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)

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.
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PMID:Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma. 861 Jan 51

A number of protein-tyrosine kinases have been shown to be important in T cell activation. One such kinase, Lck, has been demonstrated genetically to be essential for T cell receptor (TcR) signaling, and the SH2 and SH3 (src homology 2 and 3) domains of Lck have been shown to be indispensable for T cell activation. We have sought substrates with which the SH2,3 domain would interact following T cell activation, using fusion proteins containing the Lck SH2 and SH3 domains linked to glutathione S-transferase. We demonstrate that the SH2,3 region interacts specifically and directly with numerous tyrosine-phosphorylated molecules following TcR cross-linking, including constitutively with mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase and inducibly with the zeta chain of the TcR. The interaction with MAPK/extracellular-regulated kinase was via the SH3 domain. The interaction with the tyrosine-phosphorylated zeta chain, while phosphotyrosine-dependent, required both the SH3 and SH2 domains. These interactions were specific as molecules known to be tyrosine-phosphorylated following TcR cross-linking, phospholipase C-gamma1 and Fyn, were not bound. Thus, we suggest that during TcR signaling, Lck interacts with numerous molecules, including MAPK and TcR-zeta, via its SH2,3 domain. The interaction with MAPK would place Lck in a position to be involved in the complex resulting in the activation of MAPK. In addition, the binding of Lck to the tyrosine-phosphorylated zeta chain of the TcR would serve to strengthen the interaction of the associated CD4 and the TcR complex, leading to increased avidity for the antigen-major histocompatibility protein complex.
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PMID:Association between mitogen-activated protein kinase and the zeta chain of the T cell receptor (TcR) with the SH2,3 domain of p56lck. Differential regulation by TcR cross-linking. 862 61

We have investigated the roles of the phosphotyrosine phosphatase Syp (also called SH-PTP2), phospholipase C (PLC) gamma1, rasGTPase Activating Protein (rasGAP) and the adapter molecules Nck and Shc in the mitogenic response induced by PDGF in fibroblasts. Two separate approaches were used to inhibit the biological activity of these signalling proteins in vivo. Either glutathione S-transferase (GST) fusion proteins containing the SH2 domains of these proteins, or antibodies specific for these polypeptides, were microinjected into cells. GST-SH2 fusion proteins are expected to act as dominant inhibitors by competing for physiological SH2-mediated interactions, while microinjected antibodies can directly block protein functions. Inhibition of PLCgamma, Syp, Shc and Nck signals blocked PDGF-stimulated cells in G1 showing a requirement for these proteins for S-phase entry. Inhibition of rasGAP, in contrast, had no effect on S-phase entry. We next examined which of these signals were required for PDGF-induced cFos expression, a Ras-dependent event important for signalling. By using the same approaches with cells expressing beta-galactosidase under the control of a c-fos promoter, we showed that PLCgamma, Syp and Shc were necessary for ligand-induced cFos expression whereas Nck and phosphatidylinositol 3-kinase alpha were not. From these results we concluded that PDGF generates Ras-dependent and Ras-independent pathways important for DNA synthesis.
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PMID:Requirement of phospholipase C gamma, the tyrosine phosphatase Syp and the adaptor proteins Shc and Nck for PDGF-induced DNA synthesis: evidence for the existence of Ras-dependent and Ras-independent pathways. 889 Jan 67

Transfection of NMU (rat mammary adenocarcinoma) cells with NAD:arginine ADP-ribosyltransferase cDNAs from Yac-1 murine lymphoma cells or rabbit muscle increased NAD glycohydrolase and ADP-ribosyltransferase activities. The ADP-ribosyltransferase activity was released from transformed NMU cells by phosphatidylinositol-specific phospholipase C (PI-PLC) and hence glycosylphosphatidylinositol (GPI)-anchored, whereas the NAD glycohydrolase (NADase) activity remained cell-associated. By gel permeation chromatography, the size of the PI-PLC-released transferase was approximately 40 kDa and that of the detergent-solubilized NADase was approximately 100 kDa. Using polyclonal antibodies against rabbit muscle transferase on Western blots, approximately 18- and approximately 30-kDa band were visualized among proteins from the NADase fractions and 38-40-kDa bands with protein from the transferase fractions. Incubation of blots with [32P]NAD led to the incorporation of radioactivity into the immunoreactive transferase bands of 38 kDa and the immunoreactive NADase band of approximately 18 kDa. These data suggest that proteolysis of ADP-ribosyltransferase synthesized in transformed NMU cells might result in the formation of aggregates of an 18-kDa NAD glycohydrolase. A fusion protein with glutathione S-transferase linked to the amino terminus of Yac-1 transferase, from which the amino-terminal 121 amino acids had been deleted (GST-Yac-1-delta121), exhibited NADase, but not transferase, activity. The size of the recombinant fusion protein was similar to that of the proteolytic fragment seen in NMU cells transformed with transferase cDNA. These results are compatible with the conclusion that the NAD glycohydrolase activity was generated in NMU cells by proteolysis of ADP-ribosyltransferase, with release of a carboxyl-terminal fragment that possesses glycohydrolase but not transferase activity, i.e. the carboxyl-terminal portion of the transferase can exist as a catalytically active NADase.
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PMID:An 18-kDa domain of a glycosylphosphatidylinositol-linked NAD:arginine ADP-ribosyltransferase possesses NAD glycohydrolase activity. 908 12

