Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two novel sites of autophosphorylation were localized to the C-terminal tail of the PDGF beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) compared to the wild type PDGF beta-receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC-gamma could be detected. These data show that tyrosine phosphorylation of PLC-gamma is dependent on autophosphorylation of the PDGF beta-receptor at Tyr1009 and Tyr1021.
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PMID:Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma. 139 85

We have purified to homogeneity the 33-kDa phosphatidylinositol-specific phospholipase C (PI-PLC) from the culture fluid of Listeria monocytogenes, a facultative intracellular pathogen. The protein was overexpressed, and secretion of PI-PLC was further enhanced by the addition of divalent cations to the culture medium. The basic protein (pI, approximately 9.4) was complexed with anionic proteins in the crude culture fluid. It bound to DEAE-Sepharose and was eluted from Sephacryl S-200 near the void volume in low-ionic-strength buffer, suggesting aggregates of greater than or equal to 150 kDa. Gel filtration chromatography on Sephacryl S-200 in the presence of 1 M ammonium sulfate resulted in disaggregation and complete separation of PI-PLC, which interacted with the column matrix. Amino-terminal sequencing of the pure protein gave results consistent with the previously deduced sequence and showed that the signal cleavage site was between alanine 29 and tyrosine 30. The enzyme was specific for PI and showed no activity with phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It did not cleave PI-4-phosphate or PI-4,5-bisphosphate, but it was active on the membrane form of the variable surface glycoprotein from Trypanosoma brucei, a PI-glycan-anchored protein. When assayed with deoxycholate-mixed micelles of PI, activity was highly dependent on added salt. Activation by salt was also observed with Triton X-100-mixed micelles. The optimal concentration of CaCl2 or MgCl2 was lower than that of KCl or (NH4)2SO4, but activity was not specifically dependent on divalent cations and was not inhibited by addition of EDTA. With deoxycholate, the optimum pH was 7.0. A broader pH optimum ranging from 5.5 to 6.5 was observed with Triton X-100-mixed micelles. These results are consistent with a postulated role for secreted PI-PLC in the acidified primary phagocytic vesicle of infected cells.
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PMID:Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C. 139 18

The cellular actions of nerve growth factor (NGF) involve changes in protein phosphorylation, initiated by the binding and subsequent activation of its tyrosine kinase receptor, the trk protooncogene (pp140c-trk). Upon exposure to NGF, a 38-kDa tyrosine-phosphorylated protein (pp38) is identified in both PC-12 pheochromocytoma cells and NIH3T3 cells transfected with the full-length human pp140c-trk cDNA (3T3-c-trk) that is specifically coimmunoprecipitated with pp140c-trk or phosphatidylinositol-phospholipase C (PLC)-gamma 1. In both PC-12 and 3T3-c-trk cells, NGF rapidly stimulates the association of pp140c-trk and pp38 with a fusion protein containing the src homology (SH) domains of PLC gamma 1. This phosphorylation and subsequent association are specific for NGF, since epidermal growth factor, platelet-derived growth factor, and insulin do not stimulate the tyrosine phosphorylation of these proteins or their association with the PLC gamma 1 SH domains, although the receptors for these growth factors do undergo tyrosine phosphorylation and association with the PLC-gamma 1 fusion protein under these conditions. Furthermore, the NGF-dependent pp38-SH binding is specific for the SH2 domains of PLC-gamma 1, since the phosphoprotein does not bind to fusion proteins containing SH domains of ras GTPase-activating protein or the p85 subunit of phosphatidylinositol 3 kinase. Both amino- and carboxyl-terminal SH2 domains of PLC-gamma 1 are necessary for the association of pp38 with PLC-gamma 1, although each SH2 domain is sufficient for the association of pp140c-trk with PLC-gamma 1. In both PC-12 and 3T3-c-trk cells, the phosphorylation and association of pp38 with PLC gamma 1 is rapid, occurring maximally at 1 min and declining thereafter. Moreover, this effect of NGF is dose-dependent over a physiological concentration of the growth factor. The specificity and rapidity of pp38 phosphorylation and its association with PLC-gamma 1 suggest that it may be an important component in signal transduction for NGF.
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PMID:Nerve growth factor stimulates the tyrosine phosphorylation of a 38-kDa protein that specifically associates with the src homology domain of phospholipase C-gamma 1. 140 Apr 71

