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• W2016155391 abstract "Inflammation contributes to insulin resistance in diabetes and obesity. Mouse Pelle-like kinase (mPLK, homolog of human IL-1 receptor-associated kinase (IRAK)) participates in inflammatory signaling. We evaluated IRS-1 as a novel substrate for mPLK that may contribute to linking inflammation with insulin resistance. Wild-type mPLK, but not a kinase-inactive mutant (mPLK-KD), directly phosphorylated full-length IRS-1 in vitro. This in vitro phosphorylation was increased when mPLK was immunoprecipitated from tumor necrosis factor (TNF)-α-treated cells. In NIH-3T3IR cells, wild-type mPLK (but not mPLK-KD) co-immunoprecipitated with IRS-1. This association was increased by treatment of cells with TNF-α. Using mass spectrometry, we identified Ser24 in the pleckstrin homology (PH) domain of IRS-1 as a specific phosphorylation site for mPLK. IRS-1 mutants S24D or S24E (mimicking phosphorylation at Ser24) had impaired ability to associate with insulin receptors resulting in diminished tyrosine phosphorylation of IRS-1 and impaired ability of IRS-1 to bind and activate PI-3 kinase in response to insulin. IRS-1-S24D also had an impaired ability to mediate insulin-stimulated translocation of GLUT4 in rat adipose cells. Importantly, endogenous mPLK/IRAK was activated in response to TNF-α or interleukin 1 treatment of primary adipose cells. In addition, using a phospho-specific antibody against IRS-1 phosphorylated at Ser24, we found that interleukin-1 or TNF-α treatment of Fao cells stimulated increased phosphorylation of endogenous IRS-1 at Ser24. We conclude that IRS-1 is a novel physiological substrate for mPLK. TNF-α-regulated phosphorylation at Ser24 in the pleckstrin homology domain of IRS-1 by mPLK/IRAK represents an additional mechanism for cross-talk between inflammatory signaling and insulin signaling that may contribute to metabolic insulin resistance. Inflammation contributes to insulin resistance in diabetes and obesity. Mouse Pelle-like kinase (mPLK, homolog of human IL-1 receptor-associated kinase (IRAK)) participates in inflammatory signaling. We evaluated IRS-1 as a novel substrate for mPLK that may contribute to linking inflammation with insulin resistance. Wild-type mPLK, but not a kinase-inactive mutant (mPLK-KD), directly phosphorylated full-length IRS-1 in vitro. This in vitro phosphorylation was increased when mPLK was immunoprecipitated from tumor necrosis factor (TNF)-α-treated cells. In NIH-3T3IR cells, wild-type mPLK (but not mPLK-KD) co-immunoprecipitated with IRS-1. This association was increased by treatment of cells with TNF-α. Using mass spectrometry, we identified Ser24 in the pleckstrin homology (PH) domain of IRS-1 as a specific phosphorylation site for mPLK. IRS-1 mutants S24D or S24E (mimicking phosphorylation at Ser24) had impaired ability to associate with insulin receptors resulting in diminished tyrosine phosphorylation of IRS-1 and impaired ability of IRS-1 to bind and activate PI-3 kinase in response to insulin. IRS-1-S24D also had an impaired ability to mediate insulin-stimulated translocation of GLUT4 in rat adipose cells. Importantly, endogenous mPLK/IRAK was activated in response to TNF-α or interleukin 1 treatment of primary adipose cells. In addition, using a phospho-specific antibody against IRS-1 phosphorylated at Ser24, we found that interleukin-1 or TNF-α treatment of Fao cells stimulated increased phosphorylation of endogenous IRS-1 at Ser24. We conclude that IRS-1 is a novel physiological substrate for mPLK. TNF-α-regulated phosphorylation at Ser24 in the pleckstrin homology domain of IRS-1 by mPLK/IRAK represents an additional mechanism for cross-talk between inflammatory signaling and insulin signaling that may contribute to metabolic insulin resistance. Biochemical, physiological, and epidemiological studies implicate pro-inflammatory cytokines (e.g. TNF-α, 1The abbreviations used are: TNF, tumor necrosis factor; IL, interleukin; IKKβ, IκB kinase β; PI, phosphatidylinositol; PH, pleckstrin homology; mPLK, Mouse Pelle-like kinase; IRAK, IL-1 receptor-associated kinase; JNK, c-Jun N-terminal kinase; KD, kinase dead; WT, wild type; GST, glutathione S-transferase; aa, amino acids; HA, hemagglutinin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PTB, phosphotyrosine binding; Ab, antibody; Tx, transfection. IL-1β, and IL-6) in the development of insulin resistance and the pathophysiology of type 2 diabetes and obesity (1Xu H. Barnes G.T. Yang Q. Tan G. Yang D. 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Haffner S.M. Circulation. 2000; 102: 42-47Crossref PubMed Scopus (2042) Google Scholar). These studies suggest an intriguing link between inflammation and metabolic dysregulation. Indeed, IκB kinase β (IKKβ), a critical mediator of inflammatory signaling pathways activating NF-κB, has been identified as an important inhibitor of metabolic insulin signaling pathways (9Yuan M. Konstantopoulos N. Lee J. Hansen L. Li Z.W. Karin M. Shoelson S.E. Science. 2001; 293: 1673-1677Crossref PubMed Scopus (1628) Google Scholar, 10Shoelson S.E. Lee J. Yuan M. Int. J. Obes. Relat. Metab. Disord. 2003; 27: 49-52Crossref PubMed Scopus (393) Google Scholar, 11Hundal R.S. Petersen K.F. Mayerson A.B. Randhawa P.S. Inzucchi S. Shoelson S.E. Shulman G.I. J. Clin. Investig. 2002; 109: 1321-1326Crossref PubMed Scopus (591) Google Scholar, 12Kim J.K. Kim Y.J. Fillmore J.J. Chen Y. Moore I. Lee J. Yuan M. Li Z.W. Karin M. Perret P. Shoelson S.E. Shulman G.I. J. Clin. Investig. 2001; 108: 437-446Crossref PubMed Scopus (620) Google Scholar). Inactivation of IKKβ signaling increases insulin sensitivity, whereas overexpression of IKKβ or activation of IKKβ by pro-inflammatory cytokines (e.g. TNF-α) leads to insulin resistance (9Yuan M. Konstantopoulos N. Lee J. Hansen L. Li Z.W. Karin M. Shoelson S.E. Science. 2001; 293: 1673-1677Crossref PubMed Scopus (1628) Google Scholar, 12Kim J.K. Kim Y.J. Fillmore J.J. Chen Y. Moore I. Lee J. Yuan M. Li Z.W. Karin M. Perret P. Shoelson S.E. Shulman G.I. J. Clin. Investig. 2001; 108: 437-446Crossref PubMed Scopus (620) Google Scholar). Similarly, JNK is another inflammatory signaling molecule that may play a role in the insulin resistance of obesity (13Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2634) Google Scholar). One potential explanation for these observations is cross-talk between inflammatory signaling and metabolic insulin signaling pathways. Metabolic actions of insulin such as enhanced glucose uptake into skeletal muscle and adipose tissue are regulated by activation of the insulin receptor tyrosine kinase and subsequent tyrosine phosphorylation of IRS-1. Although other IRS family members including IRS-2, -3, and -4 share similar structures, IRS isoforms have both overlapping and distinct functions (14Nystrom F.H. Quon M.J. Cell. Signal. 1999; 11: 563-574Crossref PubMed Scopus (157) Google Scholar). For example, IRS-1 knock-out mice have a phenotype of growth retardation and insulin resistance, whereas IRS-2 knock-out mice have a phenotype of frank diabetes due to β-cell failure (15Araki E. Lipes M.A. Patti M.E. Bruning J.C. Haag III, B. Johnson R.S. Kahn C.R. Nature. 1994; 372: 186-190Crossref PubMed Scopus (1095) Google Scholar, 16Withers 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 (1339) Google Scholar). Thus, IRS-1 seems to be the most important IRS isoform for mediating the metabolic effects of insulin in skeletal muscle and fat. Specific tyrosine-phosphorylated motifs on IRS-1 serve as docking sites for binding and activation of PI-3 kinase. Lipid products of PI 3-kinase stimulate activation of the serine kinase phosphoinositide-dependent protein kinase 1 that then phosphorylates and activates downstream serine kinases including Akt (protein kinase B) and protein kinase Cζ. These downstream PI 3-kinase-dependent signaling molecules promote translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface, resulting in increased glucose uptake into the cell (for review of metabolic insulin signaling pathways see Ref. 14Nystrom F.H. Quon M.J. Cell. Signal. 