| Phosphoinositide-specific phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), generating two second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3). Five PLC families have been found to regulate diverse cell signaling processes. PLCepsilon, one of these five families, was first identified in C. elegans as a 210kDa Ras-interacting protein. The PIP2 hydrolysis activity of PLCepsilon has been shown to be activated by multiple signaling proteins including Ras, Rap1, Rap2, Rho A, Ral A, Galpha 12/13, and Gbetagamma. PLCepsilon has a guanine nucleotide exchange homology domain (GEF) at its N-terminus, which does not exist in any of the other PLC families.; This work aims to characterize the exchange activity of PLCepsilon and explore the physiological function of this PLC in vivo.; We hypothesized that PLCepsilonGEF has exchange activity with some small GTPases. We find that PLCepsilon activates Rap1 but not Ras in vitro . The lipid modification at the C-terminus of Rap1 is necessary for the interaction with the GEF domain. Based on this observation, we found a new substrate, Rap2, for the GEF activity. These results clearly indicate that PLCepsilon is a bifunctional enzyme.; We hypothesized that PLCepsilon has a specific function in vivo . To study the physiological function of PLCepsilon, two types of gene-targeted mice were created: A conventional gene knockout mouse (PLCepsilon -/-) and a conditional gene knockout mouse. The later one enabled us to study the physiological function of PLCepsilon in a specific time or tissue context.; PLCepsilon-/- mice develop normally to adulthood and appear to have no gross morphological or pathological phenotype. Echocardiography indicates these mice have reduced cardiac output. The mice also had an impaired cardiac response to acute isoproterenol. PLCepsilon protein and mRNA levels were upregulated in cardiac stress models while expression of other PLC isoforms was unchanged. In one model of cardiac stress, PLCepsilon null mice develop more severe hypertrophy than the wild type littermates. Our data suggests PLCepsilon has a specific physiological function positively regulating cardiac contractility. It may be protective against the stress-induced cardiac hypertrophy. |
