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Control of protein phosphorylation with a genetically encoded photocaged amino acid

Nature Chemical Biology volume 3, pages 769–772 (2007)Cite this article

Abstract

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.

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Figure 1: Incorporation of 1 into hSOD-33TAG and Pho4 phosphorylation scheme.
Figure 2: In vivo photolysis of the DMNB-Ser Pho4-GFP mutants.
Figure 3: Real-time analysis of in vivo photolysis of DMNB-Ser Pho4-GFP mutants.

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Acknowledgements

We thank E. O'Shea (Howard Hughes Medical Institute, Harvard University) for the PRS-Pho4WT-GFP plasmid, the mass spectrometry facility of the Genomics Institute of the Novartis Research Foundation for protein mass measurements and E. Peters for helpful discussions. E.A.L. and D.S. acknowledge a postdoctoral scholarship from the Alexander von Humboldt Foundation. This work was also supported by the US Department of Energy (DE-FG03-00ER46051) and the Skaggs Institute for Chemical Biology.

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Author notes

  1. Edward A Lemke and Daniel Summerer: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road SR202, La Jolla, 92037, California, USA

    Edward A Lemke, Daniel Summerer & Peter G Schultz

  2. Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, 92121-1125, California, USA

    Bernhard H Geierstanger, Scott M Brittain & Peter G Schultz

Authors
  1. Edward A Lemke
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Contributions

E.A.L. designed experiments, performed biological experiments, imaging and data analysis, and wrote the manuscript; D.S. designed experiments, evolved the synthetase, performed biological experiments and wrote the manuscript; B.H.G. and S.M.B. provided mass spectrometric analysis; B.H.G. assisted in editing the manuscript; P.G.S. designed experiments and wrote the manuscript.

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Correspondence to Peter G Schultz.

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Lemke, E., Summerer, D., Geierstanger, B. et al. Control of protein phosphorylation with a genetically encoded photocaged amino acid. Nat Chem Biol 3, 769–772 (2007). https://doi.org/10.1038/nchembio.2007.44

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