1999; 11: 563-574Crossref PubMed Scopus (157) Google Scholar). In addition to tyrosine phosphorylation sites on IRS-1 that are necessary for propagation of metabolic insulin signaling, IRS-1 contains numerous serine residues that are phosphorylated in response to treatment of cells with a variety of agents including insulin and TNF-α (17Tanti J.F. Gremeaux T. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 18Ricort J.M. Tanti J.F. Van Obberghen E. March Le -Brustel Y. Diabetologia. 1995; 38: 1148-1156Crossref PubMed Scopus (75) Google Scholar, 19Kroder G. Bossenmaier B. Kellerer M. Capp E. Stoyanov B. Muhlhofer A. Berti L. Horikoshi H. Ullrich A. Haring H. J. Clin. Investig. 1996; 97: 1471-1477Crossref PubMed Scopus (201) Google Scholar, 20Kanety H. Feinstein R. Papa M.Z. Hemi R. Karasik A. J. Biol. Chem. 1995; 270: 23780-23784Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 21Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2203) Google Scholar, 22Paz K. Hemi R. LeRoith D. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Increased serine phosphorylation of IRS-1 is generally associated with impaired tyrosine phosphorylation of IRS-1 by the insulin receptor, decreased IRS-1-associated PI 3-kinase binding and activity, and insulin resistance (17Tanti J.F. Gremeaux T. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 20Kanety H. Feinstein R. Papa M.Z. Hemi R. Karasik A. J. Biol. Chem. 1995; 270: 23780-23784Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 21Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2203) Google Scholar, 23Mothe I. Van Obberghen E. J. Biol. Chem. 1996; 271: 11222-11227Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 24Qiao L.Y. Zhande R. Jetton T.L. Zhou G. Sun X.J. J. Biol. Chem. 2002; 277: 26530-26539Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Interestingly, a number of PI 3-kinase-dependent serine kinases including Akt, protein kinase Cζ, and glycogen synthase kinase 3 can phosphorylate IRS-1 on serine residues and may participate in feedback regulation of insulin signal transduction (25Paz K. Liu Y.F. Shorer H. Hemi R. LeRoith D. Quon M.J. Kanety H. Seger R. Zick Y. J. Biol. Chem. 1999; 274: 28816-28822Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 26Li J. DeFea K. Roth R.A. J. Biol. Chem. 1999; 274: 9351-9356Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 27Ravichandran L.V. Esposito D.L. Chen J. Quon M.J. J. Biol. Chem. 2001; 276: 3543-3549Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 28Eldar-Finkelman H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9660-9664Crossref PubMed Scopus (283) Google Scholar). Moreover, activation of pro-inflammatory kinases IKKβ and JNK leads to increased serine phosphorylation of IRS-1 at Ser307 and insulin resistance (29Aguirre V. Uchida T. Yenush L. Davis R. White M.F. J. Biol. Chem. 2000; 275: 9047-9054Abstract Full Text Full Text PDF PubMed Scopus (1172) Google Scholar, 30Rui L. Aguirre V. Kim J.K. Shulman G.I. Lee A. Corbould A. Dunaif A. White M.F. J. Clin. Investig. 2001; 107: 181-189Crossref PubMed Scopus (486) Google Scholar, 31Gao Z. Hwang D. Bataille F. Lefevre M. York D. Quon M.J. Ye J. J. Biol. Chem. 2002; 277: 48115-48121Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, 32Aguirre V. Werner E.D. Giraud J. Lee Y.H. Shoelson S.E. White M.F. J. Biol. Chem. 2002; 277: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar). Other serine residues in the C-terminal region of IRS-1 (e.g. Ser612) also undergo phosphorylation in response to pro-inflammatory cytokines (33Rotter V. Nagaev I. Smith U. J. Biol. Chem. 2003; 278: 45777-45784Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). The identity of kinases that directly phosphorylate these specific serine residues on IRS-1 in response to activation of IKKβ or