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)

Activation of human platelets by cross-linking of the platelet low-affinity IgG receptor, the Fc gamma receptor IIA (Fc gamma-RIIA), or by collagen is associated with rapid phosphorylation on tyrosine of the non-receptor tyrosine kinase syk. Phosphorylation is still observed, albeit sometimes reduced, in the presence of a combination of a protein kinase C inhibitor, Ro 31-8220, and the intracellular calcium chelator, BAPTA-AM, demonstrating independence from phosphoinositide-specific phospholipase C (PLC) activity. In contrast, the combination of Ro 31-8220 and BAPTA-AM completely inhibits phosphorylation of syk in thrombin-stimulated platelets. Phosphorylation of syk increases its autophosphorylation activity measured in a kinase assay performed on syk immunoprecipitates. Fc gamma-RIIA also undergoes phosphorylation in syk immunoprecipitates from platelets activated by cross-linking of Fc gamma-RIIA but not by collagen, suggesting that it associates with the kinase. Consistent with this, tyrosine-phosphorylated Fc gamma-RIIA is precipitated by a glutathione S-transferase (GST) fusion protein containing the tandem src homology (SH2) domains of syk from Fc gamma-RIIA- but not collagen-activated cells. Two uncharacterized tyrosine-phosphorylated proteins of 40 and 65 kDa are uniquely precipitated by a GST fusion protein containing the tandem syk-SH2 domains in collagen-stimulated platelets. A peptide based on the antigen recognition activation motif (ARAM) of Fc gamma-RIIA, and phosphorylated on the two tyrosine residues found within this region, selectively binds syk from lysates of resting platelets; this interaction is not seen with a non-phosphorylated peptide. Kinase assays on Fc gamma-RIIA immunoprecipitates reveal the constitutive association of an unidentified kinase activity in resting cells which phosphorylates a 67 kDa protein. Syk is not detected in Fc gamma-RIIA immunoprecipitates from resting cells but associates with the receptor following activation and, together with Fc gamma-RIIA, is phosphorylated in the kinase assay in vitro. These results demonstrate that syk is activated by Fc gamma-RIIA cross-linking and collagen, independent of PLC, suggesting that it may have an important role in the early events associated with platelet activation. The association of syk with Fc gamma-RIIA appears to be mediated through the tandem SH2 domains in syk and the ARAM motif of Fc gamma-RIIA. A similar interaction may underlie the response to collagen, suggesting that its signalling receptor contains an ARAM motif.
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PMID:Syk interacts with tyrosine-phosphorylated proteins in human platelets activated by collagen and cross-linking of the Fc gamma-IIA receptor. 748 83

We have cloned a protein tyrosine kinase, MATK, which is expressed abundantly in megakaryocytes and the brain. We investigated whether MATK participates in the c-Kit ligand/stem cell factor (KL/SCF) signaling pathway in the megakaryocytic cell line CMK. After KL/SCF stimulation, five major proteins of molecular masses of 145, 113, 92, 76, and 63 kDa were rapidly and transiently tyrosine-phosphorylated in a time-dependent manner, peaking within 5 min, and returning to basal levels within 60 min. To study the role of MATK in the KL/SCF signaling pathway, glutathione S-transferase (GST) fusion proteins containing SH2 and SH3 domains of MATK were cloned, expressed in Escherichia coli, and purified. MATK-SH2, but not MATK-SH3, precipitated the tyrosine-phosphorylated c-Kit (molecular mass of 145 kDa) in KL/SCF-stimulated CMK cells. Other GST fusion proteins containing the SH2 domain of p85 of phosphatidylinositol 3-kinase, phospholipase C gamma-1, and ras-GAP also precipitated c-Kit. The tyrosine-phosphorylated c-Kit was co-immunoprecipitated with anti-MATK and anti-p85 antibodies in KL/SCF-stimulated CMK cells, but not in granulocyte-macrophage colony stimulating factor or interleukin-6-stimulated cells, suggesting receptor specificity. These results indicate that MATK associates with the c-Kit receptor following specific stimulation by KL/SCF via its SH2 domain and likely participates in transduction of growth signals induced by this cytokine in megakaryocytes.
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PMID:The MATK tyrosine kinase interacts in a specific and SH2-dependent manner with c-Kit. 753 44

