Calcineurin Potentiates the Activation of Procaspase-3 by Accelerating Its Proteolytic Maturation

Abstract

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 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, membranebound receptors are oligomerized in response to ligand binding and directly activate the initiator caspases (5). The intrinsic pathway involves release of cytochrome c from mitochondria into the cytosol. In the presence of cytochrome c, apoptotic protease activation factor-1 (Apaf-1), 2 a human homologue of the C. elegans CED-4, interacts with caspase-9 and produces a multiprotein complex, termed apoptosome (6 -10). The assembly of the apoptosome complex represents the initiating step for the activation of the intrinsic caspase cascade. Once activated in the apoptosome, caspase-9 cleaves procaspase-3.Multiple lines of evidence indicate that Apaf-1, cytochrome c, and caspase-9 are crucial components of the intrinsic pathway. Two other classes of regulators have been reported. The best known of these is the Bcl-2 family, a human homologue of the C. elegans CED-9. The Bcl-2 family consists of both antiapoptotic and proapoptotic members. Antiapoptotic proteins, such as Bcl-2, prevent the release of cytochrome c and thereby inhibit the subsequent activation of caspase (11). However, there is some evidence that Bcl-2 may prevent apoptosis downstream of cytochrome c. In C. elegans, CED-9 directly binds to CED-4 and inhibits the activation of CED-3 (12). It has remained unclear whether Bcl-2 inhibits the function of Apaf-1 by direct binding (13). Another family is the inhibitor of apoptosis (IAP) proteins, such as X-linked IAP. IAPs directly bind to active caspases and either suppress their protease activity or target them for destruction by ubiquitination and subsequent proteasome-mediated degradation (14, 15). X-linked IAP blocks the mitochondrial apoptosis pathway by inhibiting caspase activity directly (16). The activity of X-linked IAP is regulated by release of other factors, such as Smac/Diablo, from mitochondria (17, 18).To date, no activators have been identified that functi...

The Calcineurin B Subunit (CnB) Is a New Ligand of Integrin αM That Mediates CnB-Induced Apo2L/TRAIL Expression in Macrophages

“…However, in recent years, it has been shown that the CnB is not just a simple protein as the regulatory subunit of CnA. Cytosolic CnB can interact with tubulin, heat shock protein 60 (13), procaspase-3 (14), and PSMA7 (15) and therefore plays very important role in apoptosis and the proteasome pathway, which are not dependent on the phosphatase activity of CnA. In previous work, we found that i.p.…”

Exosome-Bound WD Repeat Protein Monad Inhibits Breast Cancer Cell Invasion by Degrading Amphiregulin mRNA

“…Total protein was mixed with Laemmli buffer, separated by SDS-PAGE and transferred to PVDF membranes (Millipore Corporation). Immunoblotting and immunoprecipitaion were carried out as described previously [24], [25]. Briefly, equal protein concentrations of lysates were incubated with antibody for 16 h, followed by incubation with Protein G Sepharose (Amersham Biosciences) for 1 h. The Sepharose beads were washed five times with the buffer described above, associated proteins were recovered by boiling in Laemmli buffer.…”

Novel Ser/Thr Protein Phosphatases in Cell Death Regulation

“…Dephosphorylation of caspase-3 at Ser150 site by PP2A increased caspase-3 activity, which was essential to trigger apoptosis in neutrophils (3). In addition, it has been shown that PP2B interacted with procaspase-3 and promoted caspase-3 maturation (100). On the other hand, protein phosphatases can be cleaved and regulated by caspases (66).…”