Protein kinase C isoenzymes in airway smooth muscle

Benjamin L.J. Webb, Mark A. Lindsay, Peter J. Barnes, Mark A. Giembycz

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22 Citations (SciVal)


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 PKCα, PKCβ(I) and PKCβ(II). In some experiments immunoreactive bands corresponding to PKCδ, PKCε and PKCθ were also labelled, whereas the γ, η and ζ 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 PKCδ, PKCε and PKCζ, 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 ε-peptide as the substrate, the first peak of activity was dependent upon Ca2+ and 4β-PDBu (PDBu = phorbol 12,13-dibutyrate), and represented a mixture of PKCs α, β(I) and β(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 PKCα', PKCβ(I)' and PKCβ(II)'. 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 α', β(I)' and β(II)' 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 ε-peptide is used as the substrate.

Original languageEnglish
Pages (from-to)167-175
Number of pages9
JournalBiochemical Journal
Issue number1
Publication statusPublished - 15 May 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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