Mammalian Phospholipase C

Cardiovascular disease is the leading cause of death in the world. The phospholipase C (PLC) family of enzymes, in particular the PLCε subfamily, are essential for normal cardiovascular function. PLCε hydrolyzes phosphatidylinositol phosphates at cellular membranes, producing inositol phosphates (IPx) and diacylglycerol (DAG). These crucial secondary messengers activate multiple downstream pathways, including cardiac contractility and the expression of hypertrophic genes. In the cardiovascular system, PLCε is regulated through direct interactions with the RhoA and Rap1A small GTPases, which in turn are activated downstream of G protein‐coupled receptors (GPCRs). RhoA is reported to activate PLCε at the plasma membrane, whereas Rap1A translocates and activates PLCε at the perinuclear membrane. However, the elements within PLCε that regulate basal activity and membrane association have not been fully identified. Similarly, the domains involved in Rap1A versus RhoA binding, activation and translocation to different subcellular membranes have not been mapped. In this work, we use a structure‐guided approach, together with cell‐based activity assays, epifluorescence, and confocal microscopy to identify the roles of PLCε regulatory elements and domains in basal activity, subcellular localization, and regulation by RhoA and Rap1A GTPases. Functional studies show N‐ and C‐terminal regulatory domains of PLCε dictate its location within the cell, and contribute differently to basal and G protein‐dependent activity. We also show that regulatory insertions within the catalytic TIM barrel, including the X–Y linker and Y‐box, aid in interfacial activation and membrane association. These studies provide much needed insights into the molecular determinants of PLCε that regulate its localization and activity in cells, which is critical for elucidating its roles in cardiovascular function.

Backgroundα-isopropylmalate synthase (MtαIPMS), an enzyme that catalyzes the first committed step of the leucine biosynthetic pathway of Mycobacterium tuberculosis is a potential drug target for the anti-tuberculosis drugs. Cations induce differential effect of activation and inhibition of MtαIPMS. To date no concrete mechanism for such an opposite effect of similarly charged cations on the functional activity of enzyme has been presented.ResultsEffect of cations on the structure and function of the MtαIPMS has been studied in detail. The studies for the first time demonstrate that different cations interact specifically at different sites in the enzyme and modulate the enzyme structure differentially. The inhibitors Zn2+ and Cd2+ ions interact directly with the catalytic domain of the enzyme and induce unfolding/denaturation of the domain. The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure. Studies with isolated catalytic TIM barrel domain showed that it can carry out the catalytic function on its own but probably requires the non-catalytic C-terminal domain for optimum functioning. An important observation was that divalent cations induce significant interaction between the regulatory and the catalytic domain of MtαIPMS thus inducing structural cooperativity in the enzyme. This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.ConclusionThe studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.

The physiological effects of many extracellular stimuli are initiated through receptor-promoted activation of phospholipase C and inositol lipid signaling pathways. The historical view that phospholipase C-promoted signaling primarily occurs through activation of heterotrimeric G proteins or tyrosine kinases has expanded in recent years with the realization that at least three different mammalian phospholipase C isozymes are directly activated by members of the Ras superfamily of GTPases. Thus, Ras, Rap, Rac, and Rho GTPases all specifically regulate certain phospholipase C isozymes, and insight into the physiological significance of these signaling responses is beginning to accrue. High resolution three-dimensional structures of phospholipase C isozymes also are beginning to shed light on their mechanism of activation.

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.

The serine/threonine kinase protein kinase D1 (PKD1) is a protein kinase C (PKC) substrate that mediates antigen receptor signal transduction in lymphocytes. PKC phosphorylates serines 744/748 within the PKD1 catalytic domain, and this is proposed to be necessary and sufficient for enzyme activation. Hence, a PKD1 mutant with alanine substituted at positions 744 and 748 (PKD-S744A/S748A) is catalytically inactive. Conversely, a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active when expressed in Cos7 or HeLa cells. The present study reveals that Ser-744/Ser-748 phosphorylation is required for PKD1 activation in lymphocytes. However, PKD-S744E/S748E is not constitutively active but, like the wild type enzyme, requires antigen receptor triggering or phorbol ester stimulation. Antigen receptor activation of wild type PKD is dependent on phospholipase C (PLC)/diacylglycerol (DAG) and PKC, whereas PKD-S744E/S748E is only dependent on PLC/DAG but no longer requires PKC. Hence, substitution of serines 744 and 748 with glutamic residues as phospho-mimics bypasses the PKC requirement for PKD1 activation but does not bypass the need for antigen receptors, PLC, or DAG. In lymphocytes, PKD1 is, thus, not regulated by PLC and PKC in a linear pathway; rather, PKD1 activation has more stringent requirements for integration of dual PLC signals, one mediated by PKCs and one that is PKC-independent.

