Antibodies against c-Jun N-terminal peptide cross-react with neo-epitopes emerging after caspase-mediated proteolysis during apoptosis.
In previous studies it has been shown that neural cells undergoing programmed cell death display strongly positive cytoplasmic immunoreactivity to polyclonal antibodies directed against a c-Jun N-terminal peptide. It was later found that c-Jun-like immunoreactivity in apoptosis was due to cross-reactivity with proteins other than c-JUN: We have analysed the biochemical counterpart of this property in neuroblastoma cell lines treated to induce apoptosis. Using the c-Jun/sc-45 antibody, several bands with apparent molecular masses distinct from c-Jun were detected in extracts in parallel with both the degree of apoptosis and the appearance of the cytoplasmic signal after immunostaining. c-Jun/sc-45 immunostaining was prevented by caspase inhibitors and did not require de novo protein synthesis. One of the antigens recognized by the c-Jun/sc-45 antibody was identified as seryl-tRNA synthetase. We provide evidence that seryl-tRNA synthetase is a substrate of caspase-3 in vitro and that the digested form turns highly immunoreactive towards the antibody. A carboxy-terminus epitope of the protein that constitutes a consensus site for caspase-3 is involved in c-Jun/sc-45 recognition. This epitope shares some amino acids with the peptide used as the immunogen and this could explain the cross-reactivity observed. In conclusion, we demonstrate here that cytoplasmic c-Jun/sc-45-like immunoreactivity specific to apoptosis is due to post-translational changes which occur in seryl-tRNA synthetase and probably also in other proteins as a consequence of caspase mediated proteolysis.
Casas
C
C
Universitat de Lleida, Facultat de Medicina, Department of Ciències Mèdiques Bàsiques, Unitat de Neurobiologia Cellular, Lleida, Spain.
Ribera
J
J
Esquerda
J E
JE
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D017209:Apoptosis; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D007163:Immunosorbent Techniques; D008856:Microscopy, Fluorescence; D009447:Neuroblastoma / Q000737:chemistry / Q000473:pathology; D010446:Peptide Fragments / Q000276:immunology*; D016755:Proto-Oncogene Proteins c-jun / Q000032:analysis / Q000037:antagonists & inhibitors / Q000276:immunology*; D012714:Serine-tRNA Ligase / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000590:Amino Acid Chloromethyl Ketones; D000906:Antibodies; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D000939:Epitopes; D010446:Peptide Fragments; D016755:Proto-Oncogene Proteins c-jun; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D003513:Cycloheximide; D020169:Caspases; D012714:Serine-tRNA Ligase