Dissection of protein linkage between keratins and pinin, a protein with dual location at desmosome-intermediate filament complex and in the nucleus

Pinin is a cell adhesion-associated and nuclear protein that has been shown to localize in the vicinity of intermediate filament (IF) convergence upon the cytoplasmic face of the desmosomal plaque as well as in the nucleus. The localization of pinin to the desmosomes has been correlated with the reinforcement of intercellular adhesion and increased LF organization. In this study, keratins 18, 8, and 19 were identified to interact with the amino end domain of pinin in a two-hybrid screening. Further truncation analyses indicated that the 2B domain of keratin contains the sequence responsible for interacting with pinin, The amino end of pinin (residues 1-98) is sufficient to bind to keratin, Point mutation analyses revealed two essential residues within the pinin fragment 1-98, leucine 8 and leucine 19, for the interaction with keratin, Finally, in vitro protein overlay binding assays confirmed the direct interaction of the amino end domain of pinin with keratins, while pinin mutant L8P GST fusion protein failed to bind to keratins in the overlay assay. Coupled with our previous morphological observations and transfection studies, these data suggest that pinin may play a role in epithelial cell adhesion and the IF complex through a direct interaction with the keratin filaments.