Research Themes

• CG-NAP function in ciliogenesis through regulation of centriolar satellite formation
  Grant-in-Aid for Scientific Research (C)
  Teikyo Heisei University
  01 Apr. 2019 - 31 Mar. 2023
• Study of initiation process of the centrosome duplication.
  Grant-in-Aid for Young Scientists (B)
  01 Apr. 2013 - 31 Mar. 2016
  In this study I got following three results which indicated that kendrin and Cep152 may regulate the centrosome duplication. At first, kendrin and Cep152 can interact via their carboxyl terminals. Second, although Cep152 may not localize at the centrosome during G2/M phase they were recruited to the centrosome again during cytokinesis. Third, by siRNA mediated- kendrin depletion, expression level of Cep152 were increased and Cep152 could localize at the centrosome efficiently rather than normal condition. Taken together I found that Cep152 might be recruited efficiently to the centrosome after kendrin was released and that this mechanism could facilitate centrosome duplication at next cell cycle.
• タウ蛋白とリン酸化酵素複合体の相互作用を介した認知症脳神経細胞死の病態解析
  28 Apr. 2011 - 31 Mar. 2015
• タウ蛋白リン酸化シグナル巨大複合体を介する変性認知症の神経細胞死に関する病態解析
  2014 - 2015
• Functional analysis of separase and its novel substrate in the licensing of centrosome duplication
  Grant-in-Aid for Scientific Research (C)
  Teikyo Heisei University
  2011 - 2013
  Centrosome assembles the bipolar spindle during mitosis to ensure the equal segregation of replicated chromosomes. It is thereby assumed that the licensing mechanism tightly regulates the initiation of centrosome duplication only once per cell cycle, though precise process remains largely unknown. Separase, a cysteine protease that triggers sister chromatid separation, is known to be involved in this licensing, however, its centrosomal substrate remains unidentified. In this study, we found that the centrosomal protein kendrin is cleaved by separase at a consensus site. Moreover, expression of a non-cleavable kendrin mutant suppresses the initiation of centrosome duplication. Our finding provides the first evidence that kendrin is a novel and crucial substrate for separase at the centrosome involved in the licensing of centrosome duplication.
• Pathological study for a protein anchoring tau kinases and tau phosphatases in Alzheimer brain tissues
  Grant-in-Aid for Scientific Research (C)
  Kobe University
  2008 - 2010
  We investigated possible roles of abnormal intracellular signaling in the tau pathology in Alzheimer's disease (AD) for FKBP12 and the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a peptidyl-prolyl cis-trans isomerase known to be important in protein assembly, folding and transportation and a tumor-suppressor respectively by using Western blotting and microscopic analyses in postmortem brain tissues from elderly controls and the patients with AD. FKBP12-positive or PTEN-positive granules were located in the nucleus, the cytoplasm of cell bodies and the proximal portion of dendrites and axons in control brains. Reduced expression of FKBP12 or PTEN was seen in the remaining neurons, especially in the tangle-bearing neurons in AD. In addition, these molecules were redistributed in damaged neurons from the nucleus to the cytoplasm or to the neuritic pathology such as neuropil threads, degenerative neurites and intracellular tangles. Labeling of some reactive astrocytes around senile plaques was seen for FKBP12 or PTEN. Double labeling of tangles was observed either for PTEN and GSK3β or for PTEN and MEK. Thus, our results suggest that FKBP12 or PTEN delocalized from the nucleus to the cytoplasm and to the tangles may cause a deregulation of PI3K pathway in the cytoplasm and may induce the nuclear dysfunction in AD neurons.
• Research of signal transduction to neuronal degeneration mediated by tau-kinases complex in dementia
  Grant-in-Aid for Scientific Research (C)
  Kobe University
  2006 - 2007
  Neuronal localization of two large scaffold proteins interacting with some protein kineses and phosphateses, which phosphorylate or dephosphorylate tau protein directly, was studied to identify the pathology underlying neuronal death in the brain tissues of the patients with degenerative dementia including Alzheinier's disease (AD) and frontotanporal dementia (FTD).
