CASPASE CLEAVAGE OF KERATIN-18 AND REORGANIZATION OF INTERMEDIATE FILAMENTS DURING EPITHELIAL-CELL APOPTOSIS

Keratins 8 (K8) and 18 (K18) are major components of intermediate fila ments (IFs) of simple epithelial cells and tumors derived from such ce lls. Structural cell changes during apoptosis are mediated by protease s of the caspase family. During apoptosis, K18 Ifs reorganize into gra nular structures enriched for K18 phosphorylated on serine 53. K18, bu t not K8, generates a proteolytic fragment during drug-and UV light-in duced apoptosis; this fragment comigrates with K18 cleaved in vitro by caspase-6, -3, and -7. K18 is cleaved by caspase-6 into NH2-terminal, 26-kD and COOH-terminal, 22-kD fragments; caspase-3 and -7 additional ly cleave the 22-kD fragment into a 19-kD fragment. The cleavage site common for the three caspases was the sequence VEVD/A, located in the conserved L1-2 linker region of K18. The additional site for caspases- 3 and -7 that is not cleaved efficiently by caspase-6 is located in th e COOH-terminal tail domain of K18. Expression of K18 with alanine ins tead of serine at position 53 demonstrated that cleavage during apopto sis does not require phosphorylation of serine 53. However, K18 with a glutamate instead of aspartate at position 238 was resistant to prote olysis during apoptosis. Furthermore, this cleavage site mutant appear s to cause keratin filament reorganization in stably transfected clone s. The identification of the L1-2 caspase cleavage site, and the conse rvation of the same or very similar sites in multiple other intermedia te filament proteins, suggests that the processing of Ifs during apopt osis may be initiated by a similar caspase cleavage.