Department of Physiology, Kyung Hee University School of Medicine, Seoul, South Korea.
Biochem Biophys Res Commun. 2012 Jan 20;417(3):1030-4. doi: 10.1016/j.bbrc.2011.12.085. Epub 2011 Dec 26.
Transient receptor potential melastatin 7 (TRPM7) channels are divalent cation-selective ion channels that are permeable to Ca(2+) and Mg(2+). TRPM7 is ubiquitously expressed in vertebrate cells and contains both an ion channel and a kinase domain. TRPM7 plays an important role in regulating cellular homeostatic levels of Ca(2+) and Mg(2+) in mammalian cells. Although studies have shown that the kinase domain of TRPM7 is required for channel activation and can phosphorylate other target proteins, a systematic analysis of intact TRPM7 channel phosphorylation sites expressed in mammalian cells is lacking. We applied mass spectrometric proteomic techniques to identify and characterize the key phosphorylation sites in TRPM7 channels. We identified 14 phosphorylation sites in the cytoplasmic domain of TRPM7, eight of which have not been previously reported. The identification of phosphorylation sites using antibody-based immunopurification and mass spectrometry is an effective approach for defining the phosphorylation status of TRPM7 channels. The present results show that TRPM7 channels are phosphorylated at multiple sites, which serves as a mechanism to modulate the dynamic functions of TRPM7 channels in mammalian cells.
瞬时受体电位 melastatin 7（TRPM7）通道是二价阳离子选择性离子通道，对 Ca（2+）和 Mg（2+）具有通透性。TRPM7 在脊椎动物细胞中广泛表达，包含离子通道和激酶结构域。TRPM7 在调节哺乳动物细胞中 Ca（2+）和 Mg（2+）的细胞内稳态水平方面发挥着重要作用。尽管研究表明 TRPM7 的激酶结构域对于通道激活是必需的，并且可以磷酸化其他靶蛋白，但对表达在哺乳动物细胞中的完整 TRPM7 通道磷酸化位点的系统分析尚缺乏。我们应用质谱蛋白质组学技术来鉴定和描述 TRPM7 通道中的关键磷酸化位点。我们在 TRPM7 的细胞质结构域中鉴定出 14 个磷酸化位点，其中 8 个以前没有报道过。使用基于抗体的免疫沉淀和质谱法鉴定磷酸化位点是确定 TRPM7 通道磷酸化状态的有效方法。本研究结果表明，TRPM7 通道在多个位点发生磷酸化，这作为一种机制调节了 TRPM7 通道在哺乳动物细胞中的动态功能。
