Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan.
PLoS One. 2013 Apr 18;8(4):e61697. doi: 10.1371/journal.pone.0061697. Print 2013.
Turnover of mRNA is a critical step in the regulation of gene expression, and an important step in mRNA decay is removal of the 5' cap. We previously demonstrated that the expression of some immediate early gene mRNAs is controlled by RNA stability during early differentiation of 3T3-L1 preadipocytes.
METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the mouse decapping protein Dcp1a is phosphorylated via the ERK signaling pathway during early differentiation of preadipocytes. Mass spectrometry analysis and site-directed mutagenesis combined with a kinase assay identified ERK pathway-mediated dual phosphorylation at Ser 315 and Ser 319 of Dcp1a. To understand the functional effects of Dcp1a phosphorylation, we examined protein-protein interactions between Dcp1a and other decapping components with co-immunoprecipitation. Dcp1a interacted with Ddx6 and Edc3 through its proline-rich C-terminal extension, whereas the conserved EVH1 (enabled vasodilator-stimulated protein homology 1) domain in the N terminus of Dcp1a showed a stronger interaction with Dcp2. Once ERK signaling was activated, the interaction between Dcp1a and Ddx6, Edc3, or Edc4 was not affected by Dcp1a phosphorylation. Phosphorylated Dcp1a did, however, enhanced interaction with Dcp2. Protein complexes immunoprecipitated with the recombinant phosphomimetic Dcp1a(S315D/S319D) mutant contained more Dcp2 than did those immunoprecipitated with the nonphosphorylated Dcp1a(S315A/S319A) mutant. In addition, Dcp1a associated with AU-rich element (ARE)-containing mRNAs such as MAPK phosphatase-1 (MKP-1), whose mRNA stability was analyzed under the overexpression of Dcp1a constructs in the Dcp1a knockdown 3T3-L1 cells.
CONCLUSIONS/SIGNIFICANCE: Our findings suggest that ERK-phosphorylated Dcp1a enhances its interaction with the decapping enzyme Dcp2 during early differentiation of 3T3-L1 cells.
mRNA 的周转率是基因表达调控的关键步骤，而 mRNA 衰变的一个重要步骤是去除 5'帽。我们之前证明，在 3T3-L1 前脂肪细胞的早期分化过程中，一些即时早期基因 mRNA 的表达受到 RNA 稳定性的控制。
方法/主要发现：在这里，我们表明在脂肪前体细胞的早期分化过程中，鼠脱帽酶 Dcp1a 通过 ERK 信号通路磷酸化。质谱分析和定点突变结合激酶测定法鉴定了 Dcp1a 的 ERK 通路介导的丝氨酸 315 和丝氨酸 319 的双重磷酸化。为了了解 Dcp1a 磷酸化的功能影响，我们通过共免疫沉淀检查了 Dcp1a 与其他脱帽成分之间的蛋白质-蛋白质相互作用。Dcp1a 通过其富含脯氨酸的 C 端延伸与 Ddx6 和 Edc3 相互作用，而 Dcp1a 中 N 端的保守 EVH1（血管扩张刺激蛋白同源 1）结构域与 Dcp2 的相互作用更强。一旦 ERK 信号被激活，Dcp1a 与 Ddx6、Edc3 或 Edc4 的相互作用不受 Dcp1a 磷酸化的影响。然而，磷酸化的 Dcp1a 增强了与 Dcp2 的相互作用。用重组磷酸模拟 Dcp1a(S315D/S319D)突变体免疫沉淀的蛋白质复合物比用非磷酸化的 Dcp1a(S315A/S319A)突变体免疫沉淀的复合物含有更多的 Dcp2。此外，Dcp1a 与含有 AU 丰富元件 (ARE) 的 mRNA 结合，如 MAPK 磷酸酶-1 (MKP-1)，在 Dcp1a 敲低的 3T3-L1 细胞中转录本的表达分析中，Dcp1a 过表达的构建物。
结论/意义：我们的研究结果表明，在 3T3-L1 细胞的早期分化过程中，ERK 磷酸化的 Dcp1a 增强了与脱帽酶 Dcp2 的相互作用。
