Src promotes destruction of c-Cbl: implications for oncogenic synergy between Src and growth factor receptors

Abstract

Cellular Src and epidermal growth factor receptor (EGFR) collaborate in the progression of certain human malignancies, and their cooverexpression characterizes relatively aggressive animal tumors. Our study addressed the mode of oncogenic cooperation and reports that overexpression of c-Src in model cellular systems results in the accumulation of EGFR at the cell surface. The underlying mechanism involves inhibition of the normal, c-Cbl-regulated process of ligand-induced receptor down-regulation. In response to activation of c-Src, c-Cbl proteins undergo tyrosine phosphorylation that promotes their ubiquitylation and proteasomal destruction. Consequently, ubiquitylation of EGFR by c-Cbl is restrained in Src-transformed cells, and receptor sorting to endocytosis is impaired. In conclusion, by promoting destruction of c-Cbl, c-Src enables EGFR to evade desensitization, which explains Src-EGFR collaboration in oncogenesis.

Figure 1

Src up-regulates EGFR expression by inhibiting c-Cbl-induced receptor ubiquitylation. (A) Chinese hamster ovary cells coexpressing EGFR and the indicated Src proteins were untreated or treated with EGF (100 ng/ml) for 15 min; thereafter, cell extracts were analyzed by immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies. (B) SYF cells coexpressing EGFR, HA-tagged ubiquitin, c-Cbl, and the indicated forms of c-Src were untreated or treated with EGF (100 ng/ml) for 15 min; thereafter, cell extracts were analyzed as indicated. (C) Down-regulation of EGFR was tested in SYF cells ectopically expressing EGFR alone (●), in combination with c-Cbl (■), or in combination with both c-Cbl and an active c-Src (Y529F; ▴). Cells were incubated at 37°C with an unlabeled EGF (100 ng/ml) for the indicated time intervals, and the residual level of surface EGFR was determined by binding of a radioactive EGF at 4°C. Averages and the corresponding SDs of triplicates (bars) are shown.

Figure 2

c-Src inhibits the interaction between c-Cbl and EGFR by down-regulating c-Cbl. (A) SYF cells expressing EGFR and Y529F-Src were subjected to a 4-h-long metabolic labeling with ³⁵S-labeled amino acids. Thereafter, cells were chased for the indicated time intervals in the absence or presence of EGF (100 ng/ml). (B) SYF cells were transfected (in duplicates) with plasmids encoding EGFR or c-Cbl, in the presence of a control vector (pUSE) or the Y529F-Src expression vector. Forty-eight hours later, cells were harvested and total cellular RNA was extracted. Transcripts corresponding to EGFR and c-Cbl were detected in Northern blots by hybridization to the respective cDNA probes. For normalization, we used a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe. (C) SYF cells transfected with vectors encoding EGFR, HA-tagged c-Cbl, and either a Y529F-Src vector or an empty plasmid, were treated for 15 min at 37°C with EGF (100 ng/ml), or they were left untreated. Cell extracts were tested for Cbl-EGFR association by using the indicated antibodies. (D) SYF cells were transfected and treated with EGF as in C. The state of c-Cbl was analyzed by immunoblotting with anti-Cbl and anti-phosphotyrosine (P-Tyr) antibodies. IP, immunoprecipitation; IB, immunoblotting.

Figure 3

Down-regulation of c-Cbl involves the catalytic activity of c-Src and colocalization to vesicular structures. (A) SYF cells were cotransfected with a c-Cbl expression vector along with increasing amounts of plasmids encoding the indicated mutants of Src or an empty vector. Whole-cell lysates were probed 48 h later with antibodies to c-Cbl or to tubulin. IP, immunoprecipitation; IB, immunoblotting.(B) SYF cells grown on glass slides were transfected with plasmids encoding Y529F-Src (a–c), HA-tagged c-Cbl (d–f), or their combination (g–i). After 24 h, cells were fixed, permeabilized, and incubated with murine antibodies to c-Src and a rat antibody to HA. FITC-conjugated antibody specific to mouse immunoglobulins (green) and Cy3-conjugated antibodies to rat immunoglobulins (red) were used for fluorescent confocal microscopy.

Figure 4

c-Src elevates phosphorylation and self-ubiquitylation of c-Cbl. (A and B) SYF cells were transfected with plasmids encoding the indicated HA-tagged forms of c-Cbl, together with a plasmid encoding Y529F-Src, or the respective empty plasmid. Forty-eight hours after transfection, cell extracts were analyzed as indicated. (C) SYF cells were transfected with plasmids encoding EGFR and a Flag-tagged ubiquitin, along with the indicated forms of Src and Cbl. Forty-eight hours after transfection, cells were untreated or treated for 15 min with EGF (100 ng/ml), and cell extracts were analyzed as indicated. IP, immunoprecipitation; IB, immunoblotting.

Figure 5

Proposed mode of interactions between c-Src and c-Cbl and their effect on EGFR trafficking. Normally, EGF promotes receptor phosphorylation, followed by recruitment of c-Cbl, receptor ubiquitylation, and sorting of EGFR to lysosomal degradation. In the presence of an active/overexpressed c-Src, both phosphorylation and ubiquitylation of c-Cbl are enhanced, and the protein is subsequently degraded by the 26S proteasome. Because of Src-induced elimination of c-Cbl, sorting of EGFR to lysosomal degradation is reduced, and the receptor is diverted to the recycling pathway. This regulatory loop may explain the association between Src activation and EGFR over-expression in tumor cells.