Calcineurin Potentiates the Activation of Procaspase-3 by Accelerating Its Proteolytic Maturation
Journal of Biological Chemistry
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
... rin (calcineurin B). Overexpression of calcineurin B in HEK293 cells potentiated processing of caspase-3 and apoptosis triggered by tumor necrosis factor-␣ and cycloheximide treatment. In a cellfree 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. Apoptosis is a type of cell death resulting from the activation of a genetically regulated cell suicide program. In the nematode Caenorhabditis elegans, a genetic pathway of apoptosis has been identified. Two genes, ced-3 and ced-4, are essential for the execution of apoptosis (1), and ced-9 negatively regulates apoptosis by preventing activation of ced-3 and ced-4 (2). Caspases, human homologues of the C. elegans CED-3, are the key effectors in the execution of apoptosis (3, 4). Caspases are synthesized in cells as inactive zymogens (procaspases), which become proteolytically processed to generate active caspase. Although the exact mechanism that activates caspases is not fully understood, at least two major pathways have been described. In the case of the extrinsic pathway, membrane-. 2 The abbreviations used are: Apaf-1, apoptotic protease activation factor-1; AMC, aminomethylcoumarin; TNF-␣, tumor necrosis factor-␣; CHX, cycloheximide; Hsp, heat shock protein; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid; IAP, inhibitor of apoptosis; DTT, dithiothreitol; PIPES, 1,4-piperazinediethanesulfonic acid; DSS, disuccinimidyl suberate; siRNA, small interference RNA.