M-Ras/R-Ras3, a transforming Ras protein regulated by Sos1, GRF1, and p120Ras GTPase-activating protein, interacts with the putative Ras effector AF6

M-Ras is a Ras-related protein that shares similar to 55% identity with K-R as and TC21. The M-Ras message was widely expressed but was most predominan t in ovary and brain. Similarly to Ha-Ras, expression of mutationally activ ated M-Ras in NIH 3T3 mouse fibroblasts or C2 myoblasts resulted in cellula r transformation or inhibition of differentiation, respectively. M-Ras only weakly activated extracellular signal-regulated kinase 2 (ERK2), but it co operated with Raf, Rac, and Rho to induce transforming foci in NIH 3T3 cell s, suggesting that M-Ras signaled via alternate pathways to these effecters . Although the mitogen-activated protein kinase/ERK kinase inhibitor, PD980 59, blocked M-Ras-induced transformation, M-Ras was more effective than an activated mitogen-activated protein kinase/ERK kinase mutant at inducing fo cus formation. These data indicate that multiple pathways must contribute t o M-Ras-induced transformation. M-Ras interacted poorly in a yeast two-hybr id assay with multiple Res effecters, including c-Raf-1, A-Raf, B-Raf, phos phoinositol-3 kinase delta, RalGDS, and Rin1. Although M-Ras coimmunoprecip itated with AF6, a putative regulator of cell junction formation, overexpre ssion of AF6 did not contribute to fibroblast transformation, suggesting th e possibility of novel effector proteins. The M-Ras GTP/GDP cycle was sensi tive to the Ras GEFs, Sos1, and GRF1 and to p120 Ras GAP. Together, these f indings suggest that while M-Ras is regulated by similar upstream stimuli t o Ha-Ras, novel targets may be responsible for its effects on cellular tran sformation and differentiation.