Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.
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PMID:Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence. 748 87

We identified a putative Saccharomyces cerevisiae homolog of a phosphoinositide-specific phospholipase C (PI-PLC) gene, PLC1, which encodes a protein most similar to the delta class of PI-PLC enzymes. The PLC1 gene was isolated during a study of yeast strains that exhibit defects in chromosome segregation. plc1-1 cells showed a 10-fold increase in aberrant chromosome segregation compared with the wild type. Molecular analysis revealed that PLC1 encodes a predicted protein of 101 kDa with approximately 50 and 26% identity to the highly conserved X and Y domains of PI-PLC isozymes from humans, bovines, rats, and Drosophila melanogaster. The putative yeast protein also contains a consensus EF-hand domain that is predicted to bind calcium. Interestingly, the temperature-sensitive and chromosome missegregation phenotypes exhibited by plc1-1 cells were partially suppressed by exogenous calcium.
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PMID:A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation. 839 35

Recent structural studies of mammalian phosphoinositide-specific phospholipase C (PI-PLC) have begun to shed light on the mechanism whereby this family of effector enzymes is able to hydrolyze phospholipid substrates to yield second messengers. PI-PLC isozymes employ a variety of modules (PH domain, EF-hand domain, SH2 domain, SH3 domain and C2 domain) that are common in proteins involved in signal transduction to reversibly interact with membranes and protein components of the signalling pathways.
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PMID:Structural views of phosphoinositide-specific phospholipase C: signalling the way ahead. 899 65

The pleckstrin homology (PH) domains of phospholipase C (PLC)-delta1 and a related catalytically inactive protein, p130, both bind inositol phosphates and inositol lipids. The binding to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by PLC-delta1 is proposed to be the critical interaction required for membrane localization to where the substrate resides; it is also required for the Ca(2+)-dependent activation of PLC-delta1 observed in the permeabilized cells. In the proximity of the PH domain, both PLC-delta1 and p130 possess the EF-hand domain, containing classical motifs implicated in calcium binding. Therefore, in the present study we examined whether the binding of the PH domain to PtdIns(4,5)P2 is regulated by changes in free Ca2+ concentration within the physiological range. A Ca2+ dependent increase in the binding to PtdIns(4,5)P2 was observed with a full-length PLC-delta1, while the isolated PH domain did not show any Ca2+ dependence. However, the connection of the EF-hand motifs to the PH domain restored the Ca2+ dependent increase in binding, even in the absence of the C2 domain. The p130 protein showed similar properties to PLC-delta1, and the EF-hand motifs were again required for the PH domain to exhibit a Ca2+ dependent increase in the binding to PtdIns(4,5)P2. The isolated PH domains from several other proteins which have been demonstrated to bind PtdIns(4,5)P2 showed no Ca2+ dependent enhancement of binding. However, when present within a chimera also containing PLC-delta1 EF-hand motifs, the Ca2+ dependent binding was again observed. These results suggest that the binding of Ca2+ to the EF-hand motifs can modulate binding to PtdIns(4,5)P2 mediated by the PH domain.
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PMID:Involvement of EF hand motifs in the Ca(2+)-dependent binding of the pleckstrin homology domain to phosphoinositides. 1049 Dec 7

Phosphoinositide-specific phospholipase C's (PI-PLCs) are ubiquitous in eukaryotes, from plants to animals, and catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In animals, four distinct subfamilies of PI-PLCs have been identified, and the three-dimensional structure of one rat isozyme, PLC-delta1, determined. Plants appear to contain only one gene family encoding PI-PLCs. The catalytic properties of plant PI-PLCs are very similar to those of animal enzymes. However, very little is known about the regulation of plant PI-PLCs. All plant PI-PLCs comprise three domains, X, Y and C2, which are also conserved in isoforms from animals and yeast. We here show that one PI-PLC isozyme from Arabidopsis thaliana, AtPLC2, is predominantly localized in the plasma membrane, and that the conserved N-terminal domain may represent an EF-hand domain that is required for catalytic activity but not for lipid binding.
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PMID:N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana. 1137 33

A thiol-reactive membrane-associated protein (TRAP) binds covalently to the cytoplasmic domain of the human insulin receptor (IR) beta-subunit when cells are treated with the homobifunctional cross-linker reagent 1,6-bismaleimidohexane. Here, TRAP was found to be phospholipase C gamma1 (PLCgamma1) by mass spectrometry analysis. PLCgamma1 associated with the IR both in cultured cell lines and in a primary culture of rat hepatocytes. Insulin increased PLCgamma1 tyrosine phosphorylation at Tyr-783 and its colocalization with the IR in punctated structures enriched in cortical actin at the dorsal plasma membrane. This association was found to be independent of PLCgamma1 Src homology 2 domains, and instead required the pleckstrin homology (PH)-EF-hand domain. Expression of the PH-EF construct blocked endogenous PLCgamma1 binding to the IR and inhibited insulin-dependent phosphorylation of mitogen-activated protein kinase (MAPK), but not AKT. Silencing PLCgamma1 expression using small interfering RNA markedly reduced insulin-dependent MAPK regulation in HepG2 cells. Conversely, reconstitution of PLCgamma1 in PLCgamma1-/- fibroblasts improved MAPK activation by insulin. Our results show that PLCgamma1 is a thiol-reactive protein whose association with the IR could contribute to the activation of MAPK signaling by insulin.
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PMID:Role of the pleckstrin homology domain of PLCgamma1 in its interaction with the insulin receptor. 1456 90

Phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate and diacylglycerol, both of which act as secondary messengers in animal cells. In this report, we identified in Vigna radiata L. (mung bean) three distinct partial cDNAs (pVr-PLC1, pVr-PLC2, and pVr-PLC3), which encode forms of putative PI-PLC. All three Vr-PLC genes were transcriptionally active and displayed unique patterns of expression. The Vr-PLC1 and Vr-PLC2 transcripts were constitutively expressed to varying degrees in every tissue of mung bean plants examined. In contrast, the Vr-PLC3 mRNA level was very low under normal growth conditions and was rapidly induced in an abscisic acid-independent manner under environmental stress conditions (drought and high salinity). An isolated genomic clone, about 8.2 kb in length, showed that Vr-PLC1 and Vr-PLC3 are in tandem array in the mung bean genome. The predicted primary sequence of Vr-PLC3 (M(r)=67.4 kDa) is reminiscent of the delta-isoform of animal enzymes which contain core sequences found in typical PI-PLCs, such as the catalytic domain comprising X and Y motifs, a lipid-binding C2 domain, and the less conserved EF-hand domain. Results of in vivo targeting experiment using a green fluorescent protein (GFP) showed that the GFP-Vr-PLC3 fusion protein was localized primarily to the plasma membrane of the Arabidopsis protoplast. The C2 domain was essential for Vr-PLC3 to be targeted to the plasma membrane. The possible biological functions of stress-responsive Vr-PLC3 in mung bean plants are discussed.
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PMID:The Vr-PLC3 gene encodes a putative plasma membrane-localized phosphoinositide-specific phospholipase C whose expression is induced by abiotic stress in mung bean (Vigna radiata L.). 1470 39

Lipids are well recognized ligands that bind to proteins in a specific manner and regulate their function. Most attention has been placed on the headgroup of phospholipids, and little is known about the role of the acyl chains in mediating any effects of lipids on proteins. In this report, free fatty acids (FFA) were found to bind and activate phospholipase C delta1(PLC delta1). The unsaturated FFA arachidonic acid (AA) and oleic acid were able to stimulate PLC delta1 up to 30-fold in a dose-dependent manner. The saturated FFA stearic acid and palmitic acid were less efficacious than unsaturated FFA, activating the enzyme up to 8-fold. The mechanism of activation appears to be due to a change in K(m) for substrate; 50 microM arachidonate reduced the K(m) for the soluble PLC substrate diC(4)PI from 1.7 +/- 0.6 mM to 0.24 +/- 0.04 mM (7-fold reduction). V(max) was not significantly altered. PLC delta1 bound to sucrose-loaded vesicles that contained AA in a concentration-dependent manner. A fragment of PLC delta1 that encompasses the EF-hand domain also bound to micelles containing AA using nondenaturing PAGE. This same fragment also inhibited AA activation of PLC delta1 in a competition assay. These results suggest that the function of the EF-hand domain of PLC delta1 is to bind lipid and to allosterically regulate catalysis. These results also suggest that esterified and nonesterified fatty acids can bind to and regulate protein function, identifying a functional role for hydrophobic interactions between lipids and proteins.
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PMID:Identification of hydrophobic interactions between proteins and lipids: free fatty acids activate phospholipase C delta1 via allosterism. 1518 94

The interaction of the isolated EF-hand domain of phospholipase C delta1 with arachidonic acid (AA) was characterized using circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectral changes indicate that AA binds to the EF domain. The near-UV CD spectra suggest that the orientations of aromatic residues in the peptide are affected when AA binds to the protein. The fluorescence of the single intrinsic tryptophan located in EF1 was enhanced by the addition of dodecylmaltoside (DDM) and AA suggesting that this region of the protein is involved in hydrophobic interactions. In the presence of a low concentration of DDM it was found that AA induced a change in fluorescence resonance energy transfer, which is indicative of a conformational change. The lipid induced conformational change may play a role in calcium binding because the isolated EF-hand domain did not bind Ca2+ in the absence of lipids, but Ca2+-dependent changes in the intrinsic tryptophan emission were observed when free fatty acids were present. These studies identify specific EF-hand domains as allosteric regulatory domains that require hydrophobic ligands such as lipids.
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PMID:Spectroscopic characterization of the EF-hand domain of phospholipase C delta1: identification of a lipid interacting domain. 1605 86

Members of the phosphoinositide-specific phospholipase C (PLC) family have key roles in cell signalling. In response to many extracellular stimuli, such as hormones, neurotransmitters, antigens and growth factors, PLCs catalyse the hydrolysis of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)], thereby generating two well-established second messengers, inositol (1,4,5)-trisphosphate and diacylglycerol. Eleven PLC isozymes encoded by different genes have been identified in mammals and, on the basis of their structure and sequence relationships, have been classified into five families designated PLCbeta (1-4), PLCgamma (1 and 2), PLCdelta (1, 3 and 4), PLCepsilon (1) and PLCzeta (1). All PLCs contain the catalytic X and Y domain, in addition to other regulatory domains including the C2 domain and the EF-hand domain. In 2005, four groups independently identified an entirely new family of PLCs--eta1 and eta2--using data mining of mammalian genomes. The properties of the PLCeta enzyme suggest that it might act as a Ca(2+) sensor, in particular, functioning during formation and maintenance of the neuronal network in the postnatal brain.
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PMID:The latest phospholipase C, PLCeta, is implicated in neuronal function. 1631 Mar 57


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