Protein kinase C (PKC), originally identified as a novel cytoplasmic protein kinase, then recognized as the major phorbol ester receptor in mammalian cells is now defined as a family of at least seven related polypeptides encoded by separate genes and expressed in a tissue-specific manner (reviewed in: Stabel, 1990). This protein kinase has gained a central role in the analysis of signal transduction through numerous experiments providing evidence for activation of protein kinase C by the second messenger diacylglycerols produced mainly from the membrane lipids phosphatidylinositol and phosphatidylcholine. With the elucidation of the structural organization of the PKC molecule it became clear that the entity "PKC" comprises more than one polypeptide (Parker et al., 1986). The complexity of this enzyme family suggests that the individual members may serve distinct roles in signal transduction pathways. As an approach to analyse the characteristics of the PKC subtypes we have used the baculovirus - insect cell expression system to produce and isolate large amounts of the polypeptides encoded by the different genes (Patel and Stabel, 1989). The mammalian enzymes expressed in the insect cells are produced as phosphoproteins which show authentic protein kinase C properties and are amenable to biochemical characterization (Stabel et al., submitted). We have also used this expression system to express another serine/threonine kinase involved in signal transduction, the protooncogene product c-Raf, in order to analyse its activation and substrate requirements. Site-directed mutagenesis has proved a useful tool to dissect functional domains of polypeptides. The construction of a kinasenegative mutant of PKC type γ has allowed us to investigate the role of the kinase domain in post-translational modification of the PKC polypeptide and its role in defined events involving PKC. One such extensively studied event is the "downregulation" of PKC after prolonged phorbol ester treatment of mammalian cells, mainly fibroblasts, epithelial cells and hematopoietic cells, which mostly contain the α and/or β types of PKC (Kischel et al., 1990). We have introduced the PKC γ type which is exclusively expressed in nervous tissue into fibroblasts and shown that it is activated and downregulated by phorbol esters in vivo. The downregulation of the PKC-γ polypeptide by proteolytic degradation also proceeds if the molecule carries an inactivated kinase domain. This suggests that downregulation of PKC-γ is not a consequence of constitutive signalling through the kinase domain but rather due to an increased proteolytic susceptibility of the polypeptide after contact with phorbol esters (Freisewinkel et al., submitted). The kinase-deficient mutant is hypophosphorylated suggesting that a substantial proportion of the post-translational modification on PKC-γ is due to inter- or intramolecular autophosphorylation.