  Morphological colocalization of one platform protein but not of another molecule both with the kinases such as PKN, PKC, PKA and casein kinase 1δ and with the phoshatases such as PP1, PP2A and PP2B was found in primary cultured neurons and in neurons of the brains from mice, rats, human controls and the demented patients. Subcellular localizations of the complex was seen in the perinuclear cytoplasm and the proximal neurites of control neurons. By contrast, it was accumulated in intracellular neurofibrillary tangles and degenerative neurites in AD brains, and also accumulated in ballooned neurons in FTD brains. In addition, the interaction of a FK-bincling protein, which is known to be associated with the conformational modification or the transport of cellular proteins and is reported to interact with the intracellular domain of amyloid precursor protein, was identified with the intraneuronal tangles in AD brains. Colocalization of an important enzyme in sphingolipid biosynthesis with tangles was also found in AD brains. Moreover, we reported the cognitive changes in the patients with Parkinson's disease, and the altered expression of a novel susceptibility gene in the schizophrenia patients with cognitive symptoms.
  Thus, our results suggest that a specific role of a scaffold protein with tau-kinases, tau-phosphatases and their interacting molecules in damaged neurons in the dementia including AD and FTD.
• Functional regulation of centrosome and Golgi apparatus by signal-anchoring proteins.
  Grant-in-Aid for Scientific Research (B)
  Kobe University
  2005 - 2007
  CG-NAP (centrosome and Golgi localized PKN-associated protein) is a coiled-coil protein identified as a binding protein for the regulatory domain of PKN that has a catalytic domain highly homologous to PKC in the carboxyl-terminal region and a unique regulatory domain in the amino-terminal region. In this study, we have analyzed the function of CG-NAP in the Golgi formation and centrosome splitting and obtained the results as follows.
  1. CG-NAP was found to interact with both microtubules and a cytoplasmic dynein subunit p150^ and localize to the Golgi apparatus in a microtubule-dependent manner. By examining the recovery process from the depolymerizing microtubules or inhibiting cytoplasmic dynein, it was revealed that CG-NAP is recruited to the minus ends of microtubules by interacting with cytoplasmic dynein, thereby localizes to the Golgi apparatus.
  2. The Golgi apparatus in mammalian cells forms a continuous ribbon of interconnected stacks of flat cisternae that are positioned close to the centrosome. Neither the molecular requirements for, nor the purpose of, Golgi ribbon formation are known. It was revealed that the Golgi apparatus is fragmented in the cells lacking CG-NAP at the Golgi. In these cells transport and glycosylation of membrane proteins occurred normally with some delay, indicating that these stacks are functional. These results suggest that CG-NAP is required for the lateral fusion of the Golgi stacks to form fully functional apparatus.
  3. The centrosome splitting occurs between duplicated centrosomes at late G2 phase, which is caused by phosphorylation of cohesion proteins between two centrosomes by a protein kinase Nek2A. The ratio of the cells with split centrosomes was increased when CG-NAP or kendrin was suppressed by siRNA. Further, these proteins were found to associate specifically with hyper-phosphorylated inactive Nek2A, suggesting their role in the suppression of Nek2A activity at centrosomes. Possible mechanisms of Nek2A inhibition, such as phosphorylation and binding, are being examined.
