Phosphorylation modulates AfsR transcriptional activation.

Abstract

(A) LC-MS/MS analysis showing that S22, T337, S391, T506, and S953 are phosphorylated in AfsR by AfsKΔC. The lowercase letter in the peptide sequence indicates phosphorylated residue. “b” and “y” denote peptide fragment ions retaining charges at the N and C terminus, respectively. The subscript numbers indicate their positions in the identified peptide. (B) Sequence alignment of Streptomyces AfsR family members highlighting the consensuses sequences neighboring phosphorylation sites S22, T337, S391, T506, and S953. Orange boxes represent phosphorylation sites. (C) Transcription assays with increasing concentrations of phosphorylated AfsR by AfsKΔC (phos-AfsR) and untreated AfsR. (D) Transcription assays of 500 nM AfsR mutants mimicking dephosphorylation (T337A) and phosphorylation (S22E, T337E, T506E, S953E, and S391E/T337E/T506E/S953E (4E)). Data are presented as mean ± SEM from 3 independent assays. n.s. means no significance; *P P T337A and AfsR4E. (F) Fluorescence polarization assay of AfsR4E and dephosphorylated AfsR (dephos-AfsR) with afs box. The concentration of afs box was 10 nM. Error bars represent mean ± SEM of n = 3 experiments. The data underlying A, C, D, E, and F are provided in S1 Data. (TIF)</p