PLC Delta Research Articles

Calmodulin Interacts and Regulates Enzyme Activity of the Mammalian Sperm Phospholipase C

Sperm-specific Phospholipase C zeta (PLCζ) is widely considered to be the sole, physiological stimulus responsible for the generation of Ca2+ oscillations that induce egg activation and early embryo development during mammalian fertilization. PLCζ, which is delivered from the fertilizing sperm into the egg cytoplasm, catalyzes the hydrolysis of its membrane-bound phospholipid substrate phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], triggering the cytoplasmic Ca2+ oscillations through the inositol 1,4,5-trisphosphate (InsP3) signaling pathway. Despite the recent advances the detailed regulatory mechanism of PLCζ is still unclear, as binding partners of this protein within the sperm or the fertilizing egg have not yet been identified. Calmodulin (CaM) is a ubiquitous Ca2+ sensor in eukaryotic cells. A previous study has reported that CaM directly interacts and regulates the activity of PLC delta 1 protein, a somatic PLC isoform with structural similarities to sperm PLCζ. Bioinformatics analysis revealed putative CaM-binding sites on PLCζ sequence. In the present study, we have used co-immunoprecipitation analysis and we show that in the presence of Ca2+, human PLCζ directly interacts with CaM. Isothermal titration calorimetry (ITC) experiments were performed to map the interaction. Three different peptides corresponding to disparate sequences within human PLCζ were used and it was shown that PLCζ interacts with CaM via one region of the molecule. In addition, recombinant proteins corresponding to the N- and C-lobe of human CaM were used for ITC experiments, which revealed that CaM interacts with PLCζ in the presence of Ca2+, only through one of its lobe domains. In vitro PIP2 hydrolysis assays revealed that CaM alters PLCζ PIP2 hydrolytic activity at high Ca2+ concentrations and, as suggested by liposome binding assays, this appears to be due to CaM binding to PLCζ affecting proper access of the enzyme active site to its substrate PI(4,5)P2.

Characterization and Functional Validation of Tobacco PLC Delta for Abiotic Stress Tolerance

The role of plant phospholipase C-mediated signaling has been implicated in various phases of plant growth and development. In this study, we report on the isolation and characterization of phospholipase C from tobacco and demonstrate that transcripts of phospholipase C are up-regulated in responses to drought and salt stress. These responses are likely by abscisic acid (ABA). Transgenic tobacco plants overexpressing the phospholipase C protein were found to tolerate higher levels of drought and also salinity stress. This tolerance could be mediated by the regulation of genes downstream to phospholipase mediated signaling. As a demonstration, when tested the transgenic plants showed higher transcript of heat shock factor NtHSF2, heat shock protein HSP70-3 and an AP2 domain transcription factor. Also the transgenic plants showed higher accumulation of sodium in older leaves compared to the young leaves. The present report is the first to demonstrate the role of phospholipase C in salinity stress tolerance.

The Trophic Effect of Ouabain on Retinal Ganglion Cell is Mediated by EGF Receptor and PKC δ Activation

It was already shown that ouabain treatment can stimulate PKC isoenzymes leading to the activation of intracellular pathways involved in cell survival, growth and proliferation. We have previously demonstrated that ouabain or PMA treatment increases retinal ganglion cell survival, an effect mediated by PKC activation. The aim of this work was to investigate the role of EGF receptors in the ouabain effect and also to study which PKC isoform is activated by treatment with ouabain and PMA. Our results show that 2.5 microM tyrphostin, 1.0 microM PP1, 4.0 microM U73122, 1.0 microM JNK inhibitor V and 2.0 microM rottlerin blocked the ouabain effect indicating an involvement of receptors for EGF, Src, PLC, JNK and PKC delta respectively. The effect of PMA was only abolished when cultures were treated with rottlerin or with the JNK inhibitor suggesting the involvement of PKC delta and JNK. These results indicate that PKC delta could be a key regulator of retinal ganglion cell survival.

