Identification of PLC210, a Caenorhabditis elegansPhospholipase C, as a Putative Effector of Ras

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.

The putative tumor suppressor RASSF1A homodimerizes and heterodimerizes with the Ras-GTP binding protein Nore1

“…The motif thereby developed predicted the presence of the previously identi®ed RA domain in the Rin polypeptides and predicted the presence of previously unknown RA domains in a number of polypeptides (including T24F1.3) several of which subsequently have been veri®ed experimentally. Thus the C. elegans phospholipase C, PLC210 (F31B12.1) (Shibatohge et al, 1998) and its human homolog PLCe do contain two functional RA domains, as predicted. Although both appear to be required for Ras-GTP activation of PLCe (Song et al, 2001;Kelley et al, 2001) in vivo, only RA-2 binds Ras-GTP4Ras-GDP; RA-1 binds Ras speci®-cally but with a much lower a nity than RA-2 and independently of GTP (Kelley et al, 2001).…”

Regulation of a Novel Human Phospholipase C, PLCε, through Membrane Targeting by Ras

“…FLAG-PLC⑀ was affinity purified with resin conjugated with anti-FLAG monoclonal antibody M2 (Sigma). The PI-PLC activities were measured essentially as described (21). Briefly, the fusion proteins were incubated in 50-l reaction mixtures containing 50 mM Mes, pH 6.8, 10 M Ca 2ϩ /EGTA, 100 mM NaCl, 0.2 mg/ml bovine serum albumin, 0.…”

“…In this report, we have identified a novel class of human PI-PLC, named PLC⑀, which is characterized by the presence of an RA domain, as a human homolog of Caenorhabditis elegans PLC210 (21). We demonstrate that PLC⑀ is regulated through direct association with Ras, adding another member to the growing list of Ras effectors.…”

Differential roles of Ras and Rap1 in growth factor-dependent activation of phospholipase Cε

“…PLCe is the most recently identified member of the PLC family. A Caenorhabditis elegans homolog of PLCe, designated PLC210, was initially characterized as a novel Ras-binding protein through yeast two-hybrid screening (Shibatohge et al, 1998), followed by the isolation of human , rat ) and mouse (Wu et al, unpublished data) cDNAs encoding PLCe. Screening of human sequence databases by the use of amino acid sequences conserved in PLC isoforms also led to the identification of the same protein (Lopez et al, 2001).…”