Ras proteins signal to a number of distinct pathways by interacting with diverse effectors. Studies of ras/effector interactions have focused on three classes, Raf kinases, ral guanylnucleotide-exchange factors, and phosphatidylinositol-3-kinases. Here we describe ras interactions with another effector, the recently identified phospholipase C epsilon (PLCepsilon). We solved structures of PLCepsilon RA domains (RA1 and RA2) by NMR and the structure of the RA2/ras complex by X-ray crystallography. Although the similarity between ubiquitin-like folds of RA1 and RA2 proves that they are homologs, only RA2 can bind ras. Some of the features of the RA2/ras interface are unique to PLCepsilon, while the ability to make contacts with both switch I and II regions of ras is shared only with phosphatidylinositol-3-kinase. Studies of PLCepsilon regulation suggest that, in a cellular context, the RA2 domain, in a mode specific to PLCepsilon, has a role in membrane targeting with further regulatory impact on PLC activity.

Phospholipase Ce (PLCe) is a novel member of phosphoinositide-specific phospholipase C enzymes with a unique regulatory link to Ras GTP-ases. In the present studies, we establish existence of two splice variants (PLCe1a and PLCe1b) derived from human PLCe1 gene. When expressed in COS or HEK293 cells, PLCe1a and PLCe1b have similar potential to be stimulated by diverse signaling pathways via tyrosine kinase and G-protein coupled receptors and share the ability to function as an effector of Ras. The expression pattern shows broader mRNA expression of PLCe1a in normal tissues; furthermore, in most cell lines expressing PLCe, PLCe1a is the only splice variant present. Analysis of normal/tumor matched pairs derived from colon and rectum demonstrates greatly reduced expression levels in tumor tissues. Further studies in a colorectal tumor cell line lacking PLCe show restoration of transcription of PLCe1a and PLCe1b by demethylating agent 5-aza-2 0 -deoxycytidine, suggesting epigenetic silencing through hypermethylation. In addition, expression of exogenous PLCe in this cell line demonstrates inhibitory effects of PLCe on cell viability and proliferation. Taken together, our findings suggest that regulatory mechanisms controlling expression of PLCe, broadened by diversity introduced by splice variants, could play important role in PLCe regulation in normal and tumor cells.