Division of Nephrology, Program in Membrane Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Am J Physiol Renal Physiol. 2023 Feb 1;324(2):F152-F167. doi: 10.1152/ajprenal.00123.2022. Epub 2022 Dec 1.
Vasopressin (VP)-regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water homeostasis. VP affects AQP2 phosphorylation at several serine residues in the COOH-terminus; among them, serine 256 (S256) appears to be a major regulator of AQP2 trafficking. Mutation of this serine to aspartic acid, which mimics phosphorylation, induces constitutive membrane expression of AQP2. However, the intracellular location(s) at which S256 phosphorylation occurs remains elusive. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells and monitored VP-stimulated phosphorylation of S256 at these sites by immunofluorescence and Western blot analysis with phospho-specific antibodies. Using methyl-β-cyclodextrin, cold block or bafilomycin, and taxol, we blocked AQP2 at the plasma membrane, in the perinuclear -Golgi network, and in scattered cytoplasmic vesicles, respectively. Regardless of its cellular location, VP induced a significant increase in S256 phosphorylation, and this effect was not dependent on a functional microtubule cytoskeleton. To further investigate whether protein kinase A (PKA) was responsible for S256 phosphorylation in these cellular compartments, we created PKA-null cells and blocked AQP2 trafficking using the same procedures. We found that S256 phosphorylation was no longer increased compared with baseline, regardless of AQP2 localization. Taken together, our data indicate that AQP2 S256 phosphorylation can occur at the plasma membrane, in the -Golgi network, or in cytoplasmic vesicles and that this event is dependent on the expression of PKA in these cells. Phosphorylation of aquaporin-2 by PKA at serine 256 (S256) occurs in various subcellular locations during its recycling itinerary, suggesting that the protein complex necessary for AQP2 S256 phosphorylation is present in these different recycling stations. Furthermore, we showed, using PKA-null cells, that PKA activity is required for vasopressin-induced AQP2 phosphorylation. Our data reveal a complex spatial pattern of intracellular AQP2 phosphorylation at S256, shedding new light on the role of phosphorylation in AQP2 membrane accumulation.
血管加压素（VP）调节肾主细胞细胞质小泡和质膜之间的水通道蛋白-2（AQP2）转运对于水稳态至关重要。VP 影响 AQP2 在羧基末端几个丝氨酸残基上的磷酸化；其中，丝氨酸 256（S256）似乎是 AQP2 转运的主要调节剂。将该丝氨酸突变为模拟磷酸化的天冬氨酸，会诱导 AQP2 的组成型膜表达。然而，S256 磷酸化发生的细胞内位置仍然难以捉摸。在这里，我们使用策略在 LLC-PK1 细胞中阻断 AQP2 在不同细胞位置的转运，并通过免疫荧光和 Western blot 分析用磷酸特异性抗体监测 VP 刺激这些位置的 S256 磷酸化。使用甲基-β-环糊精、冷阻断或巴弗洛霉素和紫杉醇，我们分别阻断 AQP2 在质膜、核周 -高尔基网络和分散的细胞质小泡中的转运。无论其细胞位置如何，VP 都会诱导 S256 磷酸化显著增加，并且这种效应不依赖于功能微管细胞骨架。为了进一步研究蛋白激酶 A（PKA）是否负责这些细胞区室中的 S256 磷酸化，我们创建了 PKA 缺失细胞并使用相同的程序阻断 AQP2 转运。我们发现，与基线相比，S256 磷酸化不再增加，无论 AQP2 定位如何。总之，我们的数据表明，AQP2 S256 磷酸化可以发生在质膜、高尔基网络或细胞质小泡中，并且该事件取决于这些细胞中 PKA 的表达。PKA 对丝氨酸 256（S256）的磷酸化在 AQP2 回收途径的各种亚细胞位置发生，这表明 AQP2 S256 磷酸化所需的蛋白质复合物存在于这些不同的回收站中。此外，我们使用 PKA 缺失细胞表明，PKA 活性是血管加压素诱导的 AQP2 磷酸化所必需的。我们的数据揭示了 AQP2 S256 磷酸化在细胞内的复杂空间模式，为磷酸化在 AQP2 膜积累中的作用提供了新的认识。
