2-HYBRID ANALYSIS REVEALS FUNDAMENTAL DIFFERENCES IN DIRECT INTERACTIONS BETWEEN DESMOPLAKIN AND CELL-TYPE-SPECIFIC INTERMEDIATE FILAMENTS

Desmosomes are cell junctions that act as sites of strong intercellula r adhesion and also serve to anchor the intermediate filament (IF) cyt oskeleton to the plasma membrane of a variety of cell types, Previous studies demonstrated that the COOH terminus of the desmosomal plaque p rotein, desmoplakin (DP), is required for the association of DP with I F networks in cultured cells and that this domain interacts directly w ith type II epidermal keratin polypeptides in vitro. However, these st udies left open the question of how desmosomes might anchor other IF t ypes known to associate with these junctions. In this report we used y east two-hybrid and in vitro dot blot assays to further examine the re quirements for direct interactions between desmoplakin and various IF types. Our results confirm the ability of the DP COOH terminus (DPCT) to interact with at least two regions of the head domain of the type I I epidermal keratin K1 and also demonstrate that DPCT can interact wit h the type III IF family members, vimentin and desmin, as well as simp le epithelial keratins. Unlike the situation for type II epidermal ker atins, the interaction between DPCT and simple epithelial keratins app ears to depend on heterodimerization of the type I and II keratin poly peptides, since both are required to detect an interaction. Furthermor e, although the interaction between DPCT and K1 requires the keratin h ead domain, deletion of this domain from the simple epithelial keratin s does not compromise interaction with DPCT. The interaction between D PCT and type III or simple epithelial keratins also appeared to be les s robust than that between DPCT and K1, In the case of K8/K18, however , the interaction as assessed by yeast two-hybrid assays increased 9-f old when a serine located in a protein kinase A consensus phosphorylat ion site 23 residues from the end of DP was altered to a glycine. Take n together, these data indicate that DP interacts directly with differ ent IF types in specific ways.