Protein kinase C isoenzymes in airway smooth muscle.
The protein kinase C (PKC) isoenzymes expressed by bovine tracheal smooth muscle (BTSM) were identified at the protein and mRNA levels. Western immunoblot analyses reliably identified PKCalpha, PKCbetaI and PKCbetaII. In some experiments immunoreactive bands corresponding to PKCdelta, PKCepsilon and PKCTheta were also labelled, whereas the gamma, eta and zeta isoforms of PKC were never detected. Reverse transcriptase PCR of RNA extracted from BTSM using oligonucleotide primer pairs designed to recognize unique sequences in the PKC genes for which protein was absent or not reproducibly identified by immunoblotting, amplified cDNA fragments that corresponded to the predicted sizes of PKCdelta, PKCepsilon and PKCzeta, which was confirmed by Southern blotting. Anion-exchange chromatography of the soluble fraction of BTSM following homogenization in Ca2+-free buffer resolved two major peaks of activity. Using epsilon-peptide as the substrate, the first peak of activity was dependent upon Ca2+ and 4beta-PDBu (PDBu=phorbol 12, 13-dibutyrate), and represented a mixture of PKCs alpha, betaI and betaII. 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 PKCalpha', PKCbetaI' and PKCbetaII'. However, these novel enzymes were cofactor-independent and did not bind [3H]PDBu, but were equally sensitive to the PKC inhibitor GF 109203X compared with bona fide conventional PKCs, and migrated on SDS/polyacrylamide gels as 81 kDa polypeptides. Taken together, these data suggest that PKCs alpha', betaI' and betaII' represent modified, but not proteolysed, forms of their respective native enzymes that retain antibody immunoreactivity and sensitivity to PKC inhibitors, but have lost their sensitivity to Ca2+ and PDBu when epsilon-peptide is used as the substrate.
Webb
B L
BL
Thoracic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, U.K.
Lindsay
M A
MA
Barnes
P J
PJ
Giembycz
M A
MA
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D017886:Durapatite; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D014132:Trachea / Q000201:enzymology*; D014158:Transcription, Genetic
D017931:DNA Primers; D007527:Isoenzymes; D012333:RNA, Messenger; D015240:Phorbol 12,13-Dibutyrate; D017886:Durapatite; D011493:Protein Kinase C