From the Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232.
J Biol Chem. 2010 Apr 23;285(17):12655-61. doi: 10.1074/jbc.M110.105759. Epub 2010 Mar 5.
Phosphorylation regulates transcription factor activity by influencing dimerization, cellular localization, activation potential, and/or DNA binding. Nevertheless, precisely how this post-translation modification mediates these processes is poorly understood. Here, we examined the role of phosphorylation on the DNA-binding properties of MafA and MafB, closely related transcriptional activators of the basic-leucine zipper (b-Zip) family associated with cell differentiation and oncogenesis. Many common phosphorylation sites were identified by mass spectrometry. However, dephosphorylation only precluded the detection of MafA dimers and consequently dramatically reduced DNA-binding ability. Analysis of MafA/B chimeras revealed that sensitivity to the phosphorylation status of MafA was imparted by sequences spanning the C-terminal dimerization region (amino acids (aa) 279-359), whereas the homologous MafB region (aa 257-323) conveyed phosphorylation-independent DNA binding. Mutational analysis showed that formation of MafA dimers capable of DNA binding required phosphorylation within the distinct N-terminal transactivation domain (aa 1-72) and not the C-terminal b-Zip region. These results demonstrate a novel relationship between the phosphoamino acid-rich transactivation and b-Zip domains in controlling MafA DNA-binding activity.
磷酸化通过影响二聚化、细胞定位、激活潜力和/或 DNA 结合来调节转录因子活性。然而，这种翻译后修饰如何介导这些过程还知之甚少。在这里，我们研究了磷酸化对 MafA 和 MafB 的 DNA 结合特性的作用，MafA 和 MafB 是与细胞分化和致癌作用相关的基本亮氨酸拉链 (b-Zip) 家族的密切相关的转录激活因子。通过质谱鉴定了许多常见的磷酸化位点。然而，去磷酸化仅排除了 MafA 二聚体的检测，从而显著降低了 DNA 结合能力。对 MafA/B 嵌合体的分析表明，对 MafA 磷酸化状态的敏感性是由跨越 C 端二聚化区域（氨基酸 279-359）的序列赋予的，而同源的 MafB 区域（氨基酸 257-323）赋予了与磷酸化无关的 DNA 结合。突变分析表明，形成能够进行 DNA 结合的 MafA 二聚体需要在独特的 N 端转录激活域（氨基酸 1-72）内进行磷酸化，而不是 C 端 b-Zip 区域。这些结果表明，富含磷酸氨基酸的转录激活和 b-Zip 结构域之间存在一种新的关系，控制 MafA 的 DNA 结合活性。
