Protein Kinase C-ζ-induced Phosphorylation of Ser318 in Insulin Receptor Substrate-1 (IRS-1) Attenuates the Interaction with the Insulin Receptor and the Tyrosine Phosphorylation of IRS-1

Abstract

Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-, we identified Ser 318 of rat IRS-1 (Ser 323 in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser 318 to alanine). To monitor phosphorylation of Ser 318 in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK IR ). Using the phospho-Ser 318 -specific antibody we observed that insulin stimulates phosphorylation of Ser 318 in IRS-1, which is mediated, at least partially, by PKC-. Moreover, we found that the previously described insulin-stimulated, PKC--mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser 318 to alanine. These results, generated in BHK IR cells, suggest that phosphorylation of Ser 318 by PKC-might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.Insulin resistance is associated with a variety of physiological and patho-physiological states, including type 2 diabetes, hypertension, glucose intolerance, and obesity; however, the molecular mechanisms that modulate insulin signaling under these conditions are difficult to resolve. One important early site of divergence in insulin signaling, which seems to be a relevant target for modulation of the signal, is insulin receptor substrate 1 (IRS-1) 1 (1). IRS-1 is a hydrophilic protein with an apparent molecular mass of 180 kDa containing a conserved pleckstrin homology domain located at the amino terminus, adjacent to a phosphotyrosine binding (PTB) domain (2). After insulin-stimulation, this domain interacts with the activated insulin receptor (IR), IRS-1 is subsequently tyrosine-phosphorylated, and the signal is transmitted further (1, 2).In addition to tyrosine phosphorylation, IRS-1 undergoes Ser/Thr phosphorylation at multiple sites (3, 4). Many in vitro and in vivo studies (5-13) have shown that increased Ser/Thr phosphorylation of IRS-1, e.g. after treatment of cells with either activators of protein kinase C (PKC), Ser/Thr phosphatase inhibitors, high insulin concentrations, or activation of cellular stress pathways by tumor necrosis factor ␣ and other cytokines, inhibits the IR-mediated tyrosine phosphorylation of IRS-1, thereby affecting insulin signal transduction.The atypical PKC-, which is activated by insulin, has been identified to transduce insulin signaling downstream from IRS-1 and phosphatidylinositol 3-kinase in mediating glucose uptake. However, as reported recently, pr...

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.…”

Serine 332 Phosphorylation of Insulin Receptor Substrate-1 by Glycogen Synthase Kinase-3 Attenuates Insulin Signaling

“…The mechanisms by which phosphorylation of serines 332 and 336 inhibits IRS-1 tyrosine phosphorylation is not fully clear. Given that those sites are found in close proximity to the PTB domain, which is important in facilitating IR-IRS-1 interaction, their phosphorylation may disrupt this interactions and thus inhibit subsequent IRS-mediated signaling (28,56,57). Alternatively, phosphorylation of those sites might recruit signaling molecules, which sterically inhibit interactions between the PTB domain of IRS-1 and the NPEY motif of the IR, preventing tyrosine phosphorylation of IRS-1 by the IR kinase (58,59).…”

“…PKC isoforms were shown to enhance serine phosphorylation of IRS-1 as well (24 -27). Specifically, phorbol 12-myristate 13-acetate-sensitive PKC isoforms were implicated in the enhanced phosphorylation of Ser 612 (24), and PKC-was shown to phosphorylate serines 570 and 318 (28,29). Phosphorylation of serines 632, 662, and 731 was shown to be associated with desensitization of insulin signaling (30,31), and nutrient-dependent stimulation enhanced the phosphorylation of Ser 302 (32); subsequently, increased phosphorylation of this serine was detected in diabetic liver (19).…”

Oxidative Stress Inhibits Insulin-like Growth Factor-I Induction of Chondrocyte Proteoglycan Synthesis through Differential Regulation of Phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK Signaling Pathways

“…Activation of IRS-1 requires tyrosine phosphorylation (in particular Tyr-612) (31), whereas serine phosphorylation at a variety of sites has been shown to be inhibitory (19,20). Depending on the specific serine residue, phosphorylation can be mediated through a variety of kinases, including protein kinase C (40,41), IB kinase (42), mTOR (43), MEK-ERK (44,45), and JNK1/2 (19,46).…”