We have isolated a novel member of the mammalian PAK (p21 activated kinase) and yeast Ste20 serine/threonine kinase family from a mouse fibroblast cDNA library, designated mPAK-3. Expression of mPAK-3 in Saccharomyces cerevisiae partially restores mating function in ste20 null cells. Like other PAKs, mPAK-3 contains a putative Cdc42Hs/Rac binding sequence and when transiently expressed in COS cells, full-length mPAK-3 binds activated (GTP gamma S (guanosine 5'-3-O-(thio-triphosphate)-bound) glutathione S-transferase (GST)-Cdc42Hs and GST-Rac1 but not GST-RhoA. As expected for a putative target molecule, mPAK-3 does not bind to an effector domain mutant of Cdc42Hs. Furthermore, activated His-tagged Cdc42Hs and His-tagged Rac stimulate mPAK-3 autophosphorylation and phosphorylation of myelin basic protein by mPAK-3 in vitro. Interestingly, the amino-terminal region of mPAK-3 contains potential SH3-binding sites and we find that mPAK-3, expressed in vitro and in vivo, shows highly specific binding to the SH3 domain of phospholipase C-gamma and at least one SH3 domain in the adapter protein Nck. These results raise the possibility of an additional level of regulation of the PAK family in vivo.
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PMID:Identification of a mouse p21Cdc42/Rac activated kinase. 755 98

cDNA clones encoding proteins related to the aggrecan/versican family of proteoglycan core proteins have been isolated with antisera against rat brain synaptic junctions. Two sets of overlapping cDNAs have been characterized that differ in their 3'-terminal regions. Northern analyses with probes derived from unique regions of each set were found to hybridize with two brain-specific transcripts of 3.3 and 3.6 kilobases (kb). The 3.6-kb transcript encodes a polypeptide that exhibits 82% sequence identity with bovine brevican and is thought to be the rat ortholog of brevican. Interestingly, the polypeptide deduced from the open reading frame of the 3.3-kb transcript is truncated just carboxyl-terminal of the central domain of brevican and instead contains a putative glypiation signal. Antibodies raised against a bacterially expressed glutathione S-transferase-brevican fusion protein have been used to show that both soluble and membrane-bound brevican isoforms exist. Treatment of the crude membrane fraction and purified synaptic plasma membranes with phosphatidylinositol-specific phospholipase C revealed that isoforms of brevican are indeed glycosylphosphatidylinositol-anchored to the plasma membrane. Moreover, digestions with chondroitinase ABC have indicated that rat brevican, like its bovine ortholog, is a conditional chondroitin sulfate proteoglycan. Immunohistochemical studies have shown that brevican is widely distributed in the brain and is localized extracellularly. During postnatal development, amounts of both soluble and phosphatidylinositol-specific phospholipase C-sensitive isoforms increase, suggesting a role for brevican in the terminally differentiating and the adult nervous system.
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PMID:Brevican, a chondroitin sulfate proteoglycan of rat brain, occurs as secreted and cell surface glycosylphosphatidylinositol-anchored isoforms. 759 78

