Identification of an RNA-binding protein that is phosphorylated by PTH and potentially mediates PTH-induced destabilization of Npt2a mRNA

Rebecca D. Murray, Michael L. Merchant, Ericka Hardin, Barbara Clark, Syed J. Khundmiri, Eleanor D. Lederer

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

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 PTHinduced 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 that 1) PTH is a major regulator of both transcription and translation, and 2) KSRP binds Npt2a mRNA but its role in PTH regulation of Npt2a mRNA is not clear.

Original languageEnglish (US)
Pages (from-to)C205-C215
JournalAmerican Journal of Physiology - Cell Physiology
Volume310
Issue number3
DOIs
StatePublished - 2016
Externally publishedYes

Keywords

  • HnRNPK-homology-type-splicing regulatory protein
  • Messenger ribonucleic acid
  • Messenger ribonucleic acid stability
  • Parathyroid hormone
  • Phosphoproteome
  • Proximal tubule
  • Ribonucleic acidbinding proteins
  • Type IIa sodium-phosphate cotransporter

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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