Figure 4. Identification of sites within NKCC2 that are phosphorylated by AMPK.

(A) GFP–NKCC2^N−term was incubated with purified AMPK in the presence of [γ-³²P]ATP and subjected to digestion by trypsin, the resulting phosphopeptides were analysed by two-dimensional peptide mapping and autoradiography. Major radiolabelled phosphopeptide spots are indicated by arrows and labelled a, b and c. The direction of electrophoresis and chromatography are indicated on the axes of the autoradiograph. (B) GFP–NKCC2^N−term was incubated with purified AMPK in the presence of [γ-³²P]ATP and subjected to digestion by trypsin, and the resulting peptides were analysed by reverse-phase HPLC. The dotted line indicates the percentage (v/v) of CH₃CN. (C) The radioactive fractions corresponding to peak 1 were subjected to solid-phase Edman sequencing, and the release of radioactivity was determined at each cycle. (D) MALDI-TOF spectrometry was used to analyse peptides contained in peak 1. Wild-type GFP–NKCC2^N−term (E) or GFP–NKCC2^N−term in which Ser¹²⁶ had been mutated to alanine (F) were incubated with purified AMPK in the presence of [γ-³²P]ATP and subjected to digestion by trypsin, the resulting phosphopeptides were analysed by two-dimensional peptide mapping and autoradiography. Major radiolabelled phosphopeptides obtained from the GFP–NKCC2^N−term are indicated by arrows and labelled. This was superimposed on the corresponding GFP–NKCC2^N−term S126A mutant phosphopeptide map. The direction of electrophoresis and chromatography are indicated on the axes of the autoradiograph. (G) ClustalW alignment of NKCC2 sequences obtained from different species and other members of the cation chloride co-transporter family. Ser¹²⁶ (rabbit NKCC2 numbering) is identified and corresponding residues are highlighted.