Elucidation of the protein phosphorylation signaling and the regulatory mechanism of cytoskeleton

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We recently reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase(Cdk) 1 during mitosis. In order to elucidate functional consequences, we produced a rat monoclonal antibody which specifically recoginzes Chk1 phosphorylated at Ser301. Immunocytochemical analyses using this antibody revealed Chk1-Ser301 phosphorylation from entry into mitosis(prophase). The level of phosphorylation appeared to peak at metaphase and to gradually decrease after anaphase. In prophase when chromosome condensation occurs without nuclear envelope breakdown, we observed two phosphorylation patterns. At earlier stages, the phophorylation tended to be observed mainly in the nucleus. However, at later stages of prophase, Chk1 phosphorylated at Ser301 appealed to exist mainly in the cytoplasm. This translocation was inhibited by treatment with leptomycin B, which blocks Crm1-mediated nuclear export. Induced expression of S286A/S301A mutant caused significant delay in mitotic entry, com … More pared with the WT case. In S286A/S301A mutant prophase cells, staining was observed mainly in the nucleus although in WT cells location was also found in the cytoplasm. These results suggested that Cdk1 controls the cytoplasmic sequestration of Chk1 through the phosphorylation of Chk1.We recently identified two novel keratin filament-binding proteins : trichoplein and Albatross. Trichoplein was found to localize not only on keratin filaments but also on mother centnoles. Knockdown in HeLa cells revealed that trichoplein is essential for anchoring of microtubules and ninein on appendages of mother centrioles. Albatross localizes not only on keratin filaments but also in the vicinity of the apical junctional complex(AJC), a cell-cell adhesive apparatus composed of tight junctions, adherens junctions and desmosomes. Knockdown in A549, lung adenocarcinoma, cells revealed that Albatross regulates the formation of AJC and lateral domains in cells. In addition, we found that keratin promotes this function, using keratin-rescued SW13 cells. Less