Serine Phosphorylation Proximal to Its Phosphotyrosine Binding Domain Inhibits Insulin Receptor Substrate 1 Function and Promotes Insulin Resistance

Abstract

Ser/Thr phosphorylation of insulin receptor substrate (IRS) proteins negatively modulates insulin signaling. Therefore, the identification of serine sites whose phosphorylation inhibit IRS protein functions is of physiological importance. Here we mutated seven Ser sites located proximal to the phosphotyrosine binding domain of insulin receptor substrate 1 (IRS-1) (S265, S302, S325, S336, S358, S407, and S408) into Ala. When overexpressed in rat hepatoma Fao or CHO cells, the mutated IRS-1 protein in which the seven Ser sites were mutated to Ala (IRS-1^7A), unlike wild-type IRS-1 (IRS-1^WT), maintained its Tyr-phosphorylated active conformation after prolonged insulin treatment or when the cells were challenged with inducers of insulin resistance prior to acute insulin treatment. This was due to the ability of IRS-1^7A to remain complexed with the insulin receptor (IR), unlike IRS-1^WT, which underwent Ser phosphorylation, resulting in its dissociation from IR. Studies of truncated forms of IRS-1 revealed that the region between amino acids 365 to 430 is a main insulin-stimulated Ser phosphorylation domain. Indeed, IRS-1 mutated only at S408, which undergoes phosphorylation in vivo, partially maintained the properties of IRS-1^7A and conferred protection against selected inducers of insulin resistance. These findings suggest that S408 and additional Ser sites among the seven mutated Ser sites are targets for IRS-1 kinases that play a key negative regulatory role in IRS-1 function and insulin action. These sites presumably serve as points of convergence, where physiological feedback control mechanisms, which are triggered by insulin-stimulated IRS kinases, overlap with IRS kinases triggered by inducers of insulin resistance to terminate insulin signaling.

We thank Ronit Sagi-Eisenberg and Kenneth Siddle for helpful comments and discussions.

This work was supported in part by research grants from the Juvenile Diabetes Foundation International, the European Foundation for the Study of Diabetes, the United States-Israel Binational Fund, the Israel Science Foundation (founded by the Israel Academy of Sciences and Humanities), and the Mitchel Kaplan Fund for Diabetes Research. Y.Z. is an incumbent of the Marte R. Gomez Professorial Chair.