The C-terminal regulatory domain is required for catalysis by Neisseria meningitidis α-isopropylmalate synthase

General and Versatile Autoinhibition of PLC Isozymes

The field of inositol signaling has expanded greatly in recent years. Given the many reviews on phosphoinositide kinases, we have chosen to restrict our discussion to inositol lipid hydrolysis focused on the phosphatases and a brief mention of the lipase isoforms. We also discuss recent discoveries that link mutations in phosphoinositide phosphatases to disease.

A fragment coding for a putative extracellular alpha-amylase, from the genomic library of the yeast Saccharomycopsis fibuligera KZ, has been subcloned into yeast expression vector pVT100L and sequenced. The nucleotide sequence revealed an ORF of 1,485 bp coding for a 494 amino acid residues long protein with 99% identity to the alpha-amylase Sfamy from S. fibuligera HUT 7212. The S. fibuligera KZ alpha-amylase (Sfamy KZ) belongs to typical extracellular fungal alpha-amylases classified in the glycoside hydrolase family 13, subfamily 1, as supported also by clustering observed in the evolutionary tree. Sfamy KZ, in addition to the essential GH13 alpha-amylase three-domain arrangement (catalytic TIM barrel plus domains B and C), does not contain any distinct starch-binding domain. Sfamy KZ was expressed as a recombinant protein in Saccharomyces cerevisiae and purified to electrophoretic homogeneity. The enzyme had a molecular mass 53 kDa and contained about 2.5% of carbohydrate. The enzyme exhibited pH and temperature optima in the range of 5-6 and 40-50 degrees C, respectively. Stable adsorption of the enzyme to starch granules was not detected but a low degradation of raw starch in a concentration-dependent manner was observed.

Background: The Bruton agammaglobulinemia tyrosine kinase (BTK) activates B-cell receptor signaling through activation of phospholipase C gamma 2 (PLCG2). Clinical resistance to the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) is highly associated with emergence of the BTK C481 mutations that prevent ibrutinib covalent binding. PLCG2 mutations also occur in these ibrutinib-resistant samples but the spectrum of mutations and their occurrence with BTK changes have not been fully delineated. Materials and Methods: All peripheral blood samples with adequate depth of sequencing coverage were included from CLL patients receiving ibrutinib (with or without other therapies) that were submitted from Ohio State University (OSU) to the OSU James Polaris Molecular Laboratory. Genomic DNA was extracted from negatively selected B cells and deep sequencing of the entire coding regions of BTK and PLCG2 performed using a custom Ion Torrent Ampliseq panel. A mean depth of greater than 1000X was obtained with hotspot mutations validated down to 1% variant allele fraction (VAF) in the B cell preparations using orthogonal mutation-specific detection methods. Results: Among 1063 CLL samples from 380 patients who received ibrutinib, BTK C481 resistance mutations were identified in 79 (20.8%) patients including 20 patients that also had co-occurring PLCG2 mutations. 11 patients (2.9%) had PLCG2 mutations without accompanying BTK C481 alterations for a cumulative incidence of PLCG2 mutations in 8.2% of ibrutinib-treated patients. These included previously described mutations in the SH2 and SH3 domain of PLCG2 (R665W, S707F, A708P and L845F) but also previously uncharacterized mutations in the PLCG2 C2 domain that were seen in 12 patients (3.2%). C2 domain mutations, always seen in association with another PLCG2 and/or BTK resistance mutation, affected codons 1140-1144 that include the highly conserved aspartic acid residues that bind calcium and mediate membrane localization in other C2-domain containing proteins. In sequential samples, PLCG2 C2-domain mutations tracked at similar levels to the co-occurring BTK and PLCG2 resistance mutations indicating their presence in the same population of CLL cells. Conclusions: Mutations in three different PLCG2 structural domains commonly co-occur with BTK C481 mutations. The identification of PLCG2 mutations in the calcium-regulated C2 domain expands the possible mechanisms that can produce PLCG2 activation following ibrutinib treatment. The diversity of recurrent mutations observed supports the need for complete PLCG2 sequencing for full characterization of ibrutinib-treated CLL samples. Citation Format: Dan Jones, Jennifer A. Woyach, Weiqiang Zhao, Sean Caruthers, Huolin Tu, Joshua Coleman, John C. Byrd, Amy J. Johnson, Gerard Lozanski. PLCG2 C2-domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing treatment with the BTK inhibitor ibrutinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2017-3150