B lymphocytes from patients expressing the X chromosome-linked immune deficiency disorder, Wiskott-Aldrich syndrome (WAS), fail to produce antibodies in response to stimulation with polysaccharides and other type-2 T cell-independent antigens. To investigate whether this abnormality reflects a defect in the signal transduction cascade normally triggered by ligation of surface immunoglobulin (sIg) on B cells, we have examined early signaling events induced by anti-Ig antibody stimulation of EBV B lymphoblastoid cell lines from WAS patients and healthy controls. Despite the expression of comparable levels of sIg and sIgM on WAS and control EBV B cells, WAS cells failed to manifest the increased proliferation in response to anti-Ig treatment observed in the control cell lines. WAS and control EBV B cells also differed in the magnitude of the change in cytosolic free calcium ([Ca2+]i) induced by sIg ligation; WAS cells showed either markedly diminished or no changes in [Ca2+]i levels whereas control EBV B cells consistently showed increases in [Ca2+]i. Anti-Ig-induced changes in inositol phosphate release were also markedly reduced in WAS compared with control cells. As protein tyrosine phosphorylation is thought to represent a proximal event in the activation of B cells, inducing increases in [Ca2+]i by virtue of tyrosine phosphorylation of phospholipase C (PLC)-gamma, profiles of protein tyrosine phosphorylation and expression of tyrosine-phosphorylated PLC-gamma 1 were compared between WAS and normal EBV B cells before and after sIg cross-linking. These studies revealed that in addition to defective mobilization of Ca2+, the WAS cells manifested little or no increase in tyrosine phosphorylation of PLC-gamma 1 or other intracellular proteins after sIg ligation. Together these results indicate the association of WAS with a defect in the coupling of sIg to signal transduction pathways considered prerequisite for B cell activation, likely at the level of tyrosine phosphorylation. The abnormalities observed in these early transmembrane signaling events in WAS EBV B cells may play a role not only in the nonresponsiveness of WAS patient B cells to certain T independent antigens, but also in the genesis of some of the other cellular deficits exhibited by these patients.
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PMID:Evidence for defective transmembrane signaling in B cells from patients with Wiskott-Aldrich syndrome. 140 Oct 74

Chimeric receptors composed of the human epidermal growth factor receptor (EGF-R) extracellular domain fused to wild-type and truncated platelet-derived growth factor receptor (PDGF-R) intracellular sequences were stably expressed in NIH 3T3 cells devoid of endogenous EGF-Rs. This experimental system allowed us to investigate the biological activity of PDGF-R cytoplasmic-domain mutants in PDGF-R-responsive NIH 3T3 cells by activating PDGF-specific signaling pathways with EGF. Deletion of 74 carboxy-terminal amino acids severely impaired the ability of the PDGF-R cytoplasmic domain to associate with cellular substrates in vitro. This deletion also inhibited receptor and substrate phosphorylation, reduced the receptor's mitogenic activity, and completely abolished its oncogenic signaling potential. Surprisingly, removal of only six additional amino acids, including Tyr-989, restored substantial receptor and substrate phosphorylation capacity as well as transforming potential and yielded a receptor with wild-type levels of ligand-induced mitogenic activity. However, the ability of this chimera to bind phospholipase C gamma was severely impaired in comparison with the ability of the wild-type receptor, while the association with other cellular proteins was not affected. Further deletion of 35 residues, including Tyr-977, nearly abolished all PDGF-R cytoplasmic-domain biological signaling activities. None of the three C-terminal truncations completely abolished the mitogenic potential of the receptors or had any influence on ligand binding or receptor down regulation. Together, these data implicate the 80 C-terminal-most residues of the PDGF-R, and possibly Tyr-989, in phospholipase C gamma binding, while receptor sequences upstream from Asp-988 appear to be essential for specific interactions with other cellular polypeptides such as ras GTPase-activating protein and phosphatidylinositol 3-kinase. Thus, the mutants described here allow the separation of distinct PDGF-activated signaling pathways and demonstrate that phospholipase C gamma phosphorylation is not required for mitogenesis and transformation.
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PMID:Differential effects of carboxy-terminal sequence deletions on platelet-derived growth factor receptor signaling activities and interactions with cellular substrates. 140 26

