Ohmori S, Shirai Y, Sakai N, Fujii M, Konishi H, Kikkawa U, Saito N
Laboratories of Molecular Pharmacology, Biosignal Research Center, Kobe University, Nada-ku, Kobe 657-8501, Japan.
Mol Cell Biol. 1998 Sep;18(9):5263-71. doi: 10.1128/MCB.18.9.5263.
We expressed delta subspecies of protein kinase C (delta-PKC) fused with green fluorescent protein (GFP) in CHO-K1 cells and observed the movement of this fusion protein in living cells after three different stimulations. The delta-PKC-GFP fusion protein had enzymological characteristics very similar to those of the native delta-PKC and was present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM caused a transient translocation of delta-PKC-GFP to the plasma membrane approximately 30 s after the stimulation and a sequent retranslocation to the cytoplasm within 3 min. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 microM), induced a slower translocation of delta-PKC-GFP, and the translocation was unidirectional. Concomitantly, the kinase activity of delta-PKC-GFP was increased by these two stimulations, when the kinase activity of the immunoprecipitated delta-PKC-GFP was measured in vitro in the absence of PKC activators such as phosphatidylserine and diacylglycerol. Hydrogen peroxide (H2O2; 5 mM) failed to translocate delta-PKC-GFP but increased its kinase activity more than threefold. delta-PKC-GFP was strongly tyrosine phosphorylated when treated with H2O2 but was tyrosine phosphorylated not at all by ATP stimulation and only slightly by TPA treatment. Both TPA and ATP induced the translocation of delta-PKC-GFP even after treatment with H2O2. Simultaneous treatment with TPA and H2O2 further activated delta-PKC-GFP up to more than fivefold. TPA treatment of cells overexpressing delta-PKC-GFP led to an increase in the number of cells in G2/M phase and of dikaryons, while stimulation with H2O2 increased the number of cells in S phase and induced no significant change in cell morphology. These results indicate that at least three different mechanisms are involved in the translocation and activation of delta-PKC.
我们在CHO-K1细胞中表达了与绿色荧光蛋白（GFP）融合的蛋白激酶C的δ亚型（δ-PKC），并观察了这种融合蛋白在三种不同刺激后在活细胞中的移动情况。δ-PKC-GFP融合蛋白具有与天然δ-PKC非常相似的酶学特性，并且在CHO-K1细胞的整个细胞质中都有分布。1 mM的ATP在刺激后约30秒导致δ-PKC-GFP短暂转位至质膜，并在3分钟内随后再转位至细胞质。一种促肿瘤佛波酯，12-O-十四酰佛波醇13-乙酸酯（TPA；1 μM），诱导δ-PKC-GFP的转位较慢，且该转位是单向的。同时，当在体外没有PKC激活剂如磷脂酰丝氨酸和二酰甘油的情况下测量免疫沉淀的δ-PKC-GFP的激酶活性时，这两种刺激均增加了δ-PKC-GFP的激酶活性。过氧化氢（H2O2；5 mM）未能使δ-PKC-GFP转位，但使其激酶活性增加了三倍以上。用H2O2处理时，δ-PKC-GFP被强烈酪氨酸磷酸化，但ATP刺激时完全不发生酪氨酸磷酸化，TPA处理时仅轻微发生酪氨酸磷酸化。即使在用H2O2处理后，TPA和ATP也均诱导了δ-PKC-GFP的转位。TPA和H2O2同时处理进一步激活δ-PKC-GFP达五倍以上。用TPA处理过表达δ-PKC-GFP的细胞导致G2/M期细胞和双核细胞数量增加，而用H2O2刺激增加了S期细胞数量，并且未诱导细胞形态发生显著变化。这些结果表明，至少三种不同的机制参与了δ-PKC的转位和激活。
