Intracellular sites of AQP2 S256 phosphorylation identified using inhibitors of the AQP2 recycling itinerary.

IF 3.7 2区 医学 Q1 PHYSIOLOGY American Journal of Physiology-renal Physiology Pub Date : 2023-02-01 Epub Date: 2022-12-01 DOI:10.1152/ajprenal.00123.2022
Pui W Cheung, Mey Boukenna, Richard S E Babicz, Shimontini Mitra, Anna Kay, Theodor C Paunescu, Noah Baylor, Chen-Chung Steven Liu, Anil V Nair, Richard Bouley, Dennis Brown
{"title":"Intracellular sites of AQP2 S256 phosphorylation identified using inhibitors of the AQP2 recycling itinerary.","authors":"Pui W Cheung, Mey Boukenna, Richard S E Babicz, Shimontini Mitra, Anna Kay, Theodor C Paunescu, Noah Baylor, Chen-Chung Steven Liu, Anil V Nair, Richard Bouley, Dennis Brown","doi":"10.1152/ajprenal.00123.2022","DOIUrl":null,"url":null,"abstract":"<p><p>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 <i>trans</i>-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 <i>trans</i>-Golgi network, or in cytoplasmic vesicles and that this event is dependent on the expression of PKA in these cells.<b>NEW & NOTEWORTHY</b> 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.</p>","PeriodicalId":7588,"journal":{"name":"American Journal of Physiology-renal Physiology","volume":"324 2","pages":"F152-F167"},"PeriodicalIF":3.7000,"publicationDate":"2023-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9844975/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"American Journal of Physiology-renal Physiology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1152/ajprenal.00123.2022","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2022/12/1 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"PHYSIOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

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 trans-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 trans-Golgi network, or in cytoplasmic vesicles and that this event is dependent on the expression of PKA in these cells.NEW & NOTEWORTHY 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.

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
使用 AQP2 循环行程抑制剂确定的 AQP2 S256 磷酸化胞内位点。
由血管加压素(VP)调控的水蒸发素-2(AQP2)在细胞质囊泡和肾主细胞质膜之间的运输对水稳态至关重要。VP会影响AQP2在COOH末端的几个丝氨酸残基上的磷酸化;其中,丝氨酸256(S256)似乎是AQP2迁移的主要调节因子。将该丝氨酸突变为天冬氨酸可模拟磷酸化,从而诱导 AQP2 的组成型膜表达。然而,S256 发生磷酸化的细胞内位置仍然难以确定。在这里,我们采用了阻断 LLC-PK1 细胞中不同细胞位置的 AQP2 运输的策略,并通过免疫荧光和使用磷酸特异性抗体进行 Western 印迹分析来监测 VP 刺激的这些位置的 S256 磷酸化。我们使用甲基-β-环糊精、冷阻断或巴佛洛霉素和紫杉醇,分别阻断了质膜、核周跨高尔基体网络和散在细胞质小泡中的 AQP2。无论其位于哪个细胞位置,VP 都会诱导 S256 磷酸化显著增加,而且这种效应并不依赖于功能性微管细胞骨架。为了进一步研究蛋白激酶A(PKA)是否是这些细胞区室中S256磷酸化的原因,我们创建了PKA无效细胞,并使用相同的程序阻断了AQP2的贩运。我们发现,与基线相比,无论 AQP2 定位如何,S256 磷酸化都不再增加。总之,我们的数据表明,AQP2 S256 磷酸化可发生在质膜、跨高尔基体网络或细胞质囊泡中,而且这一事件依赖于这些细胞中 PKA 的表达。 新进展和注意事项 PKA 在丝氨酸 256(S256)处磷酸化水通道蛋白-2,在其循环行程中发生在不同的亚细胞位置,这表明 AQP2 S256 磷酸化所需的蛋白复合物存在于这些不同的循环站。此外,我们使用 PKA 缺失的细胞表明,加压素诱导的 AQP2 磷酸化需要 PKA 的活性。我们的数据揭示了细胞内 AQP2 在 S256 处磷酸化的复杂空间模式,为磷酸化在 AQP2 膜积累中的作用提供了新的线索。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
CiteScore
8.40
自引率
7.10%
发文量
154
审稿时长
2-4 weeks
期刊介绍: The American Journal of Physiology - Renal Physiology publishes original manuscripts on timely topics in both basic science and clinical research. Published articles address a broad range of subjects relating to the kidney and urinary tract, and may involve human or animal models, individual cell types, and isolated membrane systems. Also covered are the pathophysiological basis of renal disease processes, regulation of body fluids, and clinical research that provides mechanistic insights. Studies of renal function may be conducted using a wide range of approaches, such as biochemistry, immunology, genetics, mathematical modeling, molecular biology, as well as physiological and clinical methodologies.
期刊最新文献
Sex-specific epigenetic programming in renal fibrosis and inflammation. Why is chronic kidney disease progressive? Evolutionary adaptations and maladaptations. Intracellular signaling pathways of muscarinic acetylcholine receptor-mediated detrusor muscle contractions. Role of the SLC22A17/lipocalin-2 receptor in renal endocytosis of proteins/metalloproteins: a focus on iron- and cadmium-binding proteins. Renal tubular SGK1 is required to achieve blood pressure surge and circadian rhythm.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1