Borowski P, Resch K, Schmitz H, Heiland M
Bernhard-Nocht-Institut für Tropenmedizin, Hamburg, Germany.
Biol Chem. 2000 Jan;381(1):19-27. doi: 10.1515/BC.2000.003.
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.
一种与丙型肝炎病毒（HCV）多聚蛋白氨基酸序列Arg1487 - Arg - Gly - Arg - Thr - Gly - Arg - Gly - Arg - Arg - Gly - Ile - Tyr - Arg1500相对应的合成肽被发现是蛋白激酶C（PKC）的选择性底物。在Ca2 +、佛波酯（TPA）和磷脂存在的情况下，PKC使该肽[称为HCV(1487 - 1500)]磷酸化，其米氏常数（Km）为11微摩尔，最大反应速度（Vmax）为24微摩尔·分钟-1·毫克-1。HCV(1487 - 1500)作为PKC对其他肽或蛋白质底物的竞争性抑制剂，以与底物测得的Km值相对应的半数抑制浓度（IC50）抑制激酶活性。HCV(1487 - 1500)的N端或C端缺失类似物未显示出抑制作用，且仅能被微弱磷酸化或不能被磷酸化。我们设计了另一种肽，其中酪氨酸残基被苯丙氨酸取代（[Phe1499]HCV(1487 - 1500)）。该肽既不被所测试的其他丝氨酸/苏氨酸激酶磷酸化，也不被从PKC缺失细胞制备的全细胞提取物磷酸化。[Phe1499]HCV(1487 - 1500)用于监测TPA诱导的PKC活性向JB6细胞颗粒部分的转位。使用十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）将肽与三磷酸腺苷（ATP）和无机磷酸（Pi）分离，能够同时监测PKC的自身磷酸化和肽的磷酸化。此处呈现的数据表明，[Phe1499]HCV(1487 - 1500)也可作为在组织或粗细胞提取物中存在其他激酶的情况下研究PKC活性的便捷工具。
