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:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase Cgamma, phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-Bbeta, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF. TrkA bound to glutathione S-transferase fusion proteins containing SH2-Bbeta, and NGF stimulation dramatically increased that binding. In contrast, NGF was unable to stimulate the association of TrkA with a glutathione S-transferase fusion protein containing a mutant SH2-Bbeta(R555E) with a defective SH2 domain. When overexpressed in PC12 cells, SH2-Bbeta co-immunoprecipitated with TrkA in response to NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-Bbeta as well as exogenously expressed GFP-SH2-Bbeta but not GFP-SH2-Bbeta(R555E). Overexpression of SH2-Bbeta(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-Bbeta enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or mutant SH2-Bbeta(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholipase Cgamma in response to NGF or NGF-induced activation of ERK1/2, suggesting that SH2-Bbeta may initiate a previously unknown pathway(s) that is essential for NGF-induced neurite outgrowth. Taken together, these data indicate that SH2-Bbeta is a novel signaling molecule required for NGF-induced neuronal differentiation.
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PMID:SH2-B is required for nerve growth factor-induced neuronal differentiation. 1018 54

It has been proposed that phosphoinositides and inositol phosphates serve as general ligands for members of the structurally related pleckstrin homology (PH) domain family. The N-terminal PH domain of pleckstrin (N-PH), in contrast with other PH domains, does not bind to any of these ligands with the high affinity expected for a physiological interaction. To examine whether N-PH might instead mediate protein-protein interaction, a fusion protein with glutathione S-transferase (GST) expressing N-PH (GST-N-PH) was used to screen [(35)S]methionine metabolically labelled HL-60 and Bac1. 2F5 cell lysates for potential binding partners. A 30 kDa binding protein was identified in both cell lines. Binding to N-PH demonstrated specificity, because binding was approx. 10-fold higher than when an equimolar amount of pleckstrin C-terminal PH domain (GST-C-PH) was used as probe. The 30 kDa protein could also be metabolically labelled with [(32)P]P(i) and proved to be a tyrosine-phosphorylated protein. Binding to N-PH could be specifically inhibited with phosphotyrosine but not with phosphothreonine; the inhibition was concentration-dependent. Site-directed mutagenesis indicated that a positively charged region previously identified as the phosphoinositide-binding site in N-PH and other PH domains, rather than a putative phosphotyrosine-binding region previously identified in structurally similar phosphotyrosine-binding (PTB) domains, served as the binding site. These results suggest that the positively charged region of N-PH has the potential to interact with a protein ligand that contains phosphotyrosine.
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PMID:Phosphotyrosine protein of molecular mass 30 kDa binds specifically to the positively charged region of the pleckstrin N-terminal pleckstrin homology domain. 1045 30

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, X-chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCmu, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B-cells with phorbol ester or H(2)O(2) affected Btk/PKCmu interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCmu. Transient overexpression of PKCmu deletion mutants as well as expression of selected PKCmu domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCmu/Btk complex in vivo and identify two PKCmu domains that participate in Btk interaction.
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PMID:Bruton's tyrosine kinase (Btk) associates with protein kinase C mu. 1056 98

We cloned human and murine cDNAs of a gene (designated PHR1), expressed preferentially in retina and brain. In both species, PHR1 utilizes two promoters and alternative splicing to produce four PHR1 transcripts, encoding isoforms of 243, 224, 208, and 189 amino acids, each with a pleckstrin homology domain at their N terminus and a transmembrane domain at their C terminus. Transcript 1 originates from a 5'-photoreceptor-specific promoter with at least three Crx elements ((C/T)TAATCC). Transcript 2 originates from the same promoter but lacks exon 7, which encodes 35 amino acids immediately C-terminal to the pleckstrin homology domain. Transcripts 3 and 4 originate from an internal promoter in intron 2 and either include or lack exon 7, respectively. In situ hybridization shows that PHR1 is highly expressed in photoreceptors, with lower expression in retinal ganglion cells. Immunohistochemistry localizes the PHR1 protein to photoreceptor outer segments where chemical extraction studies confirm it is an integral membrane protein. Using a series of PHR1 glutathione S-transferase fusion proteins to perform in vitro binding assays, we found PHR1 binds transducin betagamma subunits but not inositol phosphates. This activity and subcellular location suggests that PHR1 may function as a previously unrecognized modulator of the phototransduction pathway.
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PMID:PHR1 encodes an abundant, pleckstrin homology domain-containing integral membrane protein in the photoreceptor outer segments. 1058 47

The proinflammatory mediator leukotriene D(4) (LTD(4)) binds to the seven-transmembrane receptor CYSLT(1). Although this leukotriene plays an important biological role, its intracellular signaling pathways are only partly known. In previous experiments, we found that LTD(4) induced tyrosine phosphorylation and translocation of phospholipase (PLC)-gamma1 to a plasma membrane fraction in a human epithelial cell line (Int 407). In the present study, we further examined these signaling events and found that LTD(4) induced a rapid interaction between Gbetagamma subunits and PLC-gamma1; results obtained with GST fusion proteins of PLC-gamma1 suggest that this interaction is mediated via the pleckstrin homology domain of PLC-gamma1. Moreover, LTD(4) induced an increased association of c-Src with PLC-gamma1, and the selective Src family tyrosine kinase inhibitor PP1 blocked both LTD(4)-induced tyrosine phosphorylation of PLC-gamma1 and the association of PLC-gamma1 with Gbetagamma subunits. The relevance of these observations in intracellular calcium signaling was investigated by microinjecting cells with anti-Gbeta, anti-PLC-gamma1, or anti-c-Src antibodies and by pretreatment with PP1. LTD(4)-induced calcium mobilization was blocked by each of the indicated antibodies (but not isotype-matched control antibodies) and by PP1. Our data suggest that Gbetagamma subunits can, directly or indirectly, serve as membrane-bound partners for PLC-gamma1 and c-Src and that each of these proteins is essential for LTD(4)-induced downstream PLC-gamma1 signaling.
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PMID:Leukotriene D(4) triggers an association between gbetagamma subunits and phospholipase C-gamma1 in intestinal epithelial cells. 1073 40

SH2-Bbeta has been shown to bind via its SH2 (Src homology 2) domain to tyrosyl-phosphorylated JAK2 and strongly activate JAK2. In this study, we demonstrate the existence of an additional binding site(s) for JAK2 within the N-terminal region of SH2-Bbeta (amino acids 1 to 555) and the ability of this region of SH2-B to inhibit JAK2. Four lines of evidence support the existence of this additional binding site(s). In a glutathione S-transferase pull-down assay, wild-type SH2-Bbeta and SH2-Bbeta(R555E) with a defective SH2 domain bind to both tyrosyl-phosphorylated JAK2 from growth hormone (GH)-treated cells and non-tyrosyl-phosphorylated JAK2 from control cells, whereas the SH2 domain of SH2-Bbeta binds only to tyrosyl-phosphorylated JAK2 from GH-treated cells. Similarly, JAK2 is present in alphaSH2-B immunoprecipitates in the absence and presence of GH, with GH substantially increasing the coprecipitation of JAK2 with SH2-B. When coexpressed in COS cells, SH2-Bbeta coimmunoprecipitates not only wild-type, tyrosyl-phosphorylated JAK2 but also kinase-inactive, non-tyrosyl-phosphorylated JAK2(K882E), although to a lesser extent. DeltaC555 (amino acids 1 to 555 of SH2-Bbeta) that lacks most of the SH2 domain binds similarly to wild-type JAK2 and kinase-inactive JAK2(K882E). Experiments using a series of N- and C-terminally truncated SH2-Bbeta constructs indicate that the pleckstrin homology (PH) domain (amino acids 269 to 410) and amino acids 410 to 555 are necessary for maximal binding of SH2-Bbeta to inactive JAK2, but neither region alone is sufficient for maximal binding. The SH2 domain of SH2-Bbeta is necessary and sufficient for the stimulatory effect of SH2-Bbeta on JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B. In contrast, DeltaC555 lacking the SH2 domain, and to a lesser extent the PH domain alone, inhibits JAK2. DeltaC555 also blocks JAK2-mediated tyrosyl phosphorylation of Stat5B in COS cells and GH-stimulated nuclear accumulation of Stat5B in 3T3-F442A cells. These data indicate that in addition to the SH2 domain, SH2-Bbeta has one or more lower-affinity binding sites for JAK2 within amino acids 269 to 555. The interaction via this site(s) in SH2-B with inactive JAK2 seems likely to increase the local concentration of SH2-Bbeta around JAK2, thereby facilitating binding of the SH2 domain to ligand-activated JAK2. This would result in a more rapid and robust cellular response to hormones and cytokines that activate JAK2. This interaction between inactive JAK2 and SH2-B may also help prevent abnormal activation of JAK2.
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PMID:Differential binding to and regulation of JAK2 by the SH2 domain and N-terminal region of SH2-bbeta. 1075 1

