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.