Phosphoinositide-specific Phospholipase C Isoforms Research Articles

Phosphoinositide-specific phospholipase C isoforms are conveyed by osteosarcoma-derived extracellular vesicles.

Cancer cells are able to release high amounts of extracellular vesicles, thereby conditioning the normal cells in the surrounding tissue and/or in distant target organs. In the context of bone cancers, previous studies suggested that osteosarcoma cancer cells produce transforming extracellular vesicles able to induce a tumour-like phenotype in normal recipient cells. Indeed, phosphoinositide-specific phospholipase C (PI-PLC) enzymes are differentially expressed in osteosarcoma cell lines with increasing aggressiveness, thus providing helpful insights to better define their role and functions in this bone tumour. By confocal microscopy analysis, we demonstrated that osteosarcoma-derived extracellular vesicles convey all the assessed PI-PLC isoforms, and that they localize into cell membrane bubble-like structures, resembling extracellular vesicles about to be released, as conveyed and/or membrane protein. Cytofluorimetric analysis confirmed the presence of PI-PLC isoforms in the extracellular vesicles collected from conditioned media of osteosarcoma cells. These findings suggest the feasibility to use circulating extracellular vesicles as biomarkers of osteosarcoma progression and/or the monitoring of this distressing disease.

Ezrin-related Phosphoinositide pathway modifies RhoA and Rac1 in human osteosarcoma cell lines.

Selected Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes occupy the convergence point of the broad range of pathways that promote Rho and Ras GTPase mediated signalling, which also regulate the activation of ezrin, a member of the ezrin-radixin-moesin (ERM) proteins family involved in the metastatic osteosarcoma spread. Previous studies described that in distinct human osteosarcoma cell lines ezrin networks the PI-PLC with complex interplay controlling the expression of the PLC genes, which codify for PI-PLC enzymes. In the present study, we analyzed the expression and the sub-cellular distribution of RhoA and Rac1 respectively after ezrin silencing and after PI-PLC ε silencing, in order to investigate whether ezrin-RhoGTPAses signalling might involve one or more specific PI-PLC isoforms in cultured 143B and Hs888 human osteosarcoma cell lines. In the present experiments, both ezrin and PLCE gene silencing had different effects upon RhoA and Rac1 expression and sub-cellular localization. Displacements of Ezrin and of RhoA localization were observed, probably playing functional roles.

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca(2+) that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane.

Expression of Phosphoinositide-specific phospholipase C enzymes in human osteosarcoma cell lines

The definition of the number and nature of signal transduction pathways networking in the pathogenesis of osteosarcoma raised great interest. Intracellular calcium ions are important second messengers implicated in the control of cell death. The calcium concentration is regulated by signal transduction pathways, including the Phosphoinositides (PI) signaling. Phosphatydil inositol (4,5) bisphosphate (PIP2) is critical for many cellular activities. The levels of PIP2 are regulated by means of Phosphoinositide-specific Phospholipase C (PI-PLC) family of enzymes. We delineated the panel of expression of PI-PLC enzymes in four human osteosarcoma cell lines. In MG-63 cell line, PI-PLC β1, β2, β3, β4, γ1, γ2, δ1, δ3 and ε resulted expressed. In 143B cell line, PI-PLC β1, β2, β3, β4, γ1, γ2, δ1, δ3 and ε were expressed. In SaOS-2 cell line, PI-PLC β1, β3, β4, γ1, γ2, δ1, δ3, ε and η1. In Hs888 cell line, PI-PLC β1, β3, β4, γ1, δ1, δ3, δ4, ε and η1 the administration of U-73122 to cultures briefly modifies the levels of PI-PLC transcripts. The obtained complete expression panel of PI-PLC isoforms will be a useful tool for further functional studies about the role of the PI signal transduction pathway in osteosarcoma.

