Control of protein phosphorylation with a genetically encoded photocaged amino acid
Control of protein phosphorylation with a genetically encoded photocaged amino acid
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DOI：
10.1038/nchembio.2007.44
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发表时间：
2007-12-01
影响因子：
14.8
通讯作者：
Schultz, Peter G.
中科院分区：
文献类型：
--
作者：
Lemke, Edward A.;Summerer, Daniel;Schultz, Peter G.
我们在酿酒酵母中响应琥珀无义密码子TAG，遗传编码了光笼氨基酸4，5-二甲氧基-2-硝基苄基丝氨酸（DMNB-Ser，1）。这种氨基酸在活细胞中通过相对低能量的蓝光照射转化为丝氨酸，并用于非侵入性地光活化转录因子Pho 4的磷酸化，其控制细胞对无机磷酸盐的反应1。当在控制Pho 4核输出的磷酸丝氨酸位点被取代时，1阻断磷酸化和随后通过受体Msn 5的输出（参考文献2）。我们用可见激光脉冲触发单个丝氨酸残基的磷酸化，并在真实的时间内监测Pho 4-GFP融合构建体的核输出。我们观察到不同的磷酸化的Pho 4突变体，这表明动态调节Pho 4功能的独特的出口动力学。这种方法也应该有助于分析其他细胞过程，涉及游离丝氨酸残基，包括催化，生物分子识别和离子转运。
We genetically encoded the photocaged amino acid 4,5-dimethoxy-2-nitrobenzylserine (DMNB-Ser, 1) in Saccharomyces cerevisiae in response to the amber nonsense codon TAG. This amino acid was converted to serine in living cells by irradiation with relatively low-energy blue light and was used to noninvasively photoactivate phosphorylation of the transcription factor Pho4, which controls the cellular response to inorganic phosphate1. When substituted at phosphoserine sites that control nuclear export of Pho4, 1 blocks phosphorylation and subsequent export by the receptor Msn5 (ref. 2). We triggered phosphorylation of individual serine residues with a visible laser pulse and monitored nuclear export of Pho4-GFP fusion constructs in real time. We observed distinct export kinetics for differentially phosphorylated Pho4 mutants, which demonstrates dynamic regulation of Pho4 function. This methodology should also facilitate the analysis of other cellular processes involving free serine residues, including catalysis, biomolecular recognition and ion transport.