Phosphorylation and dephosphorylation of Ca2+/calmodulin-dependent protein kinase kinase β at Thr144 in HeLa cells

Abstract

Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases including CaMKI, CaMKIV, and 5'AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca 2+-dependent cellular processes including neuronal, metabolic, and pathophysiological pathways. Herein we developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analog (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and β) with a similar potency (Ki = 0.35 µM for CaMKKα and Ki = 0.2 µM for CaMKKβ) in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV and endogenous AMPKα in HeLa cells with an IC 50 of ~0.3 µM, and it suppressed the CaMKK isoforms-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases.

Molecular Mechanisms Underlying Ca2+/Calmodulin-Dependent Protein Kinase Kinase Signal Transduction

“…Phosphorylation at multiple sites in CaMKKβ/2 likely disrupts its N-terminal regulatory function to generate an autonomous activity, thereby holding the kinase tightly regulated by Ca 2+ /CaM, in agreement with the finding that CaMKKβ/2–AMPK pathway activation requires Ca 2+ /CaM signaling ( Figure 2 B,C) [ 33 , 34 , 35 ]. Analogous to Thr108 phosphorylation in rat CaMKKα/1 by PKA, Thr144 in rat CaMKKβ/2 was phosphorylated by cAMP/PKA signaling in transfected HeLa cells [ 79 ] and was dynamically regulated by protein phosphatases [ 91 ]. Schumacher et al reported that death-associated protein kinase-mediated phosphorylation of human CaMKKβ/2 at Ser511 (Ser510 in rat CaMKKβ/2) attenuates Ca 2+ /CaM-stimulated CaMKK autophosphorylation [ 92 ], although the effect of Ser511 phosphorylation on CaMKK activity is still unclear.…”