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| 1.
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Uchida, Sanae ; Yoshioka, Katsuji ; Kizu, Ryoichi ; Nakagama, Hitoshi ; Matsunaga, Tsukasa ; Ishizaka, Yukihito ; Poon, Randy Y. C. ; Yamashita, Katsumi
概要:
金沢大学医薬保健研究域薬学系<br />Cdc25 dual specificity phosphatases positively regulate the cell cycle by activating cyclin-dependen
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t kinase/cyclin complexes. Of the three mammalian Cdc25 isoforms, Cdc25A is phosphorylated by genotoxic stress-activated Chk1 or Chk2, which triggers its SCFβ-TrCP-mediated degradation. However, the roles of Cdc25B and Cdc25C in cell stress checkpoints remain inconclusive. We herein report that c-Jun NH2-terminal kinase (JNK) induces the degradation of Cdc25B. Nongenotoxic stress induced by anisomycin caused rapid degradation of Cdc25B as well as Cdc25A. Cdc25B degradation was dependent mainly on JNK and partially on p38 mitogen-activated protein kinase (p38). Accordingly, cotransfection with JNK1, JNK2, or p38 destabilized Cdc25B. In vitro kinase assays and site-directed mutagenesis experiments revealed that the critical JNK and p38 phosphorylation site in Cdc25B was Ser101. Cdc25B with Ser101 mutated to alanine was refractory to anisomycin-induced degradation, and cells expressing such mutant Cdc25B proteins were able to override the anisomycin-induced G2 arrest. These results highlight the importance of a novel JNK/p38-Cdc25B axis for a nongenotoxic stress-induced cell cycle checkpoint. ©2009 American Association for Cancer Research.
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| 2.
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Uchida, Sanae ; Watanabe, Nobumoto ; Kudo, Yasusei ; Yoshioka, Katsuji ; Matsunaga, Tsukasa ; Ishizaka, Yukihito ; Nakagama, Hitoshi ; Poon, Randy Y.C. ; Yamashita, Katsumi
概要:
Cdc25A, which is one of the three mammalian CDK-activating Cdc25 protein phosphatases (Cdc25A, B and C), is degraded thr
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ough SCFβTrCP-mediated ubiquitylation following genomic insult; however, the regulation of the stability of the other two Cdc25 proteins is not well understood. Previously, we showed that Cdc25B is primarily degraded by cellular stresses that activate stress-activated MAPKs, such as Jun NH2-terminal kinase (JNK) and p38. Here, we report that Cdc25B was ubiquitylated by SCFβTrCP E3 ligase upon phosphorylation at two Ser residues in the βTrCP-binding-motif-like sequence D94AGLCMDSPSP104. Point mutation of these Ser residues to alanine (Ala) abolished the JNK-induced ubiquitylation by SCFβTrCP, and point mutation of DAG to AAG or DAA eradicated both βTrCP binding and ubiquitylation. Further analysis of the mode of βTrCP binding to this region revealed that the PEST-like sequence from E82SS to D94AG is crucially involved in both the βTrCP binding and ubiquitylation of Cdc25B. Furthermore, the phospho-mimetic replacement of all 10 Ser residues in the E82SS to SPSP104 region with Asp resulted in βTrCP binding. Collectively, these results indicate that stress-induced Cdc25B ubiquitylation by SCFβTrCP requires the phosphorylation of S101PS103P in the βTrCP-binding-motif-like and adjacent PEST-like sequences. © 2011. Published by The Company of Biologists Ltd.
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| 3.
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Sato, Tokiharu ; Torashima, Takashi ; Sugihara, Kazushi ; Hirai, Hirokazu ; Asano, Masahide ; Yoshioka, Katsuji
概要:
金沢大学がん研究所がん分子細胞制御<br />Cerebellar granule cell precursors (GCPs) proliferate in the outer part of the external granular
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layer (EGL). They begin their differentiation by exiting the cell cycle and migrating into the inner part of the EGL. Here we report that JSAP1, a scaffold protein for JNK signaling pathways, is expressed predominantly in the post-mitotic GCPs of the inner EGL. JSAP1 knockdown or treatment with a JNK inhibitor enhances the proliferation of cultured GCPs, but the overexpression of wild-type JSAP1 leads to increased proportions of p27Kip1- and NeuN-positive cells, even with saturating concentrations of Sonic hedgehog (Shh), a potent GCP mitogen. However, these differentiation-promoting effects on GCPs are attenuated significantly in cells overexpressing a mutant JSAP1 that lacks the JNK-binding domain. Together, these data suggest that JSAP1 antagonizes the mitogenic effect of Shh on GCPs and promotes their exit from the cell cycle and differentiation, by modulating JNK activity. © 2008 Elsevier Inc. All rights reserved.
