Requirements for calcium and calmodulin in the calmodulin kinase activation cascade
Requirements for calcium and calmodulin in the calmodulin kinase activation cascade
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DOI：
10.1074/jbc.271.10.5617
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发表时间：
1996-03-08
影响因子：
4.8
通讯作者：
Soderling, TR
中科院分区：
文献类型：
--
作者：
Tokumitsu, H;Soderling, TR
我们之前纯化并克隆了大鼠脑 Ca2+/钙调蛋白依赖性蛋白激酶激酶 (CaM-KK)，68 kDa 重组 CaM-KK 在体外激活 CaM 激酶 IV (CaM-K IV) 和 CaM-K I (Tokumitsu, H.、Enslen, H. 和 Soderling, T. R. (1995) J. Biol. Chem. 270， 19320-19324)，在本研究中，我们已经确定，CaM-KK 通过磷酸化 Thr(196) 来激活 CaM-K IV 是由完整细胞中胞内 Ca2+ 升高触发的，并且需要 Ca2+/CaM 与两种酶结合。 CaM-K IV 的表达片段 (CaM-K IV178-246) 包含激活磷酸化位点 (Thr(196))，但不包含自抑制结构域或 CaM 结合结构域，仍然需要 Ca2+/CaM 才能被野生型 CaM-KK 磷酸化。 CaM-KK (CaM-KK1-434) 的截短突变体以不依赖 Ca2+/CaM 的方式磷酸化 CaM-K IV178-246，但这种组成型活性 CaM-KK1-434 需要 Ca2+/CaM 才能磷酸化和激活野生型 CaM-K IV。这些结果表明，Ca2+/CaM 与 CaM-K IV 和 CaM-KK 的结合是CaM-激酶级联，CaM-KK 和 CaM-K IV 似乎具有相似的 Ca2+/CaM 要求，EC(50) 值约为 100 nM。在 COS-7 细胞中使用 CaM-K IV 与 CaM-KK 共表达的研究表明，在离子霉素刺激下，CaM-KK 快速激活野生型 CaM-K IV 的总活性和 Ca2+/CaM 独立活性，但不能激活 Thr(196) --> Ala 突变体。
We have previously purified and cloned rat brain Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), and the 68-kDa recombinant CaM-KK activates in vitro both CaM-kinase IV (CaM-K IV) and CaM-K I (Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324), In the present study we have determined that activation of CaM-K IV through phosphorylation of Thr(196) by CaM-KK is triggered by elevated intracellular Ca2+ in intact cells and requires binding of Ca2+/CaM to both enzymes. An expressed fragment of CaM-K IV (CaM-K IV178-246), which contains the activating phosphorylation site (Thr(196)) but not the autoinhibitory domain or the CaM-binding domain, still required Ca2+/CaM for phosphorylation by wild-type CaM-KK. A truncated mutant of CaM-KK (CaM-KK1-434) phosphorylated CaM-K IV178-246 in a Ca2+/CaM-independent manner, but this constitutively active CaM-KK1-434 required Ca2+/CaM for phosphorylation and activation of wild-type CaM-K IV, These results demonstrate that binding of Ca2+/CaM to both CaM-K IV and CaM-KK is required for the CaM-kinase cascade, Both CaM-KK and CaM-K IV appear to have similar Ca2+/CaM requirements with EC(50) values of approximately 100 nM. Studies using co-expression of CaM-K IV with CaM-KK in COS-7 cells demonstrated that CaM-KK rapidly activated both total and Ca2+/CaM-independent activities of wild-type CaM-K IV, but not the Thr(196) --> Ala mutant, upon ionomycin stimulation.