Novel antibodies specific for proteolyzed forms of protein kinase C: production of anti-peptide antibodies available for in situ analysis of intracellular limited proteolysis.
We show here a novel method for the in situ analysis of proteolyzed proteins in a cell. As a model, we focused on protein kinase C (PKC) beta, which is cleaved at a specific site between the catalytic and regulatory domains by calpain, the intracellular calcium-activated neutral proteinase. To detect proteolyzed PKC beta 'cleavage-site-directed antibodies', which specifically recognize the amino-terminal region of the catalytic fragment but do not cross-react with the unproteolyzed enzymes, were raised using synthetic peptide. The synthetic peptide used in this study was QGTKVPEEKTT, corresponding to the amino-terminal region of the catalytic fragment from human PKC beta generated by calpain. Rabbits were immunized with the synthetic peptide after conjugation with a carrier protein. Antibodies obtained reacted with the 46-kDa catalytic fragment of PKC beta, whereas they did not cross-react with unproteolyzed enzyme nor other fragments with different amino-termini. Thus, our antibody is specific to the amino-terminal sequence QGTKVPEEKTT, but does not recognize the same sequence located internally in native PKC beta. When human monoblast U937 cells were treated with calcium ionophore, the catalytic fragment of PKC beta was detected in the cytosol by immunoblotting with the antibody. However, this antibody did not bind unproteolyzed 80-kDa PKC beta, although this form was dominant in the cytosol of the calcium ionophore-treated cells. We could also detect comparable amounts of catalytic fragment in the calcium ionophore-treated cells by immunocytochemical staining with the same antibody. Our method was applied to examine the proteolysis of PKC beta in neutrophils stimulated with various reagents.
Kikuchi
H
H
Institute of Medical Science, University of Tokyo, Japan.
Imajoh-Ohmi
S
S
Kanegasaki
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000917:Antibody Formation; D000001:Calcimycin / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism; D002460:Cell Line; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis* / Q000138:chemical synthesis; D011493:Protein Kinase C / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000906:Antibodies; D010446:Peptide Fragments; D000001:Calcimycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D002154:Calpain; D013755:Tetradecanoylphorbol Acetate