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.
Physiological Chemistry, Biozentrum, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
Viviane
V
Hoppe
Jürgen
J
Hoppe
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D020169:Caspases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017354:Point Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D016895:Culture Media, Serum-Free; D020169:Caspases