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Kensaku Mizuno - One of the best experts on this subject based on the ideXlab platform.
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CYTOPLASMIC LOCALIZATION OF LIM-Kinase 1 IS DIRECTED BY A SHORT SEQUENCE WITHIN THE PDZ Domain
Experimental cell research, 1998Co-Authors: Neng Yang, Osamu Higuchi, Kensaku MizunoAbstract: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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Identification of testis-specific (Limk2t) and brain-specific (Limk2c) isoforms of mouse LIM-Kinase 2 gene transcripts.
Biochemical and biophysical research communications, 1998Co-Authors: Chiho Ikebe, Kazumasa Ohashi, Kensaku MizunoAbstract:LIM-Kinase 1 (LIMK1) and LIM-Kinase 2 (LIMK2) are members of a novel serine/threonine Kinase subfamily with structural features composed of N-terminal two LIM Domains, an internal PDZ-like Domain, and a C-terminal Protein Kinase Domain. We recently identified and characterized the mouse Limk2 gene and two Limk2 transcripts (Limk2a and Limk2b) coding for Proteins with distinct N-terminal LIM structures. Here we describe two additional transcripts of the mouse Limk2 gene. One is a 1.7-kb transcript, termed Limk2t, which is specifically expressed in the testis and codes for an N-terminally truncated form of LIMK2 consisting of only a part of a PDZ-like Domain and a Protein Kinase Domain. The other is a transcript, termed Limk2c, which is specifically expressed in the brain and codes for a Protein with a 6-amino-acid insert within the Protein Kinase Domain. Exons specific to the 5'-terminal extra sequence of Limk2t and the insert sequence of Limk2c locate between exons 5-6 and exons 8-9 in the mouse Limk2 gene, respectively. Testis- and brain-specific expression of Limk2t and Limk2c suggests specific roles in these tissues.
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Identification and characterization of a novel Protein Kinase, TESK1, specifically expressed in testicular germ cells
The Journal of biological chemistry, 1995Co-Authors: Jiro Toshima, Kazumasa Ohashi, Ichiro Okano, Koh Nunoue, Miki Kishioka, Kei-ichi Kuma, Takashi Miyata, Momoki Hirai, Tadashi Baba, Kensaku MizunoAbstract:Abstract We have isolated cDNA clones encoding the rat and human forms of a novel Protein Kinase, termed TESK1 (testis-specific Protein Kinase 1). Sequence analysis indicates that rat TESK1 contains 628 amino acid residues, composed of an N-terminal Protein Kinase consensus sequence followed by a C-terminal proline-rich region. Human TESK1 contains 626 amino acids, sharing 92% amino acid identity with its rat counterpart. The Protein Kinase Domain of TESK1 is structurally similar to those of LIMK (LIM motif-containing Protein Kinase)-1 and LIMK2, with 49-50% sequence identity. Phylogenetic analysis of the Protein Kinase Domains revealed that TESK1 is most closely related to a LIMK subfamily. Chromosomal localization of human TESK1 gene was assigned to 9p13. Anti-TESK1 antibody raised against the C-terminal peptide of TESK1 recognized two polypeptides of 68 and 80 kDa in cell lysates of COS cells transfected with human TESK1 cDNA expression plasmid. TESK1 Protein expressed in COS cells exhibited serine/threonine Kinase activity, when myelin basic Protein was used as a substrate. Northern blot analysis revealed that TESK1 mRNA was specifically expressed in rat and mouse testicular germ cells. The TESK1 mRNA in the testis was detectable only after the 18th day of postnatal development of mice and was mainly expressed in the round spermatids. These observations suggest that TESK1 has a specific function in spermatogenesis.
