Interplay and Effects of Temporal Changes in the Phosphorylation State of Serine-302, -307, and -318 of Insulin Receptor Substrate-1 on Insulin Action in Skeletal Muscle Cells

Abstract

Transduction of the insulin signal is mediated by multisite Tyr and Ser/Thr phosphorylation of the insulin receptor substrates (IRSs). Previous studies on the function of single-site phosphorylation, particularly phosphorylation of Ser-302, -307, and -318 of IRS-1, showed attenuating as well as enhancing effects on insulin action. In this study we investigated a possible cross talk of these opposedly acting serine residues in insulin-stimulated skeletal muscle cells by monitoring phosphorylation kinetics, and applying loss of function, gain of function, and combination mutants of IRS-1. The phosphorylation at Ser-302 was rapid and transient, followed first by Ser-318 phosphorylation and later by phosphorylation of Ser-307, which remained elevated for 120 min. Mutation of Ser-302 to alanine clearly reduced the subsequent protein kinase C-zeta-mediated Ser-318 phosphorylation. The Ser-307 phosphorylation was independent of Ser-302 and/or Ser-318 phosphorylation status. The functional consequences of these phosphorylation patterns were studied by the expression of IRS-1 mutants. The E302A307E318 mutant simulating the early phosphorylation pattern resulted in a significant increase in Akt and glycogen synthase kinase 3 phosphorylation. Furthermore, glucose uptake was enhanced. Because the down-regulation of the insulin signal was not affected, this phosphorylation pattern seems to be involved in the enhancement but not in the termination of the insulin signal. This enhancing effect was completely absent when Ser-302 was unphosphorylated and Ser-307 was phosphorylated as simulated by the A302E307E318 mutant. Phospho-Ser-318, sequentially phosphorylated at least by protein kinase C-zeta and a mammalian target of rapamycin/raptor-dependent kinase, was part of the positive as well as of the subsequent negative phosphorylation pattern. Thus we conclude that insulin stimulation temporally generates different phosphorylation statuses of the same residues that exert different functions in insulin signaling.

Increased Phosphoenolpyruvate Carboxykinase Gene Expression and Steatosis during Hepatitis C Virus Subgenome Replication

“…Increased IRS-1 serine phosphorylation has been shown to interfere with both tyrosine phosphorylation of IRS-1 (50) and the association between IRS-1 and p85␣ subunit of PI3K necessary for full Akt activation (51). Whether this is due to the increase in phosphorylation of IRS-1 (Ser-312) or a combination of serine sites in IRS-1 is not proven here.…”

A Single Mechanism Can Explain Network-wide Insulin Resistance in Adipocytes from Obese Patients with Type 2 Diabetes

“…There is, however, no a priori reason to expect that the same mechanism of insulin resistance that we have identified in adipocytes from human beings is operational in other tissues or even in adipocytes from other species. We (3, 56, 58 -60) and others (61,62) have previously found that phosphorylation of IRS1 at serine 307 in response to insulin is coupled to a positive feedback. In particular, our earlier mathematical model analysis showed that a positive feedback best explained data for the whole system (3).…”

Insulin Signaling in Type 2 Diabetes

“…The importance of cell type and experimental conditions implicating negative or positive effects is illustrated by the phosphorylation of IRS1 at serine 312 (human sequence, corresponding to serine 307 in murine sequence), an established negative effect in different experimental setups (34 -36) that in a knock-in experiment eventually was found to have a positive effect on insulin signaling in mice in vivo (37). Examining different aspects of insulin signaling we (10 -13) and others (14,15) have previously found that phosphorylation of IRS1 at serine 307 in response to insulin is associated with a positive feedback. The systems approach we used herein demonstrates that a positive feedback to IRS1 can best explain the experimental data for the whole system.…”