Control of protein phosphorylation with a genetically encoded photocaged amino acid.
We genetically encoded the photocaged amino acid 4,5-dimethoxy-2-nitrobenzylserine (DMNB-Ser) 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 phosphate. When substituted at phosphoserine sites that control nuclear export of Pho4, 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.
Lemke
Edward A
EA
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road SR202, La Jolla, California 92037, USA.
Summerer
Daniel
D
Geierstanger
Bernhard H
BH
Brittain
Scott M
SM
Schultz
Peter G
PG
D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D009578:Nitrobenzenes / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D010777:Photochemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C525105:4,5-dimethoxy-2-nitrobenzylserine; D004268:DNA-Binding Proteins; D009578:Nitrobenzenes; C060292:PHO4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012694:Serine