School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK, Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, University of Colorado at Boulder, Boulder, CO 80309, USA, Department of Neurology, Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, CA 90089, USA and Department of Pharmacology, Ningxia Medical University, Yinchuan 750004, China.
Nucleic Acids Res. 2013 Dec;41(22):10241-53. doi: 10.1093/nar/gkt837. Epub 2013 Sep 17.
The ETS domain transcription factor Elk-1 stimulates expression of immediate early genes (IEGs) in response to mitogens. These events require phosphorylation of Elk-1 by extracellular signal-regulated kinase (ERK) and phosphorylation-dependent interaction of Elk-1 with co-activators, including histone acetyltransferases and the Mediator complex. Elk-1 also recruits ERK to the promoters of its target genes, suggesting that ERK phosphorylates additional substrates in transcription complexes at mitogen-responsive promoters. Here we report that MED14, a core subunit of the Mediator, is a bona fide ERK substrate and identify serine 986 (S986) within a serine-proline rich region of MED14 as the major ERK phosphorylation site. Mitogens induced phosphorylation of MED14 on S986 at IEG promoters; RNAi knockdown of MED14 reduced CDK8 and RNA polymerase II (RNAPII) recruitment, RNAPII C-terminal domain phosphorylation and impaired activation of IEG transcription. A single alanine substitution at S986 reduced activation of an E26 (ETS)-responsive reporter by oncogenic Ras and mitogen-induced, Elk-1-dependent transcription, whereas activities of other transcriptional activators were unaffected. We also demonstrate that Elk-1 can associate with MED14 independently of MED23, which may facilitate phosphorylation of MED14 by ERK to impart a positive and selective impact on mitogen-responsive gene expression.
ETS 结构域转录因子 Elk-1 可刺激丝裂原诱导的即刻早期基因 (IEGs) 的表达。这些事件需要细胞外信号调节激酶 (ERK) 对 Elk-1 的磷酸化，以及 Elk-1 与共激活因子（包括组蛋白乙酰转移酶和 Mediator 复合物）的磷酸化依赖性相互作用。Elk-1 还将 ERK 募集到其靶基因的启动子，这表明 ERK 在有丝分裂原响应启动子处的转录复合物中磷酸化额外的底物。在这里，我们报告 Mediator 的核心亚基 MED14 是一种真正的 ERK 底物，并鉴定出 MED14 丝氨酸-脯氨酸丰富区内的丝氨酸 986 (S986) 是 ERK 的主要磷酸化位点。丝裂原诱导 IEG 启动子上 MED14 的 S986 磷酸化；MED14 的 RNAi 敲低降低了 CDK8 和 RNA 聚合酶 II (RNAPII) 的募集、RNAPII C 末端结构域磷酸化，并损害了 IEG 转录的激活。S986 处的单个丙氨酸取代降低了致癌性 Ras 和有丝分裂原诱导的 Elk-1 依赖性转录对 E26（ETS）反应性报告基因的激活作用，而其他转录激活因子的活性不受影响。我们还证明 Elk-1 可以独立于 MED23 与 MED14 结合，这可能促进 ERK 对 MED14 的磷酸化，从而对有丝分裂原反应基因表达产生积极和选择性的影响。
