Abstract
The Ser/Thr phosphorylation of insulin receptor substrate 1 (IRS) is one key mechanism to stimulate and/or attenuate insulin signal transduction. Using a phospho-specific polyclonal antibody directed against phosphorylated Ser(318) of IRS-1, we found a rapid and transient insulin-stimulated phosphorylation of Ser(318) in human and rodent skeletal muscle cell models and in muscle tissue of insulin-treated mice. None of the investigated insulin resistance-associated factors (e.g. high glucose, tumor necrosis factor-alpha, adrenaline) stimulated the phosphorylation of Ser(318). Studying the function of this phosphorylation, we found that replacing Ser(318) by alanine completely prevented both the attenuation of insulin-stimulated Akt/protein kinase B Ser(473) phosphorylation and glucose uptake after 60 min of insulin stimulation. Unexpectedly, after acute insulin stimulation, we observed that phosphorylation of Ser(318) is not inhibitory but rather enhances insulin signal transduction because introduction of Ala(318) led to a reduction of the insulin-stimulated Akt/protein kinase B phosphorylation. Furthermore, replacing Ser(318) by glutamate, i.e. mimicking phosphorylation, improved glucose uptake after acute insulin stimulation. These data suggest that phosphorylation of Ser(318) is not per se inhibitory but is necessary to trigger the attenuation of the insulin-stimulated signal in skeletal muscle cells. Investigating the molecular mechanism of insulin-stimulated Ser(318) phosphorylation, we found that phosphatidylinositol 3-kinase-mediated activation of atypical protein kinase C-zeta and recruitment of protein kinase C-zeta to IRS-1 was responsible for this phosphorylation. We conclude that Ser(318) phosphorylation of IRS-1 is an early physiological event in insulin-stimulated signal transduction, which attenuates the continuing action of insulin.
Highlights
Termination of this cascade can be achieved by mechanisms whereby kinases modify elements of the signaling cascade, leading to an initial enhancement of signal transduction and to a sequential attenuation
This suggests that insulin action, besides an initial insulin receptor substrate 1 (IRS)-1 tyrosine phosphorylation, may lead to a rapid phosphorylation of serine residues of insulin receptor substrate 1 (IRS-1) possibly necessary to contribute to a maximal stimulation of the insulin signal transduction
We investigated the effects of different substances known to affect insulin signal transduction on the phosphorylation of Ser318 using human myotubes obtained from biopsies from quadriceps femoris and L6 myotubes
Summary
Termination of this cascade can be achieved by mechanisms whereby kinases modify elements of the signaling cascade, leading to an initial enhancement of signal transduction and to a sequential attenuation. Reports investigating pathological insulin resistance-associated stimuli pointed to the involvement of Ser phosphorylation of IRS-1 in the desensitization of insulin action [2,3,4], but there is increasing evidence that Ser phosphorylation of IRS-1 may serve as a tool to positively modulate insulin signaling under physiological conditions [5,6,7,8] This suggests that insulin action, besides an initial IRS-1 tyrosine phosphorylation, may lead to a rapid phosphorylation of serine residues of IRS-1 possibly necessary to contribute to a maximal stimulation of the insulin signal transduction. And Sequential Role of Ser(P)318 of IRS-1 stimuli; 2) the rapid phosphorylation of Ser318 exerts an enhancing effect on insulin signal transduction and glucose uptake and is necessary for the subsequent attenuation of insulin signaling and action as assessed by Ser473 phosphorylation of Akt/PKB and glucose uptake; 3) the insulin-stimulated phosphorylation in muscle cells is mediated by atypical PKC-
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