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. Henkemeye r et at, Nature (Lond.), 377. 695-701, 1995). In this study, using fibrobla sts derived from these mouse embryos (Gap(-/-); P. van der Geer et al., Mol . Cell Biol., 17. 1840-1847, 1997), we demonstrate that mitogen-activated p rotein kinase (MAPK) activation is prolonged after epidermal growth factor (EGF), but not lysophosphatidic acid, stimulation as compared with wild-typ e cells. Furthermore, these cells exhibited a moderate increase in their pr oliferative rate and saturation density, as well as a limited ability to fo rm colonies in soft agar. Stable cell lines expressing full-length p120(GAP ) not only restored the ability to downregulate MAPK after EGF stimulation but also lowered their saturation densities. Similarly, expression of p120( GAP), 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 r esults, therefore, suggest that p120(GAP) functions not only by downregulat ing the Ras/MAPK pathway after growth factor stimulation but is also import ant in regulating cell proliferation that involves its PH and CaLB domains.