The expression of mutant epidermal keratin cDNAs transfected in simple epithelial and squamous cell carcinoma lines.
We have deleted cDNA sequences encoding portions of the carboxy-terminal end of a human type I epidermal keratin K14, and examined the molecular consequences of forcing the expression of these mutants in simple epithelial and squamous cell carcinoma lines. To follow the expression of our mutant products in transfected cells, we have tagged the 3' end of the K14 coding sequence with a sequence encoding an antigenic domain of the neuropeptide substance P. Using DNA transfection and immunohistochemistry (with an antibody against substance P), we have identified a collection of mutants that have a wide range of morphological effects on the endogenous keratin filament networks of transfected cells. Mutants that are missing most of the nonhelical carboxy-terminal domain of K14 incorporate into the endogenous keratin filaments without any visible perturbations on the network. In contrast, mutants that are missing as few as 10 of the 310 amino acids of the central alpha-helical domain of the polypeptide cause gross alterations in the keratin network. In some cases, the entire cytoskeletal network of keratins was disrupted, leaving no evidence of 8-nm filaments. These results reveal the existence of a dynamic exchange between newly synthesized subunits and preexisting keratin filaments.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002872:Chromosome Deletion; D004247:DNA / Q000378:metabolism*; D004848:Epithelium; D005455:Fluorescent Antibody Technique; D006801:Humans; D007633:Keratins / Q000235:genetics*; D009154:Mutation; D010957:Plasmids; D012867:Skin / Q000378:metabolism*; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D007633:Keratins; D004247:DNA