Novel phosphorylation of aquaporin-5 at its threonine 259 through cAMP signaling in salivary gland cells
Novel phosphorylation of aquaporin-5 at its threonine 259 through cAMP signaling in salivary gland cells
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DOI：
10.1152/ajpcell.00058.2011
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发表时间：
2011-09-01
影响因子：
5.5
通讯作者：
Hosoi, Kazuo
中科院分区：
文献类型：
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作者：
Hasegawa, Takahiro;Azlina, Ahmad;Hosoi, Kazuo
Hasekawa T、Azlina A、Javkhlan P、Yao C、Akamatsu T、Hosoi K。通过唾液腺细胞中的 cAMP 信号传导，aquaporin-5 在苏氨酸 259 处的新型磷酸化。 Am J Physiol Cell Physiol 301：C667-C678，2011。首次发表于 2011 年 6 月 1 日； doi: 10.1152/ajpcell.00058.2011.-Aquaporin-5 (AQP5) 是一种水通道，在唾液分泌中发挥关键作用。使用人唾液腺 (HSG) 细胞和小鼠唾液腺研究了 AQP5 的新型磷酸化。在用野生型小鼠 AQP5 构建体稳定转染的 HSG 细胞中，在 AQP5 免疫沉淀样品中检测到与磷酸化 PKA 底物的抗体发生免疫反应的蛋白条带，并且用 8-bromo-cAMP、毛喉素或佛波醇 12-肉豆蔻酸 13-乙酸酯短期处理细胞可增强其强度，但用 A23187 钙离子载体则不会增强。这种增强作用在 PKA 抑制剂 H-89 的存在下受到抑制。即使用蛋白激酶激活剂刺激细胞，抗磷酸化 PKA 底物抗体也不能识别通过 HSG 细胞转染表达的 AQP5 突变体 (AQP5-T259A)。使用检测 AQP5 Thr259 位点磷酸化的特异性抗体进行的免疫印迹和免疫荧光研究表明，在给予异丙肾上腺素（而非毛果芸香碱）后，AQP5 在下颌下腺和腮腺的腺泡细胞顶膜处快速且短暂地磷酸化。此外，在静息和毛喉素刺激条件下，AQP5 和 AQP5-T259A 均持续定位于 HSG 细胞的质膜上。这些结果表明，AQP5 在 Thr259 处被 PKA 通过 cAMP 而不是 Ca2+ 信号通路磷酸化，并且这种磷酸化不会促进 AQP5 在唾液腺细胞中的运输。
Hasegawa T, Azlina A, Javkhlan P, Yao C, Akamatsu T, Hosoi K. Novel phosphorylation of aquaporin-5 at its threonine 259 through cAMP signaling in salivary gland cells. Am J Physiol Cell Physiol 301: C667-C678, 2011. First published June 1, 2011; doi: 10.1152/ajpcell.00058.2011.-Aquaporin-5 (AQP5), a water channel, plays key roles in salivary secretion. The novel phosphorylation of AQP5 was investigated by using human salivary gland (HSG) cells and mouse salivary glands. In the HSG cells stably transfected with a wild-type mouse AQP5 construct, a protein band immunoreactive with antibody against phosphorylated PKA substrate was detected in the AQP5 immunoprecipitated sample, and its intensity was enhanced by short-term treatment of the cells with 8-bromo-cAMP, forskolin, or phorbol 12-myristate 13-acetate, but not by that with A23187 calcium ionophore. Such enhancement was inhibited in the presence of H-89, a PKA inhibitor. An AQP5 mutant (AQP5-T259A) expressed by transfection of HSG cells was not recognized by anti-phosphorylated PKA substrate antibody, even when the cells were stimulated with the protein kinase activators. Immunoblotting and immunofluorescence studies using a specific antibody detecting AQP5 phosphorylated at its Thr259 demonstrated that AQP5 was rapidly and transiently phosphorylated at the apical membrane of acinar cells in the submandibular and parotid glands after administration of isoproterenol, but not pilocarpine. Furthermore, both AQP5 and AQP5-T259A were constitutively localized at the plasma membrane in HSG cells under the resting and forskolin-stimulated conditions. These results suggest that AQP5 is phosphorylated at its Thr259 by PKA through cAMP, but not Ca2+, signaling pathways, and that this phosphorylation does not contribute to AQP5 trafficking in the salivary gland cells.