The autonomous activity of calcium/calmodulin-dependent protein kinase IV is required for its role in transcription
The autonomous activity of calcium/calmodulin-dependent protein kinase IV is required for its role in transcription
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DOI：
10.1074/jbc.m500067200
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发表时间：
2005-05-27
影响因子：
4.8
通讯作者：
Means, AR
中科院分区：
文献类型：
--
作者：
Chow, FA;Anderson, KA;Means, AR
钙/钙调蛋白依赖性激酶 IV (CaMKIV) 是一种多功能丝氨酸/苏氨酸激酶，受两个主要事件的正向调节。第一个是钙/钙调蛋白 (Ca2+/CaM) 的结合，可解除酶的分子内自抑制并导致基础激酶活性。第二种是由上游激酶 Ca2+/钙调蛋白依赖性激酶激活。 Ca2+/CaM 结合的 CaMKIV 在其激活环苏氨酸（人 CaMKIV 中的残基 Thr(200)）上被 Ca2+/钙调蛋白依赖性激酶激酶磷酸化，导致 CaMKIV 激酶活性增加。人们也多次注意到，CaMKIV 的激活伴随着 Ca2+/CaM 独立或自主活性的产生，尽管这一事件的意义尚不清楚。在这里，我们证明了自主活动对 CaMKIV 生物学功能的重要性。我们发现 CaMKIV 在 Thr(200) 上的磷酸化导致产生完全不依赖 Ca2+/CaM 的酶。通过分析野生型和突变型 CaMKIV 蛋白在生化实验和细胞转录测定中的行为，我们证明 CaMKIV 自主活性对于 CaMKIV 介导的转录是必要且充分的。野生型 CaMKIV 驱动 cAMP 反应元件结合蛋白介导的转录的能力严格依赖于起始 Ca2+ 刺激，从而导致激酶激活和细胞自主活动的发展。无法在细胞环境中产生自主活性的突变 CaMKIV 蛋白无法驱动转录，而某些具有组成型自主活性的 CaMKIV 突变体在缺乏 Ca2+ 刺激且独立于 Ca2+/CaM 结合或 Thr(200) 磷酸化的情况下驱动转录。
Calcium/calmodulin-dependent kinase IV (CaMKIV) is a multifunctional serine/threonine kinase that is positively regulated by two main events. The first is the binding of calcium/calmodulin (Ca2+/CaM), which relieves intramolecular autoinhibition of the enzyme and leads to basal kinase activity. The second is activation by the upstream kinase, Ca2+/calmodulin-dependent kinase kinase. Phosphorylation of Ca2+/CaM-bound CaMKIV on its activation loop threonine ( residue Thr(200) in human CaMKIV) by Ca2+/calmodulin-dependent kinase kinase leads to increased CaMKIV kinase activity. It has also been repeatedly noted that activation of CaMKIV is accompanied by the generation of Ca2+/CaM-independent or autonomous activity, although the significance of this event has been unclear. Here we demonstrate the importance of autonomous activity to CaMKIV biological function. We show that phosphorylation of CaMKIV on Thr(200) leads to the generation of a fully Ca2+/CaM-independent enzyme. By analyzing the behavior of wild-type and mutant CaMKIV proteins in biochemical experiments and cellular transcriptional assays, we demonstrate that CaMKIV autonomous activity is necessary and sufficient for CaMKIV-mediated transcription. The ability of wild-type CaMKIV to drive cAMP response element-binding protein-mediated transcription is strictly dependent upon an initiating Ca2+ stimulus, which leads to kinase activation and development of autonomous activity in cells. Mutant CaMKIV proteins that are incapable of developing autonomous activity within a cellular context fail to drive transcription, whereas certain CaMKIV mutants that possess constitutive autonomous activity drive transcription in the absence of a Ca2+ stimulus and independent of Ca2+/ CaM binding or Thr(200) phosphorylation.