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

我们最近报道了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。为了阐明功能结果，我们制备了一种大鼠单抗，它特异性地识别在Ser301处磷酸化的Chk1。免疫细胞化学分析表明，Chk1-Ser301从进入有丝分裂前期就被磷酸化了。磷酸化水平在中期达到峰值，后期逐渐下降。在染色体凝聚而没有核膜破裂的早期，我们观察到两种磷酸化模式。在早期，磷酸化倾向于主要在细胞核内观察到。然而，在前期后期，Ser301处的Chk1磷酸化主要存在于细胞质中。这种转位可被软霉素B抑制，后者可阻断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著延迟，COM…与WT案相比，情况更糟。在S286A/S301a突变型前期细胞中，染色主要位于胞核，而在WT细胞中也可见胞浆染色。这些结果表明，CDK1通过对Chk1的磷酸化来控制Chk1的细胞质隔离。我们最近发现了两种新的角蛋白微丝结合蛋白：Trichoplein和Albatross。研究发现，Trichoplein不仅定位于角蛋白细丝上，还定位于母卵圆细胞上。在HeLa细胞中的基因敲除揭示了Trichoplein对于微管的锚定和Ninein在母中心粒的附属物上的固定是必不可少的。信天翁不仅定位于角蛋白细丝上，也定位于顶端连接复合体(AJC)附近，AJC是由紧密连接、粘连连接和桥粒组成的细胞-细胞黏附装置。在肺腺癌A549细胞中的基因敲除表明，信天翁调节细胞内AJC和侧区的形成。此外，我们发现角蛋白通过角蛋白拯救的SW13细胞促进这一功能。较少

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