Publications (12)48.94 Total impact
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Article: Trichoplein and Aurora A block aberrant primary cilia assembly in proliferating cells.
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ABSTRACT: The primary cilium is an antenna-like organelle that modulates differentiation, sensory functions, and signal transduction. After cilia are disassembled at the G0/G1 transition, formation of cilia is strictly inhibited in proliferating cells. However, the mechanisms of this inhibition are unknown. In this paper, we show that trichoplein disappeared from the basal body in quiescent cells, whereas it localized to mother and daughter centrioles in proliferating cells. Exogenous expression of trichoplein inhibited primary cilia assembly in serum-starved cells, whereas ribonucleic acid interference-mediated depletion induced primary cilia assembly upon cultivation with serum. Trichoplein controlled Aurora A (AurA) activation at the centrioles predominantly in G1 phase. In vitro analyses confirmed that trichoplein bound and activated AurA directly. Using trichoplein mutants, we demonstrate that the suppression of primary cilia assembly by trichoplein required its ability not only to localize to centrioles but also to bind and activate AurA. Trichoplein or AurA knockdown also induced G0/G1 arrest, but this phenotype was reversed when cilia formation was prevented by simultaneous knockdown of IFT-20. These data suggest that the trichoplein-AurA pathway is required for G1 progression through a key role in the continuous suppression of primary cilia assembly.The Journal of Cell Biology 04/2012; 197(3):391-405. · 10.26 Impact Factor -
Article: Deciphering tumor-suppressor signaling in flies: genetic link between Scribble/Dlg/Lgl and the Hippo pathways.
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ABSTRACT: Loss of apico-basal polarity is one of the crucial factors that drives epithelial tumor progression. scribble/discs large/lethal giant larvae (scrib/dlg/lgl), a group of apico-basal polarity genes, were initially identified as members of "neoplastic" tumor-suppressors in flies. The components of the Hippo signaling pathway, which is crucial for organ size control and cancer development, were also identified through Drosophila genetic screens as members of "hyperplastic" tumor-suppressors. Accumulating evidence in recent studies implies that these two tumor-suppressor signaling pathways are not mutually exclusive but rather cooperatively act to give rise to highly malignant tumors. The interaction of these tumor-suppressor pathways could include deregulations of actin cytoskeleton, cell-cell contact, and apical-domain size of the epithelial cell.Journal of Genetics and Genomics 10/2011; 38(10):461-70. · 1.88 Impact Factor -
Article: Trichoplein controls microtubule anchoring at the centrosome by binding to Odf2 and ninein.
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ABSTRACT: The keratin cytoskeleton performs several functions in epithelial cells and provides regulated interaction sites for scaffold proteins, including trichoplein. Previously, we found that trichoplein was localized on keratin intermediate filaments and desmosomes in well-differentiated, non-dividing epithelia. Here, we report that trichoplein is widely expressed and has a major function in the correct localization of the centrosomal protein ninein in epithelial and non-epithelial cells. Immunocytochemical analysis also revealed that this protein is concentrated at the subdistal to medial zone of both mother and daughter centrioles. Trichoplein binds the centrosomal proteins Odf2 and ninein, which are localized at the distal to subdistal ends of the mother centriole. Trichoplein depletion abolished the recruitment of ninein, but not Odf2, specifically at the subdistal end. However, Odf2 depletion inhibited the recruitment of trichoplein to a mother centriole, whereas ninein depletion did not. In addition, the depletion of each molecule impaired MT anchoring at the centrosome. These results suggest that trichoplein has a crucial role in MT-anchoring activity at the centrosome in proliferating cells, probably through its complex formation with Odf2 and ninein.Journal of Cell Science 02/2011; 124(Pt 6):857-64. · 6.11 Impact Factor -
Article: 14-3-3gamma mediates Cdc25A proteolysis to block premature mitotic entry after DNA damage.
