Keratin 8/18 breakdown and reorganization during apoptosis.
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 trade mark 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.
Schutte
Bert
B
Department of Molecular Cell Biology (Box 17), Research Institute Growth and Development (GROW), University of Maastricht, The Netherlands. bert.schutte@molcelb.unimaas.nl
Henfling
Mieke
M
Kölgen
Wendy
W
Bouman
Maartje
M
Meex
Stephan
S
Leers
Mathie P G
MP
Nap
Marius
M
Björklund
Viveka
V
Björklund
Peter
P
Björklund
Bertil
B
Lane
E Birgitte
EB
Omary
M Bishr
MB
Jörnvall
Hans
H
Ramaekers
Frans C S
FC
D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D053179:Caspase 7; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D020134:Catalytic Domain / Q000502:physiology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D006801:Humans; D053538:Keratin-18; D053533:Keratin-8; D007633:Keratins / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000502:physiology
C488421:KRT18 protein, human; C426203:KRT8 protein, human; D053538:Keratin-18; D053533:Keratin-8; D007633:Keratins; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053179:Caspase 7; D053453:Caspase 9; D020169:Caspases