Abstract
Tumor necrosis factor alpha (TNFalpha) or chronic hyperinsulinemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (IR) and of its major insulin receptor substrates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupling in insulin signaling, we undertook to study the interaction of Ser/Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We could demonstrate that, similar to IRS-1, IRS-2 also interacts with the juxtamembrane (JM) domain (amino acids 943-984) but not with the carboxyl-terminal region (amino acids 1245-1331) of IR expressed in bacteria as His6 fusion peptides. Moreover, incubation of rat hepatoma Fao cells with TNFalpha, bacterial sphingomyelinase, or other Ser(P)/Thr(P)-elevating agents reduced insulin-induced Tyr phosphorylation of IRS-1 and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and significantly reduced their ability to interact with the JM region of IR. Withdrawal of TNFalpha for periods as short as 30 min reversed its inhibitory effects on IR-IRS interactions. Similar inhibitory effects were obtained when Fao cells were subjected to prolonged (20-60 min) pretreatment with insulin. Incubation of the cell extracts with alkaline phosphatase reversed the inhibitory effects of insulin. These findings suggest that insulin resistance is associated with enhanced Ser/Thr phosphorylation of IRS-1 and IRS-2, which impairs their interaction with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate the insulin receptor signal. Moreover, the reversibility of the TNFalpha effects and the ability to mimic its action by exogenously added sphingomyelinase argue against the involvement of a proteolytic cascade in mediating the acute inhibitory effects of TNFalpha on insulin action.
Highlights
From the ‡Department of Molecular Cell Biology, the Weizmann Institute of Science, Rehovot 76100, Israel, the §Institute of Endocrinology, Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel, the ¶Diabetes Branch, National Institutes of Health, Bethesda, Maryland 20982, and the ʈDepartment of Biochemistry, Israel Institute for Biological Research, Ness Ziona, 70400, Israel
IRS-2 Interacts with the JM Peptide—We have previously shown that IRS-1 selectively interacts with an immobilized polypeptide corresponding to 41 amino acids of the juxtamembrane region of insulin receptor (IR) {(His)6-JM} [18], it fails to interact with an 86-amino acid polypeptide, corresponding to the carboxyl-terminal region of this receptor ((His)6-CT)
In this study we present evidence that insulin resistance at the molecular level is induced at least in part by enhanced Ser/Thr phosphorylation of both IRS-1 and IRS-2, which impedes their interaction with the juxtamembrane region of the insulin receptor, turning them into poorer receptor substrates that fail to undergo appropriate Tyr phosphorylation
Summary
ELEVATED SERINE/THREONINE PHOSPHORYLATION OF IRS-1 AND IRS-2 INHIBITS THEIR BINDING TO. PHOSPHORYLATION Keren Paz, Rina Hemi, Derek LeRoith, Avraham Karasik, Eytan Elhanany, Hannah. Access the most updated version of this article at http://www.jbc.org/content/272/47/29911. Alerts: When this article is cited When a correction for this article is posted. This article cites 42 references, 22 of which can be accessed free at http://www.jbc.org/content/272/47/29911.full.html#ref-list-1
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