mihindukulasuriya K.A. MacCorkle-Chosnek R.A. Van Hooser A. Hu M.C.-T. Brinkley B.R. Tan T.-H. J. Biol. Chem. 2002; 277: 6391-6398Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Recently, PP4 has been found to interact with the survival of motor neurons complex and enhances the temporal localization of small nuclear ribonucleoproteins (49Carnegie G.K. Sleeman J.E. Morrice N. Hastie C.J. Peggie M.W. Philp A. Lamond A.I. Cohen P.T. J. Cell Sci. 2003; 116: 1905-1913Crossref PubMed Scopus (52) Google Scholar). Given the high degree of conservation of PP4 during evolution (33Brewis N.D. Street A.J. Prescott A.R. Cohen P.T. EMBO J. 1993; 12: 987-996Crossref PubMed Scopus (200) Google Scholar, 35Brewis N.D. Cohen P.T. Biochim. Biophys. Acta. 1992; 1171: 231-233Crossref PubMed Scopus (38) Google Scholar, 50Hu M.C.-T. Shui J.W. Mihindukulasuriya K.A. Tan T-H. Gene (Amst.). 2001; 278: 89-99Crossref PubMed Scopus (16) Google Scholar), it is likely that PP4 may participate in many essential cellular processes. In this study, by applying a functional proteomic approach, we identified IRS-4 as a novel PP4-interacting protein. Moreover, we found that the stability of IRS-4 was subject to regulation by TNF-α and that PP4 mediated the degradation of IRS-4 by TNF-α. Reagents—The SuperSignal chemiluminescence system was purchased from Pierce. TNF-α was purchased from R&D Systems (Minneapolis, MN). The mixture of [35S]methionine-cysteine was purchased from ICN Biomedicals (Irvine, CA). Anti-HA antibody (12CA5) was purchased from Roche Applied Science. Anti-FLAG (M2) and anti-γ-tubulin antibodies were purchased from Sigma. Anti-α4 antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-IRS-4 antibody (Ab 1640), anti-PP4-R2 antibody (Ab 1641), and anti-PP2A antibody (Ab 1643) were raised against the C-terminal regions of IRS-4 (CDFARRDNQFDSPKRGR1242–1257), PP4-R2 (CESFMT-SREMIPERK388–401), and PP2A (CDTHPRGYGNRQNMG326–339), respectively. Anti-PP4-R1 antibody (Ab 1650) was raised against the N-terminal region of PP4-R1 (CASENIFNRQMVARS39–52). Rabbit anti-PP4 polyclonal antibodies (Ab 104 and 6101) were previously described (48Zhou G. Mihindukulasuriya K.A. MacCorkle-Chosnek R.A. Van Hooser A. Hu M.C.-T. Brinkley B.R. Tan T.-H. J. Biol. Chem. 2002; 277: 6391-6398Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Ab 1640 (anti-IRS-4) and Ab 6101 (anti-PP4) were peptide-purified using the Sulfolink Kit from Pierce. The Sepharose beads were purchased from Amersham Biosciences. All other chemical reagents were purchased from Sigma unless otherwise noted. Plasmids—pEF-HA-IRS-4 was provided by Dr. D. LeRoith (National Institutes of Health, Bethesda, MD) (18Qu B.H. Karas M. Koval A. LeRoith D. J. Biol. Chem. 1999; 274: 31179-31184Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). pBJF-FLAG-PP2A was kindly provided by Dr. J. Chen (University of Illinois at Urbana-Champaign) (37Nanahoshi M. Tsujishita Y. Tokunaga C. Inui S. Sakaguchi N. Hara K. Yonezawa K. FEBS Lett. 1999; 446: 108-112Crossref PubMed Scopus (59) Google Scholar). pCI-neo-FLAG-PP4 (48Zhou G. Mihindukulasuriya K.A. MacCorkle-Chosnek R.A. Van Hooser A. Hu M.C.-T. Brinkley B.R. Tan T.-H. J. Biol. Chem. 2002; 277: 6391-6398Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), PP4, and HA-PP4-RL (46Hu M.C.-T. Tang-Oxley Q. Qui W.R. Wang Y.-P. Mihindukulasuriya K.A. Afshar R. Tan T-H. J. Biol. Chem. 1998; 273: 33561-33565Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) were previously described. Cells and Transfection—HEK293 and HEK293T cells were grown and maintained as previously described (48Zhou G. Mihindukulasuriya K.A. MacCorkle-Chosnek R.A. Van Hooser A. Hu M.C.-T. Brinkley B.R. Tan T.-H. J. Biol. Chem. 2002; 277: 6391-6398Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). HEK293 and HEK293T cells were plated at a density of either 2.5 × 105 cells/35-mm plate well or 1.6 × 106 cells/100-mm dish and transfected the next day using the modified calcium phosphate precipitation protocol (Specialty Media, Inc., Lavallette, NJ). Cells were lysed in 1% Nonidet P-40 lysis buffer (50 mm Tris (pH 8.0), 150 mm NaCl, 2 mm EDTA, 1% Nonidet P-40) freshly supplemented with 6.6 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm dithiothreitol, 50 μmp-amidinophenylmethanesulfonyl fluoride, 50 mm NaF, and 1 mm Na3VO4. Co-immunoprecipitation and Western Blot Analysis—Co-immunoprecipitation assays were performed as previously described (51Ling P. Yao Z. Meyer C.F. Wang X.S. Oehl W. Feller S.M. Tan T.