Analysis of Intermediate Filament Binding Proteins

1.我们确定Aurora-B作为一个切割沟（CF）激酶。Aurora-B磷酸化III型中间丝（Ifs）、波形蛋白、GFAP和结蛋白，磷酸化导致它们的丝形成能力降低。我们接下来通过Aurora-B确定这些Ifs的磷酸化位点。IF突变体，其中Aurora-B和/或Rho-激酶的磷酸化位点改变为Ala或Gly，导致胞质分裂中子细胞之间的丝分离的显著缺陷。这些结果表明Aurora-B通过III型Ifs的切割沟特异性磷酸化与Rho激酶协调调节胞质分裂。我们鉴定了人TNF受体1（TNFR 1）相关死亡结构域蛋白（TRADD）是角蛋白18（K18）相互作用蛋白。内源性TRADD与K18共免疫沉淀，并与人乳腺上皮细胞中的K8/18细丝共定位。在不含角蛋白的SW 13细胞中过表达含有TRADD结合结构域的K18的NH_2-末端（aa 1-270）以及过表达K8/18，使细胞对TNF的杀伤更有抵抗力。这些结果表明K18可能通过隔离TRADD来减弱TRADD与活化的TNFR 1的相互作用，从而减轻TNF诱导的单纯上皮细胞凋亡.我们分离了δ-catenin/neural plakophilin-related armadillo-repeat protein（NPRAP）作为Densin- 180相互作用蛋白。Densin-180在体内与δ-catenin/NPRAP相关，并可能通过与δ-catenin/NPRAP-N-钙粘蛋白复合物的相互作用参与突触细胞-细胞连接的组织。

1. We identified Aurora-B as a cleavage furrow (CF) kinase. Aurora-B phosphorylates type III intermediate filaments (Ifs), vimentin, GFAP, and desmin, and the phosphorylation causes reduction of their filament forming ability. We next determined the phosphorylation sites of these Ifs by Aurora-B. IF mutants, in which phosphorylation sites by Aurora-B and/or Rho-kinase are changed to Ala or Gly, cause dramatic defect of filament separation between daughter cells in cytokinesis. These results indicate that Aurora-B coordinately regulates cytokinesis with Rho-kinase through the cleavage furrow-specific phosphorylation of type III Ifs.2. We identified human TNF receptor 1 (TNFR1)-associated death domain protein (TRADD) to be the keratin 18 (K18)-interacting protein. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH_2- terminus (aa 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. These results suggest that K18 may sequester TRADD to attenuate interactions of TRADD with activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.3. We isolated δ-catenin/neural plakophilin-related armadillo-repeat protein (NPRAP) as a Densin- 180-interacting protein. Densin-180 is associated in vivo with δ-catenin/NPRAP and may be involved in organization of the synaptic cell-cell junction through interaction with theδ-catenin/NPRAP-N- cadherin complex.