Expression of rat protein kinase C‐δ (PKC‐δ ) and PKC‐ξ in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype‐specific antisera. Although the PKC‐ξ cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC‐δ were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC‐α. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC‐ξ. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC‐δ or PKC‐ξ when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC‐δ and PKC‐ξ. In contrast to PKC‐ξ, the PKC‐δ enzyme activity phosphorylated MBP or histone in a phosphatidylserine‐(PS)/diacylglycerol(DG)‐dependent manner, albeit not to the same extent as PKC‐α. Lack of stimulation of the enzyme activity of PKC‐ξ by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC‐ξ, whereas several insect cell proteins were phosphorylated by PKC‐δ in a PS/DG‐dependent manner, including a protein of 78 kD.

Our data demonstrate that the 76 kD PKC‐ξ, in contrast to PKC‐δ, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS of PS/DG. In addition, staurosporine was about 2–4 order of magnitudes less effective in inhibiting the protein kinase activities of PKC‐δ and PKC‐δ when compared to PKC‐ξ.