Enhanced EGF mitogenic response is associated with enhanced tyrosine phosphorylation of specific cellular proteins in fibroblasts overexpressing c-src.
In previous studies examining the role of pp60c-src in cellular proliferation, we demonstrated that overexpression of wild type (wt) but not mutated c-src in C3H10T1/2 murine embryo fibroblasts resulted in a 2 to 5 fold enhancement of DNA synthesis in response to epidermal growth factor (EGF). Using phosphotyrosine antiserum to probe Western immunoblots of whole cell lysates, we determined in the present study that proteins migrating at approximately 170 (EGF receptor), 125, 100, 75, 63 and 57 kDa exhibited EGF-dependent tyrosine phosphorylations that were approximately 3 fold higher in the wt c-src overexpressors than in control cells, while proteins of 125, 100 and 75 kDa displayed basal, unstimulated levels that were approximately 2 fold higher. Pp60c-src-associated hyperphosphorylation of all but the 63 and 57 kDa proteins was reduced or ablated in cells overproducing structurally-altered forms of c-src. Furthermore, in contrast to control cells, wt c-src overexpressors retained EGF-induced tyrosine phosphorylations on the 125, 100 and 75 kDa proteins for up to 9 h of EGF treatment. These results illuminate potential substrates for pp60c-src and suggest that overproduced pp60c-src potentiates the EGF mitogenic response by increasing the extent and duration of tyrosine phosphorylations on proteins involved in EGF mitogenic signalling.
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908.
L K
LK
Wilson
S J
SJ
Parsons
D000818:Animals; D002460:Cell Line; D004261:DNA Replication / Q000187:drug effects*; D004815:Epidermal Growth Factor / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D016391:Genes, src; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D019000:Phosphotyrosine; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D016392:Proto-Oncogene Proteins pp60(c-src)