ABSTRACT
Phosphorylation-dependent protein ubiquitylation and degradation provides an irreversible mechanism to terminate protein kinase signaling. Here, we report that mammary epithelial cells require cullin-5–RING–E3-ubiquitin-ligase complexes (Cul5-CRLs) to prevent transformation by a Src–Cas signaling pathway. Removal of Cul5 stimulates growth-factor-independent growth and migration, membrane dynamics and colony dysmorphogenesis, which are all dependent on the endogenous tyrosine kinase Src. Src is activated in Cul5-deficient cells, but Src activation alone is not sufficient to cause transformation. We found that Cul5 and Src together stimulate degradation of the Src substrate p130Cas (Crk-associated substrate). Phosphorylation stimulates Cas binding to the Cul5-CRL adaptor protein SOCS6 and consequent proteasome-dependent degradation. Cas is necessary for the transformation of Cul5-deficient cells. Either knockdown of SOCS6 or use of a degradation-resistant Cas mutant stimulates membrane ruffling, but not other aspects of transformation. Our results show that endogenous Cul5 suppresses epithelial cell transformation by several pathways, including inhibition of Src–Cas-induced ruffling through SOCS6.
Footnotes
↵* Present address: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
↵‡ Present address: Biocontrol Systems, 12822 SE 32nd Street, Bellevue, WA 98005, USA.
↵§ Present address: Biomedical Sciences Program, University of Colorado, Denver, Colorado 80217, USA.
↵¶ These authors contributed equally to this work
Competing interests
The authors declare no competing financial interests.
Author contributions
All authors designed, performed and interpreted experiments. A.T., G.S.L. and J.A.C. wrote the paper.
Funding
This work was supported the National Institutes of Health [grant numbers R01-CA41072, R01-NS080194]; a pilot grant from the Hutchinson Center Synergy Fund; the Breast Cancer Research Program Pilot Project Fund at FHCRC; and the Safeway Foundation, postdoctoral fellowship from the American Cancer Society [grant number PF-07-287-01-MGO to G.L.]; and a National Science Foundation Graduate Research Fellowship to C.P. Deposited in PMC for release after 12 months.
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.127829/-/DC1
- Received January 24, 2013.
- Accepted October 30, 2013.
- © 2014. Published by The Company of Biologists Ltd