Identification and characterization of M-Ras, a novel member of the Ras-superfamily of small GTPases.

摘要

Ras proteins have been implicated in up to 30% of all human cancers. However, in some tissues the incidence of mutations in p21 Ras proteins is very low. This prompted us to screen the EST-database for new Ras-family members. Here we describe the identification and characterization of M-Ras, a novel and highly conserved member of the Ras family. M-Ras is a widely expressed protein with an apparent molecular weight of 29 kDa. Activated mutants of M-Ras transformed NIH 3T3 fibroblasts and led to factor-independent growth of an IL-3-dependent cell line. A dominant negative mutant of M-Ras was able to block activation of the c-fos promoter by an activated Src Y527F. Importantly, M-Ras was recognized by the monoclonal anti-Ras antibody Y13-259, a widely used tool to assay p21 Ras activation. In contrast to p21 Ras, M-Ras interacted only weakly with the Ras binding domains of Raf-1 and RalGDS. This led us to screen a cDNA library prepared from NIH 3T3 cells for novel effectors of M-Ras using the yeast 2-hybrid system. Several known Ras effectors were identified, thus allowing us to establish a subset of p21 Ras effectors that also interacted with M-Ras. We also identified a novel effector protein that interacted strongly with activated mutants of M-Ras and p21 Ras. Sequence analysis of this novel protein revealed a new member of the RalGDS-family of exchange factors, which we termed RPM. Murine RPM mRNA was widely expressed. In contrast to other RalGDS proteins, which have been shown to synergize with p21 Ras in activating the c-fos promoter, RPM displayed a strong inhibitory effect on an Elk-1 dependent reporter system. This inhibitory activity was dependent on a second signal that could be provided by activated p21 Ras or by MEKK-1 but not by Raf-1. Furthermore, RPM also displayed a strong negative regulatory effect on the growth of NIH 3T3 fibroblasts that were transformed by an activated Src Y527F. Since RPM did not inhibit the MEKK-1-dependent activation of the MAP-kinases Erk, JNK and p38, we conclude that expression of RPM uncouples Elk-1-dependent gene induction from MAP-kinase activation.