Calmodulin-dependent protein kinase kinase-β activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP

Activation of AMPK (AMP-activated protein kinase) by phosphorylation at Thr(172) is catalysed by at least two distinct upstream kinases, i.e. the tumour suppressor LKB1, and CaMKK beta (Ca2+/calmodulin-dependent protein kinase kinase-beta). The sequence around Thr(172) is highly conserved between the two catalytic subunit isoforms of AMPK and the 12 AMPK-related kinases, and LKB1 has been shown to act upstream of all of them. In the present paper we report that none of the AMPK-related kinases tested could be phosphorylated or activated in intact cells or cell-free assays by CaMKK. although we did observe a slow phosphorylation and activation of BRSK1 (brain-specific kinase 1) by CaMKK alpha. Despite recent reports, We could not find any evidence that the alpha and/or beta subunits of AMPK formed a stable complex with CaMKK beta. We also showed that increasing AMP concentrations in HeLa cells (which lack LKB1) had no effect oil basal AMPK phosphorylation, but enhanced the ability of agents that increase intracellular Ca2+ to activate AMPK. This is consistent with the effect of AMP oil phosphorylation of Thr(172) being due to inhibition of dephosphorylation, and confirms that the effect of AMP is independent of the upstream kinase utilized.