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ABSTRACT: 14-3-3 proteins control various cellular processes, including cell cycle progression and DNA damage checkpoint. At the DNA damage checkpoint, some subtypes of 14-3-3 (beta and zeta isoforms in mammalian cells and Rad24 in fission yeast) bind to Ser345-phosphorylated Chk1 and promote its nuclear retention. Here, we report that 14-3-3gamma forms a complex with Chk1 phosphorylated at Ser296, but not at ATR sites (Ser317 and Ser345). Ser296 phosphorylation is catalysed by Chk1 itself after Chk1 phosphorylation by ATR, and then ATR sites are rapidly dephosphorylated on Ser296-phosphorylated Chk1. Although Ser345 phosphorylation is observed at nuclear DNA damage foci, it occurs more diffusely in the nucleus. The replacement of endogenous Chk1 with Chk1 mutated at Ser296 to Ala induces premature mitotic entry after ultraviolet irradiation, suggesting the importance of Ser296 phosphorylation in the DNA damage response. Although Ser296 phosphorylation induces the only marginal change in Chk1 catalytic activity, 14-3-3gamma mediates the interaction between Chk1 and Cdc25A. This ternary complex formation has an essential function in Cdc25A phosphorylation and degradation to block premature mitotic entry after DNA damage.The EMBO Journal 08/2010; 29(16):2802-12. · 9.20 Impact Factor -
Article: Novel Positive Feedback Loop between Cdk1 and Chk1 in the Nucleus during G2/M Transition
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ABSTRACT: Chk1, one of the critical transducers in DNA damage/replication checkpoints, prevents entry into mitosis through inhibition of Cdk1 activity. However, it has remained unclear how this inhibition is cancelled at the G2/M transition. We reported recently that Chk1 is phosphorylated at Ser286 and Ser301 by Cdk1 during mitosis. Here, we show that mitotic Chk1 phosphorylation is accompanied by Chk1 translocation from the nucleus to the cytoplasm in prophase. This translocation advanced in accordance with prophase progression and was regulated by Crm-1-dependent nuclear export. Exogenous Chk1 mutated at Ser286 and Ser301 to Ala (S286A/S301A) was observed mainly in the nuclei of prophase cells, although such nuclear accumulation was hardly observed in wild-type Chk1. Induction of S286A/S301A resulted in the delay of mitotic entry. Biochemical analyses using immunoprecipitated cyclin B1-Cdk1 complexes revealed S286A/S301A expression to block the adequate activation of Cdk1. In support of this, S286A/S301A expression retained Wee1 at higher levels and Cdk1-induced phosphorylation of cyclin B1 and vimentin at lower levels. A kinase-dead version of S286A/S301A also localized predominantly in the nucleus but lost the ability to delay mitotic entry. These results indicate that Chk1 phosphorylation by Cdk1 participates in cytoplasmic sequestration of Chk1 activity, which releases Cdk1 inhibition in the nucleus and promotes mitotic entry.Journal of Biological Chemistry 12/2009; 284(49):34223-34230. · 4.77 Impact Factor -
Article: Novel positive feedback loop between Cdk1 and Chk1 in the nucleus during G2/M transition.
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ABSTRACT: Chk1, one of the critical transducers in DNA damage/replication checkpoints, prevents entry into mitosis through inhibition of Cdk1 activity. However, it has remained unclear how this inhibition is cancelled at the G(2)/M transition. We reported recently that Chk1 is phosphorylated at Ser(286) and Ser(301) by Cdk1 during mitosis. Here, we show that mitotic Chk1 phosphorylation is accompanied by Chk1 translocation from the nucleus to the cytoplasm in prophase. This translocation advanced in accordance with prophase progression and was regulated by Crm-1-dependent nuclear export. Exogenous Chk1 mutated at Ser(286) and Ser(301) to Ala (S286A/S301A) was observed mainly in the nuclei of prophase cells, although such nuclear accumulation was hardly observed in wild-type Chk1. Induction of S286A/S301A resulted in the delay of mitotic entry. Biochemical analyses using immunoprecipitated cyclin B(1)-Cdk1 complexes revealed S286A/S301A expression to block the adequate activation of Cdk1. In support of this, S286A/S301A expression retained Wee1 at higher levels and Cdk1-induced phosphorylation of cyclin B(1) and vimentin at lower levels. A kinase-dead version of S286A/S301A also localized predominantly in the nucleus but lost the ability to delay mitotic entry. These results indicate that Chk1 phosphorylation by Cdk1 participates in cytoplasmic sequestration of Chk1 activity, which releases Cdk1 inhibition in the nucleus and promotes mitotic entry.Journal of Biological Chemistry 10/2009; 284(49):34223-30. · 4.77 Impact Factor -
Article: Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation.
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ABSTRACT: We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.Biochemical and Biophysical Research Communications 12/2008; 377(4):1227-31. · 2.48 Impact Factor -
Article: Release of arachidonic acid induced by tumor necrosis factor-alpha in the presence of caspase inhibition: evidence for a cytosolic phospholipase A2alpha-independent pathway.
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ABSTRACT: Stimulation of L929 cells with tumor necrosis factor-alpha (TNFalpha) caused cell death accompanied by a release of arachidonic acid (AA). Although the inhibition of caspases has been shown to cause necrosis in TNFalpha-treated L929 cells, its role in the TNFalpha-induced release of AA has not been elucidated. The release of AA is tightly regulated by phospholipase A(2) (PLA(2)). To find out the mechanisms underlying the TNFalpha-induced release of AA, we investigated the relationship between TNFalpha stimulation and PLA(2) regulation with and without zVAD, an inhibitor of caspases. In the present study, we found that treatment with TNFalpha and zVAD stimulated release of AA and cell death in C12 cells (a variant of L929 cells lacking alpha type of cytosolic PLA(2) (cPLA(2)alpha)). Stimulation with TNFalpha/zVAD also caused the release of AA from L929-cPLA(2)alpha-siRNA cells. Treatment with pyrrophenone (a selective inhibitor of cPLA(2)alpha) completely inhibited the TNFalpha-induced release of AA, but only partially inhibited the TNFalpha/zVAD-induced response in L929 cells. The TNFalpha/zVAD-induced release of AA from C12 and L929-cPLA(2)alpha-siRNA cells was pyrrophenone-insensitive, but inhibited by treatment with butylated hydroxyanisole (BHA, an antioxidant). Treatment with dithiothreitol, which inactivates secretory PLA(2) activity, decreased the amount of AA released by TNFalpha/zVAD. TNFalpha/zVAD appears to stimulate release of AA from C12 cells in a cPLA(2)alpha-independent, BHA-sensitive manner. The possible roles of secretory PLA(2) and reactive oxygen species from different pools in the release of AA and cell death were discussed.Biochemical pharmacology 04/2008; 75(6):1358-69. · 4.25 Impact Factor -
Article: Biochemical characterization of suramin as a selective inhibitor for the PKA-mediated phosphorylation of HBV core protein in vitro.
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ABSTRACT: The inhibitory effect of suramin on the phosphorylation of GST-HBV core fusion protein (GST-Hcore) and two GST-Hcore fusion polypeptides (Hcore157B and Hcore164B) by two alpha-type cAMP-dependent protein kinases (PKAIalpha and PKAIIalpha) was biochemically investigated in vitro. It was found that (i) this phosphorylation was inhibited by suramin at a low concentration (IC(50)=approx. 10 nM); (ii) a relative high dose of suramin was required to inhibit an autophosphorylation of PKAIIalpha (IC(50)=approx. 0.7 muM) and the PKAIIalpha-mediated phosphorylation of histone H2B (IC(50)=approx. 0.4 muM); (iii) the PKAIIalpha-mediated phosphorylation of Hcore157B was more sensitive to suramin than the phosphorylation of Hcore157B by Ca(2+)-dependent protein kinase (PKC); and (iv) suramin had a high binding affinity for Hcore157B, but not for histone H2B in vitro. These results suggest that suramin selectively inhibits the PKA-mediated phosphorylation of HBV-CP through the direct binding in vitro of suramin to the Arg-rich C-terminal region (containing three potential phosphorylation sites for PKA) on HBV-CP.Biological & Pharmaceutical Bulletin 10/2006; 29(9):1810-4. · 1.66 Impact Factor -
Article: High phosphorylation of HBV core protein by two alpha-type CK2-activated cAMP-dependent protein kinases in vitro.
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ABSTRACT: Two alpha-type CK2-activated PKAs (CK2-aPKAIalpha and CK2-aPKAIIalpha) were biochemically characterized in vitro using GST-HBV core fusion protein (GST-Hcore) and GST-Hcore157B as phosphate acceptors. It was found that (i), in the absence of cAMP, these two CK2-aPKAs phosphorylated both Ser-170 and Ser-178 on GST-Hcore and Hcore157B; (ii) this phosphorylation was approx. 4-fold higher than their phosphorylation by cAMP-activated PKAs; and (iii) suramin effectively inhibited the phosphorylation of Hcore157B by CK2-aPKAIIalpha through its direct binding to Hcore157B in vitro. These results suggest that high phosphorylation of HBV-CP by two CK2-aPKAs, in the absence of cAMP, may be involved in the pregenomic RNA (pgRNA) encapsidation and DNA-replication in HBV-infected cells.FEBS Letters 03/2006; 580(3):894-9. · 3.54 Impact Factor -
Article: Release of arachidonic acid induced by tumor necrosis factor-α in the presence of caspase inhibition: Evidence for a cytosolic phospholipase A2α-independent pathway
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ABSTRACT: Stimulation of L929 cells with tumor necrosis factor-α (TNFα) caused cell death accompanied by a release of arachidonic acid (AA). Although the inhibition of caspases has been shown to cause necrosis in TNFα-treated L929 cells, its role in the TNFα-induced release of AA has not been elucidated. The release of AA is tightly regulated by phospholipase A2 (PLA2). To find out the mechanisms underlying the TNFα-induced release of AA, we investigated the relationship between TNFα stimulation and PLA2 regulation with and without zVAD, an inhibitor of caspases. In the present study, we found that treatment with TNFα and zVAD stimulated release of AA and cell death in C12 cells (a variant of L929 cells lacking α type of cytosolic PLA2 (cPLA2α)). Stimulation with TNFα/zVAD also caused the release of AA from L929-cPLA2α-siRNA cells. Treatment with pyrrophenone (a selective inhibitor of cPLA2α) completely inhibited the TNFα-induced release of AA, but only partially inhibited the TNFα/zVAD-induced response in L929 cells. The TNFα/zVAD-induced release of AA from C12 and L929-cPLA2α-siRNA cells was pyrrophenone-insensitive, but inhibited by treatment with butylated hydroxyanisole (BHA, an antioxidant). Treatment with dithiothreitol, which inactivates secretory PLA2 activity, decreased the amount of AA released by TNFα/zVAD. TNFα/zVAD appears to stimulate release of AA from C12 cells in a cPLA2α-independent, BHA-sensitive manner. The possible roles of secretory PLA2 and reactive oxygen species from different pools in the release of AA and cell death were discussed.Biochemical Pharmacology. -
Article: 14-3-3γ mediates Cdc25A proteolysis to block premature mitotic entry after DNA damage
Top co-authors
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Institutions
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2008–2012
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Aichi Cancer Center
Ōsaka-shi, Osaka-fu, Japan
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2006
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Kitasato University
- Graduate School of Medical Sciences
Tokyo, Tokyo-to, Japan
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