-H. Mol. Cell. Biol. 1999; 19: 1359-1368Crossref PubMed Scopus (78) Google Scholar), except that the immunoprecipitates were washed three times with ice-cold NETN buffer (20 mm Tris-HCl (pH 8.0), 0.1 m NaCl, 1 mm EDTA, 0.5% Nonidet P-40). Western blot analysis was performed using an enhanced chemiluminescence detection kit according to the manufacturer's protocols (Pierce). Phosphatase Assay—HEK293T cells were lysed in buffer containing 50 mm Tris-HCl (pH 8.0), 1% Nonidet P-40, 120 mm NaCl, 1 mm EDTA, 6 mm EGTA, 1 mm dithiothreitol, 50 μmp-amidinophenylmethanesulfonyl fluoride, and 2 μg/ml aprotinin. PP4 was immunoprecipitated with an anti-PP4 (Ab 104) antibody. FLAG-PP2A was immunoprecipitated with anti-FLAG (M2) antibody. The immunoprecipitates were washed three times with buffer containing 50 mm HEPES (pH 7.4), 0.1% Triton X-100, and 500 mm NaCl. Phosphatase assays were performed using Ser/Thr phosphatase assay kit 1, according to the manufacturer's protocol (Upstate Biotechnology). In brief, the immunoprecipitates were incubated with 4 μm KTpIRR peptide in 40 μl of assay buffer (50 mm Tris (pH 7.0), 0.1 mm CaCl2, and 1 mm MnCl2) at 30 °Cfor 30 min. Buffer plus peptide was used as a negative control. The immunoprecipitates were then pelleted, and the assay buffer was transferred to a 96-well, half-volume plate. The assay was terminated by the addition of 100 μl of Malachite Green solution (1 volume of 4.2% (w/v) ammonium molybdate in 4 m HCl, 3 volumes of 0.045% (w/v) Malachite Green in water, and 1 μl/ml 10% Tween 20 added fresh). After 15 min at room temperature, the assay was read at 650 nm on a PerkinElmer bioassay reader (HTS 7000 plus). Establishment of the HEK293 Cell Clone (10F1) Stably Transfected with FLAG-PP4—HEK293 cells were grown in complete Dulbecco's modified Eagle's medium containing 10% fetal calf serum supplemented with 12.5 mm HEPES, 50 μg/ml gentamycin, and 100 units/ml penicillin/streptomycin (Invitrogen). We transfected the HEK293 cells with FLAG-PP4 by the Fugene 6 method according to the manufacturer's protocol (Roche Applied Science). The transfected cells were selected by Geneticin (G418; Invitrogen) at a concentration of 750 μg/ml. The cells were replated at a 1:15 dilution whenever they reached 80% confluence. After 10–14 days, the T-75 flasks were trypsinized, and the drug-resistant cells were replated at a limiting dilution to obtain independent clones. Each clone was tested for FLAG-PP4 expression by Western blotting. Isolation of FLAG-PP4-interacting Protein Complexes—HEK293 cells stably expressing FLAG-PP4, designated as 10F1 clone, were left unstimulated or stimulated with TNF-α (10 ng/ml) for 10 min. Fifteen confluent T175 flasks of 10F1 or parental HEK293 cells (5.175 × 108 cells) were used per purification. The cells were trypsinized, washed twice with ice-cold phosphate-buffered saline, and stored at –80 °C until needed. The cells were lysed in 5 ml of PIP lysis buffer (20 mm HEPES (pH 7.4), 2 mm EDTA, 1% Triton X-100, 150 mm NaCl) freshly supplemented with 6.6 μg/ml aprotinin, 10 μg/ml leupeptin, and 50 μmp-amidinophenylmethanesulfonyl fluoride. The lysate was centrifuged at 20,817 × g (14,000 rpm) for 15 min to remove cellular debris. Chromosomal DNA was sheared with a 1-ml syringe. The whole cell extract (105 mg) was incubated with either anti-FLAG-Sepharose or unconjugated Sepharose at 4 °C for 3 h with continuous rotation, packed into a column, and washed three times with 10 ml of NETN buffer. FLAG-PP4 was eluted four times with 1× column volume of 100 μg/ml FLAG peptide in TBS (50 mm Tris-HCl (pH 7.4) and 150 mm NaCl). 0.1 μl of each 100-μl fraction was used for anti-FLAG Western blotting to determine which fraction(s) contained FLAG-PP4. The fraction containing the most FLAG-PP4 and the corresponding fractions for the negative controls were separated by 10% SDS-PAGE, and the gel was stained with Coomassie Blue. Identification of Proteins by Mass Spectrometry—Identification of proteins by mass spectrometry was performed as previously described (52Humphrey G.W. Wang Y. Russanova V.R. Hirai T. Qin J. Nakatani Y. Howard B.H. J. Biol. Chem. 2001; 276: 6817-6824Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 53Wu R.C. Qin J. Hashimoto Y. Wong J. Xu J. