Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade

AMPK (AMP-activated protein kinase) is activated allosterically by AMP and by phosphorylation of Thr(172) within the catalytic a subunit. Here we show that mutations in the regulatory gamma subunit reduce allosteric activation of the kinase by AMP. In addition to its allosteric effect, AMP significantly reduces the dephosphorylation of Thr(172) by PP (protein phosphatase)2C alpha. Moreover, a mutation in the gamma subunit almost completely abolishes the inhibitory effect of AMP on dephosphorylation. We were unable to detect any effect of AMP on Thr(172) phosphorylation by either LKB1 or CaMKK beta (Ca2+/calmodulin-dependent protein kinase kinase beta) using recombinant preparations of the proteins. However, using partially purified AMPK from rat liver, there was an apparent AMP-stimulation of Thr(172) phosphorylation by LKB1, but this was blocked by the addition of NaF, a PP inhibitor. Western blotting of partially purified rat liver AMPK and LKB1 revealed the presence of PP2C alpha in the preparations. We suggest that previous studies reporting that AMP promotes phosphorylation of Thr(172) were misinterpreted. A plausible explanation for this effect of AMP is inhibition of dephosphorylation by PP2C alpha, present in the preparations of the kinases used in the earlier studies. Taken together, our results demonstrate that AMP activates AMPK via two mechanisms: by direct allosteric activation and by protecting Thr(172). from dephosphorylation. On the basis of our new findings, we propose a simple model for the regulation of AMPK in mammalian cells by LKB1 and CaMKK beta. This model accounts for activation of AMPK by two distinct signals: a Ca2+-dependent pathway, mediated by CaMKK and an AMP-dependent pathway, mediated by LKB1.