Antibodies specific for proteolyzed forms of protein kinase C α

Abstract

The activation of protein kinase C (PKC) is irreversibly regulated by limited proteolysis catalyzed by a calcium-activated neutral cysteine protease, calpain. Calpain cleaves PKC α at specific sites in the hinge region between the catalytic and the regulatory domains of this kinase. Here we show a novel method for production of antibodies that bind specifically to the catalytic fragment of PKC α but not to the unproteolyzed protein. To detect proteolyzed PKC α ‘cleavage site-directed antibodies,’ which recognize amino-terminal regions in the nascent catalytic fragments and do not cross-react with the unproteolyzed enzymes, were raised using synthetic peptides corresponding to the amino-terminal sequences. The synthetic peptides used in this study were the sequences of human PKC α at the cleavage sites by m- and μ-types of calpains (LGPAGNKV and VISPSEDRKQPSNNLDRVKLT, respectively) and they are designated as CFα2, CFα4, in this order. Each synthetic peptide was injected into rabbit after conjugation with a carrier protein. The antibodies thus obtained (anti-CFα2 or -CFα4) specifically reacted with either the 46- or 45-kDa catalytic fragment of PKCα, respectively, whereas they did not cross-react with other fragments. Furthermore, the antibodies did not bind to the unproteolyzed enzyme nor fragments of PKCα obtained by treatment with other proteinases unless the fragment carried the same amino-terminal sequence. When human platelets were treated with calcium ionophore, the catalytic fragments of PKCα (45- and 46-kDa) were detected in the cytosol by immunoblotting with the antibodies. However, these antibodies did not bind unproteolyzed 80-kDa PKCα, although this form was dominant in the cytosol of the calcium ionophore-treated human platelets. In addition, the 45-kDa catalytic fragment of PKCα was detected in an apoptotic human fibroblast TIG-3 cells cultured in serum-free medium. Our method is applicable for analysis of proteolysis in various cellular states.