The Pleckstrin Homology Domains of Phospholipases C-β and -δ Confer Activation through a Common Site

Mammalian inositol-specific phospholipase C-beta2 (PLC beta 2) and PLC delta 1 differ in their cellular activators. PLC beta 2 can be activated by G beta gamma subunits, whereas PLC delta 1 can be activated by phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2). For both proteins, the N-terminal pleckstrin homology (PH) domain appears to mediate activation. Here, we have constructed a chimera in which we placed the N-terminal PH domain of PLC delta 1 into remaining C-terminal regions of PLC beta 2. The PH delta PLC beta chimera showed PI(4,5)P2-dependent membrane binding similar to PLC delta 1 and a G beta gamma interaction energy close to that of PLC delta 1. Like PLC delta 1, the chimera was activated by PI(4,5)P2 through the PH domain but not by G beta gamma. Because these and previous results indicate a common site of contact between the PH and catalytic domains in these two enzymes, we computationally docked the known structures of the PH and catalytic domains of PLC delta 1. A synthetic peptide whose sequence matches a potential interaction site between the two domains inhibited the basal activity of PLC beta 2, PLC delta 1, and a G beta gamma-activable PH beta 2-PLC delta 1 chimera. Also, the peptide was able to inhibit PI(4,5)P2 and G beta gamma activation of the PH-PLC delta 1 PH-PLC beta 2 enzymes in a concentration-dependent manner, suggesting that this is the region responsible for PH domain-mediated activation of the catalytic core.

Isozymes delta of phosphoinositide-specific phospholipase C.

Phospholipase C (PLC, EC 3.1.4.11) is the major starting point in the phosphatidylinositol pathway, which generates intracellular signals that regulate protein kinase C and intracellular calcium concentration. To date, three major types of phosphoinositide-specific PLC species named beta, gamma and delta, have been characterized. This article reviews recent studies on isozymes delta of PLC. Four such isozymes have been cloned and termed delta1-4. Their structural organization, regulation of activity and the interaction with membrane lipid are considered. The intracellular localization of delta isozymes and distribution in various tissues are presented. Attention is given to the pathological conditions in which an abnormal protein level of PLC delta or its activity have been observed.

Localization of phospholipase C delta3 in the cell and regulation of its activity by phospholipids and calcium.

The localization of phospholipase C delta3 (PLC delta3) in the cell and its regulatory properties has been investigated. Western blotting showed that human platelet PLC delta3 is located in the membrane and cytosolic fraction. The enzyme amount in the cytosolic fraction was significantly lower than that in the membrane fraction. In rat liver, PLC delta3 was present in both the membrane and cytosolic fraction and was absent in nuclei. Examination of the effects of phospholipids on PLC delta3 revealed that this enzyme is inhibited by phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Similar inhibition was observed in the presence of sphingomyelin and phosphatidylserine (PtdSer). This is in contrast to PLC delta1, which is activated by PtdCho and PtdEtn. In a detergent assay, PLC delta1 is activated by spermine and sphingosine, whereas PLC delta3 was inhibited by both these compounds at concentrations that maximally stimulated PLC delta1. A deletion mutant of PLC delta3, lacking the entire pleckstrin homology (PH) domain (residues 1-137), was fully active in the detergent assay, and it was inhibited by spermine, sphingosine and phospholipids to the same extent as the native enzyme. PLC delta3 activation required calcium ions. The relationship between the Ca2+ concentration and enzymatic activity was almost identical for the deletion mutant and the native enzyme. However, in the liposome assay, PLC delta3 was less sensitive to Ca2+ stimulation. This is in contrast to PLC delta1, which is equally sensitive to Ca2+ stimulation in both the detergent and liposome assays. We conclude that Ca2+ is necessary to induce specific conformational changes of PLC delta3, which leads to a productive orientation of the catalytic domain relative to the membrane. The regulatory properties of PLC delta3 described in this report suggest that PLC delta3 has a relatively low activity in cellular conditions that fully activate PLC delta1.

Expression, purification and kinetic properties of human recombinant phospholipase C delta 3.

