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. CaMKKα/β 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–CaMKKα/β, which could
bind to TIM-127-sepharose 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–CaMKKα,
GST–CaMKKβ was able to weakly bind to TIM-127-sepharose
in the presence of EGTA, probably due to the partially active conformation
of recombinant GST–CaMKKβ without Ca2+/CaM-binding.
These results suggested that the regulatory domain of CaMKKα
prevented the inhibitor from interacting with the catalytic domain
as the GST–CaMKKα 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,
CaMKKα 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+.