1 PHOSPHORYLATION OF SER 24 IN THE PH DOMAIN OF IRS-1 BY mPLK/IRAK: CROSS-TALK BETWEEN INFLAMMATORY SIGNALING AND INSULIN SIGNALING THAT MAY CONTRIBUTE TO INSULIN RESISTANCE* Jeong-a Kim, Deborah C. Yeh, Marel Ver, Yunhua Li, Andrea Carranza, Thomas P. Conrads #, Timothy D. Veenstra # , Maureen A. Harrington + , and Michael J. Quon From the Diabetes Unit, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, MD 20892; #- SAIC-Frederick Inc., Laboratory of Proteomics and Analytical Technologies, Mass Spectrometry Center, National Cancer Institute at Frederick, Frederick, MD 21702-1201; + - Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and Walther Cancer Institute, Indianapolis, IN 46202 Running Title: Phosphorylation of IRS-1 by mPLK/IRAK Address correspondence to: Michael J. Quon, M.D., Ph.D., Chief, Diabetes Unit, NCCAM, NIH, Building 10, Room 6C-205, 10 Center Drive MSC 1632, Bethesda, MD 20892-1632, Tel. 301 496-6269; Fax. 301 402-1679; E-Mail: quonm@nih.gov Inflammation contributes to insulin resistance in diabetes and obesity. This involves activation of IKKβ and JNK leading to increased serine phosphorylation of IRS-1. Mouse Pelle-like kinase (mPLK, homolog of human IL-1-receptor-associated kinase (IRAK)) also 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 TNF-α-treated cells. In NIH-3T3 IR 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 Ser 24 in the PH domain of IRS-1 as a specific phosphorylation site for mPLK. IRS-1 mutants S24D or S24E (mimicking phosphorylation at Ser 24 ) 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 IL-1 treatment of primary adipose cells. In addition, using a phospho-specific antibody against IRS-1 phosphorylated at Ser 24 , we found that IL-1 or TNF-α treatment of Fao cells stimulated increased phosphorylation of endogenous IRS-1 at Ser 24 . We conclude that IRS-1 is a novel physiological substrate for mPLK. TNF-α-regulated phosphorylation at Ser 24 in the PH 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, IL-1β, and IL-6) in the development of insulin resistance and the pathophysiology of type 2 diabetes and obesity (1-8). 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 (9-12). Inactivation of IKKβ signaling increases insulin sensitivity while overexpression of IKKβ or activation of IKKβ by pro-inflammatory cytokines (e.g., TNF-α) leads to insulin resistance (9,12). Similarly, JNK is another inflammatory signaling molecule that may play a role in the insulin resistance of obesity (13). 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 strucutres, IRS isoforms have both overlapping and distinct functions (14). For example, IRS-1 knockout mice have a phenotype of growth retardation and insulin resistance while IRS-2 knockout mice have a phenotype of frank diabetes due to beta-cell failure (15,16). Thus, IRS-1 seems to be the most important IRS isoform for mediatic 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 JBC Papers in Press. Published on April 22, 2005 as Manuscript M501439200 by guest on January 26, 2017 http://www.jbc.org/ Downloaded from