Characterisation of non-structural protein 3 of hepatitis C virus as modulator of protein phosphorylation mediated by PKA and PKC: evidences for action on the level of substrate and enzyme

Generally, the maximum activities of the protein kinases A* (PKA) and C (PK C) show an optimum value for their substrate concentrations rather than a s aturation curve; at high substrate concentrations, the kinase activity is c ompletely abolished. The C- and N-truncated form of the non-structural prot ein 3 (NS3) of hepatitis C virus (HCV) (HCV-polyprotein-(1 189-1 525)) abol ishes the inhibiting effect of the substrate, yielding saturable Michaelis- Menten kinetics of PKA and its catalytical domain (C subunit). In contrast, HCV-polyprotein(1 189-1 525) activates PKC with increasing V-max, while it abolishes the substrate inhibition of its catalytical domain (M-kinase) th rough a mechanism analogous to that of PKA and C subunit. PKC isoforms alph a, beta and gamma investigated are similarly activated by HCV-polyprotein-( 1 189-1 525). Our data suggest that NS3 attenuates the substrate inhibition through a generalized mechanism operating mainly on the substrate level th at directly results from a specific protein-protein interaction. In the cas e of the PKC, an additional kinase activating mechanism operates on the enz yme level. Both actions of NS3, the attenuation of the substrate inhibition and the activation of PKC, could not be explained by classical means that predict autophosphorylation to enhance the rate of substrate phosphorylatio n. The results are discussed in view of similar activities displayed by mat chmakers and some molecular chaperones.