Molecular Interaction and Functional Regulation of ClC-3 by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) in Human Malignant Glioma
Molecular Interaction and Functional Regulation of ClC-3 by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) in Human Malignant Glioma
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DOI：
10.1074/jbc.m109.097675
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发表时间：
2010-04-09
影响因子：
4.8
通讯作者：
Sontheimer, Harald
中科院分区：
文献类型：
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作者：
Cuddapah, Vishnu Anand;Sontheimer, Harald
多形性胶质母细胞瘤是成人中最常见和致命的原发性脑癌。肿瘤细胞弥漫性浸润大脑，使局部手术和放射治疗具有挑战性。神经胶质瘤细胞侵入正常脑是由离子通道的活性促进的，有助于细胞体积的动态调节。最近的研究特别涉及ClC-3，电压门控氯离子通道，在这个过程中。然而，ClC-3活性和细胞运动之间的相互作用知之甚少。在这里，我们证明，ClC-3是高度表达的人胶质瘤细胞的质膜上，其活性通过磷酸化通过Ca 2 +/钙调蛋白依赖性蛋白激酶II（CaMKII）调节。细胞内输注自激活的CaMK II通过贴片移液管增强氯电流3倍，这种调节被autocamtide-2相关的抑制肽，CaMK II特异性抑制剂抑制。CaMKII对氯电流的调节也在稳定的小发夹RNA敲除ClC-3通道后丧失，表明ClC-3和CaMKII的特异性相互作用。在表达ClC-3的细胞中，抑制CaMKII降低胶质瘤侵袭的程度与直接抑制ClC-3相同。C1 C-3和CaMKII的分子相互作用的重要性进一步得到了我们的发现的支持，即CaMKII与C1 C-3共定位和共免疫沉淀。ClC-3和CaMK II也在来自诊断为IV级胶质母细胞瘤的患者的组织活检中共免疫沉淀。这些肿瘤样品显示出比非恶性脑组织高10倍的ClC-3蛋白表达。这些数据表明，CaMKII是一个分子链翻译细胞内钙的变化，这是内在相关的胶质瘤迁移，细胞运动所需的ClC-3电导的变化。
Glioblastoma multiforme is the most common and lethal primary brain cancer in adults. Tumor cells diffusely infiltrate the brain making focal surgical and radiation treatment challenging. The invasion of glioma cells into normal brain is facilitated by the activity of ion channels aiding dynamic regulation of cell volume. Recent studies have specifically implicated ClC-3, a voltage-gated chloride channel, in this process. However, the interaction between ClC-3 activity and cell movement is poorly understood. Here, we demonstrate that ClC-3 is highly expressed on the plasma membrane of human glioma cells where its activity is regulated through phosphorylation via Ca2+/calmodulin-dependent protein kinase II (CaMKII). Intracellular infusion of autoactivated CaMKII via patch pipette enhanced chloride currents 3-fold, and this regulation was inhibited by autocamtide-2 related inhibitory peptide, a CaMKII-specific inhibitor. CaMKII modulation of chloride currents was also lost upon stable small hairpin RNA knockdown of ClC-3 channels indicating a specific interaction of ClC-3 and CaMKII. In ClC-3-expressing cells, inhibition of CaMKII reduced glioma invasion to the same extent as direct inhibition of ClC-3. The importance of the molecular interaction of ClC-3 and CaMKII is further supported by our finding that CaMKII co-localizes and co-immunoprecipitates with ClC-3. ClC-3 and CaMKII also co-immunoprecipitate in tissue biopsies from patients diagnosed with grade IV glioblastoma. These tumor samples show 10-fold higher ClC-3 protein expression than nonmalignant brain. These data suggest that CaMKII is a molecular link translating intracellular calcium changes, which are intrinsically associated with glioma migration, to changes in ClC-3 conductance required for cell movement.