Insulin and Metabolic Stress Stimulate Multisite Serine/Threonine Phosphorylation of Insulin Receptor Substrate 1 and Inhibit Tyrosine Phosphorylation

Background: Metabolic stresses, including hyperinsulinemia, promote insulin resistance. Results: Monoclonal antibodies raised against Ser(P)/Thr(P) residues in IRS1 were used to quantify phosphorylation in response to insulin or agents that model metabolic stress. Conclusion: Similar IRS1 Ser(P)/Thr(P) residues are increased by insulin or metabolic stress, and some correlate significantly with reduced IRS1 tyrosine phosphorylation. Significance: Metabolic stress co-opts insulin-dependent IRS1 phosphorylation to aggravate insulin resistance. IRS1 and IRS2 are key substrates of the insulin receptor tyrosine kinase. Mass spectrometry reveals more than 50 phosphorylated IRS1 serine and threonine residues (Ser(P)/Thr(P) residues) in IRS1 from insulin-stimulated cells or human tissues. We investigated a subset of IRS1 Ser(P)/Thr(P) residues using a newly developed panel of 25 phospho-specific monoclonal antibodies (pS/TmAb(Irs1)). CHO cells overexpressing the human insulin receptor and rat IRS1 were stimulated with insulin in the absence or presence of inhibitors of the PI3K Akt mechanistic target of rapamycin (mTOR) S6 kinase or MEK pathways. Nearly all IRS1 Ser(P)/Thr(P) residues were stimulated by insulin and significantly suppressed by PI3K inhibition; fewer were suppressed by Akt or mTOR inhibition, and none were suppressed by MEK inhibition. Insulin-stimulated Irs1 tyrosine phosphorylation (Tyr(P)(Irs1)) was enhanced by inhibition of the PI3K Akt mTOR pathway and correlated with decreased Ser(P)-302(Irs1), Ser(P)-307(Irs1), Ser(P)-318(Irs1), Ser(P)-325(Irs1), and Ser(P)-346(Irs1). Metabolic stress modeled by anisomycin, thapsigargin, or tunicamycin increased many of the same Ser(P)/Thr(P) residues as insulin, some of which (Ser(P)-302(Irs1), Ser(P)-307(Irs1), and four others) correlated significantly with impaired insulin-stimulated Tyr(P)(Irs1). Thus, IRS1 Ser(P)/Thr(P) is an integrated response to insulin stimulation and metabolic stress, which associates with reduced Tyr(P)(Irs1) in CHOIR/IRS1 cells.