Phosphotyrosine (p-Tyr)-dependent and -independent mechanisms of p190 RhoGAP-p120 RasGAP interaction: Tyr 1105 of p190, a substrate for c-Src, is the sole p-Tyr mediator of complex formation.
p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells revealed that p190 could bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding.
Roof
R W
RW
Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Haskell
M D
MD
Dukes
B D
BD
Sherman
N
N
Kinter
M
M
Parsons
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012097:Repressor Proteins; D017421:Sequence Analysis; D015398:Signal Transduction / Q000502:physiology; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1; D019061:src-Family Kinases
C513041:Arhgap35 protein, mouse; C513042:Arhgap5 protein, mouse; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D011506:Proteins; C513037:Rasgrf1 protein, mouse; D012097:Repressor Proteins; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases