Identification of the Degradation Determinants of Insulin Receptor Substrate 1 for Signaling Cullin-RING E3 Ubiquitin Ligase 7-mediated Ubiquitination

Abstract

Background: Negative feedback regulation of insulin signaling involves ubiquitin-dependent degradation of insulin receptor substrate 1 (IRS1). Results: Cullin-RING E3 ubiquitin ligase 7 (CRL7) mediates the ubiquitination of IRS1 in hyperphosphorylated form. Conclusion: Multisite IRS1 phosphorylation triggers interactions with CRL7 for ubiquitin modification. Significance: Insulin signaling is self-restrained when its downstream effector kinases are hyperactivated to trigger the negative feedback inhibition.

Insulin and Metabolic Stress Stimulate Multisite Serine/Threonine Phosphorylation of Insulin Receptor Substrate 1 and Inhibit Tyrosine Phosphorylation

“…There is currently some uncertainty about the mechanism of mTORC2 regulation by insulin (86), because both positive and negative feedback loops involving Akt and mTORC1 3 S6K signaling have been demonstrated in different cell types (66,87). However, a growing literature connects mTORC2 to the proteasome-mediated degradation of Irs1 in cultured cells (88,89) and possibly in animal tissues (11). The mechanism described in cultured cells involves mTORC2 phosphorylation and stabilization of Fbxw8 (F-box and WD repeat domain containing 8), the substrate-targeting subunit of cullin-RING ligase CRL7 (88), but also relies upon phosphorylation of Ser-302…”

Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen

“…CRL7 was shown to be part of a negative feedback loop via mTORC1/ribosomal protein S6 kinase (S6K) that restrains IRS1 signaling (17,19). To evaluate the effect of SV40 LT on signaling pathways downstream of IRS1, full-length SV40 LT or LT Δ69-83 was ectopically expressed in U2-OS cells and the activation status of AKT and Erk MAPK was analyzed by immunoblot analyses.…”

“…Binding of insulin or IGF1 to its receptor induces tyrosine phosphorylation of IRS1 and subsequent activation of phosphatidylinositol-3-kinase (PI3K)/AKT and Erk mitogen-activated pathway kinase (MAPK) pathways (18). It was shown that CRL7-induced degradation of IRS1 is part of a negative feedback loop via mechanistic target of rapamycin complex 1 (mTORC1) to restrain IRS1 downstream signaling (17,19). A more recent study suggested an mTORC2-dependent feedback inhibition of IRS1 by direct phosphorylation of Fbw8, resulting in enhanced stability of this F-box protein that promotes IRS1 degradation (20).…”

“…To evaluate whether SV40 LT directly interferes with CRL7 E3 ligase function, we reconstituted IRS1 ubiquitination by CRL7 in vitro using purified proteins. We recently reported that CRL7 mediates efficient ubiquitination of the N-terminal IRS1 fragment (residues 1-574) (19). GST-tagged IRS1 (designated GST-IRS1 1-574 ) was expressed using the baculovirus/insect cell system and affinity-purified.…”

“…1-574 was expressed in a baculovirus/High Five insect cell system and purified as described (19). Reaction mixtures (20 μL) containing 1.1 pmol GST-IRS1 , 50 mM Tris·HCl (pH 7.4), 5 mM MgCl 2 , 0.5 mM DTT, 2 mM ATP, 2 mM NaF, 10 nM okadaic acid, 0.2 pmol CRL7, 50 μM PK-Ub, 13 nM E1, and 1 μM UbcH5c were incubated at 37°C, and increasing amounts of LT protein (0.5-9.0 pmol) were added (for isolation of SV40 LT, see ref.…”