Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks.
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 full-length 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.
Stappenbeck
T S
TS
Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611.
Bornslaeger
E A
EA
Corcoran
C M
CM
Luu
H H
HH
Virata
M L
ML
Green
K J
KJ
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D051180:Desmoplakins; D003896:Desmosomes / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D016259:Genes, myc; D006367:HeLa Cells; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism* / Q000648:ultrastructure; D007633:Keratins / Q000378:metabolism*; D007668:Kidney; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014746:Vimentin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003598:Cytoskeletal Proteins; C495294:DSP protein, human; D051180:Desmoplakins; C495295:Dsp protein, mouse; D011994:Recombinant Proteins; D014746:Vimentin; D007633:Keratins