Caspase-mediated proteolytic activation of calcineurin in thapsigargin-mediated apoptosis in SH-SY5Y neuroblastoma cells.
We previously demonstrated a loss in calmodulin (CaM)-dependent protein kinase activity in SH-SY5Y cells undergoing thapsigargin-mediated apoptosis, (K. M. McGinnis et al., 1998, J. Biol. Chem. 273, 19993-20000). Here we demonstrate that the large subunit of the CaM-dependent protein phosphatase 2B (calcineurin) is fragmented during SH-SY5Y cell apoptosis to a major fragment of 45 kDa in a caspase inhibitor-sensitive manner. A 45-kDa fragment was also produced when purified calcineurin was digested with recombinant caspase-3. The major cleavage site was identified to be DFGD* G(386)ATAA, which removes the C-terminal CaM-binding and autoinhibitory regions from the catalytic domain. Phosphatase activity increased progressively with caspase-3 digestion, coupled with the eventual loss of CaM-dependency. Calcineurin-mediated dephosphorylation of NFATc was also detected in thapsigargin-treated cells. Last, calcineurin inhibitors FK506 and cypermethrin provided partial protection against thapsigargin-mediated apoptosis, suggesting that calcineurin overactivation contributes to thapsigargin-induced apoptosis.
Mukerjee
N
N
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
McGinnis
K M
KM
Park
Y H
YH
Gnegy
M E
ME
Wang
K K
KK
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001665:Binding Sites; D019703:Calcineurin / Q000737:chemistry / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002417:Cattle; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D050778:NFATC Transcription Factors; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011994:Recombinant Proteins; D019284:Thapsigargin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D065095:Calcineurin Inhibitors; D061945:Caspase Inhibitors; D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014157:Transcription Factors; D019284:Thapsigargin; D019703:Calcineurin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases