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

摘要

The Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ)/5′-AMP–activated protein kinase (AMPK) phosphorylation cascade affects various Ca²⁺-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca²⁺ concentrations. Moreover, the Ca²⁺/CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca²⁺/CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr¹⁴⁴ phosphorylation by activated AMPK converts CaMKKβ into a Ca²⁺/CaM-dependent enzyme as shown by completely Ca²⁺/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ 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 Thr¹⁴⁴ phosphorylation–dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr¹⁴⁴ antibody revealed phosphorylation of Thr¹⁴⁴ in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca²⁺-dependent AMPK activation by CaMKKβ.