Conformation-Dependent Reversible Interaction of Ca 2+ /Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063.

Autor: Ohtsuka S; Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University, Okayama 700-8530, Japan., Okumura T; Department of Science Education, Graduate School of Education, Okayama University, Okayama 700-8530, Japan., Τabuchi Y; Department of Science Education, Graduate School of Education, Okayama University, Okayama 700-8530, Japan., Miyagawa T; Department of Science Education, Graduate School of Education, Okayama University, Okayama 700-8530, Japan., Kanayama N; Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University, Okayama 700-8530, Japan., Magari M; Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University, Okayama 700-8530, Japan., Hatano N; Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University, Okayama 700-8530, Japan., Sakagami H; Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan., Suizu F; Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan., Ishikawa T; Department of Science Education, Graduate School of Education, Okayama University, Okayama 700-8530, Japan., Tokumitsu H; Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University, Okayama 700-8530, Japan.
Jazyk: angličtina
Zdroj: Biochemistry [Biochemistry] 2022 Apr 05; Vol. 61 (7), pp. 545-553. Date of Electronic Publication: 2022 Mar 11.
DOI: 10.1021/acs.biochem.1c00796
Abstrakt: Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK), a Ca 2+ /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 Ca 2+ -signaling pathways in cells. Recently, we developed an ATP-competitive CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7 H -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 Ca 2+ /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 Ca 2+ /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 Ca 2+ /CaM. Furthermore, CaMKKα bound to TIM-127-sepharose in the presence of Ca 2+ /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 Ca 2+ .
Databáze: MEDLINE