The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.
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PMID:The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma. 923 20

We have previously demonstrated that the phospholipase C-coupled m3-muscarinic receptor is phosphorylated in an agonist-sensitive manner by a protein kinase of approximately 40 kDa purified from porcine cerebellum (Tobin, A. B., Keys, B., and Nahorski, S. R. (1996) J. Biol Chem. 271, 3907-3916). This kinase, called muscarinic receptor kinase (MRK), is distinct from second messenger-regulated protein kinases and from beta-adrenergic receptor kinase and other members of the G-protein-coupled receptor kinase family. In the present study we propose that MRK is casein kinase 1alpha (CK1alpha) based on the following evidence: 1) the amino acid sequence from two proteolytic peptide fragments derived from purified MRK corresponded exactly to sequences within CK1alpha. 2) Casein kinase activity co-eluted with MRK activity from the final two chromatography steps in the purification of porcine brain MRK. 3) Recombinant CK1alpha expressed in Sf9 cells is able to phosphorylate both casein and the bacterial fusion protein, Ex-m3, that contains a portion of the third intracellular loop of the m3-muscarinic receptor downstream of glutathione S-transferase. 4) Partially purified CK1alpha increased the level of muscarinic receptor phosphorylation in an agonist-sensitive manner when reconstituted with membranes from Chinese hamster ovary-m3 cells expressing the human recombinant m3-muscarinic receptor. 5) Partially-purified CK1alpha phosphorylated rhodopsin, contained in urea-treated bovine rod outer segment membranes, and the extent of phosphorylation was increased in the presence of light. These data demonstrate that the kinase previously called MRK is CK1alpha, and that CK1alpha offers an alternative protein kinase pathway from that of the G-protein-coupled receptor kinase family for the stimulus-dependent phosphorylation of the m3-muscarinic receptor, rhodopsin, and possibly other G-protein-coupled receptors.
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PMID:Stimulus-dependent phosphorylation of G-protein-coupled receptors by casein kinase 1alpha. 925 10

The kinetic mechanism of the trk receptor-linked tyrosine kinase was determined using a baculovirus expressed trk kinase domain and a bacterially expressed phospholipase C-gamma/glutathione S-transferase (PLC-gamma/ GST) fusion protein as substrate. Product and dead-end inhibition studies indicate an ordered association of substrates to trkA kinase with the nucleotide ATP binding prior to the exogenous substrate PLC-gamma/GST, followed by release of the phosphorylated PLC-gamma/GST product prior to release of ADP (sequential ordered bi-bi mechanism). This is in contrast to the reported kinetic mechanisms of closely related EGF receptor and insulin receptor kinases which appear to proceed via a rapid equilibrium random mechanism. The indolocarbazole K-252a, which was previously shown to be a potent and relatively selective inhibitor of trk kinase activity, acts as a competitive inhibitor with respect to ATP. The data suggest that potent and selective kinase inhibitors can be rationally designed by exploring subtle variations surrounding the nucleotide binding sites of receptor tyrosine kinases.
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PMID:Kinetics of trkA tyrosine kinase activity and inhibition by K-252a. 944 14

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by tau, a neuronal cell-specific microtubule-associated protein, in the presence of arachidonic acid. We now report that non-neuronal tissues also contain a protein that can activate PLC-gamma in the presence of arachidonic acid. Purification of this activator from bovine lung cytosol yielded several proteins with apparent molecular sizes of 70-130 kDa. They were identified as fragments derived from an unusually large protein (approximately 700 kDa) named AHNAK, which comprises about 30 repeated motifs each 128 amino acids in length. Two AHNAK fragments containing one and four of the repeated motifs, respectively, were expressed as glutathione S-transferase fusion proteins. Both recombinant proteins activated PLC-gamma1 at nanomolar concentrations in the presence of arachidonic acid, suggesting that an intact AHNAK molecule contains multiple sites for PLC-gamma activation. The role of arachidonic acid was to promote a physical interaction between AHNAK and PLC-gamma1, and the activation by AHNAK and arachidonic acid was mainly attributable to reduction in the enzyme's apparent Km toward the substrate phosphatidylinositol 4,5-bisphosphate. Our results suggest that arachidonic acid liberated by phospholipase A2 can act as an additional trigger for PLC-gamma activation, constituting an alternative mechanism that is independent of tyrosine phosphorylation.
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PMID:AHNAK, a protein that binds and activates phospholipase C-gamma1 in the presence of arachidonic acid. 1031 99

