IDENTIFICATION OF PLC210, A CAENORHABDITIS-ELEGANS PHOSPHOLIPASE-C, AS A PUTATIVE EFFECTOR OF RAS

Mammalian Ras proteins regulate multiple effecters including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinosit ide 3-kinase. In the nematode Caenorhabditis elegans, LIN-45 Raf has b een identified by genetic analyses as an effector of LET-60 Ras. To se arch for other effecters in C. elegans, we performed a yeast two-hybri d screening for LET-60-binding proteins. The screening identified two cDNA clones encoding a phosphoinositide-specific phospholipase C (PI-P LC) with a predicted molecular mass of 210 kDa, designated PLC210, PLC 210 possesses two additional functional domains unseen in any known PI -PLCs. One is the C-terminal Ras-associating domain bearing a structur al homology with those of RalGDS and AF-6. This domain, which could be narrowed down to 100 amino acid residues, associated in vitro with hu man Ha-Ras in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific muta tions within the effector region of Ha-Ras. The other functional domai n is the N-terminal CDC25-like domain, which possesses a structural ho mology to guanine nucleotide exchange proteins for Ras. These results strongly suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras.