Characterization of phosphoinositide-specific phospholipase C from human platelets.
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ABSTRACT:
SUBMITTER: Manne V
PROVIDER: S-EPMC1147923 | biostudies-other | 1987 May
REPOSITORIES: biostudies-other
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Project description:The origin and physiological significance of the multiple Mr forms of phosphoinositide-specific phospholipase C in human platelets were investigated. The higher-Mr (400,000 and 270,000) forms of the phospholipase C were converted into the 100,000-Mr form without substantial loss of activity by incubation with a Ca2+-dependent proteinase partially purified from human platelets. These three forms of the phospholipase C were purified approx. 200-500-fold from outdated human platelet supernatants. SDS/polyacrylamide-gel electrophoresis and gel-filtration analysis suggested that the higher-Mr forms of phospholipase C were complexes of 140,000-Mr subunits, whereas the lower-Mr form consisted of a single 95,000-Mr subunit. The substrate specificity of the purified phospholipase C was investigated by using 32P-labelled polyphosphoinositide substrates purified from human platelets by a new method utilizing h.p.l.c. on an amino column. Activity against all three phosphoinositides was detected at micromolar concentrations of Ca2+; this hydrolysis was markedly stimulated by phosphatidylethanolamine and inhibited by phosphatidylcholine. Comparison of the different forms of purified phospholipase C revealed no major differences in Ca2+-sensitivity or substrate specificity. Thus, although the suggestion that the high-Mr forms of human platelet phosphoinositide-specific phospholipase C were converted into a lower-Mr form by a Ca2+-dependent proteinase has been substantiated, the physiological significance of this process remains to be determined.
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Project description: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 ?1, ?2, ?3, ?4, ?1, ?2, ?1, ?3 and ? resulted expressed. In 143B cell line, PI-PLC ?1, ?2, ?3, ?4, ?1, ?2, ?1, ?3 and ? were expressed. In SaOS-2 cell line, PI-PLC ?1, ?3, ?4, ?1, ?2, ?1, ?3, ? and ?1. In Hs888 cell line, PI-PLC ?1, ?3, ?4, ?1, ?1, ?3, ?4, ? and ?1 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.
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Project description:The phosphoinositide signal transduction pathway mediates important processes in intestinal physiology, yet the key enzyme, phosphoinositide-specific phospholipase C (PI-PLC), is not well-characterized in the colon. PI-PLC activity was examined in rat colonic membranes using exogenous [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate, and beta-glycerophosphate to suppress degradation of substrate or product. The activity of membrane PI-PLC increased 6-fold with the addition of alamethicin, and a further 2-3-fold enhancement was observed with 10 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S]), suggesting the involvement of G-protein(s). The effect of GTP[S] appeared to be specific, as up to 100 microM adenosine 5'-[gamma-thio]-triphosphate failed to stimulate PI-PLC activity, and guanosine 5'-[beta-thio]diphosphate inhibited activity. The response of membrane PI-PLC to Ca2+ was biphasic, while > 0.5 mM Mg2+ was inhibitory with or without GTP[S]. Comparable total PI-PLC activities and responses to GTP[S] and Ca2+ were observed in purified brush-border and basolateral membranes. Western immunoblots probed with monoclonal antibodies to PLC isoenzymes PLC-beta 1, -gamma 1 and -delta 1 demonstrated that these antipodal plasma membranes contain predominantly the PLC-delta 1 isoform, with small amounts of PLC-gamma 1 present but no detectable PLC-beta 1. PLC-gamma 1 was the major isoform detected in cytosol.
| S-EPMC1134300 | biostudies-other
Project description:Phosphoinositide-specific phospholipase C (PLC) ?1 has been reported to be involved in cancer cell proliferation and metastasis. However, whether PLC?1 modulates autophagy and the underlying mechanism remains unclear. Here, we investigated the relationship between PLC?1 and autophagy in the human colon cancer cell line HCT116 and hepatocellular carcinoma cell line HepG2. The results indicated that PLC?1 inhibition via lentivirus-mediated transduction with shRNA/PLC?1 or transient transfection with pRK5-PLC?1 (Y783A) vector increased LC3B-II levels and the number of autophagic vacuoles and decreased p62 levels. Addition of an autophagy inhibitor led to LC3B and p62 accumulation. Furthermore, AMPK activation promoted the autophagy induced by PLC?1 inhibition by blocking the FAK/PLC?1 axis. In addition, PLC?1 inhibition either blocked the mTOR/ULK1 axis or enhanced dissociation of the Beclin1-IP3R-Bcl-2 complex to induce autophagy. Taken together, our findings revealed that PLC?1 inhibition induced autophagy and the FAK/PLC?1 axis is a potential downstream effector of the AMPK activation-dependent autophagy signalling cascade. Both blockade of the mTOR/ULK1 axis and dissociation of the Beclin1-IP3R-Bcl-2 complex contributed to the induction of autophagy by PLC?1 inhibition. Consequently, these findings provide novel insight into autophagy regulation by PLC?1 in colon cancer and hepatocellular carcinoma cells.
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Project description:Ezrin, a protein belonging to the Ezrin, radixin and moesin (ERM) family, was engaged in the metastatic spread of osteosarcoma. The Protein 4.1, Ezrin, radixin, moesin (FERM) domain of Ezrin binds the membrane Phosphatydil inositol (4,5) bisphosphate (PIP2), a crucial molecule belonging to the Phosphoinositide (PI) signal transduction pathway. The cytoskeleton cross-linker function of Ezrin largely depends on membrane PIP2 levels, and thus upon the activity of related enzymes belonging to the PI-specific phospholipase C (PI-PLC) family. Based on the role of Ezrin in tumour progression and metastasis, we silenced the expression of Vil2 (OMIM *123900), the gene which codifies for Ezrin, in cultured human osteosarcoma 143B and Hs888 cell lines. After Ezrin silencing, the growth rate of both cell lines was significantly reduced and morphogical changes were observed. We also observed moderate variations both of selected PI-PLC enzymes within the cell and of expression of the corresponding PLC genes. In 143B cell line the transcription of PLCB1 decreased, of PLCG2 increased and of PLCE differed in a time-dependent manner. In Hs888, the expression of PLCB1 and of PLCD4 significantly increased, of PLCE moderately increased in a time dependent manner; the expression of PLCG2 was up-regulated. These observations indicate that Ezrin silencing affects the transcription of selected PLC genes, suggesting that Ezrin might influence the expression regulation of PI-PLC enzymes.
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Project description:Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-?, ?, ?, ?, ? and ?) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of ?2 and ?2 (only expressed in PA), ?4 (only expressed in MCA), ?1 (expressed in all but MA) and ? (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (?3, ?2 and ?1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the native endothelium.
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Project description:The involvement of phosphoinositides (PI) signal transduction pathway and related molecules, such as the Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes, in the pathophysiology of mood disorders is corroborated by a number of recent evidences. Our previous works identified the deletion of PLCB1 gene, which codifies for the PI-PLC β1 enzyme, in 4 out 15 patients affected with schizophrenia, and no deletion both in major depression affected patients and in normal controls. By using interphase fluorescent in situ hybridization methodology, we analyzed PLCB1 in paraffin embedded samples of orbito-frontal cortex of 15 patients affected with bipolar disorder. Deletion of PLCB1 was identified in one female patient.
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Project description:Phospholipase D (PLD) is involved in many biological processes. PLD1 plays a crucial role in regulating the platelet activity of mice; however, the role of PLD in the platelet activation of humans remains unclear. Therefore, we investigated whether PLD is involved in the platelet activation of humans. Our data revealed that inhibition of PLD1 or PLD2 using pharmacological inhibitors effectively inhibits platelet aggregation in humans. However, previous studies have showed that PLD1 or PLD2 deletion did not affect mouse platelet aggregation in vitro, whereas only PLD1 deletion inhibited thrombus formation in vivo. Intriguingly, our data also showed that the pharmacological inhibition of PLD1 or PLD2 does not affect mouse platelet aggregation in vitro, whereas the inhibition of only PLD1 delayed thrombus formation in vivo. These findings indicate that PLD may play differential roles in humans and mice. In humans, PLD inhibition attenuates platelet activation, adhesion, spreading, and clot retraction. For the first time, we demonstrated that PLD1 and PLD2 are essential for platelet activation in humans, and PLD plays different roles in platelet function in humans and mice. Our findings also indicate that targeting PLD may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders.
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Project description:Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid modified in its N terminus and localizes to the outer surface of the plasma membrane of amastigotes. We show here that TcPI-PLC is developmentally regulated in amastigotes and shows two peaks of surface expression during the developmental cycle of T. cruzi, the first immediately after differentiation of trypomastigotes into amastigotes and the second before differentiation of amastigotes into trypomastigotes. Surface expression of TcPI-PLC coincides with phosphatidylinositol 4,5-bisphosphate (PIP(2)) depletion in the host cell membrane and with an increase in the levels of its product, inositol 1,4,5-trisphosphate. During extracellular differentiation, PI-PLC is secreted into the incubation medium. Maximal early expression of TcPI-PLC on the surface of amastigotes and PIP(2) depletion coincide with host cytoskeletal changes, Ca(2+) signaling, and transcriptional responses described previously. The presence of TcPI-PLC on the outer surface of the plasma membrane of the parasite and the capacity to be secreted and to alter host phospholipids are novel mechanisms of the host-parasite interaction.
| S-EPMC2950344 | biostudies-literature
Project description:Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response.
| S-EPMC4581737 | biostudies-literature