treatment with pro-inflammatory cytokines remains unclear. In addition, other unidentified serine phosphorylation sites on IRS-1 and additional pro-inflammatory kinases may also participate in cross-talk between inflammatory signaling and insulin signaling. Thus, detailed molecular mechanisms relating inflammation with insulin resistance remain incompletely understood. Mouse Pelle-like kinase (mPLK) is a Ser/Thr kinase homologous to the Drosophila innate immune kinase Pelle and is the mouse homologue of the human IL-1 receptor-associated kinase-1 (IRAK-1) (34Vigon I. Mornon J.P. Cocault L. Mitjavila M.T. Tambourin P. Gisselbrecht S. Souyri M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5640-5644Crossref PubMed Scopus (469) Google Scholar). In response to TNF-α or IL-1β stimulation, IRAK-1 is recruited to the IL-1 receptor complex through the adaptor MyD88 (35Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar), where IRAK-1 subsequently becomes phosphorylated. IRAK-1 then interacts with TNF receptor-associated factor 6, TGFβ-activating kinase, and NF-κB-inducing kinase (36Jiang Z. Ninomiya-Tsuji J. Qian Y. Matsumoto K. Li X. Mol. Cell. Biol. 2002; 22: 7158-7167Crossref PubMed Scopus (238) Google Scholar, 37Takaesu G. Ninomiya-Tsuji J. Kishida S. Li X. Stark G.R. Matsumoto K. Mol. Cell. Biol. 2001; 21: 2475-2484Crossref PubMed Scopus (159) Google Scholar, 38Wang Q. Dziarski R. Kirschning C.J. Muzio M. Gupta D. Infect. Immun. 2001; 69: 2270-2276Crossref PubMed Scopus (159) Google Scholar) resulting in activation of IKKβ and JNK and finally NF-κB (39Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (144) Google Scholar, 40Vig E. Green M. Liu Y. Yu K.Y. Kwon H.J. Tian J. Goebl M.G. Harrington M.A. J. Biol. Chem. 2001; 276: 7859-7866Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 41Vig E. Green M. Liu Y. Donner D.B. Mukaida N. Goebl M.G. Harrington M.A. J. Biol. Chem. 1999; 274: 13077-13084Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Although IRAK-1 is required for activation of NF-κB in response to IL-1 (42Croston G.E. Cao Z. Goeddel D.V. J. Biol. Chem. 1995; 270: 16514-16517Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), the requirement for IRAK-1 kinase activity in NF-κB activation is controversial (41Vig E. Green M. Liu Y. Donner D.B. Mukaida N. Goebl M.G. Harrington M.A. J. Biol. Chem. 1999; 274: 13077-13084Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 43Maschera B. Ray K. Burns K. Volpe F. Biochem. J. 1999; 339: 227-231Crossref PubMed Scopus (79) Google Scholar, 44Knop J. Martin M.U. FEBS Lett. 1999; 448: 81-85Crossref PubMed Scopus (90) Google Scholar). mPLK catalytic activity is required for full activation of the TNF-α pathway, leading to NF-κB activation. However, physiological substrates for mPLK/IRAK-1 have not been conclusively identified. Because mPLK participates in activation of IKKβ, JNK, and NF-κB, we hypothesized that IRS-1 may be a novel substrate for mPLK, which mediates cross-talk between pro-inflammatory signaling and insulin signaling pathways. In the present study we identified Ser24 in the pleckstrin homology (PH) domain of IRS-1 as a phosphorylation site for mPLK that may contribute to insulin resistance. mPLK-WT and mPLK-KD—cDNA for full-length wild type mPLK with a C-terminal Myc-epitope tag was subcloned into pcDNA3.1 as described (45Trofimova M. Sprenkle A.B. Green M. Sturgill T.W. Goebl M.G. Harrington M.A. J. Biol. Chem. 1996; 271: 17609-17612Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). mPLK-KD is a kinase-inactive point mutant of mPLK-WT (D358N) as described (41Vig E. Green M. Liu Y. Donner D.B. Mukaida N. Goebl M.G. Harrington M.A. J. Biol. Chem. 1999; 274: 13077-13084Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). GST-IRS-1N, GST-IRS-1M, and GST-IRS-1C—GST fusion constructs containing fragments of rat IRS-1 (N, aa 2–516; M, aa 526–859; C, aa 900–1235) were generous gifts from Xiao Jian Sun (46Zhande R. Mitchell J.J. Wu J. Sun