Phosphorylation of Ser24 in the Pleckstrin Homology Domain of Insulin Receptor Substrate-1 by Mouse Pelle-like Kinase/Interleukin-1 Receptor-associated Kinase

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. Wildtype 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)-␣-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-␣. Using mass spectrometry, we identified Ser 24 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 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-␣ or interleukin 1 treatment of primary adipose cells. In addition, using a phospho-specific antibody against IRS-1 phosphorylated at Ser 24 , we found that interleukin-1 or TNF-␣ 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-␣-regulated phosphorylation at Ser 24 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-␣, 1 IL-1␤, 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, IB kinase ␤ (IKK␤), a critical mediator of inflammatory signaling pathways activating NF-B, has been identified as an important inhibitor of metabolic insulin signaling pathways (9 -12). Inactivation of IKK␤ signaling increases insulin sensitivity, whereas overexpression of IKK␤ or activation of IKK␤ by pro-inflammatory cytokines (e.g. TNF-␣) 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 o...

Common Inhibitory Serine Sites Phosphorylated by IRS-1 Kinases, Triggered by Insulin and Inducers of Insulin Resistance

“…Several candidate Ser residues were identified as potential targets for IRS-1 kinases. These include Ser-24 (11), -302 (12), -307 (13), -318 (14), -408 (10), -612 (15), -636 and -639 (16), -731 (17), and -789 (18). 3 Similarly, a number of kinases, including extracellular signal-regulated kinase, protein kinase C (PKC), IKK␤, JNK, S6K1, AMP kinase, and mTOR were implicated as potential IRS kinases (reviewed in Ref.…”

Interleukin 1β Regulation of FoxO1 Protein Content and Localization

“…The IL-1␤ action has been associated with down-regulation of the insulin signaling pathway at the insulin receptor level (28). Indeed, several key mediators of IL-1␤ signaling pathway, namely ceramide (29 -34), IRAK-1 (35), and JNK (36,37) have been shown to inhibit IRS-1 and/or Akt-1 activation. These studies indicate that IL-1␤ may suppress the cellular insulin response.…”

Epigallocatechin Gallate, a Green Tea Polyphenol, Mediates NO-dependent Vasodilation Using Signaling Pathways in Vascular Endothelium Requiring Reactive Oxygen Species and Fyn

“…Cell lysates were then prepared and subjected to immunoprecipitation with specific antibodies against phosphotyrosine (4G10, Upstate, Charlottesville, VA), Cbl (BD Biosciences, San Jose, CA), or Gab-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) according to standard methods as described (14). PI 3-kinase activity in the immunoprecipitates was detected as follows: for each sample, 10 g of sonicated phosphatidylinositol substrate (Sigma) was incubated with 50 l of PI 3-kinase reaction buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.3 mM EGTA, 10 mM MgCl 2 ) and Measurement of NO Production-Production of NO was assessed using NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA; Cayman Chemical, Ann Arbor, MI) as described previously (15).…”