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.
Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce diacylglycerol (DAG) and inositol phosphates, to activate protein kinase C (PKC) and downstream signaling pathways, including cell growth and survival. The PLCɛ subfamily has emerged as a key player in cardiovascular health, where it is required for maximum contractility. However, prolonged activation of PLCɛ results in cardiac hypertrophy and heart failure through its ability to regulate hypertrophic gene expression. This process is regulated by the small GTPase Rap1A, which is activated downstream of β‐adrenergic receptors. Rap1A binds to the C‐terminal Ras association (RA) domain of PLCɛ, simultaneously translocating the complex to the perinuclear region and activating PLCɛ. However, the molecular mechanism of this process is not known. We seek to characterize the interactions between Rap1A and PLCɛ using structural and functional studies to map the Rap1A binding site on PLCɛ and determine whether activation results in conformational changes that release autoinhibition and/or increase membrane association. These studies provide the first molecular details of the Rap1A‐dependent activation of PLCɛ and opens the door to the development of new therapeutic strategies for treating cardiac hypertrophy.Support or Funding InformationAmerican Heart Association Scientist Development Grant (16SDG29920017) to A.M.L.; Purdue Center for Cancer ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phospholipase Cε (PLCe) is activated downstream of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with small GTPases, including Rap1A and Ras. While Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability, or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors (β-ARs), translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation, and identified hydrophobic residues on the surface of the RA2 domain that are also necessary for activation. Finally, small angle X-ray scattering (SAXS) showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. This data, together with the recent structure of a catalytically active fragment of PLCe, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLC core.
Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce diacylglycerol (DAG) and inositol phosphates, leading to the activation of protein kinase C (PKC) and downstream signaling pathways, including cell growth and survival. The PLCɛ subfamily is a key player in cardiovascular function, where it contributes to maximum contractility. However, prolonged activation of PLCɛ results in cardiac hypertrophy and heart failure through its ability to regulate the expression of hypertrophic genes. This process is regulated by the small GTPase Rap1A, which is activated downstream of β‐adrenergic receptors. Rap1A binds to the C‐terminal Ras association (RA) domain of PLCɛ, simultaneously translocating the complex to the perinuclear region and activating PLCɛ. PLCɛ also contains an N‐terminal CDC25 domain, which has guanine nucleotide exchange factor (GEF) activity for Rap1A resulting in a feed forward activation loop and sustained lipid hydrolysis. However, the molecular mechanism of this process is not known. In this work, we seek to characterize the interactions between Rap1A and PLCɛ using structural and functional studies to map the Rap1A binding site on PLCɛ and determine whether activation results in conformational changes that release autoinhibition and/or increase membrane association. These studies provide the first molecular details of the Rap1A‐dependent activation of PLCɛ and open the door to the development of new therapeutic strategies for treating cardiac hypertrophy.Support or Funding InformationThis research is supported by the American Heart Association Predoctoral Fellowship 18PRE33990057 to M.S., American Heart Association Grant Scientist Development Grant 16SDG29920017 to A.M.L. and the NIH NLHBI 1R01HL 141076‐01 to A.M.L. SAXS data was collected and analyzed with the assistance of S. Chakravarthy at Argonne National Laboratory.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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