A synthetic peptide derived from the non-structural protein 3 of hepatitis C virus serves as a specific substrate for PKC

A synthetic peptide corresponding to the amino acid sequence Arg(1487)-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg(1500) of the hepatitis C virus (HCV) polyprotein was found to be a selective substrate for protein kinase C (PKC). In the presence of Ca2+, TPA and phospholipid, PKC phosphorylates the peptide [termed HCV(1487-1500)] with a K-m of 11 mu M and V-max of 24 mu mol x min(-1) x mg(-1). HCV(1487-1500) acts as a competitive inhibitor of PKC towards other peptide or protein substrates and inhibits the kinase activity with an IC50 corresponding to the K-m values measured for the substrates. N- or C-terminally deleted analogs of HCV(1487-1500) did not show inhibitory effects and were only marginally or not phosphorylatable. We designed an additional peptide in which the tyrosine residue was replaced by phenylalanine ([Phe(1499)]HCV(1487-1500)). This peptide was neither phosphorylated by other serine/threonine kinases tested nor by whole cell extracts prepared from PKC-depleted cells. [Phe(1499)]HCV(1487-1500) was used to monitor the TPA-induced translocation of PKC activity to the particulate fraction in JB6 cells. The use of SDS/PAGE to separate the peptide from ATP and P-i allowed to monitor simultaneously PKC autophosphorylation and phosphorylation of the peptide. The data presented here show that [Phe(1499)]HCV(1487-1500) can serve as a convenient tool for investigations of PKC activity also in the presence of other kinases in tissues or in crude cell extracts.