Identification of an RNA-binding protein that is phosphorylated by PTH and potentially mediates PTH-induced destabilization of Npt2a mRNA.
Identification of an RNA-binding protein that is phosphorylated by PTH and potentially mediates PTH-induced destabilization of Npt2a mRNA.
复制标题
鉴定一种 RNA 结合蛋白，该蛋白被 PTH 磷酸化，并可能介导 PTH 诱导的 Npt2a mRNA 不稳定。
DOI：
10.1152/ajpcell.00192.2015
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Lederer,EleanorD
中科院分区：
文献类型：
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作者：
Murray,RebeccaD;Merchant,MichaelL;Hardin,Ericka;Clark,Barbara;Khundmiri,SyedJ;Lederer,EleanorD
甲状旁腺激素（PTH）是IIa型钠-磷酸共转运蛋白（Npt2a）表达和功能的关键调节因子，Npt2a是负责调节肾脏磷酸盐重吸收的蛋白质。我们之前发现PTH通过转录后机制诱导Npt2a mRNA的快速衰减。我们假设pth诱导的rna结合蛋白（RBP）活性的变化介导了Npt2a mRNA的降解。为了解决这一问题，我们用100 nM PTH处理负鼠肾（OK）细胞（一种PTH敏感的近端小管细胞培养模型）30分钟和2小时，然后对PTH刺激的磷酸化蛋白质组进行质谱分析。我们鉴定了1182个蛋白在PTH反应中差异磷酸化，包括68个rbp。初步分析发现了一个磷酸化rbp， hnrnpk同源型剪接调节蛋白（KSRP），与Npt2a mRNA的3 ' -未翻译区（UTR）的预测结合位点。Western blot分析证实了KSRP在OK细胞中的表达，并显示pth依赖性易位到细胞核。从对照和pth处理的细胞中免疫沉淀KSRP，然后进行RNA分离和rt -定量PCR分析，从对照和pth处理的KSRP拉下中鉴定出Npt2a mRNA。PTH处理后KSRP的下调表明，KSRP介导PTH刺激的钠/氢交换器3mrna的减少是必需的，而不是Npt2a mRNA的减少。我们得出结论：1)PTH是转录和翻译的主要调控因子；2)KSRP结合Npt2a mRNA，但其在PTH调控Npt2a mRNA中的作用尚不清楚。
Parathyroid hormone (PTH) is a key regulator of the expression and function of the type IIa sodium-phosphate cotransporter (Npt2a), the protein responsible for regulated renal phosphate reabsorption. We previously showed that PTH induces rapid decay of Npt2a mRNA through posttranscriptional mechanisms. We hypothesized that PTH-induced changes in RNA-binding protein (RBP) activity mediate the degradation of Npt2a mRNA. To address this aim, we treated opossum kidney (OK) cells, a PTH-sensitive proximal tubule cell culture model, with 100 nM PTH for 30 min and 2 h, followed by mass spectrometry characterization of the PTH-stimulated phosphoproteome. We identified 1,182 proteins differentially phosphorylated in response to PTH, including 68 RBPs. Preliminary analysis identified a phospho-RBP, hnRNPK-homology-type-splicing regulatory protein (KSRP), with predicted binding sites for the 3′-untranslated region (UTR) of Npt2a mRNA. Western blot analysis confirmed expression of KSRP in OK cells and showed PTH-dependent translocation to the nucleus. Immunoprecipitation of KSRP from control and PTH-treated cells followed by RNA isolation and RT-quantitative PCR analysis identified Npt2a mRNA from both control and PTH-treated KSRP pulldowns. Knockdown of KSRP followed by PTH treatment showed that KSRP is required for mediating PTH-stimulated reduction in sodium/hydrogen exchanger 3 mRNA, but not Npt2a mRNA. We conclude that1) PTH is a major regulator of both transcription and translation, and2) KSRP binds Npt2a mRNA but its role in PTH regulation of Npt2a mRNA is not clear.