FIGURE 5.

AMPK phosphorylates Cytc on Thr²⁸in vitro. A, in vitro AMPK kinase assay with cow heart Cytc, conducted in the absence of AMPK activator AMP, shows a time-dependent increase of threonine phosphorylation, as determined by Western blotting analysis. B, in vitro AMPK kinase assay with cow heart Cytc shows an increase of threonine phosphorylation in the presence of AMPK activator AMP. C, Cytc subjected to an in vitro AMPK kinase assay as in A is phosphorylated on Thr²⁸ as determined by the nano-LC/ESI/MS/MS spectrum of HKpTGPNLHGLFGR. The phosphorylation site was unambiguously assigned by fragment ions y10 and y11. The sequence of the peptide was assigned by b2, b8, b9, y2, y5, y6, y8, y9, and y10.