Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks.

Abstract

Abstract. We previously demonstrated that truncated desmoplakin I (DP I) molecules containing the carboxyl terminus specifically coalign with and disrupt both keratin and vimentin intermediate filament (IF) networks when overexpressed in tissue culture cells (Stappenbeck, T. S., and K. J. Green. J. Cell Biol. 116:1197-1209. These experiments suggested that the DP carboxyl-terminal domain is involved either directly or indirectly in linking IF with the desmosome. Using a similar approach, we have now investigated the behavior of ectopically expressed fulllength DP I in cultured cells. In addition, we have further dissected the functional sequences in the carboxyl terminus of DP I that facilitate the interaction with IF networks. Transient transfection of a clone encoding full-length DP I into COS-7 cells produced protein that appeared in some cells to associate with desmosomes and in others to coalign with and disrupt IF. Deletion of the carboxyl terminus from this clone resulted in protein that still appeared capable of associating with desmosomes but not interacting with IF networks. As the amino terminus appeared to be dispensable for IF interaction, we made finer deletions in the carboxyl terminus of DP based on blocks of sequence similarity with the related molecules bullous pemphigoid antigen and plectin. We found a sequence at the very carboxyl terminus of DP that was necessary for coalignment with and disruption of keratin IF but not vimentin IF. Furthermore, the coalignment of specific DP proteins along keratin IF but not vimentin IF was correlated with resistance to extraction by Triton. The striking uncoupling resulting from the deletion of specific DP sequences suggests that the carboxyl terminus of DP interacts differentially with keratin and vimentin IF networks.

Interaction of periplakin and envoplakin with intermediate filaments

“…We have previously shown that the C subdomain of envoplakin does not colocalise with intermediate filaments in transiently transfected cells (DiColandrea et al, 2000); the same is true of the isolated C box of desmoplakin (Stappenbeck et al, 1993). Different regions of the desmoplakin C-terminus interact with keratins and vimentin (Stappenbeck et al, 1993;Meng et al, 1997) and in assays with purified recombinant proteins, the desmoplakin C-terminus is reported to interact directly with keratin 5, but not with simple type II keratins, vimentin or type I keratins (Kouklis et al, 1994).…”

Characterization of human epiplakin: RNAi-mediated epiplakin depletion leads to the disruption of keratin and vimentin IF networks

“…These bands also showed different apparent molecular masses in different types of cells. Of note, the precise molecular mass of each band could not be determined since it is known that plakin proteins run aberrantly in SDS-PAGE gel (Stappenbeck et al, 1993). Nevertheless, using the rabbit skeletal myofibril, which contained titin, nebulin and myosin as reference markers, we estimated that the sizes of human epiplakin ranged from 510 kDa in NHEK cells (lower band) to 750 kDa in HaCaT cells (higher band).…”

Structure and Regulation of the Envoplakin Gene

“…The linker domain immediately C-terminal to the rod domain is the most conserved part of the plakin family (14,15,23), and some paraneoplastic pemphigus autoantibodies cross-react with C-terminal fusion proteins of envoplakin, periplakin, and desmoplakin (23). The function of this part of the plakin proteins has not been demonstrated conclusively but is likely to be involved, together with the C-terminal repeats, in the interaction of plakins with intermediate filaments (42,43). The presence of a potential protein kinase C phosphorylation site in the linker further suggests a regulatory role in protein interactions for this domain.…”