The regulation of AMP-activated protein kinase by phosphorylation

The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic s ubunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphor ylated by the AMP-activated protein kinase kinase (AMPKK) ill vitro. We hav e used site-directed mutagenesis to study the role of phosphorylation of Th r(172) On AMPK activity. Mutation of Thr(172) to an aspartic acid residue ( T172D) in either alpha 1 or alpha 2 resulted in a kinase complex with appro x. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with prot ein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost comp letely abolished kinase activity. These results indicate that phosphorylati on of Thr172 accounts for most of the activation by AMPKK, but that other s ites are involved. In support of this we have shown that AMPKK phosphorylat es at least two other sites on the alpha subunit and one site on the beta s ubunit. Furthermore, we provide evidence that phosphorylation of Thr(172) m ay be involved in the sensitivity of the AMPK complex to AMP.