STO-609, a specific inhibitor of the Ca2+/calmodulin-dependent protein kinase kinase
STO-609, a specific inhibitor of the Ca2+/calmodulin-dependent protein kinase kinase
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DOI：
10.1074/jbc.m201075200
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发表时间：
2002-05-03
影响因子：
4.8
通讯作者：
Kobayashi, R
中科院分区：
文献类型：
--
作者：
Tokumitsu, H;Inuzuka, H;Kobayashi, R
合成了一种钙/钙调素依赖性蛋白激酶激酶（CaM-KK）选择性抑制剂STO-609，并对其体内外抑制特性进行了研究。STO-609抑制重组CaM-KK α和CaM-KK β同种型的活性，Ki值分别为80和15 ng/ml，并且还抑制其自身磷酸化活性。该化合物对各种蛋白激酶的抑制效力的比较表明，STO-609对CaM-KK具有高度选择性，而对下游CaM激酶（CaM-KI和-IV）没有任何显著影响，并且该化合物对CaM-KII的IC 50值类似于10 μ g/ ml。STO-609抑制组成型活性CaM-KKa（谷胱甘肽S-转移酶（GST）-CaM-KK-（84-434））以及野生型酶。动力学分析表明，该化合物是ATP的竞争性抑制剂。在转染的HeLa细胞中，STO-609以剂量依赖性方式抑制Ca 2+诱导的CaM-KIV活化。与该观察结果一致，抑制剂在1 μ g/ml的浓度下显著降低SH-SY 5 Y神经母细胞瘤细胞中CaM-KK的内源性活性（类似于80%抑制率）。总之，这些结果表明，STO-609是一种选择性和细胞渗透性抑制剂的CaM-KK，它可能是一个有用的工具，用于评估的生理意义的CaM-KK介导的途径在体内以及在体外。
STO-609, a selective inhibitor of Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) was synthesized, and its inhibitory properties were investigated both in vitro and in vivo. STO-609 inhibits the activities of recombinant CaM-KKalpha and CaM-KKbeta isoforms, with K-i values of 80 and 15 ng/ml, respectively, and also inhibits their autophosphorylation activities. Comparison of the inhibitory potency of the compound against various protein kinases revealed that STO-609 is highly selective for CaM-KK without any significant effect on the downstream CaM kinases (CaM-KI and -IV), and the IC50 value of the compound against CaM-KII is similar to10 mug/ ml. STO-609 inhibits constitutively active CaM-KKa (glutathione S-transferase (GST)-CaM-KK-(84-434)) as well as the wild-type enzyme. Kinetic analysis indicates that the compound is a competitive inhibitor of ATP. In transfected HeLa cells, STO-609 suppresses the Ca2+- induced activation of CaM-KIV in a dose-dependent manner. In agreement with this observation, the inhibitor significantly reduces the endogenous activity of CaM-KK in SH-SY5Y neuroblastoma cells at a concentration of 1 mug/ml (similar to80% inhibitory rate). Taken together, these results indicate that STO-609 is a selective and cell-permeable inhibitor of CaM-KK and that it may be a useful tool for evaluating the physiological significance of the CaM-KK-mediated pathway in vivo as well as in vitro.