Tsai Wan-Hua, Wang Pei-Wen, Lin Shu-Yu, Wu I-Lin, Ko Ying-Chieh, Chen Yu-Lian, Li Mengtao, Lin Su-Fang
National Institute of Cancer Research, National Health Research Institutes Zhunan Town, Miaoli County, Taiwan.
Front Microbiol. 2012 Feb 22;3:60. doi: 10.3389/fmicb.2012.00060. eCollection 2012.
The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), K-RTA, is a lytic switch protein that moderates the reactivation process of KSHV latency. By mass spectrometric analysis of affinity purified K-RTA, we showed that Thr-513 or Thr-514 was the primary in vivo phosphorylation site. Thr-513 and Thr-514 are proximal to the nuclear localization signal ((527)KKRK(530)) and were previously hypothesized to be target sites of Ser/Thr kinase hKFC. However, substitutions of Thr with Ala at 513 and 514 had no effect on K-RTA subcellular localization or transactivation activity. By contrast, replacement of Ser with Ala at Ser-634 and Ser-636 located in a Ser/Pro-rich region of K-RTA, designated as S634A/S636A, produced a polypeptide with ∼10 kDa shorter in molecular weight and reduced transactivation in a luciferase reporter assay relative to the wild type. In contrast to prediction, the decrease in molecular weight was not due to lack of phosphorylation because the overall Ser and Thr phosphorylation state in K-RTA and S634A/S636A were similar, excluding that Ser-634 or Ser-636 motif served as docking sites for consecutive phosphorylation. Interestingly, S634A/S636A lost ∼30% immuno-reactivity to MPM2, an antibody specific to pSer/pThr-Pro motif, indicating that (634)SPSP(637) motif was in vivo phosphorylated. By in vitro kinase assay, we showed that K-RTA is a substrate of CDK9, a Pro-directed Ser/Thr kinase central to transcriptional regulation. Importantly, the capability of K-RTA in associating with endogenous CDK9 was reduced in S634A/S636A, which suggested that Ser-634 and Ser-636 may be involved in CDK9 recruitment. In agreement, S634A/S636A mutant exhibited ∼25% reduction in KSHV lytic cycle reactivation relative to that by the wild type K-RTA. Taken together, our data propose that Ser-634 and Ser-636 of K-RTA are phosphorylated by host transcriptional kinase CDK9 and such a process contributes to a full transcriptional potency of K-RTA.
卡波西肉瘤相关疱疹病毒（KSHV）的复制和转录激活因子（RTA），即K-RTA，是一种裂解开关蛋白，可调节KSHV潜伏期的重新激活过程。通过对亲和纯化的K-RTA进行质谱分析，我们发现苏氨酸-513或苏氨酸-514是主要的体内磷酸化位点。苏氨酸-513和苏氨酸-514靠近核定位信号（（527）KKRK（530）），此前曾被假设为丝氨酸/苏氨酸激酶hKFC的靶位点。然而，将513和514位的苏氨酸替换为丙氨酸对K-RTA的亚细胞定位或反式激活活性没有影响。相比之下，将K-RTA富含丝氨酸/脯氨酸区域中的丝氨酸-634和丝氨酸-636替换为丙氨酸，即S634A/S636A，产生了一种分子量短约10 kDa的多肽，并且在荧光素酶报告基因检测中相对于野生型其反式激活作用降低。与预测相反，分子量的降低并非由于缺乏磷酸化，因为K-RTA和S634A/S636A中的总体丝氨酸和苏氨酸磷酸化状态相似，排除了丝氨酸-634或丝氨酸-636基序作为连续磷酸化的对接位点。有趣的是，S634A/S636A对MPM2（一种对pSer/pThr-Pro基序特异的抗体）失去了约30%的免疫反应性，表明（634）SPSP（637）基序在体内被磷酸化。通过体外激酶分析，我们表明K-RTA是CDK9的底物，CDK9是转录调控中一种重要的脯氨酸导向的丝氨酸/苏氨酸激酶。重要的是，S634A/S636A中K-RTA与内源性CDK9结合的能力降低，这表明丝氨酸-634和丝氨酸-636可能参与CDK9的募集。一致的是，相对于野生型K-RTA，S634A/S636A突变体在KSHV裂解周期重新激活方面表现出约25%的降低。综上所述，我们的数据表明K-RTA的丝氨酸-634和丝氨酸-636被宿主转录激酶CDK9磷酸化，并且这一过程有助于K-RTA的完全转录活性。
