Fig. 2. Identification of residues on NCC that are phosphorylated by OSR1 in vitro. (A) E. coli-expressed NCC[1-100] was incubated with the indicated active (Act) and kinase-inactive (KI) forms of OSR1 or SPAK in the presence of Mg-[³²P]ATP for 40 minutes. Phosphorylation of NCC[1-100] was determined following electrophoresis and subsequent autoradiography of the Colloidal-blue-stained bands corresponding to NCC[1-100]. Similar results were obtained in three separate experiments. KI-SPAK, kinase-inactive SPAK[D212A]; Act-SPAK, activated-SPAK[T233E]; KI-OSR1, kinase-inactive-OSR1[D164A]; Act-OSR1, activated OSR1[T185E]. (B) E. coli-expressed NCC[1-100] was incubated with active OSR1 in the presence of Mg-[³²P]ATP for the indicated times and phosphorylation of NCC[1-100] was determined as in A. (C) The indicated forms of NCC[1-100] were phosphorylated with active OSR1 in the presence of Mg-[³²P]ATP for 60 minutes. Phosphorylated NCC[1-100] was digested with trypsin and chromatographed on a C₁₈ column. The peak fractions containing the major ³²P-labelled peptides are labelled P1, P2, P3 and P4. (D) Summary of the mass spectrometry and solid-phase Edman sequencing data obtained following analysis of the indicated phosphopeptides. The deduced amino acid sequence of each peptide is shown and the phosphorylated residue is indicated by (p). `m' in the peptide sequence indicates methionine sulphoxide. (E) The indicated forms of NCC[1-100] were phosphorylated with kinase-inactive or active OSR1 or SPAK. Phosphorylation was analysed as in A as well as by quantification of ³²P radioactivity associated with Colloidal-blue-stained bands corresponding to NCC[1-100]. Results of the latter are presented as a percentage of phosphorylation compared to wild-type (WT) NCC[1-100] by active OSR1/SPAK. Results of duplicate samples are shown and similar results were obtained in two separate experiments. AAA corresponds to a triple NCC[1-100] mutant in which Thr46, Thr55 and Thr60 are changed to Ala.