Abstract
Mammalian Ras proteins regulate multiple effectors including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinositide 3-kinase. In the nematode Caenorhabditis elegans, LIN-45 Raf has been identified by genetic analyses as an effector of LET-60 Ras. To search for other effectors in C. elegans, we performed a yeast two-hybrid screening for LET-60-binding proteins. The screening identified two cDNA clones encoding a phosphoinositide-specific phospholipase C (PI-PLC) with a predicted molecular mass of 210 kDa, designated PLC210. PLC210 possesses two additional functional domains unseen in any known PI-PLCs. One is the C-terminal Ras-associating domain bearing a structural homology with those of RalGDS and AF-6. This domain, which could be narrowed down to 100 amino acid residues, associated in vitro with human Ha-Ras in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific mutations within the effector region of Ha-Ras. The other functional domain is the N-terminal CDC25-like domain, which possesses a structural homology 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.
Highlights
Ras proteins are small guanine nucleotide-binding proteins that function as molecular switches by cycling between the active GTP-bound state and the inactive GDP-bound state
Extensive genetic studies have demonstrated that LET-60 participates in a signal transduction cascade that includes LIN-45 (Raf), MEK-2 (MEK), SUR-1/MPK-1, and other proteins, which are highly homologous to their mammalian counterparts. let-60 functions genetically downstream of let-23, a gene whose product resembles a receptor for epidermal growth factor, in post-embryonic induction of the vulva of hermaphrodites [8]
Cloning of PLC210, a Novel Phophoinositide-specific Phopholipase C—By the yeast two-hybrid screening using LET60Val-12 as a bait, we have identified 70 independent partial cDNA clones encoding LIN-45 Raf, Ral guanine nucleotide dissociation stimulator (RalGDS)-like protein, and other proteins
Summary
Ras proteins are small guanine nucleotide-binding proteins that function as molecular switches by cycling between the active GTP-bound state and the inactive GDP-bound state (for a review see Ref. 1). Cloning of PLC210, a Novel Phophoinositide-specific Phopholipase C—By the yeast two-hybrid screening using LET60Val-12 as a bait, we have identified 70 independent partial cDNA clones encoding LIN-45 Raf, RalGDS-like protein, and other proteins.2 Among them, two clones with overlapping cDNA inserts, pACT4-2 and pACT9-1, were found to represent the C-terminal portion of a novel Ras-binding protein (Fig. 1A).
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