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| 4.
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Xiang, Shuang-Lin ; Iwasaki, Shu-ichi ; Kumano, Tomoyasu ; Sun, Xiangao ; Yoshioka, Katsuji ; Yamamoto, Ken-ichi ; 善岡, 克次
概要:
金沢大学がん研究所<br />Human Rad9 is a key cell-cycle checkpoint protein that is postulated to function in the early phase of ce
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ll-cycle checkpoint control through complex formation with Rad1 and Hus1. Rad9 is also thought to be involved in controlling apoptosis through its interaction with Bcl-2. To explore the biochemical functions of Rad9 in these cellular control mechanisms, we performed two-hybrid screening and identified Tetratricopeptide repeat protein 2 (Tpr2) as a novel Rad9-binding protein. We found that Tpr2 binds not only to Rad9, but also to Radl and Hus1, through its N-terminal tetratricopeptide repeat region, as assessed by in vivo and in vitro binding assays. However, the in vivo and in vitro interactions of Tpr2 with Rad9 were greatly enhanced by the deletion of its C-terminal J domain or by a point mutation in the conserved HPD motif in the J domain, though the binding of Tpr2 to Rad1 and Hus1 was not influenced by these J-domain mutations. We further found: (1) Rad9 transiently dissociates from Tpr2 following heat-shock or UV treatments, but the mutation of the J domain abrogates this transient dissociation of the Tpr2/Rad9 complex; and (2) the J domain of Tpr2 modulates the cellular localization of both Tpr2 itself and Rad9. These results indicate that the J domain of Tpr2 plays a criticai role in the regulation of both physical and functional interactions between Tpr2 and Rad9. © 2001 Academic Press.
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| 5.
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Muraoka, Kei-ichi ; Fujimoto, Koutaro ; Sun, Xiangao ; Yoshioka, Katsuji ; Shimizu, Kou-ichi ; Yagi, Masao ; Bose, Jr. Henry ; Miyazaki, Itsuo ; Yamamoto, Ken-ichi
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金沢大学がん研究所がん分子細胞制御<br />FK506 is a powerful immunosuppressive drug currently in use that inhibits the activation of sever
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al transcription factors (nuclear factor (NF)-AT and NF-κB) critical for T cell activation. We show here that, contrary to the situation in T cells, FK506 activates transcription factor NF-κB in non-lymphoid cells such as fibroblasts and renal mesangial cells. We further show that FK506 induces NF-κB-regulated IL-6 production in vitro and in vivo, in particular in kidney. IL-6 has been shown previously to produce renal abnormalities in vivo, such as mesangioproliferative glomerulonephritis. Similar renal abnormalities were also observed in FK506-treated animals. These results thus suggest a causal relationship between FK506-induced NF-κB activation/IL-6 production and some of FK506-induced renal abnormalities.
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| 6.
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Bayarsaikhan, Munkhuu ; Takino, Takahisa ; Gantulga, Davaakhuu ; Yoshioka, Katsuji ; Sato, Hiroshi
概要:
金沢大学がん研究所がん分子細胞制御<br />金沢大学大学院医学系研究科<br />We previously reported that the level of c-Jun NH2-terminal kinase (JNK)/stres
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s-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, increases dramatically during nerve growth factor (NGF)-induced differentiation of PC12h cells. In the present study, we investigated the function of JSAP1 during PC12h cell differentiation by knocking down the level of JSAP1. The depletion of JSAP1 caused NGF-treated PC12h cells to form aggregates and impaired their differentiation. The aggregation was not observed in JSAP1-depleted cells that were untreated or treated with epidermal growth factor. Immunocytochemical studies indicated that N-cadherin, but not E-cadherin, was localized to sites of cell–cell contact in the aggregated cells. Furthermore, an inhibitory anti-N-cadherin antibody completely blocked the aggregation. Taken together, these results suggest that JSAP1 regulates cell–cell interactions in PC12h cells specifically in the NGF-induced signaling pathway, and does so by modulating N-cadherin.
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| 7.
