Phospholipase Cepsilon (PLCepsilon) generates lipid-derived second messengers in the cardiovascular system at the plasma and perinuclear membranes. It is activated in response to a wide variety of signals, such as those conveyed by Rap1A and Ras, through a mechanism that involves its C-terminal Ras association (RA) domains (RA1 and RA2). However, the complexity and size of PLCepsilon has hindered its structural and functional analysis. In this manuscript, we report the 2.7 Å crystal structure of fragment of PLCepsilon that retains catalytic activity. The strutcure includes the RA1 domain, which forms an integral part of the conserved core. In addition, a highly conserved amphipathic helix in the autoinhibitory X–Y linker is shown to modulate activity in vitro and in cells. The studies provide a structural framework for  the core of this critical cardiovascular enzyme that will allow for a better understanding of its regulation and roles in disease.

Thermal stability of the PLCepsilon variants was measured using differential scanning fluorimetry. Briefly, a final concentration of 0.5 mg/mL PLCepsilon variant protein was incubated with SYPRO Orange, and the increase in fluorescence was measured as a function of increasing temperature. The data was fit to a Boltzmann sigmoidal function, where the inflection point corresponds to the melting temperature of the protein. 

In vitro PLCepsilon activity was measured using a liposome-based activity assay. In this assay, [3H]-PIP2 is incorporated into PE/PIP2 liposomes, and the amount of free [3H]-IP3 produced is quantified by scintillation counting. Activity was measured at 30 C using a 5 point time course (2-10 min). Control reactions lacked Ca2+ and provide the background [3H]-IP3. Specific activity was calculated as described in 10.1385/1-59259-430-1:67.

Cell-based PLCepsilon activity was measured using the [3H]-inositol phosphates (IPx) accumulation assay. COS-7 cells are transfected with PLCe variants, and metabolically labelled with [3H]-myoinositol. Cells were treated with lithium chloride to inhibit IPx degradation, and [3H]-IPx species isolated by ion exchange chromatography and quantifed by scintillation counting. Protein expression was quantified using by western blot analysis (doi: 10.1126/scisignal.aan1210).