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

我们最近报道了在有丝分裂期间，Cyclin依赖性激酶（CDK）1在Ser286和Ser301上磷酸化。为了阐明功能后果，我们产生了一种大鼠单克隆抗体，该抗体是在Ser301处磷酸化的特异性CHK1。使用该抗体的免疫细胞化学分析显示，进入有丝分裂的CHK1-SER301磷酸化（Prophase）。磷酸化水平似乎在中期达到峰值，并在后期后逐渐降低。在预言中，当染色体凝结发生而没有核包膜破裂时，我们观察到了两种磷酸化模式。在较早的阶段，噬菌体倾向于主要在核用户中观察到。然而，在预言的后期，在Ser301处磷酸化的CHK1似乎主要存在于细胞质中。通过用瘦霉素B处理阻断CRM1介导的核出口的治疗抑制了这种易位。诱导的S286A/S301A突变体的表达导致有丝分裂进入，COM…对WT病例更加张紧。在S286A/S301A突变体预言细胞中，尽管在细胞质中也发现了WT细胞位置，但主要在核中观察到染色。这些结果表明，CDK1通过CHK1的磷酸化控制CHK1的细胞质隔离。我们最近确定了两种新型的角蛋白丝结合蛋白：毛状蛋白酶和信天翁。发现Trichoplein不仅可以定位于角蛋白丝上，还可以在母半元素上定位。 HeLa细胞中的敲低表明，毛状蛋白酶对于锚定微管和9inein在母centrioles的附属物上是必不可少的。信天翁不仅将其定位在角蛋白丝上，而且还位于顶端连接络合物（AJC）附近，这是一个由紧密连接，粘附连接体和脱骨体组成的细胞粘合剂。在A549，肺腺癌中敲低，细胞显示信天翁调节AJC的形成和细胞中侧域的形成。此外，我们发现角蛋白使用角蛋白反应的SW13细胞促进了该功能。较少的