Expression of phospholipase C genes in cultured endothelial cells

During inflammation, endothelial cells (EC) are the first elements to be exposed to mediators circulating in the bloodstream. EC react with finely tuned responses mediated by different pathways, including the Phosphoinositide (PI) signal transduction system. The PI pathway contributes to a variety of cell functions, including hormone secretion, neurotransmitter signal transduction, cell growth, membrane trafficking, ion channel activity, cytoskeleton regulation, cell cycle control, apoptosis, embryonic development, organogenesis, and cell/tissue polarity. The Phosphoinositide-specific phospholipase C (PI-PLC) enzymes contribute to the regulation of the spatio-temporal balance of molecules belonging to the PI system. Thirteen mammalian PI-PLC enzymes have been identified, divided into six sub-families on the basis of amino acid sequence, domain structure and mechanism of recruitment. Isoforms within sub-families share sequence similarity, common domain organization, and general regulatory mechanism. The expression of PI-PLCs is strictly tissue specific, and evidences suggest that it varies under different conditions, such as tumor progression or cell activation. We obtained the complete panel of expression of PI-PLC isoforms in human umbilical vein endothelial cells (HUVEC), a widely used experimental model for EC. Then, we analyzed the mRNA concentration of PI-PLCs in LPS treated HUVEC by using the multiliquid bioanalyzer methodology after 3, 6, 24 48 and 72 hours from LPS administration. Marked differences in the expression of most PI-PLC codifying genes were evident.

Expression of Phosphoinositide-Specific Phospholipase C Enzymes in Human Skin Fibroblasts

Fibroblasts are involved in a number of functions regulated by different signal transduction pathways, including the phosphoinositide (PI) signaling system and related converting enzymes, such as phosphoinositide-specific phospholipase C (PI-PLC). The PI-PLC family comprises crucial effector enzymes in the PI signal transduction pathway. Once activated, PI-PLC cleaves an important membrane PI, the phosphatidylinositol (4,5) bisphosphate into inositol trisphosphate and diacylglycerol—both are crucial molecules in the transduction of signals. The activity of selected PI-PLC enzymes was reported in fibroblasts, although the complete panel of expression was not available. Each cell type expresses a group of selected PI-PLC isoforms, and knowledge of the panel of expression is a necessary and preliminary tool to address further studies. In the present study, we delineated the expression panel of PI-PLC enzymes in human skin fibroblasts. PI-PLC β1, PI-PLC β3, PI-PLC β4, PI-PLC γ1, PI-PLC γ2, PI-PLC δ1, PI-PLC δ3, PI-PLC δ4, and PI-PLC ϵ were expressed. PI-PLC β1 was weakly expressed, PI-PLC δ4 was inconstantly expressed, and PI-PLC γ2 was weakly expressed.

Expression of phosphoinositide‐specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with lipopolysaccharide

Signal transduction pathways, involved in cell cycle and activities, depend on various components including lipid signalling molecules, such as phosphoinositides and related enzymes. Many evidences support the hypothesis that inositol lipid cycle is involved in astrocytes activation during neurodegeneration. Previous studies investigated the pattern of expression of phosphoinositide-specific phospholipase C (PI-PLC) family isoforms in astrocytes, individuating in cultured neonatal rat astrocytes, supposed to be quiescent cells, the absence of some isoforms, accordingly to their well known tissue specificity. The same study was conducted in cultured rat astrocytoma C6 cells and designed a different pattern of expression of PI-PLCs in the neoplastic counterpart, accordingly to literature suggesting a PI signalling involvement in tumour progression. It is not clear the role of PI-PLC isoforms in inflammation; recent data demonstrate they are involved in cytokines production, with special regard to IL-6. PI-PLCs expression in LPS treated neonatal rat astrocytes performed by using RT-PCR, observed at 3, 6, 18 and 24 h intervals, expressed: PI-PLC beta1, beta4 and gamma1 in all intervals analysed; PI-PLC delta1 at 6, 18 and 24 h; PI-PLC delta3 at 6 h after treatment. PI-PLC beta3, delta4 and epsilon, present in untreated astrocytes, were not detected after LPS treatment. Immunocytochemical analysis, performed to visualize the sub-cellular distribution of the expressed isoforms, demonstrated different patterns of localisation at different times of exposure. These observations suggest that PI-PLCs expression and distribution may play a role in ongoing inflammation process of CNS.

Phospholipase C Isoforms Are Localized at the Cleavage Furrow during Cytokinesis

It has recently been demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP2) is localized at the cleavage furrow in dividing cells and its hydrolysis is required for complete cytokinesis, suggesting a pivotal role of PIP2 in cytokinesis. Here, we report that at least three mammalian isoforms of phosphoinositide-specific phospholipase C (PLC), PLCdelta1, PLCdelta3 and PLCbeta1, are localized to the cleavage furrow during cytokinesis. Targeting of the delta1 isoform to the furrow depends on the specific interaction between the PH domain and PIP2 in the plasma membrane. The necessity of active PLC in animal cell cytokinesis was confirmed using the specific inhibitors for PIP2 hydrolysis. These results support the model that activation of selected PLC isoforms at the cleavage furrow controls progression of cytokinesis through regulation of PIP2 levels: induction of the cleavage furrow by a contractile ring consisting of actomyosin is regulated by PIP2-dependent actin-binding proteins and formation of specific lipid domains required for membrane separation is affected by alterations in the lipid composition of the furrow.

Regulation of Phospholipase C-δ1 through Direct Interactions with the Small GTPase Ral and Calmodulin

Second messengers generated from membrane lipids play a critical role in signaling and control diverse cellular processes. Despite being one of the most evolutionarily conserved of all the phosphoinositide-specific phospholipase C (PLC) isoforms, a family of enzymes responsible for hydrolysis of the membrane lipid phosphatidylinositol bisphosphate, the mechanism of PLC-delta1 activation is still poorly understood. Here we report a novel regulatory mechanism for PLC-delta1 activation that involves direct interaction of the small GTPase Ral and the universal calcium-signaling molecule calmodulin (CaM) with PLC-delta1. In addition, we have identified a novel IQ type CaM binding motif within the catalytic region of PLC-delta1 that is not found in other PLC isoforms. Binding of CaM at the IQ motif inhibits PLC-delta1 activity, while addition of Ral reverses the inhibition. The overexpression of various Ral mutants in cells potentiates PLC-delta1 activity. Thus, the Ral-CaM complex defines a multifaceted regulatory mechanism for PLC-delta1 activation.

Regulation of phosphoinositide-specific phospholipase C.

Eleven distinct isoforms of phosphoinositide-specific phospholipase C (PLC), which are grouped into four subfamilies (beta, gamma, delta, and epsilon), have been identified in mammals. These isozymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to inositol 1,4,5-trisphosphate and diacylglycerol in response to the activation of more than 100 different cell surface receptors. All PLC isoforms contain X and Y domains, which form the catalytic core, as well as various combinations of regulatory domains that are common to many other signaling proteins. These regulatory domains serve to target PLC isozymes to the vicinity of their substrate or activators through protein-protein or protein-lipid interactions. These domains (with their binding partners in parentheses or brackets) include the pleckstrin homology (PH) domain [PtdIns(3)P, beta gamma subunits of G proteins] and the COOH-terminal region including the C2 domain (GTP-bound alpha subunit of Gq) of PLC-beta; the PH domain [PtdIns(3,4,5)P3] and Src homology 2 domain [tyrosine-phosphorylated proteins, PtdIns(3,4,5)P3] of PLC-gamma; the PH domain [PtdIns(4,5)P2] and C2 domain (Ca2+) of PLC-delta; and the Ras binding domain (GTP-bound Ras) of PLC-epsilon. The presence of distinct regulatory domains in PLC isoforms renders them susceptible to different modes of activation. Given that the partners that interact with these regulatory domains of PLC isozymes are generated or eliminated in specific regions of the cell in response to changes in receptor status, the activation and deactivation of each PLC isoform are likely highly regulated processes.

