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

Matthew J Sanders , Pascal O Grondin , Bronwyn D Hegarty , Michael A Snowden , David Carling (1)

1 Cellular Stress Group

AMP-activated protein kinase (AMPK) is activated allosterically by AMP and by phosphorylation of threonine residue 172 (T172) within the catalytic subunit ({alpha}). Here we show that mutations in the {gamma} regulatory subunit reduce allosteric activation of the kinase by AMP. In addition to its allosteric effect, AMP significantly reduces the dephosphorylation of T172 by protein phosphatase 2C{alpha} (PP2C{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 T172 phosphorylation by either LKB1 or Ca 2+}/calmodulin-dependent protein kinase kinase (CaMKK) {beta} using recombinant preparations of the proteins. However, using partially purified AMPK from rat liver there was an apparent AMP-stimulation of T172 phosphorylation by LKB1, but this was blocked by the addition of NaF, a protein phosphatase 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 T172 were misinterpreted. A plausible explanation for the 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: direct allosteric activation and by protecting T172 from dephosphorylation. Based on 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 Ca 2+}-dependent pathway, mediated by CaMKK{beta} and an AMP-dependent pathway, mediated by LKB1.

Mots clés: Life Sciences

Matthew J Sanders, Pascal O Grondin, Bronwyn D Hegarty, Michael A Snowden, David Carling. Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochemical Journal, 2006, 403 (1), pp.139-148. ⟨10.1042/BJ20061520⟩. ⟨hal-00478681⟩