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. Biol. 2002; 22: 3549-3561Crossref PubMed Scopus (238) Google Scholar). A MALDI-TOF mass spectrometer with delayed extraction, a home-built MALDI-ion trap mass spectrometer, and an electrospray ion trap mass spectrometer (LCQ; Finnigan MAT, San Jose, CA) coupled on-line with a capillary high pressure liquid chromatograph (Magic 2002; Michom BioResources, Auburn, CA) were used to acquire tandem mass spectra. A 0.1 × 50-mm Magic MS C18 column (5-μm particle diameter, 200-Å pore size) with mobile phases A (methanol/water/acetic acid, 5:95:1) and B (methanol/water/acetic acid, 85:15:1) was used with a gradient of 2–98% mobile phase B over 2.5 min, followed by 98% mobile phase B for 2 min. Specific protein bands from Coomassie Blue-stained SDS-polyacrylamide gels were excised, destained, and digested with trypsin (200 ng/digestion) in 20 μl of 50 mm NH4HCO3 buffer for 2 h at 37 °C using a protein/enzyme weight ratio of 1:1. The resulting peptides were extracted, and 20–50% of the sample was used to obtain liquid chromatography/tandem mass spectra. The tandem mass spectra were used to search the compiled NCBI nonredundant protein and expressed sequence tag data bases with the program Pep-Frag to identify the proteins. Pulse-chase Analysis—HEK293T cells were plated at a density of 1.5 × 105 cells per 35-mm plate well and transfected the next day with HA-IRS-4 (2 μg) plus empty vector or PP4 (2 μg) as described above. Thirty-six h after transfection, the medium was replaced with 2 ml of prelabeling medium (Dulbecco's modified Eagle's medium lacking cysteine and methionine + 5% fetal calf serum). After 1 h of incubation, the prelabeling medium was replaced with 1 ml of labeling medium (prelabeling medium + 0.1 mCi/ml [35S]methionine and [35S]cysteine). The cells were labeled for 4 h and then chased with fresh, nonradioactive medium for the times indicated. The cells were lysed in lysis buffer (20 mm HEPES (pH 7.4), 2 mm EDTA, 1% Triton X-100, 10% glycerol, and 150 mm NaCl) freshly supplemented with 6.6 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm dithiothreitol, and 50 μmp-amidinophenylmethanesulfonyl fluoride. 100 μg of cell lysate was immunoprecipitated with an anti-HA antibody (12CA5) and protein A-Sepharose and resolved by SDS-PAGE. The gels were dried and autoradiographed. IRS-4 Is a TNF-α-inducible, PP4-interacting Protein—We have previously shown that PP4 is involved in TNF-α signaling (48Zhou G. Mihindukulasuriya K.A. MacCorkle-Chosnek R.A. Van Hooser A. Hu M.C.-T. Brinkley B.R. Tan T.-H. J. Biol. Chem. 2002; 277: 6391-6398Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In the search for novel cellular functions of PP4, we established a HEK293 cell clone (10F1) stably transfected with FLAG-PP4 and applied a functional proteomic approach to identify TNF-α-inducible, PP4-interacting protein(s) in 10F1 cells. We treated 10F1 cells with TNF-α, and the whole cell extract of 10F1 cells was incubated with anti-FLAG-Sepharose. After extensive washing, FLAG-PP4 and the proteins that complexed with PP4 were eluted with FLAG peptide, subjected to SDS-PAGE, and stained with Coomassie Blue (Fig. 1A). By mass spectrometry, a group of potential PP4-interacting proteins in response to TNF-α stimulation was identified (Fig. 1A). We selected one of these proteins, insulin receptor protein 4 (IRS-4), a docking protein that coordinates cellular responses to insulin and insulin-like growth factor 1 (IGF-1), for further study. A representative spectrum is shown for IRS-4 along with the peptide sequence and its position in the IRS-4 protein (Fig. 1B). To confirm the interaction between PP4 and IRS-4, we took a fraction of the FLAG peptide elution and performed Western blot analysis using an anti-IRS-4 antibody. As shown in Fig. 2, the presence of IRS-4 in FLAG-PP4-interacting protein complex was greatly increased upon TNF-α treatment (top panel). These data indicate that IRS-4 is a TNF-α-inducible, PP4-interacting protein. We also found that three known PP4-interacting proteins, α4, PP4-R1, and PP4-R2, co-purified with FLAG-PP4 (Fig. 2), further confirming the binding specificity of our approach.Fig. 2IRS-4 co-purifies with PP4. Equal amounts of eluate from the fractionation described in the legend to Fig. 1A were separated via SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were Western blotted with antibodies to IRS-4, α4, PP4-R1, PP4-R2, and PP4 (FLAG).View Large Image Figure ViewerDownload (PPT) To further confirm the PP4-IRS-4 interaction, we transiently transfected 10F1 cells with HA-IRS-4, immunoprecipitated FLAG-PP4, and performed" @default.
