APOPTOSIS GENERATES STABLE FRAGMENTS OF HUMAN TYPE-I KERATINS

Type I and II keratins help maintain the structural integrity of epith elial cells. Since apoptosis involves progressive cell breakdown, we e xamined its effect on human keratin polypeptides 8, 18, and 19 (K8, K1 8, K19) that are expressed in simple-type epithelia as noncovalent typ e I (K18, K19) and type II (K8) heteropolymers. Apoptosis induces rapi d hyperphosphorylation of most known K8/18 phosphorylation sites and d elayed formation of K18 and K19 stable fragments. In contrast, K8 is r esistant to proteolysis and remains associated with the K18 fragments. Transfection of phosphorylation/glycosylation-mutant K8 and K18 does not alter fragment formation. The protein domains of the keratin fragm ents were determined using epitope-defined antibodies, and microsequen cing indicated that K18 cleavage occurs at a conserved caspase-specifi c aspartic acid. The fragments are found preferentially within the det ergent-insoluble pool and can be generated, in a phosphorylation-indep endent manner, by incubating keratins with caspase-3 or with detergent lysates of apoptotic cells but not with lysates of nonapoptotic cells . Our results indicate that type I keratins are targets of apoptosis-a ctivated caspases, which is likely a general feature of keratins in mo st if not all epithelial cells undergoing apoptosis. Keratin hyperphos phorylation occurs early but does not render the keratins better subst rates of the downstream caspases.