Platelet activation by collagen is mediated by the sequential tyrosine phosphorylation of the Fc receptor gamma-chain (FcR gamma-chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phospholipase C-gamma2 (PLC-gamma2). In this study tyrosine-phosphorylated proteins that associate with PLC-gamma2 after stimulation by a collagen-related peptide (CRP) were characterized using glutathione S-transferase fusion proteins of PLC-gamma2 Src homology (SH) domains and by immunoprecipitation of endogenous PLC-gamma2. The majority of the tyrosine-phosphorylated proteins that associate with PLC-gamma2 bind to its C-terminal SH2 domain. These were found to include PLC-gamma2, Syk, SH2-domain-containing leucocyte protein of 76 kDa (SLP-76), Lyn, linker for activation of T cells (LAT) and the FcR gamma-chain. Direct association was detected between PLC-gamma2 and SLP-76, and between PLC-gamma2 and LAT upon CRP stimulation of platelets by far-Western blotting. FcR gamma-chain and Lyn were found to co-immunoprecipitate with PLC-gamma2 as well as with unidentified 110-kDa and 75-kDa phosphoproteins. The absence of an in vivo association between Syk and PLC-gamma2 in platelets is in contrast with that for PLC-gamma1 and Syk in B cells. The in vivo function of PLC-gamma2 SH2 domains was examined through measurement of Ca2+ increases in mouse megakaryocytes that had been microinjected with recombinant proteins. This revealed that the C-terminal SH2 domain is involved in the regulation of PLC-gamma2. These data indicate that the C-terminal SH2 domain of PLC-gamma2 is important for PLC-gamma2 regulation through possible interactions with SLP-76, Syk, Lyn, LAT and the FcR gamma-chain.
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PMID:Evidence that phospholipase C-gamma2 interacts with SLP-76, Syk, Lyn, LAT and the Fc receptor gamma-chain after stimulation of the collagen receptor glycoprotein VI in human platelets. 1046 24

Endothelin-1 (ET-1) can stimulate insulin-responsive glucose transporter (GLUT4) translocation in 3T3-L1 adipocytes (Wu-Wong, J. R., Berg, C. E., Wang, J., Chiou, W. J., and Fissel, B. (1999) J. Biol. Chem. 274, 8103-8110), and in the current study, we have evaluated the signaling pathway leading to this response. First, we inhibited endogenous Galpha(q/11) function by single-cell microinjection using anti-Galpha(q/11) antibody or RGS2 protein (a GTPase activating protein for Galpha(q)) followed by immunostaining to quantitate GLUT4 translocation in 3T3-L1 adipocytes. ET-1-stimulated GLUT4 translocation was markedly decreased by 70 or 75% by microinjection of Galpha(q/11) antibody or RGS2 protein, respectively. Pretreatment of cells with the Galpha(i) inhibitor (pertussis toxin) or microinjection of a Gbetagamma inhibitor (glutathione S-transferase-beta-adrenergic receptor kinase (GST-BARK)) did not inhibit ET-1-induced GLUT4 translocation, indicating that Galpha(q/11 )mediates ET-1 signaling to GLUT4 translocation. Next, we found that ET-1-induced GLUT4 translocation was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitors wortmannin or LY294002, but not by the phospholipase C inhibitor U-73122. ET-1 stimulated the PI 3-kinase activity of the p110alpha subunit (5.5-fold), and microinjection of anti-p110alpha or PKC-lambda antibodies inhibited ET-stimulated GLUT4 translocation. Finally, we found that Galpha(q/11) formed immunocomplexes with the type-A endothelin receptor and the 110alpha subunit of PI 3-kinase and that ET-1 stimulation enhances tyrosine phosphorylation of Galpha(q/11). These results indicate that: 1) ET-1 signaling to GLUT4 translocation is dependent upon Galpha(q/11) and PI 3-kinase; and 2) Galpha(q/11) can transmit signals from the ET(A) receptor to the p110alpha subunit of PI 3-kinase, as does insulin, subsequently leading to GLUT4 translocation.
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PMID:Endothelin-1-induced GLUT4 translocation is mediated via Galpha(q/11) protein and phosphatidylinositol 3-kinase in 3T3-L1 adipocytes. 1055 59


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