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 Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 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 Km of 11 microM and Vmax of 24 micromol 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 Km 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 ([Phe1499]HCV(1487-1500)). This peptide was neither phosphorylated by other serine/threonine kinases tested nor by whole cell extracts prepared from PKC-depleted cells. [Phe1499]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 Pi allowed to monitor simultaneously PKC autophosphorylation and phosphorylation of the peptide. The data presented here show that[Phe1499]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.
Borowski
P
P
Bernhard-Nocht-Institut für Tropenmedizin, Hamburg, Germany.
Resch
K
K
Schmitz
H
H
Heiland
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D010446:Peptide Fragments; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases