FIGURE 2.

NHE5 is phosphorylated in vitro by AMPK. A, GST-NHE5 fusion proteins were incubated in vitro with [γ-³²P]ATP and purified, partially activated, rat liver AMPK in the presence or absence of AMP (0.3 mm), subjected to SDS-PAGE, and analyzed by autoradiography. Protein loading was visualized by staining the gel with Coomassie Blue (CB) dye. B, GST-NHE5 fusion proteins (571–601, 602–624, and 845–871) containing simultaneous Ala substitutions of potential target Ser residues (highlighted in enlarged bold font size) in putative AMPK recognition motifs (upper panel) were incubated with purified rat liver AMPK and [γ-³²P]ATP in vitro with or without AMP, subjected to SDS-PAGE and analyzed by autoradiography (lower panel). Data are representative of at least three independent experiments. S575–7A, S575A/S577A; S618–20A, S618A/S620A; S851–6A, S851A/S853A/S855A/S856A.