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PubMed2004

Purification of phospholipase C beta and phospholipase C epsilon from Sf9 cells.

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP(3)). The PLC beta isoforms of PLCs are activated by G proteins after hormone or neurotransmitter stimulation of G protein-coupled receptors (GPCR). PLC epsilon is a recently identified PLC isoform that is activated by Ras and G beta gamma subunit although the physiological role of this enzyme is not well understood. Methods for purification of PLC beta and PLC epsilon from Sf9 cells are described. In the case of hexahistidine (6-His)-tagged PLC beta the purification involves two steps, affinity chromatography with Ni-NTA agarose followed by heparin Sepharose chromatography. 6-His-tagged PLC epsilon can be purified in a single step with nickel nitrilotriacetic acid-agarose (Ni-NTA) affinity chromatography.

Animals↗

PubMed2000

Studies on a mechanism by which cytosolic phospholipase A2 regulates the expression and function of type IIA secretory phospholipase A2.

Although it has been proposed that arachidonate release by several secretory phospholipase A2 (sPLA2) isozymes is modulated by cytosolic PLA2 (cPLA2), the cellular component(s) that intermediates between these two signaling PLA2s remains unknown. Here we provide evidence that 12- or 15-lipoxygenase (12/15-LOX), which lies downstream of cPLA2, plays a pivotal role in cytokine-induced gene expression and function of sPLA2-IIA. The sPLA2-IIA expression and associated PGE2 generation induced by cytokines in rat fibroblastic 3Y1 cells were markedly attenuated by antioxidants that possess 12/15-LOX inhibitory activity. 3Y1 cells expressed 12/15-LOX endogenously, and forcible overexpression of 12/15-LOX in these cells greatly enhanced cytokine-induced expression of sPLA2-IIA, with a concomitant increase in delayed PG generation. Moreover, studies using 293 cells stably transfected with sPLA2-IIA revealed that stimulus-dependent hydrolysis of membrane phospholipids by sPLA2-IIA was enhanced by overexpression of 12/15-LOX. These results indicate that the product(s) generated by the cPLA2-12/15-LOX pathway following cell activation may play two roles: enhancement of sPLA2-IIA gene expression and membrane sensitization that leads to accelerated sPLA2-IIA-mediated hydrolysis.

Animals↗

PubMed1999

Cytosolic phospholipase A2-mediated regulation of phospholipase D2 in leukocyte cell lines.

Phospholipase D (PLD) has been implicated in a variety of cellular processes, including inflammation, secretion, and respiratory burst. Two distinct PLD isoforms, designated PLD1 and PLD2, have been cloned; however, the regulatory mechanism for each PLD isoform is not clear. In our present study we investigated how PLD2 activity is regulated in mouse lymphocytic leukemia L1210 cells, which mainly contain PLD2, and in PLD2 -transfected COS-7 cells. Intriguingly, A23187, a calcium ionophore that induces calcium influx, potently stimulates PLD activity in these two cell lines, suggesting that Ca2+ might be implicated in the regulation of the PLD2 activity. In addition to the A23187-induced PLD2 activation, A23187 also increases PLA2-mediated arachidonic acid release, and the A23187-stimulated PLD2 and PLA2 activities could be blocked by pretreatment of the cells with cytosolic calcium-dependent PLA2 (cPLA2) inhibitors, such as arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphonate in these two cell lines. Moreover, the A23187-induced PLD2 and PLA2 activities could be inhibited by cotransfection with antisense cPLA2 oligonucleotide. These results suggest a role for cPLA2 in the regulation of PLD2 activity in vivo. The inhibitory effect of arachidonyl trifluoromethyl ketone on the A23187-induced PLD2 activity could be recovered by addition of exogenous lysophosphatidylcholine. This study is the first to demonstrate that PLD2 activity is up-regulated by Ca2+ influx and that cPLA2 may play a key role in the Ca2+-dependent regulation of PLD2 through generation of lysophosphatidylcholine.

Animals↗

PubMed1988

Immunoreactive pancreatic phospholipase A2 and catalytically active phospholipases A2 in serum from patients with acute pancreatitis.

Measuring the content of immunoreactive pancreatic phospholipase A2 (PLA2; EC 3.1.1.4) and the catalytic activity of PLA2 in serum samples from five patients with acute pancreatitis, we found no correlation between these two measurements overall. To test the specificity of the method for catalytic PLA2, we measured PLA2 activity in serum samples before and after immunoadsorption with an antiserum to human pancreatic PLA2. The results suggest the presence of at least two immunologically distinct PLA2 enzyme proteins in sera from these patients. One of the enzymes is pancreatic in origin and may exist in active, inactive, or inhibited form. The activity profile of the second PLA2 enzyme in serum during acute pancreatitis differs from that for other common pancreatic enzymes. In the present experiment, the catalytic activity was not removed by treatment with the anti-human pancreatic PLA2 antiserum. The source of this second PLA2 activity is unknown. Some samples contained increased activities of both PLA2 forms.

Acute Disease↗

PubMed1994

Activation of phospholipase D induced by platelet-derived growth factor is dependent upon the level of phospholipase C-gamma 1.

The mechanism of phospholipase D (PLD) activation by platelet-derived growth factor (PDGF) was examined using a NIH 3T3 fibroblast cell line (3T3-gamma 1) that stably overexpresses PLC-gamma 1 isozyme. Immunoblot analysis revealed that 3T3-gamma 1 cells contained about 10-fold more PLC-gamma 1 than a control cell line (3T3-C) transfected with expression vector lacking PLC-gamma 1 cDNA. PDGF-stimulated PLD activation was 10-fold greater in 3T3-gamma 1 cells than in 3T3-C cells, indicating that PLD activation is dependent upon the level of PLC-gamma 1. Phorbol 12-myristate 13-acetate (PMA) treatment increased PLD activity to a similar extent in both 3T3-gamma 1 cells and control cells. Pretreatment with tyrosine kinase inhibitors including staurosporine and genistein decreased PLD activity by 82.6% and 87.2%, respectively, and completely blocked tyrosine phosphorylation of PDGF receptor and PLC-gamma 1 in 3T3-gamma 1 cells stimulated with PDGF. Moreover, down-regulation of protein kinase C by pretreatment of PMA caused complete inhibition of PDGF- and PMA-stimulated PLD activation. Therefore, these results suggest that PDGF-induced PLD activation may be a consequence of primary stimulation of PLC-gamma 1 and that PLD may play a role downstream from PLC-gamma 1 in PDGF-triggered mitogenesis.

3T3 Cells↗

PubMed1997

Involvement of phospholipase C-gamma2 in activation of mitogen-activated protein kinase and phospholipase A2 by zooxanthellatoxin-A in rabbit platelets.

Zooxanthellatoxin-A (ZT-A), a polyhydroxypolyene isolated from a symbiotic dinoflagellate Symbiodinium sp., caused thromboxane A2-(TXA2) dependent and genistein-sensitive aggregation in rabbit platelets. Our study was performed to clarify the mechanism of the action of ZT-A. ZT-A caused an increase in tyrosine phosphorylation of 42-kDa protein, which is defined as p42 mitogen-activated protein kinase (MAPK) by immunoprecipitation. Although indomethacin (10 microM) completely inhibited ZT-A-induced TXB2 release, it partially inhibited the MAPK activation. The remained MAPK activation was completely inhibited by genistein (50 microM). Genistein (50 microM), by itself, abolished TXB2 release induced by ZT-A. ZT-A (2 microM) stimulated liberation of arachidonic acid and the subsequent metabolites such as TXB2 and 12-hydroperoxyeicosatetraenoic acid. However, ZT-A-stimulated phosphoinositide hydrolysis which was due to an increase in tyrosine phosphorylation of phospholipase C-(PLC)gamma2. The phosphorylation of PLC-gamma2 and the phosphoinositide hydrolysis were also partially inhibited by indomethacin (10 microM), and were abolished by a combined treatment of indomethacin (10 microM) and genistein (50 microM). ZT-A- (2 microM) induced MAPK activation in the presence of indomethacin (10 microM) was concentration-dependently inhibited by staurosporine and calphostin C, protein kinase C inhibitors. PD98059 (50 microM), a MAPK kinase inhibitor, also inhibited ZT-A-induced TXB2 release. Depletion of external Ca++ abolished ZT-A- (2 microM) induced MAPK activation, phosphoinositide hydrolysis, arachidonic acid liberation and TXB2 release. These results suggest that ZT-A stimulates a protein tyrosine kinase in the presence of external Ca++, resulting in the activation of MAPK probably via PLC-gamma2 and protein kinase C. The MAPK stimulated a liberation of arachidonic acid that is rapidly converted to TXA2. The released TXA2 causes aggregation accompanied with second stimulation of MAPK cascade.

Animals↗

PubMed1977

Net activity of phospholipase A2 in brain and the lack of stimulation of the phospholipase A2-acylation stem.

Certain observations reported previously from this laboratory have not proved reproducible. These are (1) the relatively rapid hydrolysis of added phosphatidylcholine by phospholipase A2 of tissue from the cerebral cortex of the guinea pig and (2) the stimulation by 10 micron-noradrenaline and by 1.0nM-cyclic AMP of the phospholipase A2-acylation system of isolated synaptic membranes.

Retraction Notice↗

PubMed1999

Purification and characterization of phospholipase B from Kluyveromyces lactis, and cloning of phospholipase B gene.

Phospholipase B (PLB) from the yeast Kluyveromyces lactis was purified to homogeneity from culture medium. The enzyme was highly glycosylated with apparent molecular mass of 160-250 kDa, and had two pH optima, at pH 2.0 and pH 7.5. At acidic pH the enzyme hydrolyzed all phospholipid substrates tested here without metal ion. On the other hand, at alkaline pH the enzyme showed substrate specificity for phosphatidylcholine and lysophosphatidylcholine and required Ca2+, Fe3+, or Al3+ for the activity. The alkaline activity was increased more than 20-fold in the presence of Al3+ compared to that in the presence of Ca2+. cDNA sequence of PLB (KlPLB) was analyzed by a combination of several PCR procedures. KlPLB encoded a protein consist of 640 amino acids and the deduced amino acid sequence showed 66.7% similarity with the T. delbrueckii PLB. The amino acid sequence contained the lipase consensus sequence (G-X-S-X-G) and the catalytic aspartic acid motif. Replacement of Arg-112 or Asp-406 with alanine caused loss of the enzymatic activity at both pH. These results suggested that PLB activity are dependent on a catalytic mechanism similar to that of cytosolic phospholipase A2.

Acyltransferases↗

PubMed2003

Interisland mutation of a novel phospholipase A2 from Trimeresurus flavoviridis venom and evolution of Crotalinae group II phospholipases A2.

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A2 (PLA2). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA2, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD50 for mice was 1.34 microg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA2's based on the amino acid sequences revealed that neurotoxic PLA2's including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA2 groups such as PLA2 type, basic [Asp49]PLA2 type, and [Lys49]PLA2 type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA2's showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA2's, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA2's evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation.

Amino Acid Sequence↗

PubMed1993

Metal ion and salt effects on the phospholipase A2, lysophospholipase, and transacylase activities of human cytosolic phospholipase A2.

Human cytosolic phospholipase A2 (cPLA2) is an arachidonic acid specific enzyme which may play a role in arachidonic acid release, eicosanoid production, and signal transduction. The PLA2 activity of this enzyme is stimulated by microM levels of Ca2+. Using a pure recombinant enzyme, we have confirmed that cPLA2 is not absolutely dependent on Ca2+, since Sr2+, Ba2+ and Mn2+ also gave full enzyme activity. Heavy metals, in contrast, inhibited enzyme catalysis suggesting the involvement of an essential cysteine residue. In the absence of Ca2+, high salt concentrations overcame the requirement for divalent metals, indicating that Ca2+ is not required for PLA2 catalytic activity. cPLA2 also displays a lysophospholipase (lyso PLA) activity with lysophosphatidylcholine micelles as a substrate. Unlike the PLA2 activity, the lyso PLA activity toward these micelles is not stimulated by Ca2+. However, upon the addition of glycerol or Triton X-100 to the assay, Ca2+ activation is observed, indicating that substrate presentation can affect the apparent Ca2+ dependence. Glycerol was found to be a potent stimulator of lyso PLA activity and specific activities up to 50 mumol min-1 mg-1 were observed. In addition to the PLA2 and lyso PLA activities, we report that cPLA2 displays a relatively low, CoA-independent transacylase activity which produces phosphatidylcholine from lysophosphatidylcholine substrate. The observation of this novel transacylase activity is consistent with the formation of an acyl-enzyme intermediate.

Acyltransferases↗

PubMed1993

Responses of purified phospholipases A2 to phospholipase A2 activating protein (PLAP) and melittin.

The role of the phospholipase A2 (PLA2) stimulating protein PLAP in the regulation of PLA2 activity was assessed by determination of the effects of PLAP on two purified PLA2s. An approx. 14 kDa enzyme was purified from mouse thymoma cells, EL-4 cells, by cation ion exchange HPLC and immunoaffinity HPLC (with antiserum to the N-terminal sequence of an inflammatory exudate PLA2). An approx. 110 kDa enzyme was purified from mouse mammary carcinoma derived cells by sequential hydrophobic, anion exchange, hydroxyapatite and gel filtration HPLC. Neither PLAP nor melittin, an immunologically related PLA2 stimulating peptide from bee venom, increased the activity of the high molecular weight enzyme. In contrast, there was more than a 20-fold stimulation of the low molecular weight PLA2 by PLAP and an approx. 5-fold stimulation by melittin. The stimulation of enzyme activity by PLAP was observed at a protein to phospholipid ratio of 1:10(6) while the ratio of melittin to phospholipid was 1:3. Thus, PLAP mediated stimulation of PLA2 activity may include an interaction between PLAP and the enzyme, in contrast to melittin stimulation, which involves interactions between melittin and phospholipid.

Animals↗

PubMed1994

Platelet secretory phospholipase A2 fails to induce rabbit platelet activation and to release arachidonic acid in contrast with venom phospholipases A2.

The ability of platelet secretory phospholipase A2 (sPLA2) to induce platelet activation was investigated. sPLA2 (group II) contained in an activated platelet supernatant, as well as high concentrations of purified recombinant platelet sPLA2, failed to induce platelet activation. Furthermore, sPLA2 did not modify platelet activation induced by various agonists. The possible relationship between the failure of this enzyme to induce platelet activation and its origin (mammalian) or its structural group (group II) was then investigated, using pancreatic PLA2s (group I) and venom PLA2s from groups I, II and III. All venom PLA2s induced platelet activation that was accompanied by the liberation of arachidonic acid and was abolished by aspirin. In contrast, as observed for platelet sPLA2, enzymes from hog or bovine pancreas were unable to induce platelet activation even when used at high concentrations. Interestingly, PLA2 able to induce platelet activation efficiently hydrolyse phosphatidylcholine, while those inactive on platelets did not. Taken together, these results suggest that the catalytic activity of added PLA2 is necessary but not sufficient to induce platelet activation. Moreover, the ability of PLA2 to induce platelet activation is not related to its structural group (I, II, III) but rather to its origin (venom vs. mammalian) and capacity to hydrolyse phosphatidylcholine, the major phospholipid of the outer leaflet of the plasma membrane.

Animals↗

PubMed1994

Immunochemical relatedness between secretory phospholipase A2 and intracellular phospholipase A2 activity linked with arachidonic acid mobilization in macrophages.

To determine whether low mol. wt phospholipase A2 (PLA2) is involved in the mechanism of arachidonic acid mobilization induced by zymosan in mouse peritoneal macrophages, we developed an immunoblot analysis and ELISA assay using the polyclonal antibodies anti-PLA2 type I and type II previously characterized. We also measured the effect of low mol. wt PLA2 inhibitors such as p-bromophenacyl bromide, aristolochic acid, nordihydroguaiaretic acid, all trans-retinal and all trans-retinoic acid on the action of these enzymes. The antibodies antitype I PLA2 bound to mouse platelet protein fraction, while the antibodies antitype I or antitype II did not recognize components of the macrophages. Furthermore, low mol. wt PLA2 inhibitors did not inhibit arachidonic acid release produced during the phagocytosis of zymosan. This suggests that low mol. wt PLA2 are not present in cells such as macrophages. These results are consistent with a role for high mol. wt PLA2 in arachidonic acid mobilization.

Acetophenones↗

PubMed1982

Investigation of the effects of phospholipase C on human platelets: evidence that aggregation induced by phospholipase C is independent of prostaglandin generation, released ADP and is modulated by cyclic AMP.

Effects and the mechanism of action of phospholipase C (PLC), from Clostridium perfringens, on washed human platelets were examined to better understand the role of PLC in platelet function. PLC caused aggregation and secretion of [14C]-5HT, without concomitant loss of cytoplasmic, LDH, in a concentration dependent manner. P-nitrophenylphosphorylcholine, a substrate for PLC, blocked these responses in a concentration dependent manner. In other experiments hirudin, alpha-1-antitrypsin and soybean trypsin inhibitor did not inhibit PLC-induced activation of human platelets. PLC-induced aggregation and [14C]-5HT secretion was not inhibited by aspirin, a known inhibitor of prostaglandin biosynthesis. PLC-induced aggregation was selectively inhibited by analogs of 7,8-dihydroxybenzazepine and 7,8-methylenedioxybenzazepine in a concentration dependent manner. These two agents had no effect on arachidonic acid-induced aggregation. PLC-induced aggregation was not inhibited by apyrase, an enzyme which hydrolyzes ADP. In other experiments, PLC-treated platelets did not exhibit any platelet activating factor-like activity. Prostaglandin E1 and trifluoperazine showed concentration dependent inhibitor effects on PLC-mediated aggregation and secretion of [14C]-5HT. These findings indicate that: a) PLC is capable of inducing aggregation and specific secretion of [14C]-5HT without causing lysis of platelets; b) mechanism of PLC-induced activation of platelets is independent of prostaglandin generation or action, released ADP, and PAF; and c) cyclic AMP plays a modulatory role in PLC-mediated secretion and aggregation of human platelets.

Adenosine Diphosphate↗

PubMed1984

Human platelet activation by bacterial phospholipase C is mediated by phosphatidylinositol hydrolysis but not generation of phosphatidic acid: inhibition by a selective inhibitor of phospholipase C.

We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes human platelet aggregation and secretion in a concentration dependent manner. The present study was undertaken to further characterize the specificity of the effects of PLC and to better understand the mechanism of the action of this inducer. A methylene-dioxybenzazepine (MDBA) analog of trimetoquinol was synthesized and tested for antiplatelet activity. MDBA (3-30 microM) inhibited PLC-induced aggregation in a concentration dependent manner. Whereas up to 200 microM MDBA did not inhibit aggregation induced by either thrombin, arachidonic acid, or U46619. Effects of PLC (0.05 U/ml) on hydrolysis of phosphatidylinositol, production of phosphatidic acid and thromboxane B2 (TXB2) synthesis were investigated using [32P]-phosphate and [14C]-arachidonic acid labeled platelets. PLC (0.05 U/ml) caused a time dependent decrease in platelet phosphatidylinositol. Up to 50% of labeled phosphatidylinositol was lost from platelets in five minutes. MDBA (3-30 microM) inhibited PLC-induced loss of phosphatidylinositol in a concentration dependent manner. An increase in phosphatidic acid was also observed in PLC-stimulated platelets. Up to 100 microM MDBA did not inhibit production of phosphatidic acid. PLC-treated platelets did not produce any TXB2. In other experiments possible protease contamination of PLC preparations was tested by incubating PLC (0.03-0.5 U/ml) with [14C]-casein. PLC in concentrations up to ten times higher than the concentrations used in aggregation studies did not cause hydrolysis of [14C]-casein, whereas more than 30% of [14C]-casein was hydrolyzed by trypsin. PLC-induced aggregation was not inhibited by up to 300 microM adenosine or ATP. In other experiments, platelet aggregation by ADP was inhibited by adenosine and ATP in a concentration dependent manner. The addition of calcium (0.5- 2.0 mM) increased aggregation by PLC in a concentration dependent manner. These findings suggest that PLC-induced activation of platelets is: (a) dependent on phosphatidylinositol hydrolysis but not on the production of phosphatidic acid, TXB2 or secretion of ADP; (b) not caused by protease contaminants; (c) calcium dependent; and (d) MDBA inhibits PLC-induced aggregation by blocking phosphatidylinositol hydrolysis.

Adenosine↗

PubMed1994

Levels and localization of group II phospholipase A2 and annexin I in interleukin- and dexamethasone-treated rat mesangial cells: evidence against annexin mediation of the dexamethasone-induced inhibition of group II phospholipases A2.

The mechanism by which glucocorticosteroids inhibit the synthesis and secretion of pro-inflammatory arachidonate metabolites is still controversial. Initially it was postulated that glucocorticoids can induce the formation of PLA2 inhibitory proteins termed annexins. We have previously shown that the cytokine-induced 14 kDa PLA2 activity and the synthesis of prostaglandin E2 in rat mesangial cells is dose-dependently blocked by pretreatment of the cells with dexamethasone (Schalkwijk et al. (1991) Biochem. Biophys. Res. Commun. 180, 46-52). Concurrently, the synthesis of 14 kDa group II PLA2 is suppressed. The regulation of PLA2 activity is complex and may well involve superimposable mechanisms. Thus, although the decrease in PLA2 protein levels could in itself explain the dexamethasone-induced decrease in PLA2 activity, a contribution of the glucocorticoid-induced anti-phospholipase A2 protein annexin cannot be ruled out a priori. To investigate this possibility we analyzed the level of annexin I by Western blotting and immunostaining in mesangial cells treated with interleukin-1 beta and/or dexamethasone. Under conditions where 14 kDa group II PLA2 activity and protein levels were dramatically affected by interleukin-1 and dexamethasone, the level of annexin I in the cells remained constant. Dexamethasone also did not induce the secretion of annexin I. In addition, no evidence for dexamethasone-induced translocation of annexin I from the cytosol to membranes, thereby possibly sequestering the substrates for PLA2, was obtained. Immunofluorescence studies localized the cytokine-induced PLA2 to the Golgi area and punctate structures in the cytoplasm. We have also studied the subcellular localization of annexin I in rat mesangial cells using confocal microscopy. These studies located annexin I mainly in the cytoplasma and the nucleus. We conclude from these experiments that the dexamethasone-induced inhibition of 14 kDa group II PLA2 in rat mesangial cells is not mediated by annexin I and is solely due to the suppression of PLA2 gene expression.

Animals↗

PubMed1995

Differential regulation of phospholipase D and phospholipase C by protein kinase C-beta and -delta in liver macrophages.

We have studied activation of phospholipase (PL) C and PLD in liver macrophages labelled with [3H]arachidonic acid. Zymosan, phorbol 12-myristate 13-acetate (PMA), A23187 and fluoride but not arachidonic acid or lipopolysaccharide (LPS) induce an activation of PLD ([3H]phosphatidylethanol (PEt) accumulation). An activation of PLC ([3H]diacylglycerol (DAG) accumulation) is measured with zymosan, PMA and fluoride but not with A23187, LPS or arachidonic acid whereas inositol phosphates are formed with zymosan, only. Removal of extracellular calcium reduces the formation of [3H]PEt and [3H]DAG while pretreatment of the cells with dexamethasone reduces [3H]PEt formation, only. PMA- and zymosan-induced activation of PLD and PMA-induced activation of PLC both seem to be mediated by protein kinase (PK) C-beta whereas zymosan-induced activation of PLC is negatively controlled by PKC-delta. We could furthermore present evidence that the release of [3H]arachidonic acid in these cells occurs independent of an activation of PLD.

Animals↗

PubMed2005

Grb2 negatively regulates epidermal growth factor-induced phospholipase C-gamma1 activity through the direct interaction with tyrosine-phosphorylated phospholipase C-gamma1.

Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation. Upon the stimulation of growth factors and hormones, PLC-gamma1 is rapidly phosphorylated at three known sites; Tyr771, Tyr783 and Tyr1254 and its enzymatic activity is up-regulated. In this study, we demonstrate for the first time that Grb2, an adaptor protein, specifically interacts with tyrosine-phosphorylated PLC-gamma1 at Tyr783. The association of Grb2 with PLC-gamma1 was induced by the treatment with epidermal growth factor (EGF). Replacement of Tyr783 with Phe completely blocked EGF-induced interaction of PLC-gamma1 with Grb2, indicating that tyrosine phosphorylation of PLC-gamma1 at Tyr783 is essential for the interaction with Grb2. Interestingly, the depletion of Grb2 from HEK-293 cells by RNA interference significantly enhanced increased EGF-induced PLC-gamma1 enzymatic activity and mobilization of the intracellular Ca2+, while it did not affect EGF-induced tyrosine phosphorylation of PLC-gamma1. Furthermore, overexpression of Grb2 inhibited PLC-gamma1 enzymatic activity. Taken together, these results suggest Grb2, in addition to its key function in signaling through Ras, may have a negatively regulatory role on EGF-induced PLC-gamma1 activation.

Adaptor Proteins, Signal Transducing↗