Habran Lionel, Bontems Sébastien, Di Valentin Emmanuel, Sadzot-Delvaux Catherine, Piette Jacques
Laboratory of Virology and Immunology, Center for Biomedical Genoproteomics, Institute of Pathology B23, University of Liège, B-4000, Liège, Belgium.
J Biol Chem. 2005 Aug 12;280(32):29135-43. doi: 10.1074/jbc.M503312200. Epub 2005 Jun 13.
During the first stage of Varicella-Zoster virus (VZV) infection, IE63 (immediate early 63 protein) is mostly expressed in the nucleus and also slightly in the cytoplasm, and during latency, IE63 localizes in the cytoplasm quite exclusively. Because phosphorylation is known to regulate various cellular mechanisms, we investigated the impact of phosphorylation by roscovitine-sensitive cyclin-dependent kinase (RSC) on the localization and functional properties of IE63. We demonstrated first that IE63 was phosphorylated on Ser-224 in vitro by CDK1 and CDK5 but not by CDK2, CDK7, or CDK9. Furthermore, by using roscovitine and CDK1 inhibitor III (CiIII), we showed that CDK1 phosphorylated IE63 on Ser-224 in vivo. By mutagenesis and the use of inhibitors, we demonstrated that phosphorylation on Ser-224 was important for the correct localization of the protein. Indeed, the substitution of these residues by alanine led to an exclusive nuclear localization of the protein, whereas mutations into glutamic acid did not modify its subcellular distribution. When transfected or VZV-infected cells were treated with roscovitine or CiIII, an exclusive nuclear localization of IE63 was also observed. By using a transfection assay, we also showed that phosphorylation on Ser-224 and Thr-222 was essential for the down-regulation of the basal activity of the VZV DNA polymerase gene promoter. Similarly, roscovitine and CiIII impaired these properties of the wild-type form of IE63. These observations clearly demonstrated the importance of CDK1-mediated IE63 phosphorylation for a correct distribution of IE63 between both cellular compartments and for its repressive activity toward the promoter tested.
在水痘-带状疱疹病毒（VZV）感染的第一阶段，IE63（立即早期63蛋白）主要在细胞核中表达，在细胞质中也有少量表达，而在潜伏期，IE63几乎完全定位于细胞质中。由于已知磷酸化可调节多种细胞机制，我们研究了对roscovitine敏感的细胞周期蛋白依赖性激酶（RSC）介导的磷酸化对IE63定位和功能特性的影响。我们首先证明，在体外，CDK1和CDK5可使IE63的Ser-224位点发生磷酸化，而CDK2、CDK7或CDK9则不能。此外，通过使用roscovitine和CDK1抑制剂III（CiIII），我们表明在体内CDK1可使IE63的Ser-224位点发生磷酸化。通过诱变和使用抑制剂，我们证明Ser-224位点的磷酸化对于该蛋白的正确定位很重要。事实上，将这些残基替换为丙氨酸会导致该蛋白仅定位于细胞核，而突变为谷氨酸则不会改变其亚细胞分布。当用roscovitine或CiIII处理转染或VZV感染的细胞时，也观察到IE63仅定位于细胞核。通过转染试验，我们还表明Ser-224和Thr-222位点的磷酸化对于VZV DNA聚合酶基因启动子基础活性的下调至关重要。同样，roscovitine和CiIII损害了野生型IE63的这些特性。这些观察结果清楚地证明了CDK1介导的IE63磷酸化对于IE63在两个细胞区室之间的正确分布及其对测试启动子的抑制活性的重要性。
