Protein phosphorylation regulates diverse processes in eukaryotic cells. Strategies for installing site-specific phosphorylation in target proteins in eukaryotic cells, through routes that are orthogonal to enzymatic post-translational modification, would provide a powerful route for defining the consequences of particular phosphorylations. Here we show that the SepRSv1.0/tRNAv1.0CUApair (created from theMethanococcus maripaludisphosphoseryl-transfer RNA synthetase [MmSepRS]/Methanococcus janaschii[Mj]tRNAGCACyspair) is orthogonal in mammalian cells. We create a eukaryotic elongation factor 1 alpha (EF-1α) variant, EF-1α-Sep, that enhances phosphoserine incorporation, and combine this with a mutant of eRF1, and manipulations of the cell’s phosphoserine biosynthetic pathway, to enable the genetically encoded incorporation of phosphoserine and its non-hydrolyzable phosphonate analog. Using this approach we demonstrate synthetic activation of a protein kinase in mammalian cells.

中文翻译：

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哺乳动物细胞中基因编码的蛋白质磷酸化

蛋白质磷酸化调节真核细胞中的多种过程。通过与酶翻译后修饰正交的途径在真核细胞中的靶蛋白中安装位点特异性磷酸化的策略将为定义特定磷酸化的后果提供强有力的途径。在这里，我们展示了 SepRSv1.0/tRNAv1.0CUA 对（由马里帕卢甲烷球菌二磷酸丝氨酰转移 RNA 合成酶 [MmSepRS]/Methanococcus janaschii[Mj]tRNAGCACyspair 创建）在哺乳动物细胞中是正交的。我们创建了一种真核延伸因子 1 α (EF-1α) 变体 EF-1α-Sep，它增强了磷酸丝氨酸的掺入，并将其与 eRF1 的突变体相结合，并操纵细胞的磷酸丝氨酸生物合成途径，以实现磷酸丝氨酸及其不可水解的膦酸盐类似物的基因编码掺入。使用这种方法，我们展示了哺乳动物细胞中蛋白激酶的合成激活。