Schubert U, Schneider T, Henklein P, Hoffmann K, Berthold E, Hauser H, Pauli G, Porstmann T
Institut für Medizinische Immunologie, Medizinische Fakultät (Charité), Humboldt-Universität zu Berlin, Federal Republic of Germany.
Eur J Biochem. 1992 Mar 1;204(2):875-83. doi: 10.1111/j.1432-1033.1992.tb16707.x.
Vpu as a human-immunodeficiency-virus-type-1-encoded 81-amino-acid integral-membrane protein was expressed in Escherichia coli using the inducible ptrc promoter of an ATG fusion vector. Recombinant Vpu is associated with membranes of E. coli and could be partially solubilized by detergents. Recombinant Vpu was phosphorylated in vitro with purified porcine casein kinase II (CKII) as well as with a CKII-related protein kinase found in cytoplasmic extracts of human and hamster cells. Recombinant Vpu associated with E. coli membranes has turned out to be the best substrate for in vitro phosphorylation with CKII. This reaction can be inhibited by heparin and the ATP analogue 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB), both known to be potent inhibitors of CKII. Radiolabelled gamma ATP and gamma GTP were used as phosphate donors in vitro phosphorylation of recombinant Vpu. In vivo phosphorylation of Vpu in HIV-1-infected H9 cells was also inhibited by DRB. We concluded therefrom that the Vpu protein is phosphorylated by the ubiquitous CKII in HIV-1-infected human host cells. Two seryl residues in the sequence of Vpu (position 52 and 56) correspond to the consensus S/TXXD/E for CKII. These potential phosphorylation sites are located within a well-conserved dodecapeptide of Vpu (residues 47-58), which is found in different HIV-1 strains as well as in a Vpu-like protein of SIVCPZ. Monoclonal and polyclonal antibodies directed against two different epitopes of Vpu were used for immunoprecipitation of Vpu from HIV-1-infected cells and for detection of Vpu in Western blot analyses. Vpu from HIV-1-infected cells as well as recombinant Vpu expressed in E. coli were determined by SDS/PAGE using 6 M urea to be 9 kDa, which corresponds to the calculated molecular mass of Vpu.
Vpu是一种由人类免疫缺陷病毒1型编码的含81个氨基酸的整合膜蛋白，利用ATG融合载体的可诱导ptrc启动子在大肠杆菌中表达。重组Vpu与大肠杆菌的膜相关，并且可以被去污剂部分溶解。重组Vpu在体外可被纯化的猪酪蛋白激酶II（CKII）以及在人和仓鼠细胞胞质提取物中发现的一种与CKII相关的蛋白激酶磷酸化。与大肠杆菌膜相关的重组Vpu已被证明是CKII体外磷酸化的最佳底物。该反应可被肝素和ATP类似物5,6-二氯-1-（β-D-呋喃核糖基）苯并咪唑（DRB）抑制，这两种物质均为已知的CKII强效抑制剂。在重组Vpu的体外磷酸化中，放射性标记的γ-ATP和γ-GTP用作磷酸盐供体。DRB也抑制HIV-1感染的H9细胞中Vpu的体内磷酸化。由此我们得出结论，在HIV-1感染的人类宿主细胞中，Vpu蛋白被普遍存在的CKII磷酸化。Vpu序列中的两个丝氨酸残基（第52位和56位）符合CKII的共有序列S/TXXD/E。这些潜在的磷酸化位点位于Vpu一个保守的十二肽内（第47-58位氨基酸残基），在不同的HIV-1毒株以及SIVCPZ的一种Vpu样蛋白中都能找到。针对Vpu两个不同表位的单克隆抗体和多克隆抗体用于从HIV-1感染的细胞中免疫沉淀Vpu以及在蛋白质印迹分析中检测Vpu。通过使用6M尿素的SDS/PAGE测定，HIV-1感染细胞中的Vpu以及在大肠杆菌中表达的重组Vpu均为9kDa，这与Vpu的计算分子量相符。
