PKCd-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function q Michael W. Greene a, * , Mary S. Ruhoff a , Richard A. Roth b , Jeong-a Kim c , Michael J. Quon c , Jean A. Krause a a Bassett Research Institute, Mary Imogene Bassett Hospital, Cooperstown, NY 13326, USA b Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, USA c Diabetes Unit, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, MD 20892, USA Received 16 August 2006 Available online 5 September 2006 Abstract The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKCd on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKCd-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKCd catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Insulin receptor substrate; PKC; Insulin resistance; Serine phosphorylation; shRNA Insulin is the principal regulator of blood glucose con- centration primarily through its combined actions on glu- cose uptake in muscle and on inhibition of hepatic glucose production. Insulin promotes the storage of fuel in muscle, liver, and fat by stimulating glycogen synthesis and lipogenesis while inhibiting glycogenolysis and lipoly- sis [1]. The inability of normal concentrations of insulin to stimulate these processes is termed insulin resistance. Insulin resistance is the single best predictor for the devel- opment of Type 2 diabetes in people with a family history of the disease [2]. Type 2 diabetes afflicts an estimated 6% of the adult population in Western societies [3]. At the molecular level, insulin action is mediated by acti- vation of the intrinsic Tyr kinase of the insulin receptor (IR) [4]. Tyr phosphorylation of IRS-1 and IRS-2 initiates an intracellular signaling cascade [1,5]. Src homology 2 (SH2) domain-containing proteins bind to the activated Tyr phosphorylated IRS proteins triggering the activation of the Ras/MAPK and phosphatidylinositol-3 (PI-3) kinase/Akt/mTOR signaling pathways [6,7]. Structurally, IRS-1 is composed of a pleckstrin homology (PH) domain and phosphotyrosine binding (PTB) domain at the N-terminus. The IRS-1 PH domain is a phosphatidylinosi- tol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol- 3,4,5-triphosphate (PI(3,4,5)P3) binding module [8] while 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.08.158 q Abbreviations: IRS, insulin receptor substrate; IR, insulin receptor; PI 3-kinase, phosphatidylinositol 3-kinase; PDK1, phosphoinositide-depen- dent kinase1; PH, pleckstrin homology; PTB, phosphotyrosine binding domain; PI, phosphatidylinositol; SH2, src homology 2; HEK, human embryonic kidney; wt, wild-type; JNK, c-Jun N-terminal kinase; mTOR, mammalian target of rapamycin; IKK, IjB kinase: S6K, S6 kinase; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; FBS, fetal bovine serum; GST, glutathi- one-S-transferase. * Corresponding author. Fax: +1 607 547 3061. E-mail address: michael.greene@bassett.org (M.W. Greene). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 349 (2006) 976–986 BBRC