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Athauda, Senarath B. P. ; Yoshioka, Katsuji ; Shiba, Tadayoshi ; Takahashi, Kenji
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金沢大学がん研究所がん分子細胞制御<br />The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia col
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i was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe–Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 °C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15-–Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.
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| 8.
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Iwanaga, Asuka ; Wang, Guangmin ; Gantulga, Davaakhuu ; Sato, Tokiharu ; Baljinnyam, Tuvshintugs ; Shimizu, Keiko ; Takumi, Ken ; Hayashi, Motoharu ; Akashi, Takuya ; Fuse, Hideki ; Sugihara, Kazushi ; Asano, Masahide ; Yoshioka, Katsuji
概要:
金沢大学がん研究所がん分子細胞制御<br />The specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling module
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s is mediated, at least in part, by scaffold proteins. c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the Gα13 and Cdo-mediated pathway, in vitro. To date, however, JLP expression has not been analyzed in detail, nor are its physiological functions well understood. Here we investigated the expression of JLP in the mouse testis during development. Of the tissues examined, JLP was strongest in the testis, with the most intense staining in the elongated spermatids. Since the anti-JLP antibody used in this study can recognize both JLP and sperm-associated antigen 9 (SPAG9), a splice variant of JLP that has been studied extensively in primates, we also examined its expression in macaque testis samples. Our results indicated that in mouse and primate testis, the isoform expressed at the highest level was JLP, not SPAG9. We also investigated the function of JLP by disrupting the Jlp gene in mice, and found that the male homozygotes were subfertile. Taken together, these observations may suggest that JLP plays an important role in testis during development, especially in the production of functionally normal spermatozoa. © 2008 Springer Science+Business Media B.V.
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| 9.
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Sato, Tokiharu ; Enkhbat, Anir ; Yoshioka, Katsuji
概要:
金沢大学がん研究所<br />We previously reported that the scaffold protein c-Jun NH2-terminal kinase (JNK)/stress-activated protein
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kinase-associated protein 1 (JSAP1) functions in cerebellar granule cell precursors (GCPs) to promote their cell-cycle exit and differentiation. In this study, we used immunocytochemistry to examine the subcellular distribution of JSAP1 in proliferating cultured GCPs. We found that when stimulated with fibroblast growth factor-2 (FGF-2), a factor that promotes GCP differentiation through JNK and extracellular signal-regulated kinase (ERK) signaling, JSAP1 translocated to the plasma membrane and colocalized with activated JNK and ERK. In transfected cells expressing a constitutively activated FGF receptor (FGFR), JSAP1 and the activated FGFR colocalized at the plasma membrane with not only activated but also unphosphorylated and inactive JNK and ERK. These colocalizations did not occur when a mutant JSAP1 lacking the JNK-binding domain was substituted for wild-type JSAP1. Biochemical analyses of transfected cells showed that activated FGFR increased JSAP1's affinity for JNK and ERK and that JSAP1 enhanced FGFR-induced JNK and ERK activation. Collectively, these results suggest that when stimulated by FGFR, JSAP1 translocates to the plasma membrane, where it recruits JNK and ERK and facilitates their activation, leading to the differentiation of cerebellar GCPs. © 2010 The Authors. Journal compilation © 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
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| 10.
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Furuya, Hirokazu ; Yoshioka, Katsuji ; Sasaki, Hiroyuki ; Sakaki, Yoshiyuki ; Nakazato, Masamitsu ; Matsuo, Hisayuki ; Nakadai, Akira ; Ikeda, Shu-ichi ; Yanagisawa, Nobuo
概要:
金沢大学がん研究所がん分子細胞制御<br />A Japanese family with atypical type I familial amyloidotic polyneuropathy (FAP) in Iiyama, Japan
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, was studied. Most of the family members have dysfunctions of the central nervous system, in addition to typical symptoms of type I FAP. The transthyretin (TTR, also called prealbumin) gene of the atypical FAP (FAP-IY) was analyzed with recombinant DNA techniques and a RIA method. FAP-IY was found to have the mutation responsible for the methionine-for-valine substitution at position 30 of TTR, as in the case of typical type I FAP. However, analysis of DNA polymorphisms in the TTR locus showed that FAP-IY has a genetic background differing from that of the typical type I FAP. These observations lead to the consideration that a genetic factor(s) involved in the dysfunction of the central nervous system may locate in a chromosome region in close proximity to the TTR gene.
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