Increased levels of p21ras-GTP and enhanced DNA synthesis accompany elevated tyrosyl phosphorylation of GAP-associated proteins, p190 and p62, in c-src overexpressors.
While examining the role of pp60c-src in cellular proliferation, we found that overexpression of c-src in C3H10T1/2 murine fibroblasts results in an augmented mitogenic response to epidermal growth factor (EGF) [Luttrell, D.K., Luttrell, L.M. & Parsons, S.J. (1988). Mol. Cell. Biol., 8, 497-501; Wilson, L.K., Luttrell, D.K., Parsons, S.J. (1989). Mol. Cell. Biol., 9, 1536-1544] and enhanced tyrosyl phosphorylation of specific cellular proteins [Wilson, L.K. & Parsons, S.J. (1990). 5, 1471-1480]. Here we identify two of these proteins as the GAP (GTPase-activating protein of p21ras)-associated proteins, p190 and p62. Evidence is presented to support the notion that, in 10T1/2 fibroblasts, p190 is a preferred substrate of pp60c-src, while p62 is preferentially phosphorylated by the EGF receptor. First, the phosphotyrosine content of p190 in quiescent cells is three- to fivefold higher in c-src overexpressors than in control cells and is not altered by growth factor treatment. In contrast, tyrosyl phosphorylation of p62 is undetectable in quiescent cells and transiently observable upon EGF addition. Second, the phosphotyrosine content of p190 in cells overexpressing defective pp60c-src is reduced in comparison with wild-type (wt) c-src overexpressors, while that of p62 is significantly less affected. Further studies revealed that tyrosyl phosphorylation of p190 and p62 is not required for GAP complex formation, as equal amounts of p190 and p62 proteins could be detected in GAP complexes from wt and variant c-src overexpressors both before and after EGF stimulation. However, analysis of GTP-bound p21ras revealed higher basal and EGF-stimulated levels in c-src overexpressors than in control cells. Taken together, these results suggest that one mechanism by which pp60c-src may contribute to early events in the EGF-induced mitogenic pathway in 10T1/2 fibroblasts is by increasing the level of GAP-associated p190 and p62 tyrosyl phosphorylation, which in turn results in higher levels of p21ras-GTP.
Chang
J H
JH
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908.
Wilson
L K
LK
Moyers
J S
JS
Zhang
K
K
Parsons
S J
SJ
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D016391:Genes, src; D006133:Growth Substances / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011506:Proteins / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012097:Repressor Proteins; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1
C513041:Arhgap35 protein, mouse; D004268:DNA-Binding Proteins; C489561:Dok1 protein, mouse; C074561:GAP-associated protein p62; D020690:GTPase-Activating Proteins; D006133:Growth Substances; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; D016601:RNA-Binding Proteins; C513037:Rasgrf1 protein, mouse; D012097:Repressor Proteins; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1; D019000:Phosphotyrosine; D014443:Tyrosine; D006160:Guanosine Triphosphate; D004247:DNA; D016392:Proto-Oncogene Proteins pp60(c-src); D016283:Proto-Oncogene Proteins p21(ras)