Fuel and hormone regulation of phospholipase C β1 and δ1 overexpressed in RINm5F pancreatic beta cells

The mechanism by which glucose and other fuels stimulate phosphoinositide-specific phospholipase C (PLC) in pancreatic islet beta cells is not known. Previous studies have suggested that glucose may couple to PLC β1 and PLC δ1. To determine directly if fuels activate these PLC isozymes, clones stably overexpressing PLC β1 or PLC δ1 were generated in the fuel-sensitive beta cell line RINm5F, and secretagogue regulation of these PLC isoforms was determined. Overexpression of PLC β1 or PLC δ1 significantly increased PLC activity in isolated cell fractions, consistent with overexpression of active PLC isoforms in these clones. In paired experiments, stimulation of inositol phosphate (IP) accumulation by the fuel glyceraldehyde was enhanced in clones overexpressing PLC β1, in parallel with the G-protein alpha subunit activator, AlF 4 −, suggesting a coupling between glyceraldehyde and this PLC isoform. In contrast, overexpression of PLC δ1 had no effect on glyceraldehyde- or AlF 4 −-stimulated IP accumulation. Similarly, IP accumulation stimulated by ionomycin was enhanced in PLC β1, but not PLC δ1 clones, indicating that increases in intracellular free calcium [Ca 2+] i can regulate PLC β1 but not PLC δ1 overexpressed in this cell line. Interestingly, [Arg 8] vasopressin-stimulated, but not carbachol-stimulated, IP accumulation was significantly increased in clones overexpressing either PLC β1 or PLC δ1. These studies illustrate unique pathways coupling diverse secretagogues to specific PLC isoforms in islet beta cells, and demonstrate that glyceraldehyde can activate PLC β1 but not PLC δ1; whereas, vasopressin, but not carbachol, can stimulate either isoform.

Regulation of a Novel Human Phospholipase C, PLCε, through Membrane Targeting by Ras

Phosphoinositide-specific phospholipase C (PI-PLC) plays a pivotal role in regulation of intracellular signal transduction from various receptor molecules. More than 10 members of human PI-PLC isoforms have been identified and classified into three classes beta, gamma, and delta, which are regulated by distinct mechanisms. Here we report identification of a novel class of human PI-PLC, named PLCepsilon, which is characterized by the presence of a Ras-associating domain at its C terminus and a CDC25-like domain at its N terminus. The Ras-associating domain of PLCepsilon specifically binds to the GTP-bound forms of Ha-Ras and Rap1A. The dissociation constant for Ha-Ras is estimated to be approximately 40 nm, comparable with those of other Ras effectors. Co-expression of an activated Ha-Ras mutant with PLCepsilon induces its translocation from the cytosol to the plasma membrane. Upon stimulation with epidermal growth factor, similar translocation of ectopically expressed PLCepsilon is observed, which is inhibited by co-expression of dominant-negative Ha-Ras. Furthermore, using a liposome-based reconstitution assay, it is shown that the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of PLCepsilon is stimulated in vitro by Ha-Ras in a GTP-dependent manner. These results indicate that Ras directly regulates phosphoinositide breakdown through membrane targeting of PLCepsilon.

Age-dependent variations in the expression of PLC isoforms upon mitogenic stimulation of peripheral blood T cells from healthy donors

The activation of phosphoinositide-specific phospholipase C (PLC) is one of the early responses to various growth factors and it is known that these functions are altered with ageing. In the present investigation, the expression and cellular distribution of PLC isoenzymes in immunocompetent T lymphocytes from a test group of healthy individuals over 65 years old and a comparison group of healthy donors below 30 years old were compared using Western blot and confocal microscopy. All lymphocyte samples responded biochemically to phytohaemagglutinin (PHA) stimulation, as shown by at least 10-fold increases in the β3 isoform and nuclear translocation of the β1, β2, β3, β4, γ1 and γ2 isoforms; however, consistent differences in the expression of the β2 isoform in unstimulated T cells and of the β2 and γ2 isoforms in stimulated T cells suggest that altered functions of the PLC pathway may have an age-dependent impact on signal transduction events.

Subcellular distribution of phospholipase C isoforms in rodent pancreas and gastric mucosa.

Phosphoinositide-specific phospholipase C (PLC) has been implicated as a participant in cell proliferation as well as enzyme and hormone secretion. Defining the subcellular distribution of PLC isoforms would possibly contribute to further understanding of their function. We investigated the intracellular distribution of four PLCs (beta1, beta2, beta3, and gamma1) in mouse pancreatic cells as well as mouse and rat gastric mucosa cells by ultrastructural immunocytochemistry. In pancreatic acinar cells, PLCbeta1 and PLCgamma1 were demonstrated in the zymogen granules while PLCbeta2 was present in the granulae as well as the endoplasmic reticulum (ER), and PLCbeta3 was prominent in the ER. In the endocrine pancreas, PLCbeta2 immunolabeling was expressed in the secretory granulae of alpha, beta, delta, and pancreatic polypeptide cells. PLCbeta3 showed a slight labeling in the nucleus and ER of all four pancreatic endocrine cell types while PLCgamma1 was prominent in alpha cell granulae. In the gastric mucosa cells, PLCbeta2 was highly expressed in the heterochromatin areas and in the ER of parietal, chief, mucous, and enterochromaffin-like cells. PLCbeta3 were expressed in a manner similar to PLCbeta2 in those cells; however, no immunoreaction was seen in the ER of parietal cell. PLCgamma1 was demonstrated in the chief cell granulae. One possible, although yet speculative, interpretation of our results is that the studied PLC isoforms may be involved in processing in pancreatic secretory granulae and that nuclear PLCbeta2 and PLCbeta3 signaling pathways may be operative in the cells of the gastric mucosa.

Identification and chromosomal localisation by fluorescence in situ hybridisation of human gene of phosphoinositide-specific phospholipase C β 1

Members of phosphoinositide-specific phospholipase C (PLC) families are central intermediary in signal transduction in response to the occupancy of receptors by many growth factors. Among PLC isoforms, the type β 1 is of particular interest because of its reported nuclear localisation in addition to its presence at the plasma membrane. It has been previously shown that both the stimulation and the inhibition of the nuclear PLCβ 1 under different stimuli implicate PLCβ 1 as an important enzyme for mitogen-activated cell growth as well as for murine erythroleukaemia cell differentiation. The above findings hinting at a direct involvement of PLCβ 1 in controlling the cell cycle in rodent cells, and the previously reported mapping of its gene in rat chromosome band 3q35–36, a region frequently rearranged in rat tumours induced by chemical carcinogenesis, prompted us to identify its human homologue. By screening a human foetal brain cDNA library with the rat PLCβ 1 cDNA probe, we have identified a clone homologous to a sequence in gene bank called KIAA 0581, which encodes a large part of the human PLCβ 1. By using this human cDNA in fluorescence in situ hybridisation on human metaphases, it has been possible to map human PLCβ 1 on chromosome 20p12, confirming the synteny between rat chromosome 3 and human chromosome 20 and providing a novel locus of homology between bands q35–36 in rat and p12 in man. Since band 20p12 has been recently reported amplified and/or deleted in several solid tumours, the identification and chromosome mapping of human PLCβ 1 could pave the way for further investigations on the role exerted both in normal human cells and in human tumours by PLCβ 1, which has been shown to behave as a key signalling intermediate in the control of the cell cycle.

Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato.

Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-trisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 microM Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.

Identification of components of a phosphoinositide signaling pathway in retinal rod outer segments.

Phototransduction in retinal rods involves a G protein-coupled signaling cascade that leads to cGMP hydrolysis and the closure of cGMP-gated cation channels that are open in darkness, producing a membrane hyperpolarization as the light response. For many years there have also been reports of the presence of a phosphoinositide pathway in the rod outer segment, though its functions and the molecular identities of its components are still unclear. Using immunocytochemistry with antibodies against various phosphoinositide-specific phospholipase C (PLC) isozymes (beta1-4, gamma1-2, and delta1-2), we have found PLCbeta4-like immunoreactivity in rod outer segments. Similar experiments with antibodies against the alpha-subunits of the G(q) family of G proteins, which are known to activate PLCbeta4, have also demonstrated G(alpha11)-like immunoreactivity in this location. Immunoblots of total proteins from whole retina or partially purified rod outer segments with anti-PLCbeta4 and anti-G(alpha11) antibodies gave, respectively, a single protein band of the expected molecular mass, suggesting specific labelings. The retinal locations of the two proteins were also supported by in situ hybridization experiments on mouse retina with probes specific for the corresponding mouse genes. These two proteins, or immunologically identical isoforms, therefore likely mediate the phosphoinositide signaling pathway in the rod outer segment. At present, G(alpha11) or a G(alpha11)-like protein represents the only G protein besides transducin (which mediates phototransduction) identified so far in the rod outer segment. Although absent in the outer segment layer, other PLC isoforms as well as G(alpha q) (another G(q) family member), are present elsewhere in the retina.

