Caspase-mediated cleavage and activation of LIM-kinase 1 and its role in apoptotic membrane blebbing.
Actin cytoskeletal reorganization plays a critical role in cell morphological changes, including membrane blebbing during apoptosis. LIM-kinase 1 (LIMK1) regulates actin cytoskeletal reorganization by phosphorylating and inactivating cofilin, an actin filament-depolymerizing and -severing protein. We now report that LIMK1 is cleaved and activated during anti-Fas antibody-induced apoptosis in Jurkat T cells. The cleavage and activation of LIMK1 were blocked by z-DEVD-fmk, an inhibitor for caspase-3 or related proteases, thus indicating that caspase-3-like proteases are responsible for LIMK1 cleavage. The caspase-mediated cleavage of LIMK1 occurs at Asp-240, a site at the N-terminal side of the protein kinase domain, which leads to the production of an N-terminally truncated, constitutively active LIMK1 fragment. Expression of an N-terminally truncated LIMK1 fragment, LIMK1(241-647), induced membrane blebbing in both Jurkat and HeLa cells, with an extent significantly higher than that of wild-type LIMK1. Down-regulation of endogenous LIMK1 expression by small interfering RNA (siRNA) reduced the Fas-induced membrane blebbing in Jurkat cells. These findings suggest that caspase-mediated cleavage and activation of LIMK1 play a role in the membrane bleb formation during apoptosis.
Tomiyoshi
Go
G
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
Horita
Yuji
Y
Nishita
Michiru
M
Ohashi
Kazumasa
K
Mizuno
Kensaku
K
D051339:Actin Depolymerizing Factors; D000911:Antibodies, Monoclonal / Q000378:metabolism; D017209:Apoptosis / Q000276:immunology / Q000502:physiology*; D020169:Caspases / Q000378:metabolism*; D002462:Cell Membrane / Q000473:pathology*; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D054461:Lim Kinases; D008840:Microfilament Proteins; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000494:pharmacology; D010957:Plasmids; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors; D016335:Zinc Fingers / Q000502:physiology*; D019014:fas Receptor / Q000276:immunology
D051339:Actin Depolymerizing Factors; D000911:Antibodies, Monoclonal; D015853:Cysteine Proteinase Inhibitors; D008840:Microfilament Proteins; D009842:Oligopeptides; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D019014:fas Receptor; D003513:Cycloheximide; D011494:Protein Kinases; C515892:LIMK1 protein, human; D054461:Lim Kinases; D020169:Caspases