Expression and partial characterization of rat protein kinase C‐δ and protein kinase C‐ξ in insect cells using recombinant baculovirus

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Abstract
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.