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Identification and Characterization of a Novel Family of Serine/Threonine Kinases Containing Two N-terminal LIM Motifs
The Journal of biological chemistry, 1995Co-Authors: Ichiro Okano, Kazumasa Ohashi, Koh Nunoue, Momoki Hirai, Junji Hiraoka, Hidenori Otera, Shintaro Iwashita, Kensaku MizunoAbstract:Abstract We previously isolated human cDNA coding for LIMK1 (LIM motif-containing Protein Kinase-1), a putative Protein Kinase containing two LIM motifs at the N terminus and an unusual Protein Kinase Domain at the C terminus. In the present study, we isolated human cDNA encoding LIMK2, a second member of a LIMK family, with a Domain structure similar to LIMK1 and 50% overall amino acid identity with LIMK1. The Protein Kinase Domains of LIMK1 and LIMK2 are unique in that they contain an unusual sequence motif Asp-Leu-Asn-Ser-His-Asn in subDomain VIB and a highly basic insert between subDomains VII and VIII. Expression patterns of LIMK1 and LIMK2 mRNAs in human tissues differ significantly. Chromosomal localization of human LIMK1 and LIMK2 genes was assigned to 7q11.23 and 22q12, respectively, by fluorescence in situ hybridization. The Myc epitope-tagged LIMK1 and LIMK2 Proteins transiently expressed in COS cells exhibited serine/threonine-specific Kinase activity toward myelin basic Protein and histone in in vitro Kinase assay. Immunofluorescence and subcellular fractionation analysis revealed that Myc-tagged LIMK1 and LIMK2 were localized mainly in the cytoplasm. The “native” LIMK1 Protein endogenously expressed in A431 epidermoid carcinoma cells also exhibited serine/threonine Kinase activity. The specific activity of native LIMK1 from A431 cells was apparently much higher than that of “recombinant” LIMK1 ectopically expressed in COS cells, hence, it is likely that there is a mechanism, by which native LIMK1 is activated. A 140-kDa tyrosine-phosphorylated Protein (pp140) was co-immunoprecipitated with native LIMK1 from A431 cell lysates; therefore, pp140 may be a LIMK1-associated Protein involved in the regulation of LIMK1 function.
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Molecular cloning of a chicken lung cDNA encoding a novel Protein Kinase with N-terminal two LIM/double zinc finger motifs.
Journal of biochemistry, 1994Co-Authors: Kazumasa Ohashi, Jiro Toshima, Katsunori Tajinda, Toshikazu Nakamura, Kensaku MizunoAbstract:Using the cDNA fragment of chicken c-sea receptor tyrosine Kinase as a probe, we isolated from a chicken lung cDNA library overlapping cDNA clones encoding a novel Protein Kinase, which we termed LIM-Kinase (LIMK). The predicted polypeptide of 642 amino acid residues contains remarkable structural features, composed of the N-terminal two tandemly arrayed LIM/double zinc finger motifs and the C-terminal unusual Protein Kinase Domain. To our knowledge, a Protein Kinase containing the LIM motif in the molecule has not heretofore been described. The Protein Kinase Domain of LIMK shares highly conserved residues with the known Protein Kinases, but LIMK is unique in that it contains the sequence DLNSHN in subDomain VIB and a short, highly basic insert sequence, which may function as a signal for nuclear localization, between subDomain VII and VIII in the Protein Kinase Domain. Northern blot analysis revealed that the single species of LIMK mRNA of 3.8 kb is expressed predominantly in the lung, and faintly in the kidney, liver, brain, spleen, gizzard, and intestine. As the LIM motif is thought to be involved in Protein-Protein interactions by binding to another LIM motif, and is often present in the homeoDomain-containing Proteins involved in cell fate determination and in the oncogenic nuclear Proteins (rhombotins), it is likely that LIMK is involved in developmental or oncogenic processes through interactions with these LIM-containing Proteins.
Laure Aurelian - One of the best experts on this subject based on the ideXlab platform.
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A truncated Protein Kinase Domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) expressed in Escherichia coli.
