ANTIBODIES SPECIFIC FOR PROTEOLYZED FORMS OF PROTEIN-KINASE-C-ALPHA

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 alpha 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 alpha but not to th e unproteolyzed protein. To detect proteolyzed PKC alpha 'cleavage sit e-directed antibodies,' which recognize amino-terminal regions in the nascent catalytic fragments and do not cross-react with the unproteoly zed enzymes, were raised using synthetic peptides corresponding to the amino-terminal sequences. The synthetic peptides used in this study w ere the sequences of human PKC alpha at the cleavage sites by m- and m u-types of calpains (LGPAGNKV and VISPSEDRKQPSNNLDRVKLT, respectively) and they are designated as CF alpha 2, CF alpha 4, in this order. Eac h synthetic peptide was injected into rabbit after conjugation with a carrier protein. The antibodies thus obtained (anti-CF alpha 2 or -CF alpha 4) specifically reacted with either the 46- or 45-kDa catalytic fragment of PKC alpha, respectively, whereas they did not cross-react with other fragments. Furthermore, the antibodies did not bind to the unproteolyzed enzyme nor fragments of PKC alpha obtained by treatment with other proteinases unless the fragment carried the same amino-term inal sequence. When human platelets were treated with calcium ionophor e, the catalytic fragments of PKC alpha (45- and 46-kDa) were detected in the cytosol by immunoblotting with the antibodies. However, these antibodies did not bind unproteolyzed 80-kDa PKC alpha, although this form was dominant in the cytosol of the calcium ionophore-treated huma n platelets. In addition, the 45-kDa catalytic fragment of PKC alpha w as detected in an apoptotic human fibroblast TIG-3 cells cultured in s erum-free medium. Our method is applicable for analysis of proteolysis in various cellular states.