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. 2019 Jul 15;294(35):13106–13116. doi: 10.1074/jbc.RA119.008649

Figure 2.

Figure 2.

Phosphorylation of the NTD by AKT and AMPK. A, 2D 1H-15N HMQC NMR spectra of NTDΔ incubated for 3 h with purified AKT (orange) or AMPK (red) to assess Thr24 and Ser22 phosphorylation by AKT and AMPK, respectively. Chemical shift changes of phosphorylated amino acids are indicated by arrows. Doubling of peaks for pSer22 suggests dual conformations. B, 2D 1H-15N HMQC NMR spectra of prephosphorylated NTDΔ incubated with the indicated kinases for 4.5 h; NTDΔ-pSer22 with AKT to monitor Thr24 phosphorylation (orange) and NTDΔ-pT24 with AMPK to analyze Ser22 phosphorylation (red). Chemical shift changes of phosphorylated amino acids are indicated by arrows. C, the activities of both AKT and AMPK kinases on different substrates were analyzed using a fluorescence-based ADP biosensor. The unphosphorylated NTD as substrate for AKT and AMPK is shown in blue. The activity of AKT on NTD-pSer22 is shown in red. The activity of AMPK on NTD-pThr24 is shown in orange. Km values were determined based on the initial reaction rates. Data are mean ± S.D. from three independent experiments. ND, not detectable.