Department of Biological Science and Technology, The University of Tokushima Graduate School, 2–1 Minamijosanjima, Tokushima, Japan.
Cell Signal. 2011 Jul;23(7):1179-87. doi: 10.1016/j.cellsig.2011.03.005. Epub 2011 Mar 21.
The cGMP/cGMP-dependent protein kinase (cGK) signaling pathway is implicated in the functional regulation of intracellular calcium levels. In the present study, we investigated the regulation of transient receptor potential canonical 7 (TRPC7) by the cGMP/cGK-I pathway. TRPC7 contains three putative cGK phosphorylation sites (Arg-Arg/Lys-Xaa-Ser/Thr). However, the role of cGK-I in the regulation of TRPC7 activity remains unclear. In vitro and in vivo kinase assays have revealed that cGK-Iα phosphorylates mouse TRPC7 but not mouse TRPC3. Site-directed mutagenesis analysis revealed that TRPC7 was phosphorylated by cGK-Iα at threonine 15. Phosphorylation of TRPC7 significantly suppressed carbachol-induced calcium influx and CREB phosphorylation. Furthermore, co-immunoprecipitation assay demonstrated that cGK-Iα interacted with the ankyrin repeat domain in the N terminus of TRPC7. cGK-Iβ also bound to TRPC7, while the type II regulatory subunit of cAMP-dependent protein kinase did not bind. These data indicate that cGK-Iα interacts with and phosphorylates TRPC7, contributing to the quick and accurate regulation of calcium influx and CREB phosphorylation.
环鸟苷酸/环鸟苷酸依赖的蛋白激酶（cGK）信号通路参与细胞内钙离子水平的功能调节。在本研究中，我们研究了 cGMP/cGK-I 通路对瞬时受体电位经典型 7（TRPC7）的调节。TRPC7 包含三个潜在的 cGK 磷酸化位点（Arg-Arg/Lys-Xaa-Ser/Thr）。然而，cGK-I 在调节 TRPC7 活性中的作用尚不清楚。体外和体内激酶实验表明，cGK-Iα 可磷酸化鼠 TRPC7，但不能磷酸化鼠 TRPC3。定点突变分析表明，cGK-Iα 可使 TRPC7 的苏氨酸 15 发生磷酸化。TRPC7 的磷酸化显著抑制了乙酰胆碱诱导的钙内流和 CREB 磷酸化。此外，共免疫沉淀实验表明，cGK-Iα 与 TRPC7 的 N 端锚蛋白重复结构域相互作用。cGK-Iβ 也与 TRPC7 结合，而环腺苷酸依赖性蛋白激酶的 II 型调节亚基则不与 TRPC7 结合。这些数据表明，cGK-Iα 与 TRPC7 相互作用并磷酸化，有助于钙内流和 CREB 磷酸化的快速而准确的调节。
