Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis

Bacterially expressed heterotrimeric (alpha(1), beta(1), and gamma(1)) wild-type, catalytically inactive, and constitutively active forms of AMP-activated protein kinase ( AMPK) were used to study phosphorylation by an upstream AMPK kinase preparation. Here, we report the identification of two new phosphorylation sites in the alpha-subunit, viz. Thr(258) and Ser(485) (Ser(491) in the alpha(2)-subunit) by mass spectrometry, in addition to the previously characterized Thr(172) site. Also, autophosphorylation sites in the beta(1)-subunit were identified as Ser(96), Ser(101), and Ser(108). Mutagenesis of Thr(172), Thr(258), and Ser(485) to acidic residues to mimic phosphorylation in the recombinant proteins indicated that Thr172 was involved in AMPK activation, whereas Thr258 and Ser485 were not. Transfection of the non-phosphorylatable S485A and T258A mutants in CCL13 cells subjected to stresses known to activate AMPK either by increasing the AMP: ATP ratio ( slow lysis) or without changing adenine nucleotide concentrations (hyperosmolarity) resulted in no significant differences in AMPK activation. All three sites within the alpha-subunit were phosphorylated in vivo, as seen in AMPK immunoprecipitated from anoxic rat liver. In transfected CCL13 cells, the level of Ser(485) phosphorylation did not change upon AMPK activation. The newly identified phosphorylation sites could play a subtle role in the regulation of AMPK, e.g. in subcellular localization or substrate recognition.