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)

We have isolated the murine Limk1 gene, which is a single copy gene located at the distal end of mouse chromosome 5. Limk1 exhibits a 95% homology to the human homologue, LIMK, which contains two LIM domains and a putative protein kinase domain. Although Limk1 and LIMK contain all motifs found in catalytic kinase domains, amino acids previously described to be diagnostic of either serine/threonine- or tyrosine-kinases are not present. It is demonstrated that GST-Limk1-fusion protein can autophosphorylate on serine, tyrosine and threonine residues in vitro and that mutation of residue D460 within the IHRDL motif abolishes kinase activity. Northern blot showed preferential expression of a 3.5 kb message in adult spinal cord and brain. In situ hybridisation confirmed high expression levels in the nervous system, particularly in the spinal cord and the cranial nerve and dorsal root ganglia. Limk1 also contains two tandem LIM-domains. These zinc-finger like domains can mediate protein-protein interactions and have been described in nuclear and cytoskeletal proteins. The combination of LIM- and kinase domains may provide a novel route by which intracellular signalling can be integrated.
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PMID:Limk1 is predominantly expressed in neural tissues and phosphorylates serine, threonine and tyrosine residues in vitro. 747 47

LIM homeodomain proteins are a family of recently characterized proteins which contain, in addition to a homeodomain, two tandem repeats of conserved Cys-His motifs termed as LIM domains. We have recently isolated several clones from a chinook salmon pituitary cDNA library that encode two novel LIM homeodomain proteins, Isl-2 and Isl-3, which are structurally related to rat Isl-1. In the present study, we used the salmon Isl-2 to determine the role of LIM domains in DNA binding. Several glutathione S-transferase (GST) fusion proteins containing either full length Isl-2 or various portions of this protein were expressed in bacteria. Zinc blot analysis reveals that the LIM domains produced in bacteria are capable of binding zinc. Gel shift analysis indicates that all homeodomain-containing fusion proteins are able to bind to a TAAT target sequence while the fusion proteins containing only the LIM domain are not. In contrast to a previous observation that the LIM domains of rat Isl-1 have an inhibitory role in DNA binding, full length salmon Isl-2 containing both the LIM domains and a homeodomain can bind to a TAAT target sequence. To further examine the role of LIM domains in DNA binding, several GST fusion proteins were used to select specific target DNA sequences from a pool of randomly incorporated oligonucleotides. Specific target DNAs were selected by fusion proteins containing the homeodomain or the full length Isl-2, but not by LIM domain only fusion proteins, indicating that the LIM domain alone is not involved in DNA binding. The selected target DNAs were cloned and sequenced. They revealed two classes of consensus, C/TTAATG/TG/A and C/TTAAGTG, for both the homeodomain and full length Isl-2. The two classes of consensus competed with each other for binding to the homeodomain. The equilibrium dissociation constants for DNA binding, estimated by Scatchard analysis, were similar for the homeodomain and full length Isl-2.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Zinc and DNA binding properties of a novel LIM homeodomain protein Isl-2. 799 75

LIM domains, Cys-rich motifs containing approximately 50 amino acids found in a variety of proteins, are proposed to direct protein*protein interactions. To identify structural targets recognized by LIM domains, we have utilized random peptide library selection, the yeast two-hybrid system, and glutathione S-transferase fusions. Enigma contains three LIM domains within its carboxyl terminus and LIM3 of Enigma specifically recognizes active but not mutant endocytic codes of the insulin receptor (InsR) (Wu, R. Y., and Gill, G. N. (1994) J. Biol. Chem. 269, 25085-25090). Interaction of two random peptide libraries with glutathione S-transferase-LIM3 of Enigma indicated specific binding to Gly-Pro-Hyd-Gly-Pro-Hyd-Tyr-Ala corresponding to the major endocytic code of InsR. Peptide competition demonstrated that both Pro and Tyr residues were required for specific interaction of InsR with Enigma. In contrast to LIM3 of Enigma binding to InsR, LIM2 of Enigma associated specifically with the receptor tyrosine kinase, Ret. Ret was specific for LIM2 of Enigma and did not bind other LIM domains tested. Mutational analysis indicated that the residues responsible for binding to Enigma were localized to the carboxyl-terminal 61 amino acids of Ret. A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociated Enigma and Ret complexes, while a mutant that changed Asn-Lys-Leu-Tyr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exon16 of InsR failed to disrupt the complexes, indicating the Asn-Lys-Leu-Tyr sequence of Ret is essential to the recognition motif for LIM2 of Enigma. We conclude that LIM domains of Enigma recognize tyrosine-containing motifs with specificity residing in both the LIM domains and in the target structures.
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PMID:Specificity of LIM domain interactions with receptor tyrosine kinases. 866 33

LIM-containing protein kinase 1 (LIMK1) is a serine/threonine kinase with a structure composed of two LIM domains, a PDZ domain, and a protein kinase domain. We examined the subcellular localization of LIMK1 and its variously deleted mutants in HeLa cells by transfection with these cDNAs. Immunofluorescence analysis revealed that the full-length LIMK1 and its mutants deleted with LIM domain or protein kinase domain preferentially localized in the cytoplasm, while the mutants deleted with the PDZ domain or a 52 amino acid region (B region) within the PDZ domain localized mainly in the nucleus. When the normally nuclear cyclin A was fused with the PDZ domain or the B region of LIMK1, it was localized in the cytoplasm of transfected cells. The corresponding region of the PDZ domain of postsynaptic density protein (PSD)-95 had no such function. Additionally, the PDZ domain of LIMK1 had no potential to bind to the C-terminal S/TXV peptides, to which the PSD-95 PDZ domain can bind. Taken together these results suggest that the PDZ domain, particularly the B region, of LIMK1 has a specific function to localize the protein in the cytoplasm. When glutathione S-transferase (GST) fused with the PDZ domain of LIMK1 (GST-PDZ) or GST-PDZ deleted with the B region (GST-PDZ delta B) was microinjected into the nucleus of COS cells, GST-PDZ was almost completely excluded from the nucleus within 30 min, whereas GST-PDZ delta B remained in the nucleus. These findings suggest that the B region of LIMK1 probably has nuclear export signal activity.
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PMID:Cytoplasmic localization of LIM-kinase 1 is directed by a short sequence within the PDZ domain. 963 33

Platelets are anuclear, membrane-bounded fragments derived from megakaryocytes which, upon stimulation, assemble an actin skeleton including stress fibres and focal contacts. The focal contacts resemble those of tissue culture cells. However, they lack paxillin, a conspicuous component of these organelles. We found that instead of paxillin, platelets contain a related protein with a molecular mass of 55 kDa that crossreacts with a monoclonal antibody against paxillin. The gene for the 55 kDa protein was cloned from a bone marrow cDNA library and turned out to be identical to a recently discovered gene encoding hic-5. Like paxillin, hic-5 is a cytoskeletal protein containing four carboxy-terminal LIM domains and LD motifs in the amino-terminal half. The LIM domains of both hic-5 and paxillin are capable of targetting green fluorescent protein to focal contacts. In addition, GST-hic-5 precipitates the focal adhesion kinase pp125(FAK) and talin from platelet extracts. Only trace amounts of hic-5 occur in DAMI cells, a megakaryocytic cell line, and in megakaryocytes cultured from CD34+ cells obtained from umbilical cord blood. However, RT-polymerase chain reactions performed with RNA obtained from platelets gave a positive result when primers specific for hic-5 were used, but were negative with paxillin-specific primers, indicating that a switch from paxillin expression to hic-5 expression must occur late in the maturation of megakaryocytes into platelets.
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PMID:Recruitment of the LIM protein hic-5 to focal contacts of human platelets. 966 39

LIM-kinase 1 (LIMK1) is a serine/threonine kinase that phosphorylates cofilin and regulates actin-filament dynamics. LIMK1, which contains two LIM domains and a single PDZ domain, localizes predominantly in the cytoplasm, but its mutant, deleted with the PDZ domain, localizes mainly in the nucleus, thereby indicating that the PDZ domain plays a role in the cytoplasmic localization of LIMK1. Here we provide evidence that the PDZ domain of LIMK1 contains two functional leucine-rich nuclear-export signals (NESs). The PDZ domain of LIMK1 fused with glutathione S-transferase (GST-PDZ), when injected into the nucleus, was rapidly excluded from the nucleus, but its mutant with replacements of conserved hydrophobic residues in two putative NESs by alanines remained in the nucleus. The nuclear export of GST-PDZ was sensitive to leptomycin B (LMB), a specific inhibitor of nuclear export mediated by leucine-rich NESs. Malfunctional mutation of two NESs or LMB treatment prevented the nuclear export of full-length LIMK1 and induced its nuclear accumulation. These results suggest that the predominant localization of LIMK1 in the cytoplasm is supported by two NESs within the PDZ domain and that LIMK1 normally shuttles between the cytoplasm and the nucleus. We also provide evidence that a short basic cluster sequence within the protein-kinase domain is involved in the nuclear import of LIMK1.
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PMID:Nuclear export of LIM-kinase 1, mediated by two leucine-rich nuclear-export signals within the PDZ domain. 1005 54

