GTPase-activating protein SH2-SH3 domains induce gene expression in a Ras-dependent fashion.
The p21ras GTPase-activating protein (GAP) is thought to function as both a negative regulator and a downstream target of p21ras. Here, we have investigated the role of GAP by using a transient expression assay with a fos luciferase reporter plasmid. We used GAP deletion mutants that lack the domain involved in interaction with p21ras and encode essentially only the SH2-SH3 domains. When these GAP deletion mutants were expressed, we observed a marked induction of fos promoter activity similar to induction by activated p21ras. Expression of a full-length GAP construct had no effect on the activity of the fos promoter. Activation of the fos promoter by these GAP SH2-SH3 regions was inhibited by cotransfection of a dominant inhibitory mutant of p21ras, Ras(Asn-17). Thus, the induction of gene expression by GAP SH2-SH3 domains is dependent on p21ras activity. Moreover, induction of fos promoter activity by GAP SH2-SH3 domains is increased severalfold after cotransfection of an activated mutant of p21ras, Ras(Leu-61), or insulin stimulation of A14 cells, both leading to an increase in the levels of GTP-bound p21ras. The combined effect of Ras(Leu-61) and the GAP deletion mutants was not inhibited by Ras(Asn-17), indicating that GAP SH2-SH3 domains do not function to activate endogenous p21ras but cooperate with another signal coming from active p21ras. These data suggest that GAP SH2-SH3 domains serve to induce gene expression by p21ras but that additional signals coming from p21ras are required for them to function.
Medema
R H
RH
Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands.
de Laat
W L
WL
Martin
G A
GA
McCormick
F
F
Bos
J L
JL
D016475:3T3 Cells; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D016466:CHO Cells; D002872:Chromosome Deletion; D002999:Clone Cells; D006224:Cricetinae; D020690:GTPase-Activating Proteins; D005786:Gene Expression Regulation; D011905:Genes, ras; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D014162:Transfection; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D007328:Insulin; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D008156:Luciferases; D016283:Proto-Oncogene Proteins p21(ras)