Characterization of a specific form of protein kinase C overproduced by a C3H 10T( 1/2 ) cell line

Murine embryo fibroblasts (C3H 10T( 1/2 )) which were genetically enmgineered to overproduce the β₁ isoform of protein kinase C (PKC-β₁) were used to obtain homogeneous preparations of PKC-β₁ for the purpose of characterizing the specific structural and functional properties of this isoform. Fractionation of PKC activity from these cells by hydroxyapatite chromatography produced one major peak, which represented 93% of the total cellular PKC activity and was not detected in control cells. This major peak of activity was shown by Western-blotting analysis with a β₁-specific antiserum to be the overproduced β₁-isoform, and exhibited a band at 77 kDa. The functional properties of the overproduced PKC-β₁ were established with regard to phospholipid-dependence, Ca²⁺-dependence, responsiveness to a phorbol ester tumour promoter, activation by arachidonic acid (plus Ca²⁺), and inhibition by known PKC inhibitors. From these studies we conclude that PKC-β₁ overproduced by C3H 10T( 1/2 ) cells exhibits the structural and functional properties previously ascribed to native PKC. Furthermore, these data provide the first definitive biochemical characteristics of this isoform of PKC.