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

阐明蛋白质磷酸化信号和细胞骨架的调控机制

基本信息

批准号：
18370085
负责人：
INAGAKI Masaki
金额：
$ 11.28万
依托单位：
Aichi Cancer Center Research Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18370085/
关键词：
protein phosphorylation cell cycle cytoskeleton cell adhesion cell polarity intermediate filament centrosome ケラチン

项目摘要

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

我们最近报道 Chk1 在有丝分裂过程中被细胞周期蛋白依赖性激酶 (Cdk) 1 在 Ser286 和 Ser301 处磷酸化。为了阐明功能后果，我们生产了一种大鼠单克隆抗体，它特异性识别 Ser301 位点磷酸化的 Chk1。使用该抗体进行的免疫细胞化学分析显示，从进入有丝分裂（前期）起，Chk1-Ser301 就会磷酸化。磷酸化水平似乎在中期达到峰值，并在后期后逐渐降低。在前期，当染色体凝聚发生而核膜没有破裂时，我们观察到两种磷酸化模式。在早期阶段，磷酸化往往主要在细胞核中观察到。然而，在前期后期，Chk1在Ser301处磷酸化，主要存在于细胞质中。这种易位被瘦霉素 B 抑制，从而阻断 Crm1 介导的核输出。 S286A/S301A 突变体的诱导表达导致有丝分裂进入显着延迟，与 WT 情况相比。在S286A/S301A突变前期细胞中，主要在细胞核中观察到染色，但在WT细胞中也发现在细胞质中。这些结果表明，Cdk1 通过 Chk1 的磷酸化控制 Chk1 的细胞质隔离。我们最近鉴定了两种新型角蛋白丝结合蛋白：trichoplein 和 Albatross。人们发现 Trichoplein 不仅位于角蛋白丝上，而且还位于母中心粒上。 HeLa 细胞中的敲低表明，毛胆蛋白对于微管和母体中心粒附属物上的九蛋白的锚定至关重要。 Albatross 不仅定位于角蛋白丝，还定位于顶端连接复合体（AJC）附近，这是一种由紧密连接、粘附连接和桥粒组成的细胞间粘附装置。在 A549（肺腺癌）细胞中的敲低表明 Albatross 调节细胞中 AJC 和侧向结构域的形成。此外，我们使用角蛋白拯救的 SW13 细胞发现角蛋白可以促进这种功能。较少的