AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase

The Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)/5-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca2+-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKK that exhibits higher basal activity (autonomous activity), activation of the CaMKK/AMPK signaling pathway requires increased intracellular Ca2+ concentrations. Moreover, the Ca2+/CaM dependence of CaMKK appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKK activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKK at multiple residues by CaMKK-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca2+/CaM-activated, CaMKK activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKK indicated that Thr(144) phosphorylation by activated AMPK converts CaMKK into a Ca2+/CaM-dependent enzyme as shown by completely Ca2+/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKK mutant. CaMKK mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr(144) phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr(144) antibody revealed phosphorylation of Thr(144) in CaMKK in transfected COS-7 cells that was further enhanced by exogenous expression of AMPK. These results indicate that AMPK-mediated feedback phosphorylation of CaMKK regulates the CaMKK/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca2+-dependent AMPK activation by CaMKK.