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BioID-based identification of SCF-BTrCP1/2 E3 ligase substrates

ABSTRACT: RAW files, mzML files, search results (gpm.xml), TPP analysis files (pep.XML and prot.XML, as well as tab delimited output of pep.XML as .xls) and analyzed results Table 1 for the identification of BTRCP1/2 substrates using BioID. The identification of ubiquitin E3 ligase substrates has been extremely 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 signalling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semi-quantitative 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 approach presented here should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases. Replaces MassIVE submission MSV000078991 and ProteomeXchange PXD001667. Contains human readable pep.XML and prot.XML data in addition to previous submission.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Brian Raught

PROVIDER: MSV000079032 | MassIVE | Wed Feb 04 10:55:00 GMT 2015

SECONDARY ACCESSION(S): PXD001784

REPOSITORIES: MassIVE

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BioID-based identification of SCF-BTrCP1/2 E3 ligase substrates

Project description:RAW files, mzML files, search results (gpm.xml), TPP analysis files (pep.XML and prot.XML, as well as tab delimited output of pep.XML as .xls) and analyzed results Table 1 for the identification of BTRCP1/2 substrates using BioID. The identification of ubiquitin E3 ligase substrates has been extremely 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 signalling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semi-quantitative 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 approach presented here should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases.

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