To obtain sufficient quantities of pure phospholipase C delta 3 (PLC delta 3) necessary for structural and kinetic studies, cDNA of human fibroblast PLC delta 3 was cloned in the pPROEX-1 vector, expressed in E. coli cells as a (6 x His) fusion protein and purified to homogeneity. From 1 L of E. coli culture 8 mg of pure PLC delta 3 was obtained by a two step purification procedure, which includes phosphocellulose and Mono S cation exchange chromatography. The presence of His tag did not affect the catalytic and regulatory properties of PLC delta 3. The K(app) for PIP2 was 142 +/- 11 and 156 +/- 12 microM for His.PLC delta 3 and PLC delta 3, respectively. Recombinant PLC delta 3 showed an absolute requirement for Ca2+. Increasing the free Ca2+ concentration from 0.2 to 0.5 microM resulted in a sharp increase in enzyme activity. In comparison with human recombinant PLC delta 1 the delta 3 isoenzyme was more sensitive to low Ca2+ concentration. The Ca2+ concentration yielding maximal activation of PLC delta 1 and PLC delta 3 was 10 and 1 microM, respectively. The activity of PLC delta 3 was stimulated by polyamines and by basic proteins such as protamine, histone and mellitin. PLC delta 3 was activated most effectively by spermine and histone but the extent of this activation was lower than for PLC delta 1. The data presented indicate that the expression of PLC delta 3 in E. coli cells permits to obtain active enzyme. The catalytic and regulatory properties of PLC delta 3 are similar to those of PLC delta 1.

Cloning and Identification of Amino Acid Residues of Human Phospholipase Cδ1 Essential for Catalysis

In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC.

Dopamine D1 receptor regulation of phospholipase C.

Dopamine is an endogenous catecholamine which exerts its actions by occupancy of specific receptors. Dopamine receptors are classified into two main groups: the two cloned D1-like receptors (D1A and D1B in rats; D1B is also known as D5 in humans) are linked to stimulation of adenylyl cyclase, while the three cloned D2-like receptors (D2 or D2A, D3 or D2B, D4 or D2C) are linked to inhibition of adenylyl cyclase. All these dopamine receptors originally cloned from the brain are expressed in tissues outside the central nervous system including the kidney. Dopamine regulates many cellular activities, including transmembrane ion transport. Activation of D1-like receptor decreases sodium transport by cAMP dependent and cAMP independent mechanisms. Dopamine, via D1-like receptors, may inhibit Na+/H+ exchange activity in renal brush border membranes by a cAMP independent/Gs alpha-linked mechanism. Another cAMP independent pathway of sodium transport inhibition is mediated by phospholipase C, which has several isoforms (PLC beta, PLC gamma, and PLC delta with several members in each). Catecholamines stimulate expression and activity of phospholipase C isoforms in a concentration, time, and receptor-dependent as well as regional and subcellular compartmental-specific manner. In renal cortical membranes, intrarenal administration of norepinephrine for 3-4 h increases PLC beta expression and activity but has no effect on PLC gamma activity. In contrast, intrarenal administration of a D1 agonist for 3-4 h increases PLC beta 1 but decreases PLC gamma expression and activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin-mediated phosphoinositide hydrolysis in Chinese hamster ovary cells overexpressing phospholipase C-delta 1.

The regulatory mechanism(s) of a phosphoinositide-specific phospholipase C, PLC-delta 1, was investigated using a clone of stably overexpressed PLC-delta 1 (PLC delta 30 cells) in Chinese hamster ovary cells. Thrombin stimulation of PLC delta 30 cells exhibited 6.5-fold increase in total inositol phosphates (InsP), which was significantly higher than that in the vector-transfected (V1) cells (2.0-fold). AIF-4 increased InsP accumulation in both V1 and PLC delta 30 cells, and pertussis toxin partially blocked InsP accumulation in thrombin-stimulated PLC delta 30 cells. Guanosine thiotriphosphate (GTP gamma S) markedly potentiated thrombin-stimulated InsP generation in permeabilized PLC delta 30 cells compared with V1 cells, suggesting possible involvement of a G-protein (s) in the activation of PLC-delta 1. In PLC delta 30 cells, ionomycin-induced significant InsP generation and thrombin-stimulated InsP generation were completely inhibited by addition of EGTA. Furthermore, the stimulatory effects of thrombin plus GTP gamma S in PLC delta 30 cells were more sensitive to change in free calcium concentration than in V1 cells. Suppression by 12-O-tetradecanoylphorbol 13-acetate of thrombin-stimulated InsP accumulation was not affected by increasing Ca2+ concentration. These results indicate that thrombin-induced PLC-delta 1 activation is regulated via both G-protein(s) and calcium.

Binding of phospholipase Cδ1 to phospholipid vesicles

Binding of phospholipase C delta 1 (PLC delta) to phospholipid vesicles was studied using large, unilamellar phospholipid vesicles (LUVs). PLC delta bound weakly to vesicles composed of phosphatidylserine (PS) or phosphatidylcholine (PC) or phosphatidylethanolamine (PE) + PC, and even more weakly to vesicles composed of phosphatidylinositol. The enzyme bound strongly to LUVs composed of PE + PC and phosphatidylinositol 4,5-bisphosphate (PIP2) or sphingomyelin (SM). Binding of 50% of PLC delta occurred at 0.25 nmol/ml PIP2 when LUVs composed of PE + PC (molar ratio of 80:20), plus various amounts of PIP2, were used at a constant phospholipid concentration of 300 nmol/ml. When LUVs composed of PE + PC + PIP2 (molar ratio of 79:20:1) were tested as a function of increasing phospholipid concentration, 50% binding of PLC delta occurred at 1.2 nmol/ml PIP2 and 120 nmol/ml total phospholipid. Similar measurements were conducted with other phospholipids and PIP2 at a molar ratio of 99:1. These showed that 50% binding of PLC delta occurred at a level of 0.9 nmol/ml PIP2 with 80 nmol/ml PC; at 2.2 nmol/ml PIP2 with 170 nmol/ml PS; at 4.2 nmol/ml PIP2 with 320 nmol/ml PI; and at 0.26 nmol/ml PIP2 with 20 nmol/ml total liver phospholipids. Binding to phosphatidylinositol 4-phosphate was much weaker. When LUVs composed of PE + PC + SM (molar ratio 48:12:40) were tested as a function of increasing phospholipid concentration, 50% binding of PLC delta occurred at a level of 96 nmol/ml SM. This is well below the concentration of SM that can be calculated to face the cytosol. Binding of PLC delta to LUVs decreased as the temperature was lowered from 37 degrees C to 0 degree C. Thus PLC delta shows a high degree of specificity for binding to PIP2 and SM. Under physiological conditions a considerable fraction of PLC delta may be bound to cellular membranes, either in an inactive form if bound to PIP2 at low resting Ca2+ concentrations, or in the inhibited form if bound to SM.

The role of inositol phospholipids in the association of band 4.1 with the human erythrocyte membrane.

Band 4.1 is a major protein of the erythrocyte membrane skeleton. It promotes the binding of spectrin to F-actin and may anchor the skeletal network to the plasma membrane via its association with integral membrane proteins. Here, we have investigated the involvement of inositol phospholipids in the binding of band 4.1 to erythrocyte membranes using membrane vesicles stripped of all peripheral proteins at alkaline pH. Trypsinization of these vesicles allows the discrimination of two classes of band 4.1 binding sites: trypsin-sensitive sites (60-65% of the total), largely or exclusively on band 3, and trypsin-resistant sites (35-40% of the total), composed, at least in part, of the glycophorins. ATP depletion or activation of erythrocyte phosphoinositol phospholipase C led to a reduction in membrane phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] content by 20-70% in different experiments. The resulting decrease of band 4.1 binding to vesicles by was variable, but averaged about 15-20%. The same treatments led to an average decrease in the band 4.1 binding capacity of trypsinized vesicles of 55%. Since this is equivalent to a 20% decrease in the binding capacity of non-trypsinized vesicles (consistent with the above result), it indicates that PtdIns(4,5)P2 regulates the binding of band 4.1 only to trypsin-resistant binding sites (and to only a subset of these) accounting for about 15-20% of total band 4.1 binding sites on membranes. We found that hydrolysis of > 95% of PtdIns(4,5)P2 with exogenous phospholipase C-delta (PLC delta) resulted in no further decrease in band 4.1 binding to vesicles than did hydrolysis of 65-70% of PtdIns(4,5)P2 which is accessible to erythrocyte phosphoinositol phospholipase C. This suggests that only 65-70% of total membrane PtdIns(4,5)P2 is involved in regulating band 4.1 binding. Significantly, the pool of PtdIns(4,5)P2 involved is the same pool which can be hydrolysed by erythrocyte phosphoinositol phospholipase C, and which has been shown to be metabolically labile in erythrocytes. The membrane binding capacity for band 4.1 found in this study (averaging 1000 micrograms/mg vesicle protein) is considerably higher than that found in previous studies. The results are consistent with the existence of a binding site for band 4.1 on each copy of the major transmembrane proteins (band 3 and the glycophorins). These results provide new insights into the involvement of membrane inositol phospholipids in cytoskeletal-membrane interactions.