Conformation-Dependent Reversible Interaction of Ca2+/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063
Conformation-Dependent Reversible Interaction of Ca2+/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063
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Ca2/钙调蛋白依赖性蛋白激酶激酶与抑制剂 TIM-063 的构象依赖性可逆相互作用
DOI：
10.1021/acs.biochem.1c00796
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发表时间：
2022
期刊：
影响因子：
2.9
通讯作者：
Satomi Ohtsuka;Taisei Okumura;Yuna Τabuchi;Tomoyuki Miyagawa;Naoki Kanayama;Masaki Magari;Naoya Hatano;Hiroyuki Sakagami;Futoshi Suizu;Teruhiko Ishikawa;Hiroshi Tokumitsu
中科院分区：
文献类型：
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作者：
Araya Kibrom Desta;Shuji Ohira;Ismail Arai;Kazutoshi Fujikawa;Satomi Ohtsuka;Taisei Okumura;Yuna Τabuchi;Tomoyuki Miyagawa;Naoki Kanayama;Masaki Magari;Naoya Hatano;Hiroyuki Sakagami;Futoshi Suizu;Teruhiko Ishikawa;Hiroshi Tokumitsu
钙调蛋白依赖性蛋白激酶激酶（Ca 2 +/CaM-dependent protein kinase kinase，CaMKK）是一种钙/钙调蛋白依赖性酶，可磷酸化激活多功能激酶，包括CaMKI、CaMKIV、蛋白激酶B/Akt和5′ AMP-激活的蛋白激酶，参与细胞内多种钙信号通路。最近，我们开发了ATP竞争性CaMKK抑制剂TIM-063（2-羟基-3-硝基-7H-苯并[de]苯并[4，5]咪唑并[2，1-a]异喹啉-7-酮，Ohtsuka等人，Biochemistry 2020，59，1701-1710）。为了获得CaMKK与TIM-063相互作用的机理见解，我们制备了TIM-063偶联的琼脂糖凝胶（TIM-127-琼脂糖凝胶）用于酶/抑制剂复合物的缔合/解离分析。转染的COS-7细胞和小鼠脑提取物中的CaMKKα/β与TIM-127-琼脂糖特异性结合，并在加入TIM-063后以类似于重组GST-CaMKKα/β的方式解离，其可以以Ca 2 +/CaM依赖性方式与TIM-127-琼脂糖结合，并在加入TIM-063后以剂量依赖性方式从琼脂糖上解离。与GST-CaMKKα相反，GST-CaMKKβ能够在EGTA存在下与TIM-127-sepharose弱结合，这可能是由于重组GST-CaMKKβ的部分活性构象不具有Ca 2 +/CaM结合。这些结果表明，CaMKKα的调节结构域阻止了抑制剂与催化结构域的相互作用，因为缺乏调节结构域（残基438-463）的GST-CaMKKα突变体（残基126-434）与TIM-127-琼脂糖相互作用，无论是否存在Ca 2 +/CaM。此外，在Ca 2 +/CaM存在下，CaMKKα与TIM-127-sepharose结合，加入过量EGTA后，CaM与TIM-127-sepharose完全解离。这些结果表明，TIM-063在其活性状态下与CaMKK相互作用并抑制CaMKK，但在其自抑制状态下不相互作用，并且这种相互作用可能是可逆的，这取决于细胞内Ca 2+的浓度。
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+.