Journal
EXPERIMENTAL CELL RESEARCH
Volume 297, Issue 1, Pages 11-26Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.yexcr.2004.02.019
Keywords
M30-CytoDeath; caspase; keratin; phosphorylation; cytoskeleton
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Monoclonal antibodies that specifically recognize caspase cleaved K18 fragments or specific (phospho)epitopes on intact K8 and K18 were used for a detailed investigation of the temporal and causal relationship of proteolysis and phosphorylation in the collapse of the keratin cytoskeleton during apoptosis. Caspases involved in the specific proteolysis of keratins were analyzed biochemically using recombinant caspases and specific caspase inhibitors. Finally, the fate of the keratin aggregates was analyzed using the M30-ApoptoSense(TM) Elisa kit to measure shedding of caspase cleaved fragments into the supernatant of apoptotic cell cultures. From our studies, we conclude that C-terminal K18 cleavage at the (393)DALD/S site is an early event during apoptosis for which caspase 9 is responsible, both directly and indirectly by activating downstream caspases 3 and 7. Cleavage of the L1-2 linker region of the central alpha-helical rod domain is responsible for the final collapse of the keratin scaffold into large aggregates. Phosphorylation facilitates formation of these aggregates, but is not crucial. K8 and K18 remain associated in heteropolymeric aggregates during apoptosis. At later stages of the apoptotic process, that is, when the integrity of the cytoplasmic membrane becomes compromised, keratin aggregates are shed from the cells. (C) 2004 Elsevier Inc. All rights reserved.
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