• Research of signal transduction to neuronal degeneration mediated by tau phorphorylation in dementia
  Grant-in-Aid for Scientific Research (C)
  Kobe University
  2004 - 2005
  Neuronal localization of a large complex inteacting with some kinases and phosphatases, which phosphorylate or dephosphorylate tau protein directly, was investigated to clarify the mechanism underlying neuronal degeneration seen in brain tissues of the patients with degenerative dementia including Alzheimer's disease (AD) and fronto-temporal dementia (FTD). These kinases include PKN, PKC, PKA and Casein kinase 1 delta, and the phosphatases include PP1, PP2A and PP2B. In primary cultured brain tissues and the brains from mice, rats, human controls and the demented patients, the complex molecule was localized immunohistochemically only in neurons. Study for subcellular localization revealed that the complex was distributed in the cytoplasm within neuronal cell bodies and proximal neuritis in normal tissues. By contrast, it was accumulated within neurofibrillary tangles and degenerative neuritis in AD brains, and also accumulated within ballooned cell bodies of residual neurons in FTD brains.
  In addition, neuropathological examinations were performed in some patients with familial dementia. Neuronal loss accompanied by axonal dystrophy and glial activations was remarkable in the hippocampus and frontal, parietal and occipital lobes in the brain of a patient with hereditary sensory neuropathy type 1 with deafness and dementia, while some patients with tau P301S mutation showed pathological changes in frontal and temporal lobes and subcortical nuclei, which consisted of neuronal loss, microvacuolation, astrocytic fibrosis, neuropil threads, ballooned neurons and glial fibrillary tangles.
  Thus, our results suggest a specific role of the interacting molecule with tau-kinases and phosphatases in neurodegeneration in the dementia including AD and FTD.
• Functional Analysis of an Anchoring Protein CG-NAP in the Golgi Apparatus.
  Grant-in-Aid for Scientific Research (C)
  Kobe University
  2003 - 2004
  CG-NAP is a giant coiled-coil protein localized at centrosome and the Golgi apparatus. To investigate the role of CG-NAP in the Golgi apparatus, the Golgi-targeting mechanism was analyzed and the effects of dissociation of CG-NAP from the Golgi were examined on the Golgi morphology and on the membrane traffic.
  1.By immunofluorescence microscopy of CG-NAP in intact and semi-intact cells, it was revealed that CG-NAP begins to dissociate from the Golgi at late G2 phase and completely dissociates at prophase.
  2.The Golgi-targeting region was identified to the amino-terminal region of CG-NAP by examining the localization of various deletion mutants. This region was effectively phosphorylated by mitotically activated protein kinase plk, which might cause dissociation of CG-NAP from the Golgi.
  3.Overexpression of the Golgi-targeting region resulted in dissociation of endogenous CG-NAP from the Golgi in COS cells. In these cells, Golgi marker proteins represented fragmented localization, suggesting that the Golgi apparatus is fragmented. Further, GFP-tagged Golgi marker proteins showed unstable movements in live-cell imaging. On the other hand, the transport of VSV-Gts protein from endoplasmic reticulum to plasma membrane was not inhibited.
  4.When CG-NAP expression was suppressed by siRNA, similar effects were observed on the Golgi morphology and on the VSV-Gts traffic.
  These results suggest that CG-NAP is involved in the formation and/or maintenance of the Golgi apparatus at pericentrosomal area.
• Functional Analysis of an Anchoring Protein CG-NAP in the Centrosome
  Grant-in-Aid for Scientific Research (C)
  Biosignal Research Center, Kobe University
  2001 - 2002
  Microtubule assembly is initiated by γ-tubulin ring complex (γ-TuRC). In yeast, microtubule is nucleated from γ-TuRC anchored to the amino terminus of the spindle pole body component SpcllOp, which interacts with Cmd1p (calmodulin) at the carboxyl terminus. However, mammalian protein that anchors γ-TuRC remains to be elucidated. A giant coiled-coil protein CG-NAP (c___entrosome and G___olgi localized PKN___-a___ssociated p___rotein) was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and shares high homology with the caroboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with γ-tubulin through binding with γ-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated each other, and with endogenous GCP2 and γ-tubulin, suggesting that CG-NAP and kendrin form complex and interact with γ-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule aster formation, moreover, combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in mammalian centrosome by anchoring γ-TuRC.