A Novel Domain Mediates Insulin-Induced Proteasomal Degradation of Insulin Receptor Substrate 1 (IRS-1)

Boura-Halfon, Sigalit
Shuster-Meiseles, Timor
Beck, Avital
Petrovich, Katia
Gurevitch, Diana
Ronen, Denise
Zick, Yehiel

Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel

Address all correspondence and requests for reprints to: Yehiel Zick, Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100, Israel. E-Mail: [email protected].

This work was supported by research grants from The Juvenile Diabetes Foundation International, The European Foundation for the Study of Diabetes, The Israel Science Foundation (founded by the Israel Academy of Sciences and Humanities), The Mitchel H. Caplan Fund for Diabetes Research, D-Cure Israel, and the Minerva Foundation.

Disclosure Summary: The authors have nothing to disclose.

Received March 1, 2010. Accepted August 16, 2010.

First Published Online September 15, 2010

Abbreviations: aa, Amino acids; AS, antisense; CUL 7, cullin 7; dicumarol, 3,3′-methylene-bis (4-hydroxycoumarin); DIDI, domain involved in degradation of IRS-1; E3, ubiquitin-protein ligase; GFP, green fluorescent protein; HA, hemagglutinin; IRS, insulin receptor substrate; Mdm2, murine double minute; mTOR, mammalian target of rapamycin; PH, pleckstrin homology; PI3K, phosphatidylinositol 3 kinase; PTB, P-Tyr binding; PKB, protein kinase B; S, sense; WT, wild type.

Molecular Endocrinology 24(11):p 2179-2192, November 2010.

Insulin receptor substrate-1 (IRS-1) plays a pivotal role in insulin signaling, therefore its degradation is exquisitely regulated. Here, we show that insulin-stimulated degradation of IRS-1 requires the presence of a highly conserved Ser/Thr-rich domain that we named domain involved in degradation of IRS-1 (DIDI). DIDI (amino acids 386-430 of IRS-1) was identified by comparing the intracellular degradation rate of several truncated forms of IRS-1 transfected into CHO cells. The isolated DIDI domain underwent insulin-stimulated Ser/Thr phosphorylation, suggesting that it serves as a target for IRS-1 kinases. The effects of deletion of DIDI were studied in Fao rat hepatoma and in CHO cells expressing Myc-IRS-1^WT or Myc-IRS-1^Δ386-430. Deletion of DIDI maintained the ability of IRS-1^Δ386-434 to undergo ubiquitination while rendering it insensitive to insulin-induced proteasomal degradation, which affected IRS-1^WT (80% at 8 h). Consequently, IRS-1^Δ386-434 mediated insulin signaling (activation of Akt and glycogen synthesis) better than IRS-1^WT. IRS-1^Δ386-434 exhibited a significant greater preference for nuclear localization, compared with IRS-1^WT. Higher nuclear localization was also observed when cells expressing IRS-1^WT were incubated with the proteasome inhibitor MG-132. The sequence of DIDI is conserved more than 93% across species, from fish to mammals, as opposed to approximately 40% homology of the entire IRS-1. These findings implicate DIDI as a novel, highly conserved domain of IRS-1, which mediates its cellular localization, rate of degradation, and biological activity, with a direct impact on insulin signal transduction.