Mutations dislocate caspase‐12 from the endoplasmatic reticulum to the cytosol

Mouse AKR‐2B cells express two forms of caspase‐12: the full‐length form coding for a protein of 47.8 kDa and a new splice variant of 40.2 kDa which is devoid of the CARD domain. In addition, three point mutations were disclosed: I/L‐15, E/D‐46 and P/L‐105. A major portion of the two protein variants was found in the cytosol. Immunofluorescence studies showed an even distribution of caspase‐12 within the cell, indicative for a cytoplasmatic localization. Transfection of AKR‐2B cells with wild‐type caspase‐12 showed a colocalization of this protein with the endoplasmic reticulum (ER). Unlike mouse embryonal fibroblasts (MEF) which contain wild‐type caspase‐12, AKR‐2B cells were largely resistant against treatment with the endoplasmatic reticulum stressing reagents brefeldin and tunicamycin. In AKR‐2B cells, cytoplasmatic caspase‐12 is bound to high molecular weight complexes of >1000 kDa [Cell Death Differ. 9 (2001) 125] and serum depletion leads to cleavage and detachment of caspase‐12 from this high molecular weight complex. Cleavage of caspase‐12 and ‐3 occurred almost simultaneously reaching a maximum 3–5 h after serum deprivation at which time also maximum apoptosis is found. Analysis of caspase‐12 cleavage in vitro in comparison with fragmentation in vivo suggests that during death in AKR‐2B cells induced by starvation, cleavage was brought about by caspase‐3 at positions D24 and D94. Thus, mutated caspase‐12 is differently integrated in signaling pathways of cell death and has lost its function as initiator caspase upon ER‐stress. Instead, it is turned into a substrate of effector caspases. The implication of these findings in the pathological phenotype of ARK‐2B mice is discussed.