Gannon-Murakami Laura, Murakami Kentaro
Department of Biology, University of Vermont, Burlington, Vermont 05405, USA.
J Biol Chem. 2002 Jun 28;277(26):23116-22. doi: 10.1074/jbc.M201478200. Epub 2002 Apr 11.
The 14-3-3 protein is a family of highly conserved acidic proteins found in a wide range of eukaryotes from yeast to mammals. 14-3-3 acts as an adapter protein and interacts with signaling molecules including protein kinase C (PKC). Although 14-3-3 zeta was originally characterized as an endogenous PKC inhibitor, it was reported to activate PKC in vitro, but the in vivo regulation of PKC by 14-3-3 is still not well understood. To examine the regulation of PKC by 14-3-3 in the cell, we have generated a sub-cell line, PC12-B3, that stably expresses FLAG epitope-tagged 14-3-3 zeta isoform in PC12 cells. Here we show that PKC-alpha and PKC-epsilon become associated with 14-3-3 zeta when the cells are neuronally differentiated by nerve growth factor. We found that the immunoprecipitate by anti-FLAG antibody contains constitutive and autonomous Ca(2+)-independent non-classical PKC activity. In contrast, the FLAG immunoprecipitate has no Ca(2+)-dependent classical PKC activity despite the fact that PKC-alpha is present in the FLAG immunoprecipitate from differentiated PC12-B3 cells. Our results show that the association with 14-3-3 zeta has distinct effects on classical PKC and non-classical PKC activity.
14-3-3蛋白是一类高度保守的酸性蛋白家族，存在于从酵母到哺乳动物的广泛真核生物中。14-3-3作为一种衔接蛋白，与包括蛋白激酶C（PKC）在内的信号分子相互作用。尽管14-3-3ζ最初被鉴定为一种内源性PKC抑制剂，但据报道它在体外可激活PKC，不过14-3-3对PKC的体内调节仍未完全清楚。为了研究细胞中14-3-3对PKC的调节作用，我们构建了一个亚细胞系PC12-B3，该细胞系在PC12细胞中稳定表达带有FLAG表位标签的14-3-3ζ亚型。在此我们表明，当细胞通过神经生长因子进行神经元分化时，PKC-α和PKC-ε会与14-3-3ζ结合。我们发现，抗FLAG抗体的免疫沉淀物含有组成性且自主的不依赖Ca(2+)的非经典PKC活性。相比之下，尽管分化的PC12-B3细胞的FLAG免疫沉淀物中存在PKC-α，但该沉淀物却没有依赖Ca(2+)的经典PKC活性。我们的结果表明，与14-3-3ζ的结合对经典PKC和非经典PKC活性具有不同的影响。
