Characterisation of protein kinase C isoforms and enzymic activity from the αT3‐1 gonadotroph‐derived cell line

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Abstract
Western blots of αT3-1 cell extracts were immunostained with antibodies specific for various protein kinase C (PKC) isoforms. These revealed the presence of PKC types α, ϵ and ζ, but β, γ, δ and η were not detected. The potency with which partially-purified cytosolic PKC from αT3-1 cells was activated by phorbol 12,13-dibutyrate (PDBu), mezerein and 1,2-dioctanoyl-sn-glycerol was assessed in the presence and absence of Ca2+ The inhibitors staurosporine, K252a, H7, GF109203X and Ro 31-8220 were tested on basal activity, PDBu-induced activity and Ca2++ PDBu-induced kinase activity. Each inhibitor showed distinct differences in their IC50 values under the three conditions, suggesting that these inhibitors may exhibit different potencies on the PKC isoforms present in αT3-1 cells. Although histone IIIs was used as the phosphate acceptor for most of these experiments, the efficiency of α, ϵ and ζ, peptide and GS peptide substrates were also determined, with ϵ peptide giving the greatest activity in the presence of PDBu or Ca2+. Each substrate displayed a different pattern of activation under the conditions tested. Overall, the findings suggest that 3 or more PKC isoforms with varying specificities are present in gonadotroph-derived αT3-1 cells and that the contribution of each isofonn should be considered when these cells are used in models of anterior pituitary cell function where PKC is involved.