Conformation-Dependent Reversible Interaction of Ca2+/ Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), a Ca2+/CaM-dependent enzyme that phosphorylates and activates multifunctional kinases, including CaMKI, CaMKIV, protein kinase B/Akt, and 5'AMP-activated protein kinase, is involved in various Ca2+-signaling pathways in cells. Recently, we developed an ATP competitive CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo-[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one, Ohtsuka et al. Biochemistry 2020, 59, 1701-1710). To gain mechanistic insights into the interaction of CaMKK with TIM-063, we prepared TIM-063-coupled sepharose (TIM-127-sepharose) for association/dissociation analysis of the enzyme/inhibitor complex. CaMKK alpha/beta in transfected COS-7 cells and in mouse brain extracts specifically bound to TIM-127-sepharose and dissociated following the addition of TIM-063 in a manner similar to that of recombinant GST-CaMKK alpha/beta, which could bind to TIM-127sepharose in a Ca2+/CaM-dependent fashion and dissociate from the sepharose following the addition of TIM-063 in a dose dependent manner. In contrast to GST-CaMKK alpha, GST-CaMKK beta was able to weakly bind to TIM-127-sepharose in the presence of EGTA, probably due to the partially active conformation of recombinant GST-CaMKK beta without Ca2+/CaM-binding. These results suggested that the regulatory domain of CaMKK alpha prevented the inhibitor from interacting with the catalytic domain as the GST-CaMKK alpha mutant (residues 126-434) lacking the regulatory domain (residues 438-463) interacted with TIM-127-sepharose regardless of the presence or absence of Ca2+/CaM. Furthermore, CaMKK alpha bound to TIM-127-sepharose in the presence of Ca2+/ CaM completely dissociated from TIM-127-sepharose following the addition of excess EGTA. These results indicated that TIM-063 interacted with and inhibited CaMKK in its active state but not in its autoinhibited state and that this interaction is likely reversible, depending on the concentration of intracellular Ca2+.