Abstract
Phospholipase Cε (PLCε) generates lipid-derived second messengers at the plasma and perinuclear membranes in the cardiovascular system. 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 PLCε has hindered its structural and functional analysis. Herein, we report the 2.7 Å crystal structure of the minimal fragment of PLCε that retains basal activity. This structure includes the RA1 domain, which forms extensive interactions with other core domains. A conserved amphipathic helix in the autoregulatory X–Y linker of PLCε is also revealed, which we show modulates activity in vitro and in cells. The studies provide the structural framework for the core of this critical cardiovascular enzyme that will allow for a better understanding of its regulation and roles in disease.
Highlights
Phospholipase Cε (PLCε) generates lipid-derived second messengers at the plasma and perinuclear membranes in the cardiovascular system
Starting from this point, an additional series of catalytically active PLCε domain deletion variants were purified from baculovirus-infected insect cells (Fig. 1a): PHCOOH, the largest PLCε fragment expressed and purified to date, pleckstrin homology (PH)-COOH ΔRA1; PH-RA1; PH-C2, and EF3-RA1, which was designed based on the structure of PLCδ23
These results demonstrate that the RA1 domain, and the region connecting the C2 and RA1 domains, contribute to the structure and catalytic competency of the PLCε catalytic core
Summary
Phospholipase Cε (PLCε) generates lipid-derived second messengers at the plasma and perinuclear membranes in the cardiovascular system. We report the 2.7 Å crystal structure of the minimal fragment of PLCε that retains basal activity. This structure includes the RA1 domain, which forms extensive interactions with other core domains. Many PLC enzymes are activated in response to extracellular stimuli conveyed by G-protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), and contribute to numerous processes, including cell proliferation, differentiation, and survival[1,2,3,4]. Domains (RA1 and RA2) that have been proposed to autoinhibit basal activity[18], interact with muscle-specific A-kinase anchoring protein (mAKAP) at the perinuclear membrane[19], and bind activated Rap1A and Ras[13,20,21]
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