Complex formation between the p21ras GTPase-activating protein and phosphoproteins p62 and p190 is independent of p21ras signalling.
We have investigated whether complex formation between the p21ras GTPase-activating protein (GAP) and the phosphotyrosine-containing proteins p62 and p190 is dependent on functional p21ras, to test the hypothesis that binding of p21rasGTP to GAP enables GAP to associate with these phosphoproteins. The formation of p21rasGTP was inhibited by a dominant interfering mutant of p21ras, p21ras(Asn-17), which was introduced with a vaccinia virus expression system. We used NIH3T3 cells in which complex formation between GAP and tyrosine-phosphorylated p62 and p190 can be induced either by v-src transformation, by incubating the cells with the phosphotyrosine phosphatase inhibitor pervanadate or by activation of a growth factor receptor tyrosine kinase. In all cases, expression of p21ras(Asn-17) did not affect the presence or the formation of the GAP-phosphoprotein complexes. To monitor the effectiveness of p21ras inhibition, we measured p21ras-mediated phosphorylation of extracellular signal-regulated kinase 2 (ERK2). In all cases, expression of p21ras(Asn-17) completely blocked signalling to ERK2. From these data we conclude that p21rasGTP formation is not essential for complex formation between GAP and tyrosine-phosphorylated p62 and p190, and thus complex formation does not depend on interaction of GAP with p21rasGTP.
Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
G J
GJ
Pronk
A M
AM
de Vries-Smits
C
C
Ellis
J L
JL
Bos
D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012097:Repressor Proteins; D015398:Signal Transduction; D051758:Transcription Factor TFIIH; D019473:Transcription Factors, TFII; D014638:Vanadates / Q000494:pharmacology; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1
C513041:Arhgap35 protein, mouse; D004268:DNA-Binding Proteins; C072546:GTF2H1 protein, human; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; C513037:Rasgrf1 protein, mouse; D012097:Repressor Proteins; D019473:Transcription Factors, TFII; C076715:pervanadate; D020703:ras GTPase-Activating Proteins; D020666:ras-GRF1; D051758:Transcription Factor TFIIH; D014638:Vanadates; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D016283:Proto-Oncogene Proteins p21(ras)