C69
A tale of two Cbls: interplay of c-Cbl and Cbl-b in epidermal growth factor receptor downregulation
Steven Pennock, Zhixiang Wang
Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada
Background
The mammalian Cbl protein family consists of the three homologues c-Cbl, Cbl-b, and Cbl-3. The Cbls are oncoproteins by virtue of their prominent role in regulating signal transduction. Understanding how they regulate upstream receptors such as EGFR is vital to delineating exactly how they contribute to cancer.
Results
(a) Mutant EGFR1044 that truncated after residue 1044 did not associate with c-Cbl and was not ubiquitinated initially in response to EGF, but internalized with kinetics similar to that of wild-type EGFR. This indicates that c-Cbl-mediated ubiquitination is not required for EGF-induced EGFR endocytosis. (b) The previously identified internalization-deficient mutant EGFR (EGFR1010LL/AA) bound to c-Cbl and was fully ubiquitinated in response to EGF, which indicates that c-Cbl binding and ubiquitination are not sufficient for EGFR internalization. (c) c-Cbl disassociation from EGFR occurred well before EGFR degradation and was concurrent with selective dephosphorylation of EGFR at Y1045. (d) Cbl-b was associated with both EGFR and EGFR1044. Cbl-b association with EGFR rose markedly following a decrease in c-Cbl association, corresponding to a second peak of EGFR ubiquitination occurring later in EGFR trafficking. (e) Knockdown both c-Cbl and Cbl-b by RNAi abolished EGFR down-regulation, but had no effect on the rate of EGFR internalization.
Conclusion
Both Cbls accounted for total receptor ubiquitination. While c-Cbl and Cbl-b are each alone sufficient to effect EGFR degradation, both are involved in the physiological, EGF-mediated process of receptor downregulation. These data ultimately reveal a previously unacknowledged temporal interplay of two major Cbl homologues with the trafficking EGFR.
