Control of protein phosphorylation with a genetically encoded photocaged amino acid

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 Ph04, which controls the cellular response to inorganic phosphate!. When substituted at phosphoserine sites that control nuclear export of Ph04, 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 Ph04-GFP fusion constructs in real time. We observed distinct export kinetics for differentially phosphorylated Ph04 mutants, which demonstrates dynamic regulation of Ph04 function. This methodology should also facilitate the analysis of other cellular processes involving free serine residues, including catalysis, biomolecular recognition and ion transport.