Analysis of Intermediate Filament Binding Proteins

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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.