Potential signaling substrates for the insulin-like growth factor I (IGF-I) receptor are SH2 domain proteins including the p85 subunit of phosphatidylinositol 3-kinase, the tyrosine phosphatase Syp, GTPase activating protein (GAP), and phospholipase C-gamma (PLC-gamma). In this study, we demonstrate an association between the IGF-I receptor and p85, Syp, and GAP, but not with PLC-gamma in lysates of cells overexpressing the human IGF-I receptor. We further investigated these interactions using glutathione S-transferase (GST) fusion proteins containing the amino-terminal SH2 domains of p85 or GAP, or both SH2 domains of Syp or PLC-gamma to precipitate the IGF-I receptor from purified receptor preparations and from whole cell lysates. p85-, Syp-, and GAP-GSTs precipitated the IGF-I receptor, whereas the PLC-gamma-GST did not. Using phosphopeptides corresponding to IGF-I receptor phosphorylation sites, we determined that the p85- and Syp-GST association with the IGF-I receptor could be inhibited by a carboxyl-terminal peptide containing pY1316 and that the GAP-GST association could be inhibited by a NPXY domain peptide. The GAP-GST binding site was confirmed by showing that a mutant IGF-I receptor with a deletion of the NPXY domain including tyrosine 950 was poorly precipitated by the GAP-GST. We conclude that p85 and Syp may bind directly to the IGF-I receptor at tyrosine 1316, and that GAP may bind to the IGF-I receptor at and PLC-gamma was not evident. p85, Syp, and GAP are potential modulators of IGF-I receptor signal transduction.
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PMID:Localization of the insulin-like growth factor I receptor binding sites for the SH2 domain proteins p85, Syp, and GTPase activating protein. 764 82

The SH2 domains of cytoplasmic signaling proteins bind to autophosphorylated growth factor receptors by direct recognition of specific phosphotyrosine-containing sites. To identify the phosphotyrosine involved in association of phospholipase C (PLC)-gamma 1 with the beta platelet-derived growth factor receptor (PDGFR), and to investigate which contiguous residues confer specificity for PLC-gamma 1, phosphotyrosine-containing glutathione S-transferase (GST) fusion proteins possessing different regions of the beta-PDGFR were incubated with lysates of Rat-2 cells that overexpress PLC-gamma 1. The phosphorylated C-terminal tail of the PDGFR bound PLC-gamma 1, but did not associate with phosphatidylinositol (PI) 3'-kinase or GTPase-activating protein (GAP). High-affinity binding of PLC-gamma 1 was dependent on phosphorylation of Tyr-1021. Creation of a new phosphorylation site by replacing Asp-1018 with tyrosine did not restore binding of PLC-gamma 1 in the absence of Tyr-1021, indicating that the location of the phosphorylated tyrosine is important for PLC-gamma 1 binding. Substitution of the proline at the +3 position relative to Tyr-1021 with methionine (Y1021IIP-->Y1021IIM) in the phosphorylated PDGFR tail did not alter PLC-gamma 1 association, but conferred binding activity towards PI 3'-kinase, indicating that this residue is critical in discriminating between PLC-gamma 1 and PI 3'-kinase. Progressive conversion of the three residues C-terminal to Tyr-1021 to the consensus for PI 3'-kinase binding (YMDM) allowed PI 3'-kinase association, but did not block PLC-gamma 1 binding, suggesting that additional residues other than the three residues immediately following the phosphotyrosine may contribute to the association of PLC-gamma 1 with the PDGFR. These results indicate that phosphorylation at Tyr-1021 in the tail of the PDGFR creates a specific binding site for PLC-gamma 1. Proline at the +3 position relative to Tyr-1021 is crucial in conferring specificity for binding to PLC-gamma 1.
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PMID:Identification of residues in the beta platelet-derived growth factor receptor that confer specificity for binding to phospholipase C-gamma 1. 768 24

A short, proline-rich region spanning residues 566-577 in human 5-lipoxygenase is a binding site for the Src homology 3 (SH3) domain of growth factor receptor-bound protein 2 (Grb2), an "adaptor" protein for tyrosine kinase-mediated cell signaling. Purified 5-lipoxygenase bound to glutathione S-transferase fusion products of Grb2 and a truncated version of Grb2 containing its SH3 domain. A peptide corresponding to the proline-rich, SH3-binding motif inhibited formation of the 5-lipoxygenase.Grb2 complex in vitro. The peptide also inhibited the redistribution of 5-lipoxygenase from the cytosol to the membrane in intact or permeabilized neutrophils activated by calcium ionophore A23187. 5-Lipoxygenase did not bind to the SH3 domains of other signaling proteins, such as GTPase-activating protein and phospholipase C gamma; however, it bound to certain cytoskeletal proteins including alpha-actinin and actin. 5-Lipoxygenase contains a consensus guanine nucleotide-binding site at residues 296-299, and guanine nucleotides inhibit 5-lipoxygenase activity in vitro. Our results suggest that 5-lipoxygenase may have a previously unrecognized role in tyrosine kinase signaling, distinct from its catalysis of lipid mediator formation. Our results also clarify the molecular basis for compartmentalization and translocation of 5-lipoxygenase in myeloid cells, implying that it binds to proteins other than its activating protein.
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PMID:5-Lipoxygenase contains a functional Src homology 3-binding motif that interacts with the Src homology 3 domain of Grb2 and cytoskeletal proteins. 792 73

