PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE PROTEIN-KINASE ENCODED BY THE US3 GENE OF HERPES-SIMPLEX VIRUS TYPE-2
PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE PROTEIN-KINASE ENCODED BY THE US3 GENE OF HERPES-SIMPLEX VIRUS TYPE-2
复制标题
DOI：
10.1006/viro.1993.1644
复制
发表时间：
1993-12-01
期刊：
影响因子：
3.7
通讯作者：
NISHIYAMA, Y
中科院分区：
文献类型：
--
作者：
DAIKOKU, T;YAMASHITA, Y;NISHIYAMA, Y
用DEAE-纤维素色谱法分离模拟感染或感染野生型单纯疱疹病毒2型（HSV-2）或HSV-2的US 3基因破坏突变体的Vero细胞的核糖体后细胞质组分，并将仅在野生型病毒感染细胞提取物中可检测到的蛋白激酶峰组分进行层析。该酶从核糖体后上清液中纯化了1000倍以上，最终制剂含有一种表观分子量为66千道尔顿（K）的主要蛋白质，该蛋白质在自磷酸化反应中被磷酸化。Western印迹分析表明，对应于预测的HSV-2 US 3蛋白序列的15个氨基酸的合成肽的抗体与酶级分中的66 K蛋白强烈反应。在Superose 12 HR色谱上，蛋白激酶活性在对应于约60 K的表观分子量的位置处洗脱为单个主峰。这些结果表明，66 K蛋白是由HSV-2的US 3基因编码的蛋白激酶，并且它作为单体起作用。HSV-2蛋白激酶对高浓度的盐有较强的耐受性，但KCl浓度超过400 mM时，对蛋白激酶有明显的抑制作用。当使用合成寡肽研究底物特异性时，发现在目标丝氨酰基残基的氨基末端侧含有乙酰基残基的肽是蛋白激酶的最佳底物。然而，替换的丝氨酰残基苏氨酸显着降低了磷酸化的速度，这种酶，这表明苏氨酸是一个穷人的磷酸受体的蛋白激酶。该酶对酪蛋白激酶II的强效抑制剂肝素具有抗性，但对环核苷酸依赖性蛋白激酶和蛋白激酶C的强效抑制剂H-9（N-（2-氨乙基）-5-异喹啉磺酰胺二盐酸盐）中度敏感。槲皮素对蛋白激酶也有抑制作用，其抑制作用与ATP呈竞争性（Ki = 10 μM）。结果表明，HSV-2 US 3蛋白激酶的生化性质与HSV-1和伪狂犬病病毒编码的38-kDa蛋白激酶非常相似，但在某些方面又有所不同。
Post-ribosomal cytoplasmic fractions from Vero cells mock-infected or infected with wild-type herpes simplex virus type 2 (HSV-2) or a US3 gene-disrupted mutant of HSV-2 were fractionated with DEAE-cellulose chromatography, and the peak fraction of the protein kinase which was detectable only in the extract of wild-type virus-infected cells was subjected to succesive chromatography. The enzyme was purified more than 1000-fold from the post-ribosomal supernatant, and the final preparation contained one major protein of apparent molecular weight 66 kilodalton (K), which was phosphorylated in the autophosphorylation reaction. Western blotting analysis showed that antibodies to an synthetic peptide corresponding to the 15 amino acids of the predicted HSV-2 US3 protein sequence strongly reacted with a 66 K protein in the enzyme fractions. On Superose 12 HR chromatography, the protein kinase activity was eluted as a single major peak at a position corresponding to an apparent molecular mass of approximately 60 K. These results suggest that the 66 K protein is the protein kinase encoded by the US3 gene of HSV-2 and that it acts as a monomer. The HSV-2 protein kinase was relatively resistant to high concentrations of salt, but KCl above 400 mMexerted a significant inhibitory effect. When the substrate specificity was investigated using synthetic oligopeptides, the peptides containing arginyl residues on the amino-terminal side of the target seryl residue were found to be the best substrates for the protein kinase. However, the replacement of the seryl residue to threonine markedly reduced the rate of phosphorylation by this enzyme, suggesting that threonine is a poor phosphate acceptor of the protein kinase. The enzyme was resistant to heparin, a potent inhibitor of casein kinase II, but was moderately sensitive to H-9 (N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride), a potent inhibitor of cyclic nucleotide-dependent protein kinases and protein kinase C. Quercetin, a bioflavonoid, also inhibited the protein kinase and the inhibitory effect was competitive towards ATP (Ki = 10 μM). The results indicate that the biochemical properties of the HSV-2 US3 protein kinase are very similar to those of the HSV-1 counterpart and pseudorabies virus-encoded 38-kDa protein kinase, but are different from those in several respects.