The Cullin 5 RING ligase complex inhibits Src activity and Src-dependent transformation of MCF10A epithelial cells, in part by targeting pY proteins such as pYCas for degradation by the ubiquitin-proteasome system (Teckchandani et al., 2014). Because overexpression of Cas alone did not phenocopy CRL5 inhibition (Teckchandani et al., 2014), we infer that CRL5 down-regulates additional pY proteins that become limiting when Cas is over-expressed. We sought to identify such pY proteins by screening for pY peptides whose abundance increases when Cul5 is inhibited. To this end, control and Cul5-deficient MCF10A cells were lysed under denaturing conditions, proteins were digested with trypsin, and peptides were labeled with isobaric TMT tags for quantitative pY proteomics (Zhang et al., 2007). In one experiment, samples were prepared from control and Cul5-deficient cells that were starved for epidermal growth factor (EGF) for 0, 24 or 72 hr. Starvation time had no systematic effect on peptide abundance, so, in a second experiment, we prepared biological triplicate samples from growing control and Cul5-deficient cells. Sixteen pY peptides increased significantly in Cul5-deficient cells in both experiments, including pY128 from Cas and pY117 and pY266 from BCAR3.

Two independent experiments were performed.

141231_85_CP_Cul5_6plex_pY_psms paired samples of MCF10A shCont and shCul5 shifted to assay media for 0, 24 hr or 72 hr

160825_297_CP_10A_shCUL5     paired samples of MCF10A shCont and shCul5 in growth media, biological triplicates

Each experiment used TMT 6-plex iTRAQ labeling.

Two files are provided for each experiment. (a) The .raw files. (b) Excel spreadsheets showing calculations.

Please note that 160825_297_CP_sup.raw is the .raw file for peptides that did not bind to the anti-phosphotyrosine column.

Methods are described in the linked publication.