Phosphoinositide-specific phospholipase C (PI-PLC) plays a pivotal role in regulation of intracellular signal transduction from various receptor molecules. More than 10 members of human PI-PLC isoforms have been identified and classified into three classes ␤, ␥, and ␦, which are regulated by distinct mechanisms. Here we report identification of a novel class of human PI-PLC, named PLC⑀, which is characterized by the presence of a Ras-associating domain at its C terminus and a CDC25-like domain at its N terminus. The Ras-associating domain of PLC⑀ specifically binds to the GTP-bound forms of Ha-Ras and Rap1A. The dissociation constant for HaRas is estimated to be approximately 40 nM, comparable with those of other Ras effectors. Co-expression of an activated Ha-Ras mutant with PLC⑀ induces its translocation from the cytosol to the plasma membrane. Upon stimulation with epidermal growth factor, similar translocation of ectopically expressed PLC⑀ is observed, which is inhibited by co-expression of dominant-negative Ha-Ras. Furthermore, using a liposome-based reconstitution assay, it is shown that the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of PLC⑀ is stimulated in vitro by Ha-Ras in a GTP-dependent manner. These results indicate that Ras directly regulates phosphoinositide breakdown through membrane targeting of PLC⑀.

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.Ras proteins are small guanine nucleotide-binding proteins that serve as molecular switches in regulation of cellular proliferation and differentiation by cycling between the active GTP-bound and the inactive GDP-bound forms (for a review, see Ref. 1). In mammalian cells, the GTP-bound Ras exerts its action by physically associating with and activating effector proteins, such as the serine/threonine kinase Raf-1, through its effector region (amino acid residues 32-40 in human Ha-Ras). In addition to Raf-1 and its isoforms B-Raf and A-Raf, recent searches have identified a number of Ras effectors (or effector candidates) that associate directly with Ras in a GTP-dependent manner (for a review, see Ref.2). Two of them, RalGDS 1

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.

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