X.J. Mol. Cell. Biol. 2002; 22: 1016-1026Crossref PubMed Scopus (182) Google Scholar). IRS-1-WT—cDNA for human IRS-1 with a C-terminal HA-epitope tag was subcloned into pCIS2 mammalian expression vector as described (47Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar). IRS-1-S24A, IRS-1-S24D, IRS-1-S24E—Point mutants of IRS-1-WT were created using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. GST-IRS-1-(2–336), GST-IRS-1-(2–336)-S24A—Expression vectors for GST fusion proteins containing aa 1–336 of hIRS-1 and the corresponding S24A point mutant were constructed. Human IRS-1 cDNA template was amplified by PCR. Primers 5′-cgc gga tcc GCG AGC CCT CCG GAG AGC-3′ and 5′-AT GGT GCC TTC GCC GTC ACT-3′ were used for PCR. The PCR product was gel-purified, digested with Bam H1, and ligated into pGex 4T-1 (Amersham Biosciences) that was pre-digested with Bam H1 and SmaI. GST-IRS-1-(1–336)-S24A was constructed from GST-IRS-1-(1–336) using the QuikChange site-directed mutagenesis kit. DNA sequences for both wild type and mutants of GST-IRS-1 constructs were confirmed by direct sequencing. GLUT4-HA—An expression vector for human GLUT4 with the influenza hemagglutinin epitope (HA1) inserted in the first exofacial loop of GLUT4 was used as described (48Quon M.J. Guerre-Millo M. Zarnowski M.J. Butte A.J. Em M. Cushman S.W. Taylor S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5587-5591Crossref PubMed Scopus (87) Google Scholar). Antibodies—Anti-IRS-1, anti-phosphotyrosine (4G10), and the anti-p85 subunit of PI 3-kinase antibodies were purchased from Upstate Biotechnology, Inc. (Charlottesville, VA), anti-IRAK-1 antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and anti-GST antibody was purchased from Amersham Biosciences (Piscataway, NJ). Anti-c-Myc antibody was purchased from Roche Applied Science (Indianapolis, IN). Anti-HA antibody was purchased from Covance (Denver, PA). NIH-3T3 fibroblasts stably overexpressing human insulin receptors (NIH-3T3IR), Cos-7, HEK293, HepG2, and FaO cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine in a humidified atmosphere with 5% CO2. Bovine aortic endothelial cells (Clonetics Corp., San Diego, CA) in primary culture were grown in EGM-2 MV (Cambrex Biosciences, Walkersville, MD) as described (49Zeng G. Quon M.J. J. Clin. Investig. 1996; 98: 894-898Crossref PubMed Scopus (665) Google Scholar) and used at passages 3–4. The evening before transfection cells were seeded in 60-mm dishes at 50% confluence. Polyfect (Qiagen Inc., Valencia, CA) was used to transiently transfect cells according to the manufacturer's instructions. One day after transfection cells were serum-starved overnight and then treated without or with TNF-α or insulin as described in figure legends. Rat adipose cells in primary culture were prepared and transfected as previously described (50Quon M.J. Zarnowski M.J. Guerre-Millo M. de la Luz Sierra M. Taylor S.I. Cushman S.W. Biochem. Biophys. Res. Commun. 1993; 194: 338-346Crossref PubMed Scopus (71) Google Scholar, 51Quon M.J. Chen H. Ing B.L. Liu M.L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar). Before cell lysis, cells were briefly washed with ice-cold phosphate-buffered saline. Cells were lysed with lysis buffer containing 50 mm Tris, pH 7.2, 125 mm NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 1 mm EDTA, 1 mm NaVO3, 20 mm NaF, 1 mm sodium pyrophosphate, and complete protease inhibitor mixture (Roche Applied Science). Cell debris were removed by centrifugation at 17,000 × g for 10 min at 4 °C. Samples were then boiled with Laemmli sample buffer for 5 min, and proteins were resolved by 10% SDS-PAGE. Samples were immunoblotted by standard methods, and blots were quantified by scanning densitometry (Molecular Dynamics). Bacteria transformed with GST-IRS-1 constructs were grown in LB media containing 50 mg/ml ampicillin. This starter culture was diluted 1/10 in to 100 ml of LB medium containing 50 mg/ml ampicillin and grown at 37 °C for 2 h with vigorous shaking (300 rpm) until the culture reached A 0.6. GST-IRS-1 fusion proteins were induced with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside for 2 h at 37 °C. Bacteria were pelleted by centrifugation at 2236 × g for 10 min. The pellet was resuspended in 4 ml of ice-cold phosphate-buffered saline, and cells were lysed by 20-s/10-s-interval sonication. The lysate was centrifuged at 15,115 × g for 10 min, and the supernatant was incubated with 0.6 ml of 50% glutathione-Sepharose 4B slurry at 4 °C for 30 min. Sepharose beads were washed with ice-cold phosphate-buffered saline 3 times, and GST-IRS-1 fusion proteins were eluted with 10 mm reduced glutathione, 50 mm Tris-HCl, pH 8.0. In some cases proteins were concentrated with a YM-10 column (Millipore Corp., Bedford, MA). Cos-7 cells or NIH-3T3IR cells were transiently transfected with empty vector (control), mPLK-WT, or mPLK-KD. In some experiments cells were serum-starved overnight and then treated without or with TNF-α (10 ng/ml, 5 min). Recombinant mPLK was immunoprecipitated by incubating cell lysates (1 mg of total protein) with 1 μg of anti-Myc antibody for 2 h at 4 °C followed by incubation with protein A/G-agarose beads (Santa Cruz Biotechnology) at 4 °C for 1 h. The immunocomplex was washed once with cell lysis buffer, once with buffer B (20 mm Tris, pH 7.4, 150 mm NaCl, 0.1% Nonidet P-40), and twice with buffer C (20 mm Tris. pH 7.4, 150 mm NaCl). The immunocomplex was then incubated in kinase assay buffer (50 mm Tris, pH 8.0, 10 mm MgCl2, 1 mm dithiothreitol, 50 μm ATP) with 10 μCi of [γ32P]ATP and either purified full-length IRS-1 (1 μg) (Upstate Biotechnology) or purified GST-IRS-1 fusion proteins (2 μg) for 1 h at 37 °C. The reaction was stopped by adding Laemmli sample buffer and boiling for 5 min. Samples were then subjected to 10% SDS-PAGE, transferred to nitrocellulose membranes, and exposed to x-ray film or phosphor screens for autoradiography or PhosphorImager analysis (Storm 860, Amersham Biosciences Corp.). NIH-3T3IR cells transiently transfected with IRS-1-WT, IRS-1-S24A, IRS-1-S24D, or IRS-1-S24E were serum-starved overnight and treated without or with insulin (100 nm, 5 min). Cell lysates were then subjected to immunoprecipitation with anti-HA antibody to recover recombinant IRS-1 proteins. ⅘ of each sample was used for detection of co-immunoprecipitated p85 subunit of PI 3-kinase by immunoblotting with anti-p85 antibody. The remainder of the sample immunoprecipitate was used to detect IRS-1-associated PI 3-kinase activity. For each sample, 10 μg of sonicated phosphatidylinositol substrate (Sigma) was incubated with 50 μl of PI 3-kinase reaction buffer (20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.3 mm EGTA, 10 mm MgCl2) and with 10 μCi of [γ32P]ATP for 10 min at 37 °C. The reaction was stopped by adding 100 μl of 0.1 n HCl. Phospholipid was extracted with 200 μl of CHCl3/CH3OH (1:1). The organic phase was collected and applied to silica gel thin layer chromatography plates (Whatman) pre-activated with 1% potassium oxalate. Thin layer chromatography was performed in CHCl3/CH3OH/H2O/NH4OH (60:47:12.3:2). The plates were dried, and results were visualized by autoradiography. Quantification was done by PhosphorImager analysis, and results were normalized for the amount of IRS-1 recovered by immunoprecipitation. Cell lysates were incubated with primary antibody at 4 °C for 2 h. The immune complex was pulled down with protein A/G beads. The beads were washed three times with cell lysis buffer, and then samples were mixed with SDS-sample buffer and boiled for 5 min. The samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blotted with antibodies as indicated. Rat adipose cells were co-transfected by electroporation with GLUT4-HA and either IRS-1-WT or IRS-1-S24D as described (50Quon M.J. Zarnowski M.J. Guerre-Millo M. de la Luz Sierra M. Taylor S.I. Cushman S.W. Biochem. Biophys. Res. Commun. 