ELEVATED SERINE THREONINE PHOSPHORYLATION OF IRS-1 AND IRS-2 INHIBITSTHEIR BINDING TO THE JUXTAMEMBRANE REGION OF THE INSULIN-RECEPTOR ANDIMPAIRS THEIR ABILITY TO UNDERGO INSULIN-INDUCED TYROSINE PHOSPHORYLATION/

Tumor necrosis factor alpha (TNF alpha) or chronic hyperinsulinemia th at induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (Hi) and of its major insulin receptor substr ates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupl ing in insulin signaling, we undertook to study the interaction of Ser /Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We coul d demonstrate that, similar to IRS-1, IRS-2 also interacts with the ju xtamembrane (JM) domain (amino acids 943-984) but not with the carboxy l-terminal region (amino acids 1245-1331) of IR expressed in bacteria as His(6) fusion peptides, Moreover, incubation of rat hepatoma Fao ce lls with TNF alpha, bacterial sphingomyelinase, or other Ser(P)/Thr(P) -elevating agents reduced insulin-induced Tyr phosphorylation of IRS-I and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and signific antly reduced their ability to interact with the JM region of IR. With drawal of TNF alpha for periods as short as 30 min reversed its inhibi tory effects on IR-IRS interactions, Similar inhibitory effects were o btained when Fao cells were subjected to prolonged (20-60 min) pretrea tment with insulin, Incubation of the cell extracts with alkaline phos phatase reversed the inhibitory effects of insulin. These findings sug gest that insulin resistance is associated with enhanced Ser/Thr phosp horylation of IRS-1 and IRS-S, which impairs their interaction with th e JIVE region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and fu rther propagate the insulin receptor signal. Moreover, the reversibili ty of the TNF alpha effects and the ability to mimic its action by exo genously added sphingomyelinase argue against the involvement of a pro teolytic cascade in mediating the acute inhibitory effects of TNF alph a on insulin action.