Identification and Functional Characterization of ERK/MAPK Phosphorylation Sites in the Runx2 Transcription Factor
Identification and Functional Characterization of ERK/MAPK Phosphorylation Sites in the Runx2 Transcription Factor
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DOI：
10.1074/jbc.m109.040980
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发表时间：
2009-11-20
影响因子：
4.8
通讯作者：
Franceschi, Renny T.
中科院分区：
文献类型：
--
作者：
Ge, Chunxi;Xiao, Guozhi;Franceschi, Renny T.
Runx2转录因子是间充质细胞向骨系分化所必需的，是成骨细胞特异性基因表达的主要调节因子。Runx2受许多转录后调控，包括选择性蛋白分解和磷酸化。我们以前报道过Runx2被ERK/MAPK通路磷酸化并被激活(肖，G.，酱，D.，Thomas，P.，Benson，M.D.，Guan，K.，Karsty，G.和Franceschi，R.T.(2000)J.Biol)。化学。275、4453-4459)。在这项研究中，我们使用体外和体内磷酸化分析、质谱分析和功能分析相结合的方法，确定了Runx2的Pro/Ser/Thinine结构域中的两个位点Ser(301)和Ser(319)是这种调控所必需的。在体外和细胞培养中，这些位点被激活的ERK1磷酸化。除了确认ERK依赖于Ser(319)的磷酸化外，质谱学还发现了Ser(43)和Ser(510)的另外两个ERK磷酸化位点。此外，S301A、S319A突变的引入使Runx2对MAPK依赖的激活产生抵抗，并降低了其在转染Runx2缺失的颅骨细胞和间充质细胞后刺激成骨细胞特异性基因表达和分化的能力。相反，S301E和S319E Runx2突变体增强了转录活性，这种活性最小地依赖于MAPK信号，这与添加模拟丝氨酸磷酸化的负电荷一致。这些结果强调了Runx2磷酸化在成骨细胞基因表达调控中的重要作用，并为解释生理信号如何通过ERK/MAPK途径作用于骨骼刺激成骨细胞特异性基因表达提供了机制。
The Runx2 transcription factor is required for commitment of mesenchymal cells to bone lineages and is a major regulator of osteoblast-specific gene expression. Runx2 is subject to a number of post-transcriptional controls including selective proteolysis and phosphorylation. We previously reported that Runx2 is phosphorylated and activated by the ERK/MAPK pathway (Xiao, G., Jiang, D., Thomas, P., Benson, M. D., Guan, K., Karsenty, G., and Franceschi, R. T. (2000) J. Biol. Chem. 275, 4453-4459). In this study, we used a combination of in vitro and in vivo phosphorylation analysis, mass spectroscopy, and functional assays to identify two sites at Ser(301) and Ser(319) within the proline/serine/threonine domain of Runx2 that are required for this regulation. These sites are phosphorylated by activated ERK1 in vitro and in cell culture. In addition to confirming ERK-dependent phosphorylation at Ser(319), mass spectroscopy identified two other ERK-phosphorylated sites at Ser(43) and Ser(510). Furthermore, introduction of S301A, S319A mutations rendered Runx2 resistant to MAPK-dependent activation and reduced its ability to stimulate osteoblast-specific gene expression and differentiation after transfection into Runx2-null calvarial cells and mesenchymal cells. In contrast, S301E, S319E Runx2 mutants had enhanced transcriptional activity that was minimally dependent on MAPK signaling, consistent with the addition of a negative charge mimicking serine phosphorylation. These results emphasize the important role played by Runx2 phosphorylation in the control of osteoblast gene expression and provide a mechanism to explain how physiological signals acting on bone through the ERK/MAPK pathway can stimulate osteoblast-specific gene expression.