The amino terminus of nerve growth factor (NGF) is susceptible to proteolytic cleavage. A comparison of the bioactivity of highly purified full-length recombinant human (1-118)rhNGF and NH2-terminal truncated (10-118)rhNGF revealed lower potency of (10-118)rhNGF with regard to early NGF responses in neuron-like PC12 cells. Approximately 50 times higher concentrations of (10-118)rhNGF than (1-118)rhNGF were required to elicit the same extent of tyrosine phosphorylation of key enzymes in different second messenger pathways, i.e. the NGF receptor tyrosine kinase p140trkA, phospholipase C gamma-1, and the extracellular signal-regulated kinase ERK1. A similar reduced potency for induction of the transcription factor c-Fos was observed with (10-118)rhNGF compared to (1-118)rhNGF. The lower potency of (10-118)rhNGF in triggering early responses correlated with its 40-fold lower affinity for PC12 cells. Whereas (10-118)rhNGF had a more than 300-fold lower affinity for the high affinity receptor p140trkA than (1-118)rhNGF, amino-terminal truncation of NGF changed its affinity for the low affinity receptor p75NGFR only slightly (5-10-fold). These observations suggest that amino acids 1-9 of NGF are important for binding to the signal transducing receptor p140trkA. Proteolytic cleavage of the NGF amino terminus, therefore, reduces its potency in starting several second messenger pathways leading to neuronal differentiation of PC12 cells.
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PMID:The amino terminus of nerve growth factor is involved in the interaction with the receptor tyrosine kinase p140trkA. 142 22

Epidermal growth factor (EGF) can stimulate inositol lipid hydrolysis in rat hepatocytes and can accelerate GTP/GDP exchange in hepatic membranes. Both of these responses can be abolished by pretreatment with pertussis toxin, suggesting that EGF may regulate phospholipase C (PLC) activity via a guanine nucleotide-binding regulatory protein (G protein) in liver cells. In contrast, in A431 human epidermoid carcinoma cells EGF can induce a rapid phosphorylation of PLC-gamma on tyrosine residues that increases the activity of immunoprecipitated PLC-gamma, suggesting that tyrosine phosphorylation of PLC-gamma may be the mechanism for EGF-stimulated inositol trisphosphate production in these cells. To determine the importance of the phosphorylation of PLC-gamma on tyrosine residues in a system where the EGF receptor apparently couples to a G protein, the effect of EGF on tyrosine phosphorylation of PLC-gamma was examined in rat hepatocytes. PLC-gamma was immunoprecipitated from cell lysates with a PLC-gamma antiserum and its tyrosine phosphorylation state was determined using both Western blot analysis with phosphotyrosine antibodies and direct measurement of phosphorylated amino acids. The results were compared with analogous experiments performed with A431 cells and another cultured cell line expressing high levels of human EGF receptors, Rat1hER fibroblasts. Although the amount of PLC-gamma in rat hepatocytes is similar to that in A431 cells and slightly higher than that in Rat1hER cells, EGF causes a barely detectable increase in the phosphorylation of PLC-gamma on tyrosine in hepatocytes, whereas it stimulates a significant degree of phosphorylation of PLC-gamma on tyrosine in Rat1hER or A431 cells. Pretreatment of hepatocytes with pertussis toxin abolishes the ability of EGF to activate PLC, as determined by an increase in intracellular Ca2+, but has no effect on the small amount of phosphate incorporated into tyrosine residues on the PLC-gamma protein, demonstrating that this low level of PLC-gamma phosphorylation does not correlate with changes in PLC activity. The data suggest that phosphorylation of PLC-gamma on tyrosine is not important for EGF-enhanced PLC activity in hepatocytes. This conclusion implies that EGF may use a mechanism to regulate PLC activity in hepatocytes that is different from that used in cultured cells expressing high levels of EGF receptors.
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PMID:Epidermal growth factor activates phospholipase C in rat hepatocytes via a different mechanism from that in A431 or rat1hER cells. 143 49

In the course of our investigation of phospholipase C (PLC)-gamma 1 phosphorylation by using a set of anti-PLC-gamma 1 monoclonal antibodies (P.-G. Suh, S. H. Ryu, W. C. Choi, K.-Y. Lee, and S. G. Rhee, J. Biol. Chem. 263:14497-14504, 1988), we found that some of these antibodies directly recognize a 47-kDa protein. We show here that this 47-kDa protein is identical to the SH2/SH3-containing protein Nck (J. M. Lehmann, G. Riethmuller, and J. P. Johnson, Nucleic Acids Res. 18:1048, 1990). Nck was found to be constitutively phosphorylated on serine in resting NIH 3T3 cells. Platelet-derived growth factor (PDGF) treatment led to increased Nck phosphorylation on both tyrosine and serine. Nck was also found to be phosphorylated on tyrosine in epidermal growth factor (EGF)-treated A431 cells and in v-Src-transformed NIH 3T3 cells. Multiple sites of serine phosphorylation were detected in Nck from resting cells, and no novel sites were found upon PDGF or EGF treatment. A single major tyrosine phosphorylation site was found in Nck in both PDGF- and EGF-treated cells and in v-Src-transformed cells. This same tyrosine was phosphorylated in vitro by purified PDGF and EGF receptors and also by pp60c-src. We compared the phosphorylation of Nck and PLC-gamma 1 in several cell lines transformed by oncogenes with different modes of transformation. Although PLC-gamma 1 and Nck have significant amino acid identity, particularly in their SH3 regions, and both associate with growth factor receptors in a ligand-dependent manner, they were not always phosphorylated on tyrosine in a coincident manner.
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PMID:The SH2/SH3 domain-containing protein Nck is recognized by certain anti-phospholipase C-gamma 1 monoclonal antibodies, and its phosphorylation on tyrosine is stimulated by platelet-derived growth factor and epidermal growth factor treatment. 144 8

Several lines of evidence implicate a regulatory tyrosine phosphorylation in the activation of phospholipase C (PLC) by the T cell antigen receptor (TCR). These include studies using inhibitors of protein tyrosine kinases (PTKs). In Jurkat T cells expressing the heterologous human muscarinic receptor (HM1), PLC activity can be induced by either the TCR or HM1. HM1 activates PLC via a guanine nucleotide binding protein. We have studied the selectivity of the effects of the PTK inhibitors, herbimycin A and genistein, in this system. The results indicate that these inhibitors have different mechanisms of action, and suggest that herbimycin A, but not genistein, is a specific inhibitor of PTKs in T cells. Herbimycin A markedly inhibited both the resting and induced levels of phosphotyrosine-containing proteins, including the gamma 1 isozyme of PLC and the zeta chain of the TCR, and prevented activation of PLC by anti-TCR mAb. Herbimycin A did not inhibit activation of PLC by HM1. Genistein had a much less pronounced effect than herbimycin A on the appearance of tyrosine phosphoproteins. Moreover, genistein inhibited activation of PLC by both the TCR and HM1, and inhibition was only partial. Genistein was cytotoxic and markedly inhibited protein synthesis in both Jurkat cells and human peripheral lymphocytes. Herbimycin A was not cytotoxic. These findings confirm the role of a regulatory tyrosine phosphorylation in activation of PLC by the TCR. Herbimycin A was a selective inhibitor of a subclass of PTKs in Jurkat cells. In contrast, inhibition of signal transduction and later events in T cells by genistein may be due to effects other than direct inhibition of PTK activity.
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PMID:The protein tyrosine kinase inhibitor herbimycin A, but not genistein, specifically inhibits signal transduction by the T cell antigen receptor. 147 73

1. Activation of neutrophils results in increased tyrosine phosphorylation of several proteins that may have important roles in receptor/effector coupling. In this study, the effect of a protein tyrosine kinase inhibitor on receptor-mediated neutrophil activation by platelet-activating factor (PAF), leukotriene, B4 (LTB4) and N-formylmethionylleucylphenylalanine (FMLP) is investigated. 2. alpha-Cyano-3,4-dihydroxythiocinnamamide dose-dependently inhibited intracellular calcium release and superoxide generation from human neutrophils activated by 1 microM LTB4, PAF, and FMLP. 3. In the presence of cytochalasin B, FMLP stimulated elastase release from neutrophils was also inhibited to unstimulated levels by 5 min pretreatment with alpha-cyano-3,4-dihydroxythiocinnamamide. 4. The inhibitory action of alpha-cyano-3,4-dihydroxythiocinnamamide was found to be at or upstream of phospholipase C activation, blocking both phosphatidylinositol hydrolysis and protein kinase C activation. alpha-Cyano-3,4-dihydroxythiocinnamamide did not affect agonist receptor binding sites or receptor affinity in neutrophils. 5. Immunoblot analysis demonstrated the tyrosine phosphorylation of proteins of 41, 56, 66, and 104 kDa in neutrophils treated with agonists. Treatment of neutrophils with alpha-cyano-3,4-dihydroxythiocinnamamide prior to stimulation with chemoattractants reduced tyrosine phosphorylation of the above phosphoproteins. 6. These results indicate that alpha-cyano-3,4-dihydroxythiocinnamamide might be a useful agent in characterizing the essential proteins and biochemical pathways that regulate neutrophil activation.
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PMID:Inhibition of human neutrophil responses by alpha-cyano-3,4-dihydroxythiocinnamamide; a protein-tyrosine kinase inhibitor. 150 49


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