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 programm ed cell death display strongly positive cytoplasmic immunoreactivity to pol yclonal antibodies directed against a c-Jun N-terminal peptide. It was late r found that c-Jun-like immunoreactivity in apoptosis was due to crossreact ivity with proteins other than c-Jun. We have analysed the biochemical coun terpart of this property in neuroblastoma cell lines treated to induce apop tosis. Using the c-Jun/sc-45 antibody, several bands with apparent molecula r masses distinct from c-Jun were detected in extracts in parallel with bot h the degree of apoptosis and the appearance of the cytoplasmic signal afte r immunostaining. c-Jun/sc-45 immunostaining was prevented by caspase inhib itors and did not require de novo protein synthesis. One of the antigens re cognized by the c-Jun/sc-45 antibody was identified as seryl-tRNA synthetas e. 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 e pitope shares some amino acids with the peptide used as the immunogen and t his could explain the cross-reactivity observed. In conclusion, we demonstr ate here that cytoplasmic c-Jun/sc-45-like immunoreactivity specific to apo ptosis is due to post-translational changes which occur in seryl-tRNA synth etase and probably also in other proteins as a consequence of caspase media ted proteolysis.