Calcineurin potentiates the activation of procaspase-3 by accelerating its proteolytic maturation.
We have previously shown that procaspase-3 exists in a high molecular weight complex in neonatal rat brain. Here, we purify and identify the protein that interacts with procaspase-3 from rat neonatal cortex. We searched binding proteins to procaspase-3 from a cytosolic extract of neonatal rat brain using chromatogram, two-dimensional gel electrophoresis, and far Western immunoblot. Analysis by tandem mass spectrometry identified the protein as a regulatory subunit of calcineurin (calcineurin B). Overexpression of calcineurin B in HEK293 cells potentiated processing of caspase-3 and apoptosis triggered by tumor necrosis factor-alpha and cycloheximide treatment. In a cell-free system, overexpression of calcineurin B in HEK293 cells markedly increased processing of caspase-3 by cytochrome c. Immunodepletion of calcineurin B from cytosolic extracts from Jurkat cells decreased processing of caspase-3 by cytochrome c. Knockdown of calcineurin B by RNA interference resulted in reduced apoptosis in HEK293 cells but not in caspase-3-deficient MCF-7 cells. These results suggest that calcineurin B potentiates the activation of procaspase-3 by accelerating its proteolytic maturation.
Saeki
Makio
M
Department of Pharmacology, Graduate School of Dentistry, Osaka University, Suita, Osaka 565-0871, Japan. msaeki@dent.osaka-u.ac.jp
Irie
Yasuyuki
Y
Ni
Lin
L
Itsuki
Yuki
Y
Terao
Yutaka
Y
Kawabata
Shigetada
S
Kamisaki
Yoshinori
Y
D000818:Animals; D017209:Apoptosis; D001921:Brain / Q000378:metabolism; D019703:Calcineurin / Q000378:metabolism / Q000502:physiology*; D053148:Caspase 3 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D003513:Cycloheximide / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D051381:Rats; D017208:Rats, Wistar; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D014409:Tumor Necrosis Factor-alpha; D045304:Cytochromes c; D003513:Cycloheximide; D019703:Calcineurin; D053148:Caspase 3; D053453:Caspase 9