Proteins of the 14-3-3 family have been implicated in various physiological processes, and are thought to function as adaptors in various signal transduction pathways. In addition, 14-3-3 proteins may contribute to the reorganization of the actin cytoskeleton by interacting with as yet unidentified actin-binding proteins. Here we show that the 14-3-3 zeta isoform interacts with both the actin-depolymerizing factor cofilin and its regulatory kinase, LIM (Lin-11/Isl-1/Mec-3)-domain-containing protein kinase 1 (LIMK1). In both yeast two-hybrid assays and glutathione S-transferase pull-down experiments, these proteins bound efficiently to 14-3-3 zeta. Deletion analysis revealed consensus 14-3-3 binding sites on both cofilin and LIMK1. Furthermore, the C-terminal region of 14-3-3 zeta inhibited the binding of cofilin to actin in co-sedimentation experiments. Upon co-transfection into COS-7 cells, 14-3-3 zeta-specific immunoreactivity was redistributed into characteristic LIMK1-induced actin aggregations. Our data are consistent with 14-3-3-protein-induced changes to the actin cytoskeleton resulting from interactions with cofilin and/or LIMK1.
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PMID:Identification of cofilin and LIM-domain-containing protein kinase 1 as novel interaction partners of 14-3-3 zeta. 1232 73

The Lhx9 LIM-homeodomain transcription factor and its truncated isoform Lhx9alpha are generated by alternative splicing of the Lhx9 gene. Here we investigated the differential functional properties of these two isoforms. Lhx9alpha, which lacks parts of the homeodomain, was unable to bind DNA in EMSA experiments, but was able to associate with CLIM cofactors in GST pull-down assays. In transfection experiments in PC12 cells, Lhx9alpha fusion constructs systematically showed a nuclear localization, as opposed to Lhx9 fusion constructs, which also localized to the cytoplasm. Moreover, Lhx9 increased NGF-induced neuronal differentiation of PC12 cells. Lhx9alpha, on the other hand, did not significantly increase neuronal differentiation but had an effect on the morphology of PC12 cells. Finally, as tested by RT-PCR experiments on transfected PC12 cells, Lhx9 was not able to induce the transcription of Lhx9alpha. Our results show significantly different functional properties for Lhx9 and Lhx9alpha, and suggest that Lhx9alpha can compete away limiting amounts of nuclear CLIM cofactors. Thus, Lhx9 and Lhx9alpha isoforms could be implicated in regulating various aspects of neuronal differentiation.
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PMID:Lhx9 and lhx9alpha: differential biochemical properties and effects on neuronal differentiation. 1558 34

The DNA-binding protein recombination signal-binding protein-Jk (RBP-J) plays a key role in transcriptional regulation by targeting the intracellular domain of Notch (NIC) and the Epstein-Barr virus nuclear antigen 2 (EBNA2) to specific promoters. In the absence of the Notch signaling, RBP-J acts as a transcriptional suppressor through recruiting co-suppressors such as histone deacetylase (HDAC). KyoT2 is a LIM domain protein that suppresses the RBP-J-mediated transcriptional activation. In the current study, we show that the polycomb group (PcG) protein HPC2, which functions as a transcriptional suppressor, is a candidate of KyoT2-binding proteins. To confirm the physical and functional interaction between KyoT2 and HPC2, we carried out yeast two-hybrid, GST-pull down, co-immunoprecipitation, as well as mammalian two-hybrid assays. Our results showed HPC2 and KyoT2 interacted both in vitro and in vivo, probably through the C-terminal fragment of HPC2 and LIM domains of KyoT2. In addition, we also found that overexpression of HPC2, not only inhibited transactivation of a RBP-J-dependent promoter by NIC, but also transactivation by RBP-J-VP16, a constitutively active form of RBP-J. Taken together, our results suggested that KyoT2 might inhibit the RBP-J-mediated transactivation through NIC by recruiting co-suppressors such as HPC2.
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PMID:The PcG protein HPC2 inhibits RBP-J-mediated transcription by interacting with LIM protein KyoT2. 1571 Apr 17

Human Bex2 (brain expressed X-linked, hBex2) is highly expressed in the embryonic brain, but its function remains unknown. We have identified that LMO2, a LIM-domain containing transcriptional factor, specifically interacts with hBex2 but not with mouse Bex1 and Bex2. The interaction was confirmed both by pull-down with GST-hBex2 and by coimmunoprecipitation assays in vivo. Using electrophoretic mobility shift assay, we have demonstrated the physical interaction of hBex2 and LMO2 as part of a DNA-binding protein complex. We have also shown that hBex2 can enhance the transcriptional activity of LMO2 in vivo. Furthermore, using mammalian two-hybrid analysis, we have identified a neuronal bHLH protein, NSCL2, as a novel binding partner for LMO2. We then showed that LMO2 could up-regulate NSCL2-dependent transcriptional activity, and hBex2 augmented this effect. Thus, hBex2 may act as a specific regulator during embryonic development by modulating the transcriptional activity of a novel E-box sequence-binding complex that contains hBex2, LMO2, NSCL2 and LDB1.
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PMID:Human Bex2 interacts with LMO2 and regulates the transcriptional activity of a novel DNA-binding complex. 1631 16


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