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• W2042121553 title "Protein Phosphatase 4 Interacts with and Down-regulates Insulin Receptor Substrate 4 following Tumor Necrosis Factor-α Stimulation" @default.
• W2042121553 cites W1274298899 @default.
• W2042121553 cites W1521781692 @default.
• W2042121553 cites W1598855117 @default.
• W2042121553 cites W1697523131 @default.
• W2042121553 cites W1967675708 @default.
• W2042121553 cites W1968805130 @default.
• W2042121553 cites W1971825456 @default.
• W2042121553 cites W1974654074 @default.
• W2042121553 cites W1974740641 @default.
• W2042121553 cites W1977000651 @default.
• W2042121553 cites W1983513002 @default.
• W2042121553 cites W1985113364 @default.
• W2042121553 cites W1989324173 @default.
• W2042121553 cites W1989387009 @default.
• W2042121553 cites W2000623239 @default.
• W2042121553 cites W2009818179 @default.
• W2042121553 cites W2011210301 @default.
• W2042121553 cites W2012338495 @default.
• W2042121553 cites W2013578942 @default.
• W2042121553 cites W2015077434 @default.
• W2042121553 cites W2016403730 @default.
• W2042121553 cites W2020888229 @default.
• W2042121553 cites W2022588964 @default.
• W2042121553 cites W2026332814 @default.
• W2042121553 cites W2031504013 @default.
• W2042121553 cites W2032017374 @default.
• W2042121553 cites W2033429522 @default.
• W2042121553 cites W2039060098 @default.
• W2042121553 cites W2042824986 @default.
• W2042121553 cites W2044488792 @default.
• W2042121553 cites W2044571148 @default.
• W2042121553 cites W2045580211 @default.
• W2042121553 cites W2047329119 @default.
• W2042121553 cites W2049219071 @default.
• W2042121553 cites W2050247932 @default.
• W2042121553 cites W2052645554 @default.
• W2042121553 cites W2064801081 @default.
• W2042121553 cites W2067205516 @default.
• W2042121553 cites W2068494384 @default.
• W2042121553 cites W2069033908 @default.
• W2042121553 cites W2069853996 @default.
• W2042121553 cites W2075645766 @default.
• W2042121553 cites W2084162862 @default.
• W2042121553 cites W2092896701 @default.
• W2042121553 cites W2093880435 @default.
• W2042121553 cites W2095252238 @default.
• W2042121553 cites W2097134216 @default.
• W2042121553 cites W2097369775 @default.
• W2042121553 cites W2098324895 @default.
• W2042121553 cites W2102683312 @default.
• W2042121553 cites W2107781080 @default.
• W2042121553 cites W2107836354 @default.
• W2042121553 cites W2113520782 @default.
• W2042121553 cites W2117036763 @default.
• W2042121553 cites W2120733137 @default.
• W2042121553 cites W2123208560 @default.
• W2042121553 cites W2125367136 @default.
• W2042121553 cites W2136028935 @default.
• W2042121553 cites W2142782839 @default.
• W2042121553 cites W2143859801 @default.
• W2042121553 cites W2149340540 @default.
• W2042121553 cites W2154713680 @default.
• W2042121553 cites W2156532107 @default.
• W2042121553 cites W2156696618 @default.
• W2042121553 cites W2166515327 @default.
• W2042121553 cites W2171661317 @default.
• W2042121553 cites W2171872390 @default.
• W2042121553 cites W4253418569 @default.
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