Johnson S A, Mandavia N, Wang H D, Johnson D L
Departments of Molecular Pharmacology and Biochemistry, Norris Comprehensive Cancer Center, University of Southern California School of Pharmacy and Keck School of Medicine, Los Angeles, California 90089-9121, USA.
Mol Cell Biol. 2000 Jul;20(14):5000-9. doi: 10.1128/MCB.20.14.5000-5009.2000.
Our previous studies have demonstrated that the level of the central transcription factor TATA-binding protein (TBP) is increased in cells expressing the hepatitis B virus (HBV) X protein through the activation of the Ras signaling pathway, which serves to enhance both RNA polymerase I and III promoter activities. To understand the mechanism by which TBP is regulated, we have investigated whether enhanced expression is modulated at the transcriptional level. Nuclear run-on assays revealed that the HBV X protein increases the number of active transcription complexes on the TBP gene. In transient-transfection assays with both transformed and primary hepatocytes, the human TBP promoter was shown to be induced by expression of the HBV X protein in a Ras-dependent manner, requiring both Ral guanine nucleotide dissociation stimulator (RalGDS) and Raf signaling. Transient overexpression of TBP did not affect TBP promoter activity. To further delineate the downstream Ras-mediated events contributing to TBP promoter regulation in primary rat hepatocytes, the best-characterized Ras effectors, Raf, phosphoinositide 3-kinase (PI-3 kinase), and RalGDS, were examined. Activation of either Raf or RalGDS, but not that of PI-3 kinase, was sufficient to induce TBP promoter activity. Both Raf- and RalGDS-mediated induction required the activation of mitogen-activated protein kinase kinase (MEK). In addition, another distinct Ras-activated pathway, which does not require MEK activation, appears to induce TBP promoter activity. Analysis of the DNA sequence requirement within the TBP promoter responsible for these regulatory events defined three distinct regions that modulate the abilities of Raf, RalGDS, and the Ras-dependent, MEK-independent pathways to regulate human TBP promoter activity. Together, these results provide new evidence that TBP can be regulated at the transcriptional level and identify three distinct Ras-activated pathways that modulate this central eukaryotic transcription factor.
我们之前的研究表明，在表达乙型肝炎病毒（HBV）X蛋白的细胞中，通过Ras信号通路的激活，中心转录因子TATA结合蛋白（TBP）的水平会升高，这有助于增强RNA聚合酶I和III启动子的活性。为了了解TBP受调控的机制，我们研究了其增强的表达是否在转录水平受到调节。细胞核连续转录分析表明，HBV X蛋白增加了TBP基因上活性转录复合物的数量。在转化肝细胞和原代肝细胞的瞬时转染实验中，人TBP启动子显示出以Ras依赖的方式被HBV X蛋白的表达所诱导，这需要Ral鸟嘌呤核苷酸解离刺激因子（RalGDS）和Raf信号传导。TBP的瞬时过表达并不影响TBP启动子的活性。为了进一步阐明在原代大鼠肝细胞中导致TBP启动子调控的下游Ras介导事件，我们检测了特征最明确的Ras效应分子Raf、磷脂酰肌醇3激酶（PI-3激酶）和RalGDS。Raf或RalGDS的激活，但不是PI-3激酶的激活，足以诱导TBP启动子活性。Raf和RalGDS介导的诱导都需要丝裂原活化蛋白激酶激酶（MEK）的激活。此外，另一条不依赖MEK激活的独特的Ras激活途径似乎也能诱导TBP启动子活性。对TBP启动子内负责这些调控事件的DNA序列需求的分析确定了三个不同区域，它们调节Raf、RalGDS以及Ras依赖、MEK独立途径调节人TBP启动子活性的能力。总之，这些结果提供了新的证据，表明TBP可以在转录水平受到调控，并确定了三条不同的Ras激活途径来调节这种核心真核转录因子。
