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

我们最近报道，在有丝分裂过程中，Chk 1被细胞周期蛋白依赖性激酶（Cdk）1磷酸化在Ser 286和Ser 301。为了阐明功能的后果，我们生产了一种大鼠单克隆抗体，它特异性地抑制在Ser 301磷酸化的Chk 1。使用该抗体的免疫细胞化学分析显示Chk 1-Ser 301磷酸化从进入有丝分裂（前期）。磷酸化水平在中期达到高峰，后期后逐渐下降。在分裂前期，当染色体凝聚发生而核膜不破裂时，我们观察到两种磷酸化模式。在早期阶段，磷酸化倾向于主要在细胞核中观察到。然而，在后期阶段的前期，在Ser 301磷酸化的Chk 1呼吁主要存在于细胞质中。这种易位被抑制治疗与链霉素B，它阻止Crm 1介导的核输出。S286 A/S301 A突变体的诱导表达导致有丝分裂进入的显著延迟， ...更多信息 关于WT的案例在S286 A/S301 A突变体前期细胞中，染色主要在细胞核中观察到，尽管在WT细胞中也在细胞质中发现定位。这些结果表明，Cdk 1通过磷酸化Chk 1来控制Chk 1的胞质螯合。我们最近发现了两种新的角蛋白结合蛋白：Appllein和Albatross。Trichoplein不仅定位于角蛋白丝，而且也定位于母中心孔。在HeLa细胞中的敲除揭示了微管蛋白和ninein在母亲中心粒的附属物上的锚定是必不可少的。信天翁不仅定位于角蛋白丝上，而且定位于顶端连接复合体（AJC）附近，AJC是由紧密连接、粘附连接和桥粒组成的细胞-细胞粘附器。在肺腺癌A549细胞中的敲除揭示了Albatross调节细胞中AJC和侧结构域的形成。此外，我们发现角蛋白促进这一功能，使用角蛋白拯救的SW 13细胞。少

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