Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein fami ly, They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they o ccur in the nuclei of cells and, in epithelial cells, at the plasma membran e within the desmosomal plaques, We have shown by solid phase-binding assay s that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epitheli al cells and, to some extent, to vimentin, In line with this we show that r ecombinant PKP1a binds strongly to Ifs assembled in vitro from CKs 8/18, 5/ 14, vimentin or desmin and integrates them into thick (up to 120 nm in diam eter) IF bundles extending for several mu m. The basic amino-terminal, non- arm-repeat domain of PKP1a is necessary and sufficient for this specific in teraction as shown by blot overlay and centrifugation experiments, in parti cular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio, In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunop recipitation and sucrose density fractionation, The significance of these i nteractions of PKP1a with IF proteins on the one hand and desmoplakin on th e other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended Ifs in vivo, We postulate that (1) effective ce llular regulatory mechanisms exist that prevent plakophilins from unschedul ed IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recrui tment of plakophilins to the desmosomal plaques.