It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-immobilized matrix was the delta 1 isoform of phosphatidylinositol-specific phospholipase C (PLC). We expressed rat PLC-delta 1 in Escherichia coli as a fusion protein with glutathione S-transferase, and found that the bacterial lysate shows a significant amount of Ins(1,4,5)P3 binding. The lysate was applied to Ins(1,4,5)P3-immobilized column chromatography and the eluate with 2 M NaCl solution containing only a 100-kDa protein showed high Ins(1,4,5)P3 binding. The lysate was also purified to near homogeneity using a glutathione-Sepharose 4B affinity system. Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as the brain-derived enzyme. PLC-delta 1 consists of the amino-terminal nonconserved region and two well-conserved regions among isozymes, designated as X and Y, which are thought to constitute a catalytic core of the enzyme. Using a combination of deletion mutants and proteolytic products of the enzyme, we were able to locate an Ins(1,4,5)P3 binding domain in the molecule. Deletion of 223 residues from the amino terminus completely abolished the binding activity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affect the binding. A 76-kDa proteolytic product of the expressed PLC-delta 1 which lacked 60 amino acids at the amino terminus showed a minimal Ins(1,4,5)P3 binding activity. A peptide consists of 14 amino acids corresponding to residues 30-43 of PLC-delta 1, which contains 6 basic amino acids, binds to an Ins(1,4,5)P3-immobilized matrix. Moreover, Ins(1,4,5)P3 binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indicate that the amino-terminal domain of PLC-delta 1 is important for the binding of both Ins(1,4,5)P3 and phosphatidylinositol 4,5-bisphosphate.
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PMID:Expression and characterization of an inositol 1,4,5-trisphosphate binding domain of phosphatidylinositol-specific phospholipase C-delta 1. 805 Nov 6

Tyrosine phosphorylation of multiple cellular proteins is a critical event in T cell receptor (TCR)-mediated activation. This pathway has also been implicated in cellular transformation in multiple systems. The viral oncogene v-cbl is the transforming gene of a murine retrovirus that induces pre-B cell lymphomas and myelogenous leukemias. The product of its cellular homolog, p120cbl, is a 120-kDa cytoplasmic protein that is non-transforming when overexpressed. Here we show that the 120-kDa protein tyrosine phosphorylated in Jurkat T cells upon TCR engagement is p120cbl. Following stimulation through the TCR, this tyrosine phosphorylation is rapid and reversible. Tyrosine-phosphorylated p120cbl binds to glutathione S-transferase fusion proteins generated from SH2 domains of the Fyn, Lck, and Blk protein tyrosine kinases, GTPase-activating protein and phospholipase C gamma. The p120cbl from unactivated and activated cells also binds to full-length glutathione S-transferase-Grb2 and the Grb2 N-terminal SH3 domain, but not to the Grb2 C-terminal SH3 domain. Additionally, p120cbl binds to SH3 domains of Fyn and Lck, but not Blk. These data expand our knowledge of protein tyrosine kinase signaling pathways in T cells by identifying a prominent tyrosine kinase substrate. This protein, the product of the cellular homolog of a transforming oncogene, can interact with several known signaling molecules.
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PMID:The protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor. 808 87

Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.
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PMID:Insulin-stimulated oocyte maturation requires insulin receptor substrate 1 and interaction with the SH2 domains of phosphatidylinositol 3-kinase. 841 61


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