Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells.
Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells.
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DOI：
10.1152/ajprenal.1997.272.6.f816
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发表时间：
1997-06
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影响因子：
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通讯作者：
T. Katsura;C. Gustafson;D. Ausiello;Dennis Brown
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文献类型：
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作者：
T. Katsura;C. Gustafson;D. Ausiello;Dennis Brown
水通道蛋白 2 (AQP2) 在细胞内囊泡和质膜之间的加压素依赖性易位已在体内和体外得到证实。此外，加压素诱导的肾主细胞顶膜水渗透性的增加取决于细胞内腺苷3',5'-环单磷酸的增加和蛋白激酶A (PKA)的激活。为了确定 AQP2 的运输是否依赖于 PKA 磷酸化，我们首先检查了 PKA 抑制剂 N-(2[[3-(4-溴苯基)-2-丙烯基]-氨基]-乙基)-5-异喹啉磺酰胺 (H-89) 对转染的 LLC-PK1 细胞中 AQP2 易位的影响。用 H-89 预处理细胞 60 分钟，完全抑制加压素诱导的 AQP2 膜插入。该试剂还导致 AQP2 在高尔基体区域密集积聚。接下来，用 AQP2 cDNA 稳定转染 LLC-PK1 细胞，其中 PKA 磷酸化位点 Ser256 被丙氨酸 (S256A) 取代。 S256A-AQP2在体外不被PKA磷酸化，并且在基础条件下S256A-AQP2主要定位于细胞内囊泡，与野生型AQP2相似。然而，在用加压素或毛喉素刺激后，S256A-AQP2的细胞分布保持不变。此外，在 S256A-AQP2 转染细胞中未观察到 AQP2 转染细胞中常见的加压素诱导的内吞作用增加。这些结果表明 Ser256 PKA 磷酸化位点可能参与加压素诱导的 AQP2 从细胞内囊泡到质膜的运输以及随后刺激的内吞作用。
Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intracellular vesicles and the plasma membrane has been demonstrated in vivo and in vitro. Furthermore, the vasopressin-induced increase in apical membrane water permeability of renal principal cells is dependent on a rise in intracellular adenosine 3',5'-cyclic monophosphate and activation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effect of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89) on AQP2 translocation in transfected LLC-PK1 cells. Vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of the cells for 60 min with H-89. This reagent also caused a dense accumulation of AQP2 in the Golgi region. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in which the PKA phosphorylation site, Ser256, was replaced with alanine (S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-AQP2 was mainly localized to intracellular vesicles in the basal condition, similar to wild-type AQP2. However, after stimulation with vasopressin or forskolin, the cellular distribution of S256A-AQP2 remained unchanged. In addition, the usual vasopressin-induced increase in endocytosis seen in AQP2-transfected cells was not observed in S256A-AQP2-transfected cells. These results demonstrate that the Ser256 PKA phosphorylation site is possibly involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.