Insulin and metabolic stress stimulate multisite serine/threonine phosphorylation of insulin receptor substrate 1 and inhibit tyrosine phosphorylation [Signal Transduction]
March 20th, 2014 by Hancer, N. J., Qiu, W., Cherella, C., Li, Y., Copps, K. D., White, M. F.
IRS1 and IRS2 are key substrates of the insulin receptor tyrosine kinase. Mass spectrometry reveals more than 50 phosphorylated IRS1 serine and threonine residues (phospho-S/Ts) in IRS1 from insulin-stimulated cells or human tissues. We investigated a subset of IRS1 phospho-S/Ts using a newly developed panel of 25 phosphospecific monoclonal antibodies (αpS/TmAbIrs1). Chinese hamster ovary (CHO) cells overexpressing the human insulin receptor and rat Irs1 were stimulated with insulin in the absence or presence of inhib-itors of the PI3K→Akt→mTOR→S6 kinase or MAP kinase (MEK) pathways. Nearly all Irs1 phospho-S/Ts were stimulated by insulin and significantly suppressed by PI3K inhibition; fewer were suppressed by Akt or mTOR inhibition, and none by MEK inhibition. Insulin-stimulated Irs1 tyrosine phosphorylation (pTyrIrs1) was enhanced by inhibition of the PI3K→Akt→mTOR pathway, and correlated with decreased pS302Irs1, pS307Irs1, pS318Irs1, pS325Irs1, and pS346Irs1. Metabolic stress−modeled by anisomycin, thapsigargin or tunicamycin−increased many of the same phospho-S/Ts as insulin, some of which (pS302Irs1, pS307Irs1 and four others) correlated significantly with impaired insulin-stimulated pTyrIrs1. Thus Irs1 phospho-S/T is an integrated response to insulin stimulation and metabolic stress, which associates with reduced pTyrIrs1 in CHOIR/IRS1 cells.