Apoptosis Generates Stable Fragments of Human Type I Keratins*
- From the Veterans Administration Palo Alto Health Care System, Palo Alto, California 94304 and Digestive Disease Center, Stanford University School of Medicine, Palo Alto, California 94305, and§Clontech Laboratories Inc., Palo Alto, California 94303
Abstract
Type I and II keratins help maintain the structural integrity of epithelial cells. Since apoptosis involves progressive cell breakdown, we examined its effect on human keratin polypeptides 8, 18, and 19 (K8, K18, K19) that are expressed in simple-type epithelia as noncovalent type I (K18, K19) and type II (K8) heteropolymers. Apoptosis induces rapid hyperphosphorylation of most known K8/18 phosphorylation sites and delayed formation of K18 and K19 stable fragments. In contrast, K8 is resistant 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 fragments were determined using epitope-defined antibodies, and microsequencing indicated that K18 cleavage occurs at a conserved caspase-specific aspartic acid. The fragments are found preferentially within the detergent-insoluble pool and can be generated, in a phosphorylation-independent 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-activated caspases, which is likely a general feature of keratins in most if not all epithelial cells undergoing apoptosis. Keratin hyperphosphorylation occurs early but does not render the keratins better substrates of the downstream caspases.
Footnotes
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↵* This work was supported by Veterans Affairs Merit and Career Development Awards, NIH Grant DK47918, and Digestive Disease Center Grant DK38707.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom requests for reprints should be addressed: Palo Alto VA Medical Center, 154J, 3801 Miranda Ave., Palo Alto, CA 94304.
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↵¶ To whom other correspondence should be addressed: Palo Alto VA Medical Center, G.I. 111, 3801 Miranda Ave., Palo Alto, CA 94304.
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↵1 The abbreviations used are: IF, intermediate filament(s); An, anisomycin; Emp, Empigen BB; K, keratin; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PARP, poly(ADP-ribose) polymerase; pS, phospho-serine (Ser(P)); PVDF, polyvinylidene difluoride.
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↵2 N. O. Ku, J. Liao, and M. B. Omary, submitted for publication.
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- Received September 16, 1997.
