Limited proteolysis of filamin is catalyzed by caspase-3 in U937 and Jurkat cells.
Members of the caspase family have been implicated as key mediators of apoptosis in mammalian cells. However, few of their substrates are known to have physiological significance in the apoptotic process. We focused our screening for caspase substrates on cytoskeletal proteins. We found that an actin binding protein, filamin, was cleaved from 280 kDa to 170, 150, and 120 kDa major N-terminal fragments, and 135, 120, and 110 kDa major C-terminal fragments when apoptosis was induced by etoposide in both the human monoblastic leukemia cell line U937, and the human T lymphoblastic cell line Jurkat. The cleavage of filamin was blocked by a cell permeable inhibitor of caspase-3-like protease, Ac-DEVD-cho, but not by an inhibitor of caspase-1-like protease, Ac-YVAD-cho. These results suggest that filamin is cleaved by a caspase-3-like protease. To examine whether caspase-3 cleaves filamin in vitro, we prepared a recombinant active form of caspase-3 directly using a Pichia pastoris overexpression system. When we applied recombinant active caspase-3 to the cell lysate of U937 and Jurkat cells, filamin was cleaved into the same fragments seen in apoptosis-induced cells in vivo. Platelet filamin was also cleaved directly from 280 kDa to 170, 150, and 120 kDa N-terminal fragments, and the cleavage pattern was the same as observed in apoptotic human cells in vivo. These results suggest that filamin is an in vivo substrate of caspase-3.
Umeda
T
T
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Kouchi
Z
Z
Kawahara
H
H
Tomioka
S
S
Sasagawa
N
N
Maeda
T
T
Sorimachi
H
H
Ishiura
S
S
Suzuki
K
K
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D001792:Blood Platelets / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002467:Cell Nucleus / Q000648:ultrastructure; D003285:Contractile Proteins / Q000737:chemistry / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation; D005047:Etoposide / Q000494:pharmacology; D064448:Filamins; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010843:Pichia / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D020298:U937 Cells
D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D003285:Contractile Proteins; D015853:Cysteine Proteinase Inhibitors; D064448:Filamins; D008840:Microfilament Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D005047:Etoposide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases