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Peter Borowski, Kerstin Resch, Herbert Schmitz, Max Heiland

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 K_m of 11?M and V_max of 24 ?mol × min^?1 × 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 ([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 P_i 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.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 381, 01/2000
Pages: 19 - 27

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