PROTEIN-KINASE-C ISOENZYMES IN AIRWAY SMOOTH-MUSCLE

The protein kinase C (PKC) isoenzymes expressed by bovine tracheal smo oth muscle (BTSM) were identified at the protein and mRNA levels. West ern immunoblot analyses reliably identified PKC alpha, PKC beta(I), an d PKC beta(II). In some experiments immunoreactive bands corresponding to PKC delta, PKC epsilon and PKC theta were also labelled, whereas t he gamma, eta and zeta isoforms of PKC were never detected. Reverse tr anscriptase PCR of RNA extracted from BTSM using oligonucleotide prime r pairs designed to recognize unique sequences in the PKC genes for wh ich protein was absent or not reproducibly identified by immunoblottin g, amplified cDNA fragments that corresponded to the predicted sizes o f PKC delta, PKC epsilon and PKC zeta, which was confirmed by Southern blotting. Anion-exchange chromatography of the soluble fraction of BT SM following homogenization in Ca2+-free buffer resolved two major pea ks of activity. Using epsilon-peptide as the substrate, the first peak of activity was dependent upon Ca2+ and 4 beta-PDBu (PDBu = phorbol 1 2,13-dibutyrate), and represented a mixture of PKCs alpha, beta(I), an d beta(II). In contrast, the second peak of activity, which eluted at much higher ionic strength, also appeared to comprise a combination of conventional PKCs that were arbitrarily denoted PKC alpha', PKC beta( I)' and PKC beta(II)'. However, these novel enzymes were cofactor-inde pendent and did not bind [H-3]PDBu, but were equally sensitive to the PKC inhibitor GF 109203X compared with bona fide conventional PKCs, an d migrated on SDS/polyacrylamide gels as 81 kDa polypeptides. Taken to gether, these data suggest that PKCs alpha', beta(I)' and beta(II)' re present modified, but not proteolysed, forms of their respective nativ e enzymes that retain antibody immunoreactivity and sensitivity to PKC inhibitors, but have lost their sensitivity to Ca(2+)and PDBu when ep silon-peptide is used as the substrate.