Department of Cell and Systems Physiology University of Occupational and Environmental Health School of Medicine, Kitakyushu 807-8555, Japan.
Department of Life Science, Rikkyo (St Paul's) University, Nishi-ikebukuro, Toshima-ku, Tokyo 171-8501, Japan.
Cell Signal. 2020 Jan;65:109434. doi: 10.1016/j.cellsig.2019.109434. Epub 2019 Oct 30.
Muscarinic receptor stimulation or protein kinase C (PKC) activation in rat adrenal medullary and PC12 cells rapidly induces tyrosine phosphorylation of TWIK-related-acid-sensitive K 1 (TASK1) channels with the subsequent clathrin-dependent endocytosis. Our previous study suggested that the muscarinic signal is transmitted to the non-receptor tyrosine kinase Src through PKC and Pyk2. Although PKC activation is known to stimulate Pyk2 in certain types of cells, its molecular mechanism remains unclear. In this study, proximity ligation assay (PLA) and other molecular biological approaches were used to elucidate the details of this muscarinic signaling in PC12 cells. When green fluorescent protein (GFP)-TASK1 was expressed, the majority of GFP-TASK1 was located at the cell periphery. However, the simultaneous expression of GFP-TASK1 and PKCα, but not PKCδ, led to GFP-TASK1 internalization. Muscarinic receptor stimulation resulted in transient co-localization of Pyk2 and Src at the cell periphery, and expression of kinase dead (KD) Pyk2 and Src, but not Pyk2 and KD Src, resulted in GFP-TASK1 internalization. PLA analysis revealed that in response to muscarine, PKCαactivates Pyk2 through phosphorylating its serine residues. These results indicate that muscarinic receptor stimulation induces TASK1 channel endocytosis sequentially through PKCα, Pyk2, and Src, and PKCα activates Pyk2 through phosphorylation.
毒蕈碱型乙酰胆碱受体刺激或蛋白激酶 C（PKC）激活可迅速诱导大鼠肾上腺髓质和 PC12 细胞中 TWIK 相关酸敏感钾 1（TASK1）通道的酪氨酸磷酸化，随后发生网格蛋白依赖性内吞作用。我们之前的研究表明，毒蕈碱信号通过 PKC 和 Pyk2 传递到非受体酪氨酸激酶Src。虽然已知 PKC 激活可在某些类型的细胞中刺激 Pyk2，但其分子机制尚不清楚。在这项研究中，我们使用接近连接测定（PLA）和其他分子生物学方法来阐明 PC12 细胞中这种毒蕈碱信号的细节。当表达绿色荧光蛋白（GFP）-TASK1 时，大多数 GFP-TASK1 位于细胞边缘。然而，GFP-TASK1 和 PKCα的同时表达，但不是 PKCδ的表达，导致 GFP-TASK1 内化。毒蕈碱受体刺激导致 Pyk2 和Src 在细胞边缘的瞬时共定位，并且激酶失活（KD）Pyk2 和 Src 的表达，但不是 Pyk2 和 KD Src 的表达，导致 GFP-TASK1 内化。PLA 分析显示，在响应毒蕈碱时，PKCα 通过磷酸化其丝氨酸残基激活 Pyk2。这些结果表明，毒蕈碱受体刺激通过 PKCα、Pyk2 和 Src 依次诱导 TASK1 通道内吞作用，并且 PKCα 通过磷酸化激活 Pyk2。
