Calcium binding of ARC mediates regulation of caspase 8 and cell death.
Apoptosis repressor with CARD (ARC) possesses the ability not only to block activation of caspase 8 but to modulate caspase-independent mitochondrial events associated with cell death. However, it is not known how ARC modulates both caspase-dependent and caspase-independent cell death. Here, we report that ARC is a Ca(2+)-dependent regulator of caspase 8 and cell death. We found that in Ca(2+) overlay and Stains-all assays, ARC protein bound to Ca(2+) through the C-terminal proline/glutamate-rich (P/E-rich) domain. ARC expression reduced not only cytosolic Ca(2+) transients but also cytotoxic effects of thapsigargin, A23187, and ionomycin, for which the Ca(2+)-binding domain of ARC was indispensable. Conversely, direct interference of endogenous ARC synthesis by targeting ARC enhanced such Ca(2+)-mediated cell death. In addition, binding and immunoprecipitation analyses revealed that the protein-protein interaction between ARC and caspase 8 was decreased by the increase of Ca(2+) concentration in vitro and by the treatment of HEK293 cells with thapsigargin in vivo. Caspase 8 activation was also required for the thapsigargin-induced cell death and suppressed by the ectopic expression of ARC. These results suggest that calcium binding mediates regulation of caspase 8 and cell death by ARC.
Jo
Dong-Gyu
DG
Department of Life Science, Gwangju Institute of Science and Technology, 1 Oryongdong, Bukgu, Gwangju 500-712, South Korea.
Jun
Joon-Il
JI
Chang
Jae-Woong
JW
Hong
Yeon-Mi
YM
Song
Sungmin
S
Cho
Dong-Hyung
DH
Shim
Sang Mi
SM
Lee
Ho-June
HJ
Cho
Chunghee
C
Kim
Do Han
DH
Jung
Yong-Keun
YK
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D019169:Jurkat Cells; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
D051017:Apoptosis Regulatory Proteins; D009124:Muscle Proteins; C112246:NOL3 protein, human; D011994:Recombinant Proteins; D019284:Thapsigargin; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases; D002118:Calcium