Xu X, Keshwani M, Meyer K, Sarikas A, Taylor S, Pan ZQ

Hyper-activation of mechanistic target of rapamycin complex 1 (mTORC1) and its effector kinase s6 kinase 1 (S6K1) is known to trigger multisite seryl phosphorylation of insulin receptor substrate 1 (IRS1), leading to its ubiquitination and degradation. This negative feedback inhibition functions to restrain phospho inositide 3-kinase (PI3-K) activity, and plays ...

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critical roles in the pathogenesis of cancer and type II diabetes. Recent work has implicated a role for cullin-RING E3 ubiquitin ligase 7 (CRL7) in targeting IRS1 for mTORC1/S6K1-dependent degradation. In the present study we have employed both cell-based degradation and reconstituted ubiquitination approaches to define molecular features associated with IRS1 critical for CRL7-mediated ubiquitination and degradation. We have mapped IRS1 degradation signal sequence to its N-terminal 574 amino acid residues, of which the integrity of Ser307/Ser312 and Ser527, each constituting a S6K1 phosphorylation consensus site, was indispensible for supporting CRL7-forced degradation. In vitro, S6K1 was able to support the ubiquitination of bacterially expressed IRS1 N-terminal fragment by CRL7 but at low levels. In contrast, CRL7 supported efficient ubiquitination of IRS1 N-terminal fragment in hyper-phosphorylated form, which was isolated from infected insect cells, suggesting requirement of additional phosphorylation by kinases yet to be identified. Finally, removal of IRS1 amino acids 1-260 led to substantial reduction of ubiquitination efficiency suggesting a role for this region in mediating productive interactions with CRL7. The requirement of multisite phosphorylation and IRS1 N-terminus for its turnover may ensure that complete IRS1 degradation occurs only when mTORC1 and S6K1 reach exceedingly high levels.

Date: Oct. 08, 2012

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