1993; 194: 338-346Crossref PubMed Scopus (71) Google Scholar, 51Quon M.J. Chen H. Ing B.L. Liu M.L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar). After 20 h cells were processed and treated with insulin at final concentrations of 0, 0.07, or 60 nm for 30 min at 37 °C. Cell surface epitope-tagged GLUT4 was determined using monoclonal anti-HA antibody (HA-11, Covance) in conjunction with 125I-labeled sheep anti-mouse IgG as described (51Quon M.J. Chen H. Ing B.L. Liu M.L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar). One μl of tryptic-digested IRS-1 sample was co-crystallized with 1 μl of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 1% trifluoroacetic acid and spotted directly on a stainless steel matrix-assisted laser desorption ionization (MALDI) plate. Mass spectra were acquired using an Applied Biosystems 4700 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA). For all mass spectra the laser frequency was 200 Hz, and for collision-induced dissociation the collision energy was 1 keV (air was used as the collision gas). MALDI spectra were internally calibrated (<20 ppm) using trypsin autolysis products. Post-acquisition base-line correction and smoothing was carried out using the software provided with the instrument. Spectra were submitted to Mascot (matrixscience.com) for peptide mass fingerprinting. The phospho-peptide DVRKVGYLRKPKpSMHK (pS, phosphorylated Ser) was synthesized and conjugated to keyhole limpet hemocyanin as an antigen. New Zealand White rabbits were immunized and boosted three times with the conjugate. Anti-serum was further purified by collecting through an affinity column that was coupled with the non-phosphorylated DVRKVGYLRKPKSMHK peptide. Insulin Target Cells Express mPLK/IRAK—We first examined whether mPLK (or its orthologue such as IRAK) is endogenously expressed in cells from metabolic and vascular insulin target tissues. Endogenous IRAK was detected in lysates of both rat adipose cells and bovine aortic endothelial cells (BAEC) by immunoblotting with anti-IRAK antibody (Fig. 1A). In addition, we also detected endogenous mPLK/IRAK in COS-7 cells and NIH-3T3IR cells. Using an antibody that can detect the activated form of mPLK/IRAK (52Mamidipudi V. Lin C. Seibenhener M.L. Wooten M.W. J. Biol. Chem. 2004; 279: 4161-4165Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), we found that IL-1β or TNF-α treatment of primary adipose cells or endothelial cells activated endogenous mPLK/IRAK in insulin target cells (Fig. 1B). Interestingly, treatment of Fao hepatoma cells with insulin also activated mPLK/IRAK at levels comparable with those observed in response to IL-1β (data not shown). IRS-1 Is a Direct Substrate for mPLK in Vitro—We next tested whether mPLK can directly phosphorylate IRS-1 in vitro. An immune-complex kinase assay was performed using full-length purified IRS-1 as substrate and recombinant mPLK-WT or mPLK-KD immunoprecipitated from transiently transfected NIH-3T3IR cells treated without or with TNF-α (Fig. 2). IRS-1 underwent significant phosphorylation only in the presence of mPLK-WT (Fig. 2, A and C, lanes 3 and 4) but not when incubated with mPLK-KD (A and C, lanes 5 and 6) or control samples (A and C, lanes 1 and 2). Moreover, mPLK-WT immunoprecipitated from cells treated with TNF-α was able to phosphorylate IRS-1 to a greater extent than mPLK-WT from untreated cells (Fig. 2, A and C, cf. lanes 3 and 4). As expected, mPLK-WT (but not mPLK-KD) underwent autophosphorylation that was increased by TNF-α treatment (Fig. 2A, lower panel, lanes 3 and 4). Immunobl" @default.
• W2016155391 created "2016-06-24" @default.
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• W2016155391 date "2005-06-01" @default.
• W2016155391 modified "2023-10-01" @default.
• W2016155391 title "Phosphorylation of Ser24 in the Pleckstrin Homology Domain of Insulin Receptor Substrate-1 by Mouse Pelle-like Kinase/Interleukin-1 Receptor-associated Kinase" @default.
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