Etk/Bmx, a member of the Tec family of nonreceptor protein-tyrosine kinases, is characterized by an N-terminal pleckstrin homology domain and has been shown to be a downstream effector of phosphatidylinositol 3-kinase. P21-activated kinase 1 (Pak1), another well characterized effector of phosphatidylinositol 3-kinase, has been implicated in the progression of breast cancer cells. In this study, we characterized the role of Etk in mammary development and tumorigenesis and explored the functional interactions between Etk and Pak1. We report that Etk expression is developmentally regulated in the mammary gland. Using transient transfection, coimmunoprecipitation and glutathione S-transferase-pull down assays, we showed that Etk directly associates with Pak1 via its N-terminal pleckstrin homology domain and also phosphorylates Pak1 on tyrosine residues. The expression of wild-type Etk in a non-invasive human breast cancer MCF-7 cells significantly increased proliferation and anchorage-independent growth of epithelial cancer cells. Conversely, expression of kinase-inactive mutant Etk-KQ suppressed the proliferation, anchorage-independent growth, and tumorigenicity of human breast cancer MDA-MB435 cells. These results indicate that Pak1 is a target of Etk and that Etk controls the proliferation as well as the anchorage-independent and tumorigenic growth of mammary epithelial cancer cells.
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PMID:Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicity of breast cancer cells. 1138 70

Rkp1/Cpc2, a fission yeast RACK1 homolog, interacts with Pck2, a PKC homolog, and is involved in the regulation of pck2-mediated signaling process. The N-terminal region of split pleckstrin homology domain (nPH) in human PLC-gamma1 bound to Rkp1/Cpc2 concomitantly with Pck2. nPH inhibited kinase activity of GST-Pck2 purified from Schizosaccharomyces pombe in vitro. The lethality induced by pck2(+) overexpression was suppressed by coexpression of either rkp1(+) or nPH domain. This result suggests that Rkp1/Cpc2 interacts with PH domain-containing protein and regulates the Pck2-mediated signaling process in S. pombe.
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PMID:Pleckstrin homology domain interacts with Rkp1/Cpc2, a RACK1 homolog, to modulate Pck2-mediated signaling process in Schizosaccharomyces pombe. 1174 Dec 88

The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.
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PMID:Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity. 1188 51

The importance of the tyrosine phosphorylation cascades in the initiation and regulation of the functional responsiveness of human neutrophils is well established. On the other hand, the link between the G protein-coupled receptors (to which the receptors for chemotactic factors belong) and the activation of tyrosine kinases is very poorly characterized. Based on previous observations indicating that the stimulation of tyrosine phosphorylation was sensitive to inhibition by the phosphatidylinositol 3-kinase inhibitor wortmannin and the recent description of pleckstrin homology domain-containing tyrosine kinases (the Tec family), we have examined the potential implication of the latter in the responses of human neutrophils to chemotactic factors. The results obtained indicate firstly that several members of the Tec family of tyrosine kinases are expressed in human neutrophils, including Tec, Btk, and Bmx. Stimulation of the cells with fMet-Leu-Phe led to a rapid activation of Tec as indicated by its translocation to a membrane fraction and to increases in its in situ level of tyrosine phosphorylation and its capacity to tyrosine phosphorylate itself or an exogenous substrate (SAM68-GST) in in vitro kinase assays. The activation of Tec was inhibited by pertussis toxin as well as by wortmannin. The results of this study provide direct evidence for the implication of Tec family kinases in the responses of human neutrophils to chemotactic factors. They also suggest that one of the links between G protein-coupled receptors and tyrosine kinases depends on the activation of phosphatidylinositol 3-kinase and the generation of phosphatidylinositol 3,4,5-trisphosphate.
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PMID:Chemotactic factor-induced recruitment and activation of Tec family kinases in human neutrophils. Implication of phosphatidynositol 3-kinases. 1194 May 95


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