Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein.
p120 GTPase-activating protein (GAP) is a negative regulator of Ras that functions at a key relay point in signal transduction pathways that control cell proliferation. Among other proteins, p120 GAP associates with p190, a GAP for the Ras-related protein, Rho. To characterize the p120.p190 interaction further, we used bacterially expressed glutathione S-transferase fusion polypeptides to map the regions of p120 necessary for its interactions with p190. Our results show that both the N-terminal and the C-terminal SH2 domains of p120 are individually capable of binding p190 expressed in a baculovirus/insect cell system. Moreover, the two SH2 domains together on one polypeptide bind synergistically to p190, and this interaction is dependent on tyrosine phosphorylation of p190. In addition, mutation of the highly conserved Arg residues in the critical FLVR sequences of both SH2 domains of full-length p120 reduces binding to tyrosine-phosphorylated p190. The dependence on p190 phosphorylation for complex formation with p120 SH2 domains observed in vitro is consistent with analysis of the native p120.p190 complexes formed in vivo. These findings suggest that SH2-phosphotyrosine interaction is one mechanism by which the cell regulates p120.p190 association and thus may be a means for coordinating the Ras- and Rho-mediated signaling pathways.
Bryant
S S
SS
Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor 48109, USA.
Briggs
S
S
Smithgall
T E
TE
Martin
G A
GA
McCormick
F
F
Chang
J H
JH
Parsons
S J
SJ
Jove
R
R
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002455:Cell Division; D002460:Cell Line; D003001:Cloning, Molecular; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012097:Repressor Proteins; D015398:Signal Transduction; D018411:Spodoptera; D014443:Tyrosine / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1
C074560:ARHGAP35 protein, human; C513043:ARHGAP5 protein, human; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; C513039:RASGRF1 protein, human; D012097:Repressor Proteins; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1; D014443:Tyrosine