ACTIVATION AND SUBSTRATE-SPECIFICITY OF THE HUMAN PROTEIN-KINASE-C ALPHA-ISOENZYME AND ZETA-ISOENZYME

Protein kinase C (PKC), a class of serine/threonine kinases activated by Ca2+ and/or phospholipids, is involved in a variety of cellular processes such as proliferation, differentiation and secretion. Nine members of the PKC gene family are known; these are differentially expressed in eukaryotic cells and can be divided into two sub-groups: the Ca2+-dependent (classical) PKC isoenzymes alpha, betaI, betaII and gamma, and the Ca2+-independent neoPKC isoenzymes delta, epsilon, zeta, eta and theta. A detailed biochemical characterisation of these PKC isoenzymes is one prerequisite for the elucidation of their distinct roles within cellular signal transduction. In this study, we report the cloning of a human PKC-zeta cDNA, its expression in recombinant baculovirus-infected insect cells and the partial purification of the PKC-zeta isoenzyme. In comparison to highly purified human PKC alpha, a representative of the classical PKC subgroup, purified PKC zeta was characterised with respect to activator requirement, substrate specificity, proteolytic activation and sensitivity towards PKC inhibitors. In contrast to PKC alpha, PKC zeta exhibits a constitutive kinase activity which is independent of Ca2+, phosphatidylserine and diacylglycerol. Arachidonic acid alone or a combination of gamma-linolenic acid and phosphatidylserine slightly enhance PKC zeta activity. In the presence of the classical PKC activators phosphatidylserine/diacylglycerol, PKC alpha phosphorylates a PKC-alpha pseudosubstrate-derived peptide, an epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein to an equal extent, whilst PKC zeta phosphorylates only the PKC-alpha-derived peptide. However, arachidonic acid greatly diminishes PKC-alpha activity towards the epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein, but enhances PKC-zeta activity towards the PKC-alpha-derived peptide. These results indicate a possible modulation of substrate specificity of these two PKC isoenzymes by (the binding of) different activators (to their regulatory domains). In the case of PKC zeta, this finding is strengthened by the fact that the epidermal growth factor receptor-derived peptide, which is not a substrate for the holoenzyme, is significantly phosphorylated by a protein fragment generated by limited proteolysis and comprising only the kinase domain. Furthermore, PKC zeta, in contrast to PKC alpha, is insensitive to PKC inhibitors known to interfere either with the regulatory or the catalytic domain and cannot be activated by phorbol ester treatment of NIH 3T3 cells or insect cells, overexpressing the respective PKC isoenzyme. The potential implications of these findings on the mechanism(s) of activation and the substrate specificity of PKC zeta are discussed.