Inositol lipid phosphorylation and breakdown in rat liver nuclei is affected by hydrocortisone blood levels

The possibility that inositol lipid metabolism is related to nuclear events accompanying steroid hormone action has been investigated by comparing lipid phosphorylation and breakdown in normal rat liver nuclei and in hypo- and hypercortisolemic conditions. Lipid phosphorylation in vitro showed the presence of diacylglycerol (DAG)-, phosphatidylinositol (PI)- and phosphatidylinositol-4-phosphate (PIP)-kinase activity, with differences between total tissue homogenates and isolated nuclei, relevant to the treatment in vivo. Administration of hydrocortisone (HC) produced a marked decrease in the phosphorylated nuclear products without influencing the homogenate kinase activity. Under conditions which were optimal for the kinase activities, nuclear PIP-kinase was strongly increased in presence of a high blood level of HC whereas PI-kinase activity was reduced. From these observations it appears that the observed differences were due to specific modulation of kinase activities rather than to changes in the availability of substrates. The phosphoinositide-specific phospholipase C (PLC) activity was also investigated. In the presence of a high HC blood level, the phosphodiesteratic cleavage of PIP strongly increased, while that of phosphatidylinositol bisphosphate (PIP2) was similar in normal and hypercortisolemic conditions. Nuclear phosphoinositide hydrolysis was affected by PLC, beta and gamma isoforms, which were equally represented in all the conditions investigated, indicating that the observed changes of activity were due to a modulation rather than to a change in the amount of enzyme. These results suggest that inositol lipid metabolism plays a role in the nuclear modifications accompanying steroid hormone induction of transcriptional activity.

Purification and characterization of nuclear phospholipase C specific for phosphoinositides.

A phosphoinositide-specific phospholipase C (PLC) was solubilized from the isolated nuclei of rat ascites hepatoma AH7974 cells by ultrasonication in 2 M KCl. The extract was then subjected to five steps of column chromatographies in the order of Sephacryl S-300, phosphocellulose, Mono Q, Mono S, and Superose 6. Four forms of PLC (tentatively designated as N1, N2, N3, and N4) were purified 440-1400-fold. N1, N2, N3, and N4 showed apparent molecular masses of 85, 83, 80, and 88 kDa, respectively, on SDS-polyacrylamide gel electrophoresis. N1 cross-reacted with the antibody against the delta 1 isoform, while the other three forms did not cross-react with any of the antibodies against PLC-delta 1, -gamma 1, -gamma 2, and -beta 1. They hydrolyzed phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) but did not show any activities against phosphatidylcholine and phosphatidylethanolamine. They showed the same optimal pH:pH 6.5 for PI hydrolysis and pH 7.0 for both PIP and PIP2 hydrolyses. They absolutely required Ca2+ for activity, with optimal concentrations of 10(-3)-10(-5) M for PIP and 10(-4)-10(-5) M for PIP2. For PI hydrolysis, N1, N2, and N3 required a Ca2+ concentration higher than 10(-2) M whereas N4 revealed significant activity even at 10(-5) M Ca2+ concentrations. Two forms of plasma membrane PLC and three forms of cytosolic PLC were purified from AH7974 cells by the same procedure as for nuclear PLC. Comparative study with these three groups revealed that all of the purified PLC isoforms shared similar enzymological properties except N4, which showed an exceptionally high affinity to Mono S column and was active at low concentrations of Ca2+ for PI as substrate. Furthermore, when PLC isoforms of nuclei from adult resting rat liver were compared with those from regenerating rat liver after partial hepatectomy, a PLC isoform corresponding to N4 of AH7974 cells was found only in regenerating liver nuclei. From these results, it was suggested that the nuclei of growing liver cells possessed a unique form of PLC (N4).