The Phosphorylation of Ser318 of Insulin Receptor Substrate 1 Is Not per se Inhibitory in Skeletal Muscle Cells but Is Necessary to Trigger the Attenuation of the Insulin-stimulated Signal

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 resistanceassociated factors (e.g. high glucose, tumor necrosis factor-␣, 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 insulinstimulated 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-and recruitment of protein kinase C-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.

Phosphorylation of Ser357 of Rat Insulin Receptor Substrate-1 Mediates Adverse Effects of Protein Kinase C-δ on Insulin Action in Skeletal Muscle Cells

“…the previously published PKC-␦-dependent sites Ser 24 and Ser 318 . The phosphorylation of Ser 357 did not result in the dissociation of PKC-␦ and IRS-1, as was reported for PKC-and IRS-1 after phosphorylation of Ser 318 (22), but even appeared to result in an enhanced recruitment. This could facilitate other serine phosphorylation events on IRS-1 or its receptor, leading to the described reduced tyrosine phosphorylation and attenuation of insulin signaling (41).…”

“…The cells were starved in Dulbecco's modified Eagle's medium (5.5 mM glucose) without fetal calf serum for 24 h and then stimulated with insulin (10 nM) or TPA (0.5 M) as indicated. Human myotubes were cultured in ␣-minimum essential medium containing 5.5 mM glucose with 2% fetal calf serum and 2% antibiotic antimycotic solution (22). C2C12 cells were cultured in Dulbecco's modified Eagle's medium containing 25 mM glucose, 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin.…”

“…Although particularly the activation of atypical PKCs has been reported as positive modulation of insulin signaling (15,16), the majority of studies has demonstrated that PKCs are implicated in impaired insulin signaling including classical (17)(18)(19), novel (20), and more recently also atypical PKC isoforms (21). Activation of PKC isoforms by insulin (11,21,22), hyperglycemia (18,23), and lipids (24 -27) leads to reduced insulin action and insulin resistance. The knock-out of PKC-␣ in mice or inhibition of PKC-⑀ in rats enhances insulin signaling (28,29), and PKC--deficient mice are protected from fat-induced insulin resistance (30).…”

Role of Central Insulin-Like Growth Factor-1 Receptor Signalling in Ageing and Endocrine Regulation

“…These phosphorylations lead to specific regulation of downstream signalling. The phosphorylation of serine residues of IRS-1 contribute to positive or negative regulation of IRS-1 action (Weigert et al 2005;Weigert et al 2008). Important for this regulation are the particular phosphorylation sites (Herschkovitz et al 2007) as well as the timing of phosphorylation (Weigert et al 2005;Weigert et al 2008).…”

“…The phosphorylation of serine residues of IRS-1 contribute to positive or negative regulation of IRS-1 action (Weigert et al 2005;Weigert et al 2008). Important for this regulation are the particular phosphorylation sites (Herschkovitz et al 2007) as well as the timing of phosphorylation (Weigert et al 2005;Weigert et al 2008). Currently, the serine phosphorylation sites with positive effect on IRS-1 action are regarded to be phosphorylated at first to support IRS-1 activity protecting from phosphorylation at residues with inhibitory effect (Weigert et al 2005;Weigert et al 2008;Gual, Le Marchand-Brustel, and Tanti 2005;Luo et al 2007).…”