Regulation of extracellular signal-regulated kinase activity by p120 RasGAP does not involve its pleckstrin homology or calcium-dependent lipid binding domains but does require these domains to regulate cell proliferation.
The gene encoding for p120 RasGAP, has been disrupted in mice (M. Henkemeyer et al., Nature (Lond.), 377: 695-701, 1995). In this study, using fibroblasts derived from these mouse embryos (Gap-/-; P. van der Geer et al., Mol. Cell Biol., 17: 1840-1847, 1997), we demonstrate that mitogen-activated protein kinase (MAPK) activation is prolonged after epidermal growth factor (EGF), but not lysophosphatidic acid, stimulation as compared with wild-type cells. Furthermore, these cells exhibited a moderate increase in their proliferative rate and saturation density, as well as a limited ability to form colonies in soft agar. Stable cell lines expressing full-length p120GAP not only restored the ability to down-regulate MAPK after EGF stimulation but also lowered their saturation densities. Similarly, expression of p120GAP, missing either its pleckstrin homology (PH) or its calcium-dependent lipid binding (CaLB)/C2 domain, restored MAPK down-regulation and retained the ability to associate with p190 RhoGAP and to be phosphorylated by v-src but exhibited higher saturation densities similar to Gap-/- cells. Our results, therefore, suggest that p120GAP functions not only by down-regulating the Ras/MAPK pathway after growth factor stimulation but is also important in regulating cell proliferation that involves its PH and CaLB domains.
Koehler
J A
JA
Banting and Best Department of Medical Research, Medical Genetics, University of Toronto, Toronto, Ontario M9W 7H4, Canada.
Moran
M F
MF
D000818:Animals; D001798:Blood Proteins / Q000737:chemistry*; D002118:Calcium / Q000378:metabolism*; D002452:Cell Count; D002455:Cell Division; D002460:Cell Line; D003260:Contact Inhibition; D004268:DNA-Binding Proteins; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D020690:GTPase-Activating Proteins; D017353:Gene Deletion; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D012097:Repressor Proteins; D020729:p120 GTPase Activating Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020666:ras-GRF1
C074560:ARHGAP35 protein, human; C513043:ARHGAP5 protein, human; C513041:Arhgap35 protein, mouse; C513042:Arhgap5 protein, mouse; D001798:Blood Proteins; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; C513039:RASGRF1 protein, human; C513037:Rasgrf1 protein, mouse; D011956:Receptors, Cell Surface; D012097:Repressor Proteins; D020729:p120 GTPase Activating Protein; C039077:platelet protein P47; D020666:ras-GRF1; D004815:Epidermal Growth Factor; D020794:Receptor Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium