Journal articles: 'Phospholipase C gamma 2 [PLC 2]'

7

Lee, SB, AK Rao, KH Lee, X. Yang, YS Bae, and SG Rhee. "Decreased expression of phospholipase C-beta 2 isozyme in human platelets with impaired function." Blood 88, no. 5 (1996): 1684–91. http://dx.doi.org/10.1182/blood.v88.5.1684.bloodjournal8851684.

Full text

Abstract:

Platelets from a patient with a mild inherited bleeding disorder and abnormal platelet aggregation and secretion show reduced generation of inositol 1,4,5-trisphosphate, mobilization of intracellular Ca2+, and phosphorylation of pleckstrin in response to several G protein mediated agonists, suggesting a possible defect at the level of phospholipase C (PLC) activation (see accompanying report). A procedure was developed that allows quantitation of platelet PLC isozymes. After fractionation of platelet extracts by high-performance liquid chromatography, 7 out of 10 known PLC isoforms were detected by immunoblot analysis. The amount of these isoforms in normal platelets decreased in the order PLC- gamma 2 > PLC-beta 2 > PLC-beta 3 > PLC-beta 1 > PLC-gamma 1 > PLC- delta 1 > PLC-beta 4. Compared with normal platelets, platelets from the patient contained approximately one-third the amount of PLC-beta 2, whereas PLC-beta 4 was increased threefold. These results suggest that the impaired platelet function in the patient in response to multiple G protein mediated agonists is attributable to a deficiency of PLC-beta 2. They document for the first time a specific PLC isozyme deficiency in human platelets and provide an unique opportunity to understand the role of different PLC isozymes in normal platelet function.

APA, Harvard, Vancouver, ISO, and other styles

8

Arkinstall, S., M. Payton, and K. Maundrell. "Activation of phospholipase C gamma in Schizosaccharomyces pombe by coexpression of receptor or nonreceptor tyrosine kinases." Molecular and Cellular Biology 15, no. 3 (1995): 1431–38. http://dx.doi.org/10.1128/mcb.15.3.1431.

Full text

Abstract:

The fission yeast Schizosaccharomyces pombe has no detectable endogenous receptor tyrosine kinases or associated signalling apparatus, and we have used this cell system to reconstitute mammalian platelet-derived growth factor beta (PDGF beta) receptor-linked activation of phospholipase C gamma 2 (PLC gamma 2). The PDGF beta receptor migrates as a glycosylated protein of 165 kDa associated exclusively with membrane fractions. No tyrosine autophosphorylation was detected when PDGF beta was expressed alone. PLC gamma 2 appears as a 140-kDa protein distributed between particulate and soluble fractions which exhibits characteristic selectivity for phosphatidylinositol 4,5-bisphosphate and is sensitive to powerful activation by Ca2+. When coexpressed, both PDGF beta and PLC gamma 2 undergo tyrosine phosphorylation, and this is accompanied by a > 26-fold increase in [3H]inositol 4,5-biphosphate ([3H]IP2) and [3H]inositol 1,4,5-triphosphate [3H]IP3 production. Treatment with the tyrosine phosphatase inhibitor pervanadate further increased PLC gamma 2 tyrosine phosphorylation as well as [3H]IP2 and [3H]IP3 generation. Phosphorylated PLC gamma 2 was found predominantly in membrane fractions. To test a nonreceptor tyrosine kinase, we then expressed the human proto-oncogene c-src together with its negative regulator Csk. These were immunodetectable as bands at 60 kDa (c-Src) and 50 kDa (Csk) and distributed between membrane and cytosolic fractions. When yeast coexpressing c-Src, Csk, and PLC gamma 2 was incubated with pervanadate, PLC gamma 2 was tyrosine phosphorylated and [3H]IP2 and [3H]IP3 production increased 11.0- and 7.0-fold, respectively. Csk expressed alone with PLC gamma 2 was ineffective. Similar PLC gamma 2 activation was observed upon in vitro mixing with the extracts expressing either c-Src or the PDGF beta receptor. In summary, this is the first report of a reconstitution of mammalian tyrosine kinase-linked effector activation in yeast cells and also the first demonstration of direct PLC gamma 2 activation by the proto-oncogene c-src. These observations indicate that S. pombe provides a powerful cell system in which to study critical molecular interactions and activities underlying receptor and nonreceptor tyrosine kinase-dependent cell signaling.

APA, Harvard, Vancouver, ISO, and other styles

9

Bolt, M. J. G., B. M. Bissonnette, R. K. Wali, S. C. Hartmann, T. A. Brasitus, and M. D. Sitrin. "Characterization of phosphoinositide-specific phospholipase C in rat colonocyte membranes." Biochemical Journal 292, no. 1 (1993): 271–76. http://dx.doi.org/10.1042/bj2920271.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

10

Homma, Y., Y. Emori, F. Shibasaki, K. Suzuki та T. Takenawa. "Isolation and characterization of a γ-type phosphoinositide-specific phospholipase C (PLC-γ2)". Biochemical Journal 269, № 1 (1990): 13–18. http://dx.doi.org/10.1042/bj2690013.

Full text

Abstract:

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).

APA, Harvard, Vancouver, ISO, and other styles

11

Piiper, A., D. Stryjek-Kaminska, and S. Zeuzem. "Epidermal growth factor activates phospholipase C-gamma 1 via G(i)1-2 proteins in isolated pancreatic acinar membranes." American Journal of Physiology-Gastrointestinal and Liver Physiology 272, no. 5 (1997): G1276—G1284. http://dx.doi.org/10.1152/ajpgi.1997.272.5.g1276.

Full text

Abstract:

In the present study, isolated pancreatic acinar membranes were used to investigate the mechanism of epidermal growth factor (EGF)-induced activation of phospholipase C (PLC). The data show that EGF caused a rapid and strong increase in tyrosine phosphorylation of the EGF receptor, with a maximum 5-15 s after the beginning of the incubation followed by a decline. With use of [3H]phosphatidylinositol 4,5-bisphosphate as an exogenous substrate, PLC activity increased fourfold on exposure of the membranes to EGF (85 nM). In contrast, EGF-induced tyrosine phosphorylation of PLC-gamma 1 was rather small, indicating that tyrosine phosphorylation of PLC-gamma 1 is not proportional to changes in PLC activity. EGF-induced activation of PLC was strongly inhibited by pretreatment of the membranes with pertussis toxin, by an antibody raised against a COOH-terminal sequence shared by alpha-subunits of the inhibitory G proteins G(i)1 and G(i)2, and by an anti-PLC-gamma 1 antibody, whereas anti-G(i) alpha 3, anti-Gq/11 alpha, and anti-PLC-beta 1 antibodies had no effect. In contrast, pertussis toxin or the anti-G(i) alpha 1-2 antibody had no effect on EGF-induced tyrosine phosphorylation of PLC-gamma 1. EGF promoted association of G(i) proteins with both the EGF receptor and PLC-gamma 1 with similar kinetics as EGF-receptor autophosphorylation. All EGF-induced responses were abolished by the specific tyrosine kinase inhibitor pp60v-arc (137-157), suggesting that EGF-receptor tyrosine kinase activity is essential for G(i)1-2-mediated activation of PLC-gamma 1. However, there was no evidence of tyrosine phosphorylation of G(i) alpha 1-2. Taken together, these data show that EGF causes activation of PLC-gamma 1 by a mechanism requiring activation of G(i)1-2 and only a small increase in tyrosine phosphorylation of PLC-gamma 1.

APA, Harvard, Vancouver, ISO, and other styles

12

Ting, A. T., L. M. Karnitz, R. A. Schoon, R. T. Abraham, and P. J. Leibson. "Fc gamma receptor activation induces the tyrosine phosphorylation of both phospholipase C (PLC)-gamma 1 and PLC-gamma 2 in natural killer cells." Journal of Experimental Medicine 176, no. 6 (1992): 1751–55. http://dx.doi.org/10.1084/jem.176.6.1751.

Full text

Abstract:

Crosslinking of the low affinity immunoglobulin G (IgG) Fc receptor (Fc gamma R type III) on natural killer (NK) cells initiates antibody-dependent cellular cytotoxicity. During this process, Fc gamma R stimulation results in the rapid activation of phospholipase C (PLC), which hydrolyzes membrane phosphoinositides, generating inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers. We have recently reported that PLC activation after Fc gamma R stimulation can be inhibited by a protein tyrosine kinase (PTK) inhibitor. Based on the paradigm provided by the receptor tyrosine kinases, we investigated whether PLC-gamma 1 and/or PLC-gamma 2 are expressed in NK cells, and whether the PLC-gamma isoforms are tyrosine phosphorylated in response to Fc gamma R stimulation. Immunoblotting analyses with PLC-gamma 1- and PLC-gamma 2-specific antisera demonstrate that both isoforms are expressed in human NK cells. Furthermore, Fc gamma R crosslinking triggers the tyrosine phosphorylation of both PLC-gamma 1 and PLC-gamma 2 in these cells. Phosphorylation of both isoforms is detectable within 1 min, and returns to basal level within 30 min. Pretreatment with herbimycin A, a PTK inhibitor, blocked the Fc gamma R-induced tyrosine phosphorylation of PLC-gamma 1 and PLC-gamma 2, and the subsequent release of inositol phosphates. These results suggest that Fc gamma R-initiated phosphoinositide turnover in human NK cells is regulated by the tyrosine phosphorylation of PLC-gamma. More broadly, these observations demonstrate that nonreceptor PTK(s) activated by crosslinkage of a multisubunit receptor can phosphorylate both PLC-gamma isoforms.

APA, Harvard, Vancouver, ISO, and other styles

13

Homma, Y., H. Sakamoto, M. Tsunoda, M. Aoki, T. Takenawa та T. Ooyama. "Evidence for involvement of phospholipase C-γ2 in signal transduction of platelet-derived growth factor in vascular smooth-muscle cells". Biochemical Journal 290, № 3 (1993): 649–53. http://dx.doi.org/10.1042/bj2900649.

Full text

Abstract:

In order to examine the mechanisms underlying smooth-muscle cell proliferation, we investigated effect of platelet-derived growth factor (PDGF) dimers on proliferation of rabbit vascular smooth-muscle cells (VSMCs) and also involvement of phospholipase C (PLC) isoforms in the signal transduction. PDGF-BB and -AB, but not -AA, stimulated cell proliferation and intracellular production of inositol trisphosphate. Northern and Western analyses demonstrated that VSMCs mainly expressed PLC-gamma 2 and PLC-delta 1 among four PLC isoforms tested. A number of cellular proteins, including PLC-gamma 2, but not PLC-delta 1, were phosphorylated on a tyrosine residue by the stimulation of either PDGF-BB or -AB. These results suggest a functional association of PDGF receptor and PLC-gamma 2 that might be responsible for PDGF-dependent VSMC growth. In addition, the expression of PLC-gamma 2 was extremely low in the primary VSMC cultures and was induced during further cultivation of the primary cultures, indicating that an acquisition of PDGF-signal-transducing components, including PLC-gamma 2, may be an important step for proliferation of smooth-muscle cells.

APA, Harvard, Vancouver, ISO, and other styles

14

Takata, M., and T. Kurosaki. "A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2." Journal of Experimental Medicine 184, no. 1 (1996): 31–40. http://dx.doi.org/10.1084/jem.184.1.31.

Full text

Abstract:

Defects in the gene encoding Bruton's tyrosine kinase (Btk) result in a disease called X-linked agammaglobulinemia, in which there is a profound decrease of mature B cells due to a block in B cell development. Recent studies have shown that Btk is tyrosine phosphorylated and activated upon B cell antigen receptor (BCR) stimulation. To elucidate the functions of this kinase, we examined BCR signaling of DT40 B cells deficient in Btk. Tyrosine phosphorylation of phospholipase C (PLC)-gamma 2 upon receptor stimulation was significantly reduced in the mutant cells, leading to the loss of both BCR-coupled phosphatidylinositol hydrolysis and calcium mobilization. Pleckstrin homology and Src-homology 2 domains of Btk were required for PLC-gamma 2 activation. Since Syk is also required for the BCR-induced PLC-gamma 2 activation, our findings indicate that PLC-gamma 2 activation is regulated by Btk and Syk through their concerted actions.

APA, Harvard, Vancouver, ISO, and other styles

15

Maa, M. C., T. H. Leu, B. J. Trandel, J. H. Chang, and S. J. Parsons. "A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma." Molecular and Cellular Biology 14, no. 8 (1994): 5466–73. http://dx.doi.org/10.1128/mcb.14.8.5466.

Full text

Abstract:

p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.

APA, Harvard, Vancouver, ISO, and other styles

16

Maa, M. C., T. H. Leu, B. J. Trandel, J. H. Chang, and S. J. Parsons. "A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma." Molecular and Cellular Biology 14, no. 8 (1994): 5466–73. http://dx.doi.org/10.1128/mcb.14.8.5466-5473.1994.

Full text

Abstract:

p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.

APA, Harvard, Vancouver, ISO, and other styles

17

Hernández-Sotomayor, S. M. T., та G. Carpenter. "Non-catalytic activation of phospholipase C-γ 1 in vitro by epidermal growth factor receptor". Biochemical Journal 293, № 2 (1993): 507–11. http://dx.doi.org/10.1042/bj2930507.

Full text

Abstract:

To investigate the possible functional role of epidermal growth factor (EGF) receptor-phospholipase C-gamma 1 (PLC-gamma 1) complexes, we have measured PLC-gamma 1 activity in vitro in the absence or presence of purified EGF receptor. Immunoprecipitates of PLC-gamma 1 from control A-431 cells were incubated without or with purified EGF receptor in the absence or presence of ATP. Under these conditions the EGF receptor increased non-tyrosine-phosphorylated PLC-gamma 1 activity 3-4-fold in the absence or presence of ATP, but increased tyrosine-phosphorylated and activated PLC-gamma 1 by only 20-50%. Both basal and autophosphorylated forms of the purified EGF receptor increased the activity of the non-tyrosine-phosphorylated PLC-gamma 1, and stoichiometric levels of purified receptor were required to increase PLC activity. Other tyrosine kinases such as the platelet-derived growth factor receptor and erbB-2, but not the insulin receptor, also stimulated PLC-gamma 1 activity. PLC-gamma 1 activity could be activated with the kinase-negative EGF receptor, but a C-terminal truncated receptor was much less effective. Purified EGF receptor could also activate PLC-beta 1, but with a much decreased potency compared with PLC-gamma 1. Our results suggest that in vitro the EGF receptor can increase PLC-gamma 1 activity independently of tyrosine phosphorylation.

APA, Harvard, Vancouver, ISO, and other styles

18

Koblan, K. S., M. D. Schaber, G. Edwards, J. B. Gibbs та D. L. Pompliano. "src-homology 2 (SH2) domain ligation as an allosteric regulator: modulation of phosphoinositide-specific phospholipase Cγ1 structure and activity". Biochemical Journal 305, № 3 (1995): 745–51. http://dx.doi.org/10.1042/bj3050745.

Full text

Abstract:

Phosphoinositide-specific phospholipase C gamma 1 (PI-PLC gamma 1) catalyses the hydrolysis of PtdIns(4,5)P2 to generate the second messengers diacylglycerol and Ins(1,4,5)P3. PI-PLC gamma 1, an src-homology 2/3 (SH2/SH3)-domain-containing enzyme, is activated in response to growth-factor-induced tyrosine phosphorylation, and, in vivo, is translocated from the cytosol to the particulate cell fraction. Here we report the bacterial expression of rat brain PI-PLC gamma 1 under the control of the T7 promoter. Production of the active enzyme in amounts suitable for structure-function analysis depended on coupling the translation of PLC gamma 1 to the expression of the phage-phi 10 coat protein. Purification of the enzyme was facilitated by the presence of a three-amino-acid C-terminal antibody epitope tag (Glu-Glu-Phe) engineered into the cloned PLC gamma 1. Examination of the specific activity, pH-rate profile, [Ca2+]-dependence and substrate specificity of bacterially expressed PLC gamma indicated that it had kinetic properties similar to those of PLC gamma isolated from bovine brain. The substrate specificity was dependent on [Ca2+]: at low [Ca2+] (1-10 microM) PtdIns(4,5)P2 was a better substrate than PtdIns. Addition of phosphotyrosine-containing peptides (12-mers) with the cognate sequence of the high-affinity binding site for PLC gamma 1 on the activated epidermal-growth-factor (EGF) receptor (Tyr-992) increased enzyme activity (up to 85%) in vitro. Cognate non-phosphorylated peptides had no effect on activity. When c.d. spectroscopy was used to monitor the effect of added phosphotyrosine-containing peptide on the structure of recombinant PLC gamma 1, significant spectral shifts, indicative of a conformational change, were observed upon complexation with the EGF-receptor phosphotyrosine-containing 12-residue peptide (Tyr*-992). How SH2 domains from PLC gamma 1 can mediate structural rearrangements and modulate enzymic activity on their ligation by growth-factor receptors is discussed.

APA, Harvard, Vancouver, ISO, and other styles

19

Coorssen, J. R. "Phospholipase activation and secretion: evidence that PLA2, PLC, and PLD are not essential to exocytosis." American Journal of Physiology-Cell Physiology 270, no. 4 (1996): C1153—C1163. http://dx.doi.org/10.1152/ajpcell.1996.270.4.c1153.

Full text

Abstract:

Numerous studies have identified phospholipase metabolites as membrane fusogens, and phospholipase D (PLD) (J.R. Coorssen and R.J. Haslam. FEBS Lett. 316: 170-174, 1993), C (PLC), and A2 (PLA2) activities correlate with secretion. Do these enzymes have essential or modulatory roles? This study confirms that secretion does not require Ca2+ or PLC (Coorssen et al. Cell Regul. 1: 1027-1041, 1990). Arachidonic acid (AA), phosphatidic acid (PA) and analogues, exogenous metabolites of PLA2 and PLD, were tested in electropermeabilized human platelets. AA potentiated guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-induced secretion, and eicosanoids were not essential. Endogenous [3H]AA formation correlated with GTP gamma S-induced secretion, and phorbol 12-myristate 13-acetate (PMA) promoted these effects. Inhibitors were used to probe phospholipase influences on secretion. Only PLD inhibitors blocked secretion. However, PMA blocked inhibition of protein kinase C (PKC) and secretion by quercetin, suggesting that PA formed by PLD supports PKC activation and GTP gamma S-induced secretion. Thus PA analogues had no effect alone but enhanced GTP gamma S-induced PKC activity and secretion. Slower PLD activation compared with secretion also indicates a nonessential role. This is the first report of a Ca(2+)-independent PLA2 activity in human platelets, use of quercetin as a PLD inhibitor, and dissociation of PLA2, PLC, and PLD activities from secretion. No major phospholipase activities are essential to the final steps in exocytosis, but modulatory roles are indicated.

APA, Harvard, Vancouver, ISO, and other styles

20

Law, C. L., K. A. Chandran, S. P. Sidorenko, and E. A. Clark. "Phospholipase C-gamma1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk." Molecular and Cellular Biology 16, no. 4 (1996): 1305–15. http://dx.doi.org/10.1128/mcb.16.4.1305.

Full text

Abstract:

Antigen receptor ligation on lymphocytes activates protein tyrosine kinases and phospholipase C-gamma (PLC-gamma) isoforms. Glutathione S-transferase fusion proteins containing the C-terminal Src-homology 2 [SH2(C)] domain of PLC-gamma1 bound to tyrosyl phosphorylated Syk. Syk isolated from antigen receptor-activated B cells phosphorylated PLC-gamma1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, whereas Lyn from the same B cells phosphorylated PLC-gamma1 only on Tyr-771. The ability of Syk to phosphorylate PLC-gamma1 required antigen receptor ligation, while Lyn was constitutively active. An mCD8-Syk cDNA construct could be expressed as a tyrosyl-phosphorylated chimeric protein tyrosine kinase in COS cells, was recognized by PLC-gamma1 SH2(C) in vitro, and induced tyrosyl phosphorylation of endogenous PLC-gamma1 in vivo. Substitution of Tyr-525 and Tyr-526 at the autophosphorylation site of Syk in mCD8-Syk substantially reduced the kinase activity and the binding of this variant chimera to PLC-gamma1 SH2(C) in vitro; it also failed to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. In contrast, substitution of Tyr-348 and Tyr-352 in the linker region of Syk in mCD8-Syk did not affect the kinase activity of this variant chimera but almost completely eliminated its binding to PLC-gamma1 SH(C) and completely eliminated its ability to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. Thus, an optimal kinase activity of Syk and an interaction between the linker region of Syk with PLC-gamma1 are required for the tyrosyl phosphorylation of PLC-gamma1.

APA, Harvard, Vancouver, ISO, and other styles

21

Wu, D., C. K. Huang, and H. Jiang. "Roles of phospholipid signaling in chemoattractant-induced responses." Journal of Cell Science 113, no. 17 (2000): 2935–40. http://dx.doi.org/10.1242/jcs.113.17.2935.

Full text

Abstract:

Chemoattractants, including chemokines, play a central role in regulation of inflammatory reactions by attracting and activating leukocytes. These molecules have been found to regulate metabolism of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) via phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K). Recent studies of mouse lines that lack PLC-(beta)2, PLC-(beta)3, or PI3K(gamma) demonstrate that chemoattractants act through PLC-(beta)2 and PLC-(beta)3 to hydrolyze PtdIns(4,5)P(2) and through PI3K(gamma) to phosphorylate PtdIns(4,5)P(2) in mouse neutrophils. These studies also confirmed the importance and revealed new roles of these signaling pathways in chemoattractant-induced responses.

APA, Harvard, Vancouver, ISO, and other styles

22

Kashishian, A., and J. A. Cooper. "Phosphorylation sites at the C-terminus of the platelet-derived growth factor receptor bind phospholipase C gamma 1." Molecular Biology of the Cell 4, no. 1 (1993): 49–57. http://dx.doi.org/10.1091/mbc.4.1.49.

Full text

Abstract:

We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1.

APA, Harvard, Vancouver, ISO, and other styles

23

Daniel, J. L., C. Dangelmaier та J. B. Smith. "Evidence for a role for tyrosine phosphorylation of phospholipase Cγ2 in collagen-induced platelet cytosolic calcium mobilization". Biochemical Journal 302, № 2 (1994): 617–22. http://dx.doi.org/10.1042/bj3020617.

Full text

Abstract:

(1) The non-specific protein kinase C inhibitor, staurosporine, inhibited collagen-induced increases in cytosolic free Ca2+ while having no effect on Ca2+ mobilization by other platelet agonists. A more specific inhibitor of protein kinase C, Ro 31-8220, did not inhibit collagen-induced Ca2+ mobilization. Neither drug had an effect on platelet adhesion to collagen. (2) Staurosporine inhibited collagen-stimulated tyrosine phosphorylation, while Ro 31-8220 had no effect. (3) It also inhibited collagen-induced phosphatidic acid formation, inositol trisphosphate formation and arachidonic acid liberation. (4) Ro 31-8220 did not inhibit collagen-stimulated arachidonic acid formation, but it enhanced collagen-stimulated phosphatidic acid and inositol trisphosphate formation. (5) Immunoprecipitation of phospholipase C gamma 2 (PLC gamma 2) with a specific antibody demonstrated that PLC gamma 2 was phosphorylated on tyrosine after stimulation by collagen. (6) The phosphorylation of PLC gamma 2 was inhibited by staurosporine but not by Ro 31-8220. These results provide additional evidence that the mechanism of signal transduction for collagen is different from other platelet agonists and indicate that it involves activation of PLC gamma through a tyrosine kinase-dependent mechanism.

APA, Harvard, Vancouver, ISO, and other styles

24

Weber, J. R., G. M. Bell, M. Y. Han, T. Pawson, and J. B. Imboden. "Association of the tyrosine kinase LCK with phospholipase C-gamma 1 after stimulation of the T cell antigen receptor." Journal of Experimental Medicine 176, no. 2 (1992): 373–79. http://dx.doi.org/10.1084/jem.176.2.373.

Full text

Abstract:

Stimulation of the T cell antigen receptor (TCR) activates a protein tyrosine kinase and leads to the tyrosine phosphorylation of phosphoinositide-specific phospholipase C-gamma 1 (PLC gamma 1). The molecular interactions involved in this phosphorylation are not known. After stimulation of the TCR on Jurkat T cells, tyrosine-phosphorylated proteins of 36, 38, 58, and 63 kD coprecipitate with PLC gamma 1. An identical pattern of proteins precipitate with TrpE fusion proteins that contain the Src homology (SH) 2 domains of PLC gamma 1, indicating that these regions of PLC gamma 1 are responsible for binding. TCR stimulation leads to an association between the SH2 domains of PLC gamma 1 and a protein tyrosine kinase, which, by peptide mapping, is identical to p56lck. These studies establish that p56lck associates with PLC gamma 1 as a result of TCR stimulation of Jurkat cells, suggesting that p56lck plays a central role in coupling the TCR to the activation of PLC gamma 1.

APA, Harvard, Vancouver, ISO, and other styles

25

Fernald, A. W., G. A. Jones та G. Carpenter. "Limited proteolysis of phospholipase C-γ 1 indicates stable association of X and Y domains with enhanced catalytic activity". Biochemical Journal 302, № 2 (1994): 503–9. http://dx.doi.org/10.1042/bj3020503.

Full text

Abstract:

Phospholipase C-gamma 1 (PLC-gamma 1) was treated with Staphylococcus aureus V8 protease (V8) and the digestion products were analysed with site-specific antibodies. V8 treatment generated three immunodetectable PLC-gamma 1 fragments of 120, 97, and 39 kDa. The 39 kDa fragment is derived from the C-terminus of PLC-gamma 1 and includes the conserved Y domain present in all PLC isoenzymes. The 120 and 97 kDa fragments are derived from the N-terminus of PLC-gamma 1, possess the conserved X domain common to all PLC isoenzymes, and the src-homology domains unique to PLC-gamma 1 and -gamma 2. It is likely that the 97 kDa fragment is a V8 product of the 120 kDa fragment. As the C-terminal 39 kDa fragment, and either of the N-terminal 120 or 97 kDa fragments, were precipitable with antibody specific to a sequence present in only the 39 kDa fragment, the data indicate co-precipitation of separate polypeptide chains that remain associated after V8 proteolysis. Importantly, V8 treatment increased the activity of PLC-gamma 1 and did not alter the calcium requirement. The influence of other modulators of PLC-gamma 1 activity, however, was lost following V8 treatment. These results suggest the stable association of the X and Y domains within PLC-gamma 1, and demonstrate that proteolysis in the region of PLC-gamma 1 that is subject to tyrosine phosphorylation can enhance catalytic activity.

APA, Harvard, Vancouver, ISO, and other styles

26

Fazioli, F., U. H. Kim, S. G. Rhee, C. J. Molloy, O. Segatto, and P. P. Di Fiore. "The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency." Molecular and Cellular Biology 11, no. 4 (1991): 2040–48. http://dx.doi.org/10.1128/mcb.11.4.2040.

Full text

Abstract:

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.

APA, Harvard, Vancouver, ISO, and other styles

27

Nakano, H., H. Ohno, and T. Saito. "Activation of phospholipase C-gamma 1 through transfected platelet-derived growth factor receptor enhances interleukin 2 production upon antigen stimulation in a T-cell line." Molecular and Cellular Biology 14, no. 2 (1994): 1213–19. http://dx.doi.org/10.1128/mcb.14.2.1213.

Full text

Abstract:

Phospholipase C-gamma 1 (PLC gamma 1) plays an important role in the signal transduction pathway by producing second messengers. However, the activation mechanism of PLC gamma 1 and the role of the phosphatidylinositol pathway for interleukin 2 (IL-2) production in T lymphocytes remain to be determined. To analyze the functional role of this pathway in T cells, we expressed an epidermal growth factor receptor (EGF) or platelet-derived growth factor (PDGF) receptor (EGF-R or PDGF-R), both of which are known to directly activate PLC gamma 1 in fibroblasts, into a murine T-cell hybridoma. Both receptors were expressed on the cell surface and caused tyrosine phosphorylation of multiple substrates, including the receptor itself, upon ligand binding. While EGF stimulation did not either cause phosphorylation of PLC gamma 1 or induce Ca2+ mobilization in the EGF-R transfectant in this system, PDGF treatment induced tyrosine phosphorylation of PLC gamma 1 and Ca2+ mobilization in the PDGF-R transfectant. Stimulation through PDGF-R enhanced IL-2 production upon antigen stimulation of the transfectants, although PDGF treatment alone did not induce IL-2 production. These results suggest that activation of the phosphatidylinositol pathway affects the downstream pathway to IL-2 production but is not sufficient to produce IL-2 and that cooperation with signals from tyrosine kinase cascades is required for IL-2 production.

APA, Harvard, Vancouver, ISO, and other styles

28

Nakano, H., H. Ohno, and T. Saito. "Activation of phospholipase C-gamma 1 through transfected platelet-derived growth factor receptor enhances interleukin 2 production upon antigen stimulation in a T-cell line." Molecular and Cellular Biology 14, no. 2 (1994): 1213–19. http://dx.doi.org/10.1128/mcb.14.2.1213-1219.1994.

Full text

Abstract:

Phospholipase C-gamma 1 (PLC gamma 1) plays an important role in the signal transduction pathway by producing second messengers. However, the activation mechanism of PLC gamma 1 and the role of the phosphatidylinositol pathway for interleukin 2 (IL-2) production in T lymphocytes remain to be determined. To analyze the functional role of this pathway in T cells, we expressed an epidermal growth factor receptor (EGF) or platelet-derived growth factor (PDGF) receptor (EGF-R or PDGF-R), both of which are known to directly activate PLC gamma 1 in fibroblasts, into a murine T-cell hybridoma. Both receptors were expressed on the cell surface and caused tyrosine phosphorylation of multiple substrates, including the receptor itself, upon ligand binding. While EGF stimulation did not either cause phosphorylation of PLC gamma 1 or induce Ca2+ mobilization in the EGF-R transfectant in this system, PDGF treatment induced tyrosine phosphorylation of PLC gamma 1 and Ca2+ mobilization in the PDGF-R transfectant. Stimulation through PDGF-R enhanced IL-2 production upon antigen stimulation of the transfectants, although PDGF treatment alone did not induce IL-2 production. These results suggest that activation of the phosphatidylinositol pathway affects the downstream pathway to IL-2 production but is not sufficient to produce IL-2 and that cooperation with signals from tyrosine kinase cascades is required for IL-2 production.

APA, Harvard, Vancouver, ISO, and other styles

29

Fazioli, F., U. H. Kim, S. G. Rhee, C. J. Molloy, O. Segatto, and P. P. Di Fiore. "The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency." Molecular and Cellular Biology 11, no. 4 (1991): 2040–48. http://dx.doi.org/10.1128/mcb.11.4.2040-2048.1991.

Full text

Abstract:

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.

APA, Harvard, Vancouver, ISO, and other styles

30

Li, W., G. G. Deanin, B. Margolis, J. Schlessinger, and J. M. Oliver. "Fc epsilon R1-mediated tyrosine phosphorylation of multiple proteins, including phospholipase C gamma 1 and the receptor beta gamma 2 complex, in RBL-2H3 rat basophilic leukemia cells." Molecular and Cellular Biology 12, no. 7 (1992): 3176–82. http://dx.doi.org/10.1128/mcb.12.7.3176.

Full text

Abstract:

In basophils, mast cells, and the RBL-2H3 tumor mast cell line, cross-linking the high-affinity immunoglobulin E receptor (Fc epsilon R1) stimulates a series of responses, particularly the activation of phospholipase C (PLC), that lead to allergic and other immediate hypersensitivity reactions. The mechanism of activation of PLC, however, is not clear. Here, we show that cross-linking Fc epsilon R1 on RBL-2H3 cells causes the tyrosine phosphorylation of at least 12 cellular proteins, including PLC gamma 1 (PLC gamma 1) and the receptor beta and gamma subunits. 32P-labeled PLC gamma 1 can be detected by anti-phosphotyrosine antibody as early as 10 s after the addition of antigen. The tyrosine-phosphorylated 33-kDa beta subunit and 9- to 11-kDa gamma subunit of the Fc epsilon R1 are additionally phosphorylated on serine and theonine residues, respectively, and are found as complexes with other phosphotyrosine-containing proteins in antigen-stimulated cells. Our results indicate a means by which the Fc epsilon R1 may control PLC activity in RBL-2H3 cells and raise the possibility that other receptor-mediated signalling events in mast cells may also be controlled through protein tyrosine phosphorylation.

APA, Harvard, Vancouver, ISO, and other styles

31

Li, W., G. G. Deanin, B. Margolis, J. Schlessinger, and J. M. Oliver. "Fc epsilon R1-mediated tyrosine phosphorylation of multiple proteins, including phospholipase C gamma 1 and the receptor beta gamma 2 complex, in RBL-2H3 rat basophilic leukemia cells." Molecular and Cellular Biology 12, no. 7 (1992): 3176–82. http://dx.doi.org/10.1128/mcb.12.7.3176-3182.1992.

Full text

Abstract:

In basophils, mast cells, and the RBL-2H3 tumor mast cell line, cross-linking the high-affinity immunoglobulin E receptor (Fc epsilon R1) stimulates a series of responses, particularly the activation of phospholipase C (PLC), that lead to allergic and other immediate hypersensitivity reactions. The mechanism of activation of PLC, however, is not clear. Here, we show that cross-linking Fc epsilon R1 on RBL-2H3 cells causes the tyrosine phosphorylation of at least 12 cellular proteins, including PLC gamma 1 (PLC gamma 1) and the receptor beta and gamma subunits. 32P-labeled PLC gamma 1 can be detected by anti-phosphotyrosine antibody as early as 10 s after the addition of antigen. The tyrosine-phosphorylated 33-kDa beta subunit and 9- to 11-kDa gamma subunit of the Fc epsilon R1 are additionally phosphorylated on serine and theonine residues, respectively, and are found as complexes with other phosphotyrosine-containing proteins in antigen-stimulated cells. Our results indicate a means by which the Fc epsilon R1 may control PLC activity in RBL-2H3 cells and raise the possibility that other receptor-mediated signalling events in mast cells may also be controlled through protein tyrosine phosphorylation.

APA, Harvard, Vancouver, ISO, and other styles

32

Huynh, Minh Quang, Ulrich Kaiser, Jennifer Goßmann, and Andreas Neubauer. "Expression of Phospholipse C Gamma 2 in Diffuse Large B-Cell Lymphomas,." Blood 118, no. 21 (2011): 3667. http://dx.doi.org/10.1182/blood.v118.21.3667.3667.

Full text

Abstract:

Abstract Abstract 3667 Cell survival and proliferation of diffuse large B-cell lymphomas (DLBCL) are dependent on the NF-κB signaling pathway. Antigen binding of the B-cell receptor (BCR) leads to phosphorylation of Src tyrosine kinases, resulting in PLCg2 activation, which is important for regulation of NF-κB through the protein kinase C (PKC). The PLC-PKC cascade also activates the Ras signaling pathway. There are several clinical studies showing that inhibition of PKCβ with the specific inhibitor Enzastaurin could be a promising new therapy target for DLBCL. To our knowledge, there are little data about the role of PLCg2 in DLBCL. It has been shown that the activity of Syk and PLCg2 are well correlated with sensitivity of DLBCL to dasatinib. Our study performed immunohistological staining of PLCg2 in 86 primary DLBCL. PLCg2 was strongly expressed in 54 out of 86 cases (63%) and weakly expressed in 28 cases (32%). Only 4 cases (5%) were negative for PLCg2. In three DLBCL cell lines (SuDHL-4, SuDHL-6 and U2932) we could show an inhibitory effect of the PLC-inhibitor U73122 on cell proliferation. Treatment in combination with Enzastaurin or the Src-inhibitor pp2 had an additive effect on cell proliferation compared to U73122 alone. In conclusion, PLCg2 is strongly expressed in a great subset of DLBCL. Inhibition of PLCg2 could be, at least in these cases, a new target for lymphoma treatment. Disclosures: No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

33

Marrero, M. B., B. Schieffer, H. Ma, K. E. Bernstein, and B. N. Ling. "ANG II-induced tyrosine phosphorylation stimulates phospholipase C-gamma 1 and Cl-channels in mesangial cells." American Journal of Physiology-Cell Physiology 270, no. 6 (1996): C1834—C1842. http://dx.doi.org/10.1152/ajpcell.1996.270.6.c1834.

Full text

Abstract:

Angiotensin II (ANG II)-induced, activation of phospholipase C (PLC) and Ca(2+)-dependent Cl-channels is an important signal transduction pathway for mesangial cell contraction and growth. Although ANG II receptors are traditionally though to be G protein coupled, recent evidence suggests that they may also mediate protein tyrosine phosphorylation. In cultured rat mesangial cells, 10(-7) MANG II stimulated the tyrosine phosphorylation of PLC-gamma 1 and elevation of intracellular inositol 1,4,5-trisphosphate (IP3) and Ca2+ levels; peak response occurred within 0.5 min. In cell-attached patches, ANG II stimulated the activity of Ca(2+)-dependent, 3- to 4-pS Cl-channels (number of channels x open probability) from 0.063 +/- 0.022 to 0.77 +/- 0.20. Tyrosine kinase inhibition with genistein or herbimycin A blocked all four ANG II-induced responses. We conclude the following. 1) Stimulation of inositol phosphate hydrolysis by PLC, release of IP3-dependent intracellular Ca2+ stores, and activation of Ca(2+)-dependent C1-channels by ANG II are dependent on the tyrosine phosphorylation of PLC-gamma 1.2) This ANG II-induced signal transduction cascade provides a possible mechanism for both the contractile and growth-stimulating effects of ANG II on glomerular mesangial cells.

APA, Harvard, Vancouver, ISO, and other styles

34

Martin, T. F. J., J. E. Lewis та J. A. Kowalchyk. "Phospholipase C-β1 is regulated by a pertussis toxin-insensitive G-protein". Biochemical Journal 280, № 3 (1991): 753–60. http://dx.doi.org/10.1042/bj2800753.

Full text

Abstract:

Regulation of phospholipase C (PLC) by receptors is mediated either through protein tyrosine phosphorylation or by activation of GTP-binding proteins (Gp). For the latter, pertussis toxin (PT)-sensitive and -insensitive pathways have been described, indicating PLC regulation by at least two types of G-proteins. The identity of PLC isoenzymes which are regulated by either type of Gp remains to be determined. Thyrotropin-releasing hormone stimulates a PLC in GH3 cells via a PT-insensitive Gp. Reconstitution methods for the assay of the GH3-cell Gp were developed. Previously, the membrane PLC was found to be reversibly extracted from membranes by high salt and to be activated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) only when membrane-associated, suggesting that Gp was retained in salt-extracted membranes. In the present work, Gp was cholate-solubilized from PLC-deficient membranes and incorporated into phospholipid vesicles, which were found to confer GTP[S]- and AlF4(-)-stimulated activity on a solubilized membrane PLC. The reconstitution provided a direct assay for the GH3-cell Gp which was shown to be distinct from Gi, Go and Gs proteins by immunodepletion studies. Incorporation of G-protein beta-gamma subunits into phospholipid vesicles with Gp inhibited GTP[S]-stimulated activity in the reconstitution. The results indicated that Gp is a heterotrimeric G-protein with the properties expected for the PT-insensitive GH3-cell Gp protein. PLC-beta 1 was fully purified and shown to be regulated by Gp in the reconstitution. In contrast, PT-sensitive G-proteins failed to affect the activity of PLC-beta 1. The results indicate (1) that a PT-insensitive Gp regulates PLC-beta 1 and (2) that PT-sensitive and -insensitive pathways of PLC regulation employ different PLC isoenzymes as well as different G-proteins.

APA, Harvard, Vancouver, ISO, and other styles

35

Mitchell, C. H., J. J. Zhang, L. Wang, and T. J. Jacob. "Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases." American Journal of Physiology-Cell Physiology 272, no. 1 (1997): C212—C222. http://dx.doi.org/10.1152/ajpcell.1997.272.1.c212.

Full text

Abstract:

The whole cell recording technique was used to examine an outwardly rectifying chloride current activated by hypotonic shock in bovine pigmented ciliary epithelial (PCE) cells. Removal of internal and external Ca2+ did not affect the activation of these currents, but they were abolished by the phospholipase C inhibitor neomycin. The current was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and 4,4'-disothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner, but tamoxifen, dideoxyforskolin, and quinidine did not affect it. This blocking profile differs from that of the volume-sensitive chloride channel in neighboring nonpigmented ciliary epithelial cells (Wu, J., J. J. Zhang, H. Koppel, and T. J. C. Jacob, J. Physiol, Lond. 491: 743-755, 1996), and this difference implies that the volume responses of the two cell types are mediated by different chloride channels (Jacob, T. J. C., and J. J. Zhang. J. Physiol. Lond. In press). Intracellular administration of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to PCE cells induced a transient, time-independent, outwardly rectifying chloride current that closely resembled the current activated by hypotonic shock. DIDS produced a voltage-dependent block of the GTP gamma S-activated current similar to the block of the hypotonically activated current. Intracellular neomycin completely prevented activation of this current as did incubation of the cells in calphostin C. and inhibitor of protein kinase C (PKC). Removal of Ca2+ did not affect activation of the current by GTP gamma S but extended the duration of the response. Inhibition of phospholipase A2 (PLA2) with p-bromophenacyl bromide prevented the activation of the hypotonically induced current and also inhibited the current once activated by hypotonic solution. The findings imply that the hypotonic response in PCE cells is mediated by both phospholipase C (PLC) and PLA2. Both phospholipases generate arachidonic acid, and, in addition, the PLC pathway regulates the PLA2 pathway via a PKC-dependent phosphorylation of PLA2.

APA, Harvard, Vancouver, ISO, and other styles

36

Piiper, A., D. Stryjek-Kaminska, R. Klengel, and S. Zeuzem. "CCK, carbachol, and bombesin activate distinct PLC-beta isoenzymes via Gq/11 in rat pancreatic acinar membranes." American Journal of Physiology-Gastrointestinal and Liver Physiology 272, no. 1 (1997): G135—G140. http://dx.doi.org/10.1152/ajpgi.1997.272.1.g135.

Full text

Abstract:

Four different isoforms of phospholipase C-beta (PLC-beta 1-4) have been discovered, raising the important question of whether a distinct receptor activates a single PLC-beta isoenzyme or a subset of PLC-beta isoenzymes. The present study was designed to investigate activation of PLC-beta isoenzymes by three different PLC-activating agonists that bind to different receptor entities, i.e., cholecystokinin octapeptide (CCK-8), bombesin, and carbachol in rat pancreatic acinar membranes. PLC activity was measured using exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate. Western blot analysis of pancreatic acinar membranes revealed the presence of PLC-beta 1, -beta 3, -gamma 1, and -delta 1, but not of PLC-beta 2, -beta 4, -gamma 2, and -delta 2. Preincubation of the membranes with anti-PLC-beta 1 or -beta 3 antibody reduced agonist-induced activation of PLC. The order of sensitivity toward inhibition by anti-PLC-beta 1 antibody was CCK-8 > bombesin > carbachol. An opposite order of sensitivity was found for inhibition of PLC activity by anti-PLC-beta 3 antibody (carbachol > bombesin > CCK-8). Anti-PLC-beta 2, -beta 4, -gamma 1, -gamma 2, -delta 1, and -delta 2 antibodies had no effect. Preincubation of the membranes with an antibody raised against the COOH terminus of the alpha-subunit of Gq/11 proteins inhibited PLC activity in response to all three different receptor agonists to a similar extent, whereas anti-Gi alpha 1-2 and anti-Gi alpha 3 antibodies had no effect. In conclusion, the data of the present study indicate that CCK-8 and carbachol activate PLC-beta 1 and PLC-beta 3, respectively, whereas bombesin activates both PLC-beta 1 and PLC-beta 3. Activation of PLC-beta by these receptor agonists is mediated by Gq/11.

APA, Harvard, Vancouver, ISO, and other styles

37

Ma, H., H. Matsunaga, B. Li, M. B. Marrero, and B. N. Ling. "Regulation of PDGF-beta receptor-operated Ca2+ channels by phospholipase C-gamma 1 in glomerular mesangial cells." American Journal of Physiology-Renal Physiology 271, no. 5 (1996): F994—F1003. http://dx.doi.org/10.1152/ajprenal.1996.271.5.f994.

Full text

Abstract:

Platelet-derived growth factor (PDGF)-induced Ca2+ signaling mechanisms were examined in cultured rat glomerular mesangial cells. PDGF-BB stimulated the tyrosine phosphorylation of phospholipase C (PLC)-gamma 1, the formation of a PLC-gamma 1/PDGF-beta receptor membrane complex, and the generation of intracellular inositol 1,4,5-trisphosphate (IP3). Preincubation with a tyrosine kinase inhibitor (genistein) abolished these PDGF-induced responses. Activation of 1-pS Ca2+ channels in cell-attached patches by intrapipette PDGF-BB was also abolished by tyrosine kinase inhibition. In the absence of PDGF-BB, channels were activated in cell-attached patches exposed to intrapipette thapsigargin (IP3-independent releaser of intracellular Ca2+ stores) and in excised inside-out patches exposed to increasing “cytoplasmic” Ca2+ (10(-8) to 10(-6) M). In cell-attached patches, channel activation by PDGF-BB was abolished when extracellular Ca2+ was < 1 mM. In glomerular mesangial cells 1) PDGF-BB stimulates tyrosine phosphorylation of PLC-gamma 1, PDGF-beta receptor/PLC-gamma 1 membrane complex formation, IP3 production, and 1-pS Ca2+ channel activity; 2) all four PDGF-induced responses are abolished by tyrosine kinase inhibition; 3) PDGF receptor-operated Ca2+ channels are sensitive to both intra- and extracellular Ca2+.

APA, Harvard, Vancouver, ISO, and other styles

38

Azzoni, L., M. Kamoun, T. W. Salcedo, P. Kanakaraj, and B. Perussia. "Stimulation of Fc gamma RIIIA results in phospholipase C-gamma 1 tyrosine phosphorylation and p56lck activation." Journal of Experimental Medicine 176, no. 6 (1992): 1745–50. http://dx.doi.org/10.1084/jem.176.6.1745.

Full text

Abstract:

Binding of ligand to the alpha subunit of Fc gamma RIIIA(CD16), expressed at the natural killer (NK) cell membrane in association with homo or heterodimers of proteins of the zeta family, results in phosphorylation of several proteins on tyrosine residues. We have analyzed the role of protein tyrosine phosphorylation in the regulation of molecular events induced upon stimulation of Fc gamma RIIIA in NK cells and in T cells expressing the Fc gamma RIII alpha chain in association with endogenous zeta 2 homodimers and devoid of other (CD3, CD2) transducing molecules. Our data indicate that treatment of these cells with protein tyrosine kinase inhibitors prevents not only Fc gamma RIIIA-induced protein tyrosine phosphorylation but also phosphatidylinositol 4,5 diphosphate hydrolysis and increased intracellular Ca2+ concentration, indicating a primary role of tyrosine kinase(s) in the induction of these early activation events. Occupancy of Fc gamma RIIIA by ligand results in phospholipase C (PLC)-gamma 1 tyrosine phosphorylation in NK cells and in Fc gamma RIIIA-transfected CD3-/CD2- T cells, and induces functional activation of p56lck in Fc gamma RIIIA alpha/zeta 2-transfected T cells, suggesting the possibility that the receptor-induced PLC-gamma 1 activation occurs upon phosphorylation of its tyrosine residues mediated by this kinase and is, at least in part, responsible for the signal transduction mediated via CD16 upon ligand binding.

APA, Harvard, Vancouver, ISO, and other styles

39

Law, C. L., S. P. Sidorenko, K. A. Chandran, et al. "CD22 associates with protein tyrosine phosphatase 1C, Syk, and phospholipase C-gamma(1) upon B cell activation." Journal of Experimental Medicine 183, no. 2 (1996): 547–60. http://dx.doi.org/10.1084/jem.183.2.547.

Full text

Abstract:

Cross-linking B cell antigen receptor (BCR) elicits early signal transduction events, including activation of protein tyrosine kinases, phosphorylation of receptor components, activation of phospholipase C-gamma (PLC-gamma), and increases in intracellular free Ca2+. In this article, we report that cross-linking the BCR led to a rapid translocation of cytosolic protein tyrosine phosphatase (PTP) 1C to the particulate fraction, where it became associated with a 140-150-kD tyrosyl-phosphorylated protein. Western blotting analysis identified this 140-150-kD protein to be CD22. The association of PTP-1C with CD22 was mediated by the NH2-terminal Src homology 2 (SH2) domain of PTP-1C. Complexes of either CD22/PTP-1C/Syk/PLC-gamma(1) could be isolated from B cells stimulated by BCR engagement or a mixture of hydrogen peroxidase and sodium orthovanadate, respectively. The binding of PLC-gamma(1) and Syk to tyrosyl-phosphorylated CD22 was mediated by the NH2-terminal SH2 domain of PLC-gamma(1) and the COOH-terminal SH2 domain of Syk, respectively. These observations suggest that tyrosyl-phosphorylated CD22 may downmodulate the activity of this complex by dephosphorylation of CD22, Syk, and/or PLC-gamma(1). Transient expression of CD22 and a null mutant of PTP-1C (PTP-1CM) in COS cells resulted in an increase in tyrosyl phosphorylation of CD22 and its interaction with PTP-1CM. By contrast, CD22 was not tyrosyl phosphorylated or associated with PTP-1CM in the presence of wild-type PTP-1C. These results suggest that tyrosyl-phosphorylated CD22 may be a substrate for PTP-1C regulates tyrosyl phosphorylation of CD22.

APA, Harvard, Vancouver, ISO, and other styles

40

Li, Kaiwen, Xiaoming Ma, Qiong Wang, et al. "Phospholipase C-like protein 2 (PLC-L2) is associated with cytolytic ability of CD8+ T cells and prognosis of prostate cancer." Materials Express 10, no. 5 (2020): 725–32. http://dx.doi.org/10.1166/mex.2020.1694.

Full text

Abstract:

PLC-L2, whose related members of its family facilitate in the differentiation of T cells, is a potential epigenetic marker for PCa. The density of tumor-infiltrating CD8+ T cells in PCa is associated with a better prognosis. It remains unclear how PLC-L2 affects the TIL and PCa. A human tissue microarray (TMA) and PCa patients from the Cancer Genome Atlas were used to address the correlations between PLC-L2 and the infiltrated immune cells and to evaluate the relationship between PLC-L2 and prognosis of PCa patients. More advanced PCa expressed less PLC-L2 than less advanced PCa, in terms of pathological grade, clinical stage, and the presence of lymph node and distant metastasis. Besides, the expression of PLC-L2 had positive correlations with CD8+ T cells and antigen-presenting cells such as macrophage and dendritic cells. PLC-L2 expression was also positively correlated with local immune cytolytic ability, TNF-alpha, IFN-gamma, IL-12A, IL-12B, and IL-18. Furthermore, PLC-L2 expression was an independent factor of a favorable prognosis in PCa patients. This study reveals that PLC-L2 might recruit CD8+ T cells and promote their cytolytic ability in PCa. PLC-L2 accompanied by CD8+ cytotoxic T cells could suppress the development of PCa and subsequently increase prognosis in PCa patients.

APA, Harvard, Vancouver, ISO, and other styles

41

Taylor, S. J., and J. H. Exton. "Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements." Biochemical Journal 248, no. 3 (1987): 791–99. http://dx.doi.org/10.1042/bj2480791.

Full text

Abstract:

The effect of the GTP analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]) on the polyphosphoinositide phospholipase C (PLC) of rat liver was examined by using exogenous [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. GTP[S] stimulated the membrane-bound PLC up to 20-fold, with a half-maximal effect at approx. 100 nM. Stimulation was also observed with guanosine 5′-[beta gamma-imido]triphosphate, but not with adenosine 5′-[gamma-thio]triphosphate, and was inhibited by guanosine 5′-[beta-thio]diphosphate. Membrane-bound PLC was entirely Ca2+-dependent, and GTP[S] produced both a decrease in the Ca2+ requirement and an increase in activity at saturating [Ca2+]. The stimulatory action of GTP[S] required millimolar Mg2+. [8-arginine]Vasopressin (100 nM) stimulated the PLC activity approx. 2-fold in the presence of 10 nM-GTP[S], but had no effect in the absence of GTP[S] or at 1 microM-GTP[S]. The hydrolysis of PtdIns(4,5)P2 by membrane-bound PLC was increased when the substrate was mixed with phosphatidylethanolamine, phosphatidylcholine or various combinations of these with phosphatidylserine. With PtdIns(4,5)P2, alone or mixed with phosphatidylcholine, GTP[S] evoked little or no stimulation of the PLC activity. However, maximal stimulation by GTP[S] was observed in the presence of a 2-fold molar excess of phosphatidylserine or various combinations of phosphatidylethanolamine and phosphatidylserine. Hydrolysis of [3H]phosphatidylinositol 4-phosphate by membrane-bound PLC was also increased by GTP[S]. However, [3H]phosphatidylinositol was a poor substrate, and its hydrolysis was barely affected by GTP[S]. Cytosolic PtdIns(4,5)P2-PLC exhibited a Ca2+-dependence similar to that of the membrane-bound activity, but was unaffected by GTP[S]. It is concluded that rat liver plasma membranes possess a Ca2+-dependent polyphosphoinositide PLC that is activated by hormones and GTP analogues, depending on the Mg2+ concentration and phospholipid environment. It is proposed that GTP analogues and hormones, acting through a guanine nucleotide-binding protein, activate the enzyme mainly by lowering its Ca2+ requirement.

APA, Harvard, Vancouver, ISO, and other styles

42

Mohammadi, M., A. M. Honegger, D. Rotin, et al. "A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1." Molecular and Cellular Biology 11, no. 10 (1991): 5068–78. http://dx.doi.org/10.1128/mcb.11.10.5068.

Full text

Abstract:

Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown.

APA, Harvard, Vancouver, ISO, and other styles

43

Mohammadi, M., A. M. Honegger, D. Rotin, et al. "A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1." Molecular and Cellular Biology 11, no. 10 (1991): 5068–78. http://dx.doi.org/10.1128/mcb.11.10.5068-5078.1991.

Full text

Abstract:

Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown.

APA, Harvard, Vancouver, ISO, and other styles

44

Ren, C. L., T. Morio, S. M. Fu, and R. S. Geha. "Signal transduction via CD40 involves activation of lyn kinase and phosphatidylinositol-3-kinase, and phosphorylation of phospholipase C gamma 2." Journal of Experimental Medicine 179, no. 2 (1994): 673–80. http://dx.doi.org/10.1084/jem.179.2.673.

Full text

Abstract:

CD40 is a 50-kD glycoprotein that plays an important role in B cell survival, memory, and immunoglobulin isotype switch. Engagement of the CD40 antigen by monoclonal antibodies (mAbs) results in increased protein tyrosine kinase (PTK) activity, which plays an important role in mediating the biologic effects of CD40. We demonstrate, using an in situ phosphorylation technique, that CD40 cross-linking by the anti-CD40 mAb 626.1 resulted within 1 min in increased phosphorylation of the src type kinase, lyn, in Daudi B cell lines and remained sustained for up to 20 min. The activity of lyn kinase, as measured by immune complex kinase assay, was also increased after CD40 engagement, with similar kinetics. In contrast, the phosphorylation and activity of fyn, fgr, and lck kinases demonstrated minimal changes following stimulation of Daudi cells with mAb 626.1 over this same time period. CD40 engagement also resulted in phosphorylation of phospholipase C gamma 2 of phosphatidylinositol (PLC gamma 2) and phosphatidylinositol (PI)-3-kinase. Phosphorylation of PI-3-kinase was shown to be associated with an increase in its enzymatic activity. These results suggest that lyn plays an important role in CD40-mediated PTK activation and identify PLC gamma 2 and PI-3-kinase targets for CD40-mediated phosphorylation, suggesting a role for these two enzymes in CD40 signal transduction.

APA, Harvard, Vancouver, ISO, and other styles

45

Pleiman, C. M., M. R. Clark, L. K. Gauen, et al. "Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase." Molecular and Cellular Biology 13, no. 9 (1993): 5877–87. http://dx.doi.org/10.1128/mcb.13.9.5877.

Full text

Abstract:

Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly.

APA, Harvard, Vancouver, ISO, and other styles

46

Pleiman, C. M., M. R. Clark, L. K. Gauen, et al. "Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase." Molecular and Cellular Biology 13, no. 9 (1993): 5877–87. http://dx.doi.org/10.1128/mcb.13.9.5877-5887.1993.

Full text

Abstract:

Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly.

APA, Harvard, Vancouver, ISO, and other styles

47

Chen, P., H. Xie, M. C. Sekar, K. Gupta, and A. Wells. "Epidermal growth factor receptor-mediated cell motility: phospholipase C activity is required, but mitogen-activated protein kinase activity is not sufficient for induced cell movement." Journal of Cell Biology 127, no. 3 (1994): 847–57. http://dx.doi.org/10.1083/jcb.127.3.847.

Full text

Abstract:

We recently have demonstrated that EGF receptor (EGFR)-induced cell motility requires receptor kinase activity and autophosphorylation (P. Chen, K. Gupta, and A. Wells. 1994. J. Cell Biol. 124:547-555). This suggests that the immediate downstream effector molecule contains a src homology-2 domain. Phospholipase C gamma (PLC gamma) is among the candidate transducers of this signal because of its potential roles in modulating cytoskeletal dynamics. We utilized signaling-restricted EGFR mutants expressed in receptor devoid NR6 cells to determine if PLC activation is necessary for EGFR-mediated cell movement. Exposure to EGF (25 nM) augmented PLC activity in all five EGFR mutant cell lines which also responded by increased cell movement. Basal phosphoinositide turnover was not affected by EGF in the lines which do not present the enhanced motility response. The correlation between EGFR-mediated cell motility and PLC activity suggested, but did not prove, a causal link. A specific inhibitor of PLC, U73122 (1 microM) diminished both the EGF-induced motility and PLC responses, while its inactive analogue U73343 had no effect on these responses. Both the PLC and motility responses were decreased by expression of a dominant-negative PLC gamma-1 fragment in EGF-responsive infectant lines. Lastly, anti-sense oligonucleotides (20 microM) to PLC gamma-1 reduced both responses in NR6 cells expressing wild-type EGFR. These findings strongly support PLC gamma as the immediate post receptor effector in this motogenic pathway. We have demonstrated previously that EGFR-mediated cell motility and mitogenic signaling pathways are separable. The point of divergence is undefined. All kinase-active EGFR mutants induced the mitogenic response while only those which are autophosphorylated induced PLC activity. U73122 did not affect EGF-induced thymidine incorporation in these motility-responsive infectant cell lines. In addition, the dominant-negative PLC gamma-1 fragment did not diminish EGF-induced thymidine incorporation. All kinase active EGFR stimulated mitogen-activated protein (MAP) kinase activity, regardless of whether the receptors induced cell movement; this EGF-induced MAP kinase activity was not affected by U73122 at concentrations that depressed the motility response. Thus, the signaling pathways which lead to motility and cell proliferation diverge at the immediate post-receptor stage, and we suggest that this is accomplished by differential activation of effector molecules.

APA, Harvard, Vancouver, ISO, and other styles

48

Thomas, C. P., M. Kester, and M. J. Dunn. "A pertussis toxin-sensitive GTP-binding protein couples endothelin to phospholipase C in rat mesangial cells." American Journal of Physiology-Renal Physiology 260, no. 3 (1991): F347—F352. http://dx.doi.org/10.1152/ajprenal.1991.260.3.f347.

Full text

Abstract:

The mechanisms of stimulation of phospholipase C (PLC) by endothelin, specifically the role of guanine nucleotide-binding proteins (GTP-binding proteins) in coupling the endothelin receptor to PLC, were investigated in rat mesangial cells. Endothelin-1 (ET) synergistically released inositol polyphosphates in the presence of the stimulatory GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in permeabilized cells. In addition, in intact cells, pertussis toxin partially inhibited the stimulation of total inositol phosphates (IPn) by ET. Pertussis toxin also reduced the peak ET-stimulated intracellular free calcium level ([Ca2+]i) in these cells, both in the presence and absence of extracellular calcium. Pertussis toxin induced ADP ribosylation of a 41- to 43-kDa protein in mesangial cell membranes, and this effect was inhibited by prior exposure to ET and augmented by the inhibitory GDP analogue, guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). Thus a pertussis toxin-sensitive GTP-binding protein is involved in the activation of PLC by ET in glomerular mesangial cells.

APA, Harvard, Vancouver, ISO, and other styles

49

Reynolds, N. J., H. S. Talwar, J. J. Baldassare та ін. "Differential induction of phosphatidylcholine hydrolysis, diacylglycerol formation and protein kinase C activation by epidermal growth factor and transforming growth factor-α in normal human skin fibroblasts and keratinocytes". Biochemical Journal 294, № 2 (1993): 535–44. http://dx.doi.org/10.1042/bj2940535.

Full text

Abstract:

We have investigated coupling between the epidermal growth factor (EGF) receptor and the phospholipase C (PLC)/protein kinase C (PKC) signal-transduction system in normal skin fibroblasts and keratinocytes, for which EGF and transforming growth factor alpha (TGF-alpha) are mitogenic. EGF and TGF-alpha induced a rapid increase in tyrosine phosphorylation of the EGF receptor, in both fibroblasts and keratinocytes, but failed to induce tyrosine phosphorylation of PLC-gamma 1 or detectable phosphoinositide hydrolysis, as measured by two sensitive assays. In fibroblasts, EGF induced phosphatidylcholine (PC) hydrolysis, resulting in increased diacylglycerol (DAG). In contrast, in keratinocytes, there was no detectable PC hydrolysis or elevation of DAG in response to EGF or TGF-alpha. EGF and TGF-alpha activated PKC in fibroblasts, as evidenced by increased phosphorylation of a specific cellular PKC substrate (myristoylated alanine-rich C-kinase substrate, ‘MARCKS’). In keratinocytes, TGF-alpha and EGF induced only a modest increase in MARCKS protein phosphorylation. This apparent modest activation of PKC, in the absence of detectable DAG formation, may have been mediated by arachidonic acid, which was released from keratinocytes in response to TGF-alpha, and has been shown to stimulate PKC activity in vitro. These data demonstrate that (1) in dermal fibroblasts and keratinocytes, which express normal levels of EGF receptors, EGF receptor activation is not coupled to tyrosine phosphorylation of PLC-gamma 1 or PtdIns hydrolysis, suggesting that these events are not required for the mitogenic activity of EGF or TGF-alpha in these cells, (2) coupling of EGF receptor to PC hydrolysis is cell-type specific, and (3) in skin fibroblasts, DAG, formed through EGF-induced PC hydrolysis, is capable of activating PKC.

APA, Harvard, Vancouver, ISO, and other styles

50

Wieland, Catharina W., Britta Siegmund, Giorgio Senaldi, Michael L. Vasil, Charles A. Dinarello, and Giamila Fantuzzi. "Pulmonary Inflammation Induced by Pseudomonas aeruginosa Lipopolysaccharide, Phospholipase C, and Exotoxin A: Role of Interferon Regulatory Factor 1." Infection and Immunity 70, no. 3 (2002): 1352–58. http://dx.doi.org/10.1128/iai.70.3.1352-1358.2002.

Full text

Abstract:

ABSTRACT Chronic pulmonary infection with Pseudomonas aeruginosa is common in cystic fibrosis (CF) patients. P. aeruginosa lipopolysaccharide (LPS), phosholipase C (PLC), and exotoxin A (ETA) were evaluated for their ability to induce pulmonary inflammation in mice following intranasal inoculation. Both LPS and PLC induced high levels of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), IL-6, gamma interferon (IFN-γ), MIP-1α and MIP-2 in the lungs but did not affect IL-18 levels. ETA did not induce TNF-α and was a weak inducer of IL-1β, IL-6, macrophage inflammatory protein 1α (MIP-1α), and MIP-2. Remarkably, ETA reduced constitutive lung IL-18 levels. LPS was the only factor inducing IFN-γ. LPS, PLC, and ETA all induced cell infiltration in the lungs. The role of interferon regulatory factor-1 (IRF-1) in pulmonary inflammation induced by LPS, PLC, and ETA was evaluated. When inoculated with LPS, IRF-1 gene knockout (IRF-1 KO) mice produced lower levels of TNF-α, IL-1β, and IFN-γ than did wild-type (WT) mice. Similarly, a milder effect of ETA on IL-1β and IL-18 was observed for IRF-1 KO than for WT mice. In contrast, the cytokine response to PLC did not differ between WT and IRF-1 KO mice. Accordingly, LPS and ETA, but not PLC, induced expression of IRF-1 mRNA. IRF-1 deficiency had no effect on MIP-1α and MIP-2 levels and on cell infiltration induced by LPS, PLC, or ETA. Flow cytometric evaluation of lung mononuclear cells revealed strongly reduced percentages of CD8+ and NK cells in IRF-1 KO mice compared to percentages observed for WT mice. These data indicate that different virulence factors from P. aeruginosa induce pulmonary inflammation in vivo and that IRF-1 is involved in some of the cytokine responses to LPS and ETA.

APA, Harvard, Vancouver, ISO, and other styles

Journal articles on the topic 'Phospholipase C gamma 2 [PLC 2]'

Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles