BioID-based Identification of Skp Cullin F-box (SCF)?-TrCP1/2 E3 Ligase Substrates.

ABSTRACT: The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCF(?-TrCP1) and SCF(?-TrCP2) are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NF?B signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCF(?-TrCP1/2) interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCF(?-TrCP1/2) substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for ?-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that ?-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases.

SUBMITTER: Coyaud E 

PROVIDER: S-EPMC4587326 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature