Effect of mutation and phosphorylation of type I keratins on their caspase-mediated degradation

Type I keratins K18 and K19 undergo caspase-mediated degradation during apo ptosis, Two known K18 caspase cleavage sites are aspartates in the consensu s sequences VEVDA and DALDS, located within the rod domain and tail domain, respectively. Several K14 (another type I keratin) mutations within the ca spase cleavage motif have been described inpatients with epidermolysis bull osa simplex. Here we use extensive mutational analysis to show that K19 and K14 are caspase substrates and that the ability to undergo caspase-mediate d digestion of K18, K19, or K14 is highly dependent on the location and nat ure of the mutation within the caspase cleavage motif, Caspase cleavage of K14 occurs at the aspartate of VEMDA, a consensus sequence found in type I keratins K12-17 with similar but not identical sequences in K18 and K19. Fo r K18, apoptosis-induced cleavage occurs sequentially, first at (393)DALD a nd then at (VEVD)-V-234. Hyperphosphorylation of K18 protects from caspase- 3 in vitro digestion at (VEVD)-V-234 but not at (393)DALD. Hence, keratins K12-17 are likely caspase substrates during apoptosis. Keratin hyperphospho rylation, which occurs early in apoptosis, protects from caspase-mediated K 18 digestion in a cleavage site-specific manner. Mutations in epidermolysis bullosa simplex patients could interfere with K14 degradation during apopt osis, depending on their location.