Genomes and Genes
REGULATION OF PHOSPHOLIPASE C
Principal Investigator: JOHN E contact SONDEK
Affiliation: University of North Carolina
Abstract: Many extracellular stimuli produce their physiological effects through promotion of inositol lipid hydrolysis. These responses historically have been thought to occur either through heptahelical receptors coupled to heterotrimeric G proteins that activate phospholipase C-beta (PLC-beta) isozymes or through tyrosine kinase-activating receptors producing SH2 domain-dependent activation of PLC-gamma. However, small GTP binding proteins of the Rac family activate PLC-beta2, and we recently illustrated that this interaction occurs through GTP-dependent binding of Rac to the PH domain. PLC-epsilon was discovered as a novel phospholipase C activated by heterotrimeric G protein subunits (Galpha12/13 and Gbetagamma) as well as by interaction of Ras family GTPases with two Ras-associating domains in the C-terminus. We recently discovered that Rho family GTPases also directly activate PLC-epsilon in part through a unique insert in the conserved catalytic core of the enzyme. In addition PLC-epsilon acts as an upstream activator of small GTP binding proteins through guanine nucleotide exchange activity of a Cdc25 domain in its N-terminus. Thus, signals from both heterotrimeric and small GTPases converge on PLC-beta2, and both converge and diverge through the nexus PLC-epsilon. The goal of this research is to elucidate the mechanistic/structural bases for the regulated activities of PLC-beta and PLC-epsilon isozymes. We are defining the spectrum of small GTPases within the Rho and Ras families that bind to and activate PLC-epsilon and PLC-beta2. Consequently, we will delineate the functional interfaces responsible for these interactions by mutational analyses (Specific Aim 1). These studies will include surface plasmon resonance analyses to define direct interactions, co-transfection studies measuring inositol phosphate accumulation, and in vitro reconstitution approaches quantifying phospholipase C activity. PLC-epsilon exhibits novel downstream signaling activities through the functional interplay of its Cdc25 domain with other portions of PLC-epsilon. The multifunctional nature of this isozyme will be explored in detail (Specific Aim 2) by quantitatively assessing the guanine nucleotide exchange potential of PLC-epsilon against a broad panel of GTPases and by mutationally defining the interacting surfaces of PLC-epsilon and Ras GTPases. An ultimate goal of our research is to define the structural organization of PLC-beta and PLC-epsilon using crystallography. Accordingly, Specific Aim 3 will focus on structure determination of (i) full length and various structural domains of PLC-beta and PLC-epsilon isozymes, including high affinity complexes between: (ii) Rac and the PH domain of PLC-(2, (iii) Ras family GTPases and portions of PLC-epsilon containing the Ras-binding or Cdc25 domains, as well as the newly identified Rho-interacting region within the catalytic core. Accomplishment of these research goals will provide major new insights into the underlying mechanistic and structural bases for the diverse functions of these multifaceted signaling proteins that fulfill central roles in the signaling responses to many extracellular stimuli and that exist as potential drug targets for broadly diverse diseases.
Funding Period: 1998-05-01 - 2008-04-06
more information: NIH RePORT
- Autoinhibition and phosphorylation-induced activation of phospholipase C-γ isozymesNicole Hajicek
Department of Pharmacology and Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 7365, United States
Biochemistry 52:4810-9. 2013..This idea is supported by mutations in a complementary surface of the catalytic core that also enhanced phospholipase activity. ..
- Kinetic scaffolding mediated by a phospholipase C-beta and Gq signaling complexGary L Waldo
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
Science 330:974-80. 2010..Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs...
- Direct activation of human phospholipase C by its well known inhibitor u73122Ryan R Klein
Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599 7360, USA
J Biol Chem 286:12407-16. 2011..Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes...
- A fluorogenic, small molecule reporter for mammalian phospholipase C isozymesWeigang Huang
Division of Medicinal Chemistry and Natural Products, The University of North Carolina at Chapel Hill, 27599, United States
ACS Chem Biol 6:223-8. 2011..This novel reporter will likely find broad applications that vary from imaging PLC activity in live cells to high-throughput screening of PLC inhibitors...
- Mechanism of phosphorylation-induced activation of phospholipase C-gamma isozymesAurelie Gresset
Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 7365, USA
J Biol Chem 285:35836-47. 2010..Consequently, PLC-γ isozymes link phosphorylation to phospholipase activation by elaborating upon primordial regulatory mechanisms found in other PLCs...
- Both forward and reverse TCA cycles operate in green sulfur bacteriaKuo Hsiang Tang
Department of Biology and Chemistry, Washington University, St Louis, Missouri 63130, USA
J Biol Chem 285:35848-54. 2010..Moreover, CO(2) is essential for assimilating acetate and pyruvate through the CO(2)-anaplerotic pathway and pyruvate synthesis from acetyl-CoA...
- Prediction of protein-protein interfaces on G-protein beta subunits reveals a novel phospholipase C beta2 binding domainErin J Friedman
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
J Mol Biol 392:1044-54. 2009..Finally, this comparative evolutionary approach is suitable for application to any protein involved in a significant number of protein-protein interactions...
- Phospholipase C isozymes as effectors of Ras superfamily GTPasesT Kendall Harden
Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
J Lipid Res 50:S243-8. 2009..High resolution three-dimensional structures of phospholipase C isozymes also are beginning to shed light on their mechanism of activation...
- General and versatile autoinhibition of PLC isozymesStephanie N Hicks
Department of Pharmacology, The University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
Mol Cell 31:383-94. 2008..Similar regulation occurs in other PLC members, and a general mechanism of interfacial activation at membranes is presented that provides a unifying framework for PLC activation by diverse stimuli...
- Activation of human phospholipase C-eta2 by GbetagammaYixing Zhou
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA
Biochemistry 47:4410-7. 2008..Taken together, these studies illustrate that PLC-eta2 is a direct downstream effector of Gbetagamma and, therefore, of receptor-activated heterotrimeric G proteins...