Phospholipase C

BackgroundPhospholipases hydrolyze glycerophospholipids and generate diverse lipid-derived molecules with secondary messenger activity. Out of these, phospholipase C (PLC) specifically cleaves the phospholipids at ester linkages and yields diacylglycerol (DAG) and phosphorylated head groups. PLCs are classified further as phosphatidylinositol-specific PLCs (PI-PLCs) and non-specific PLCs with biased specificity for phosphatidylcholine (NPC/PC-PLC). ResultsIn the present report, we identified and characterized PLC genes in the genomes of three orchids, Phalaenopsis equestris (seven PePLCs), Dendrobium catenatum (eight DcPLCs), and Apostasia shenzhenica (seven AsPLCs). Multiple sequence alignment analysis confirmed the presence of conserved X and Y catalytic domains, calcium/lipid-binding domain (C2 domain) at the C terminal region, and EF-hand at the N-terminal region in PI-PLC proteins and esterase domain in PC-PLC. Systematic phylogenetic analysis established the relationship of the PLC protein sequences and clustered them into two groups (PI-PLC and PC-PLC) along with those of Arabidopsis thaliana and Oryza sativa. Gene architecture studies showed the presence of nine exons in all PI-PLC genes while the number varied from one to five in PC-PLCs. RNA-seq-based spatio-temporal expression profile for PLC genes was generated, which showed that PePC-PLC1, PePC-PLC2A, DcPC-PLC1A, DcPC-PLC1B, DcPC-PLC2, DcPC-PLC1B, and AsPC-PLC1 had significant expression in all reproductive and vegetative tissues. The expression profile is matched to their upstream cis-regulatory promoter elements, which indicates that PLC genes have a role in various growth and development processes and during stress responses. ConclusionsThe present study unwrapped the opportunity for functional characterization of selected PLC genes in planta for plant improvement.

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gained recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Like protein kinase C isozymes, chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in alpha2-chimaerin that restricts access of phorbol esters and DAG, thereby limiting its activation. Although phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type alpha2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize alpha2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the ligand binding site in the alpha2-chimaerin C1 domain, we identified key amino acids that stabilize the inactive conformation. Mutation of these sites renders alpha2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Notably, in contrast to PMA, epidermal growth factor promotes full translocation of alpha2-chimaerin in a phospholipase C-dependent manner, but not of a C1 domain mutant with reduced affinity for DAG (P216A-alpha2-chimaerin). Therefore, DAG generation and binding to the C1 domain are required but not sufficient for epidermal growth factor-induced alpha2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of alpha2-chimaerin. Like other DAG/phorbol ester receptors, including protein kinase C isozymes, alpha2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.

For signal transduction to occur with rapid temporal resolution (on the order of milliseconds), there must be mechanisms for rapid signal termination (see news and views by Montell). Cook et al. studied phototransduction in Drosophila as one example where the response to the input signal (light intensity and light duration) must be tightly controlled to allow proper temporal and intensity resolution. Phototransduction in Drosophila occurs via activation of a G protein-coupled receptor that activates G q ; that, in turn, stimulates phospholipase C (PLC). The rate-limiting step in signal termination is the rate of GTPase activity of the activated G q . The authors found that the rate of signal termination and the ability to resolve light of varying intensities were dependent on the concentration of PLC and not on the interaction of PLC with a scaffold protein. PLC acted not only to propagate the light-generated signal, but also to terminate the signal by stimulating the GTPase activity of G q . These data provide in vivo confirmation of results found in vitro with mammalian PLC: PLC is a GTPase-activating protein (GAP). Montell, C. (2000) PLC fills a GAP in G-protein-coupled signalling. Nature Cell Biol. 2 : E82-E83. [Online Journal] Cook, B., Bar-Yaacov, M., Ben-Ami, H.C., Goldstein, R.E., Paroush, Z., Selinger, Z., and Minke, B. Phospholipase C and termination of G-protein-mediated signalling in vivo . Nature Cell Biol. 2 : 296-301. [Online Journal]

Dependence on the Lazaro Phosphatidic Acid Phosphatase for the Maximum Light Response