The Journal of biological chemistry, 1991Co-Authors: Jianyuan Luo, C. C. Smith, Michael Kulka, Laure AurelianAbstract:The amino-terminal Domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) contains a serine/threonine-specific Protein Kinase that has characteristics of a growth factor receptor (Chung, T. D., Wymer, J. P., Smith, C. C., Kulka, M., and Aurelian, L. (1989) J. Virol. 63, 3389-3398; Chung, T. D., Wymer, J. P., Kulka, M. Smith, C. C., and Aurelian, L. (1990) Virology 179, 168-178). To characterize this Protein Kinase (PK) Domain further we constructed a bacterial expression vector (pJL11) containing DNA sequences encoding ICP10 amino acid residues 1-445. Bacteria containing pJL11 were induced to express a 29-kDa Protein (designated pp29la1) that represents a truncated portion of the ICP10-PK Domain (includes PK catalytic motifs I-V) as demonstrated by immunoprecipitation with antibodies that recognize different antigenic Domains, competition studies with extracts of ICP10-positive eukaryotic cells, and peptide mapping.pp29la1 has autophosphorylating and transphosphorylating activity for calmodulin. The enzyme is activated by Mn2+ but not by Mg2+ ions, and autophosphorylation is inhibited by histone. It differs from the authentic ICP10-PK in that phosphorylation is specific only for threonine.
H Marsden - One of the best experts on this subject based on the ideXlab platform.
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Identification of structural Domains within the large subunit of herpes simplex virus ribonucleotide reductase.
The Journal of general virology, 1994Co-Authors: J Conner, A Cross, J Murray, H MarsdenAbstract:The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional Protein consisting of a unique N-terminal Protein Kinase Domain and a ribonucleotide reductase Domain. Previous studies showed that the two functional Domains are linked by a protease sensitive site. Here we provide evidence for two subDomains, of 30K and 53K, within the reductase Domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact Protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 micrograms/ml) of trypsin or V8 protease, cleavage between the subDomains was prevented by the presence of the N-terminal Protein Kinase Domain. At higher protease concentrations (1 micrograms/ml) the N-terminal Domain is extensively proteolysed and the 30K and 53K Domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two Domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two Domain structure, and that the interDomain linking region is protected by the unique N terminus.
Jianyuan Luo - One of the best experts on this subject based on the ideXlab platform.
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A truncated Protein Kinase Domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) expressed in Escherichia coli.
The Journal of biological chemistry, 1991Co-Authors: Jianyuan Luo, C. C. Smith, Michael Kulka, Laure AurelianAbstract:The amino-terminal Domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) contains a serine/threonine-specific Protein Kinase that has characteristics of a growth factor receptor (Chung, T. D., Wymer, J. P., Smith, C. C., Kulka, M., and Aurelian, L. (1989) J. Virol. 63, 3389-3398; Chung, T. D., Wymer, J. P., Kulka, M. Smith, C. C., and Aurelian, L. (1990) Virology 179, 168-178). To characterize this Protein Kinase (PK) Domain further we constructed a bacterial expression vector (pJL11) containing DNA sequences encoding ICP10 amino acid residues 1-445. Bacteria containing pJL11 were induced to express a 29-kDa Protein (designated pp29la1) that represents a truncated portion of the ICP10-PK Domain (includes PK catalytic motifs I-V) as demonstrated by immunoprecipitation with antibodies that recognize different antigenic Domains, competition studies with extracts of ICP10-positive eukaryotic cells, and peptide mapping.pp29la1 has autophosphorylating and transphosphorylating activity for calmodulin. The enzyme is activated by Mn2+ but not by Mg2+ ions, and autophosphorylation is inhibited by histone. It differs from the authentic ICP10-PK in that phosphorylation is specific only for threonine.
J Conner - One of the best experts on this subject based on the ideXlab platform.
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Identification of structural Domains within the large subunit of herpes simplex virus ribonucleotide reductase.
The Journal of general virology, 1994Co-Authors: J Conner, A Cross, J Murray, H MarsdenAbstract:The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional Protein consisting of a unique N-terminal Protein Kinase Domain and a ribonucleotide reductase Domain. Previous studies showed that the two functional Domains are linked by a protease sensitive site. Here we provide evidence for two subDomains, of 30K and 53K, within the reductase Domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact Protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 micrograms/ml) of trypsin or V8 protease, cleavage between the subDomains was prevented by the presence of the N-terminal Protein Kinase Domain. At higher protease concentrations (1 micrograms/ml) the N-terminal Domain is extensively proteolysed and the 30K and 53K Domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two Domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two Domain structure, and that the interDomain linking region is protected by the unique N terminus.
