title: Identification of an RNA-binding protein that is phosphorylated by PTH and potentially mediates PTH-induced destabilization of Npt2a mRNA.
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 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 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.
authors.affiliation: Department of Physiology and Biophysics, University of Louisville, Louisville, Kentucky; Department of Medicine/Kidney Disease Program, University of Louisville, Louisville, Kentucky;
authors.forename: Rebecca D
authors.initials: RD
authors.lastname: Murray
authors.affiliation: Department of Medicine/Kidney Disease Program, University of Louisville, Louisville, Kentucky;
authors.forename: Michael L
authors.initials: ML
authors.lastname: Merchant
authors.affiliation: Western Kentucky University, Bowling Green, Kentucky; and.
authors.forename: Ericka
authors.initials: E
authors.lastname: Hardin
authors.affiliation: Department of Biochemistry, University of Louisville, Louisville, Kentucky.
authors.forename: Barbara
authors.initials: B
authors.lastname: Clark
authors.affiliation: Robley Rex Veterans Affairs Medical Center, Louisville, Kentucky; Department of Physiology and Biophysics, University of Louisville, Louisville, Kentucky; Department of Medicine/Kidney Disease Program, University of Louisville, Louisville, Kentucky;
authors.forename: Syed J
authors.initials: SJ
authors.lastname: Khundmiri
authors.affiliation: Robley Rex Veterans Affairs Medical Center, Louisville, Kentucky; Department of Physiology and Biophysics, University of Louisville, Louisville, Kentucky; Department of Medicine/Kidney Disease Program, University of Louisville, Louisville, Kentucky; e.lederer@louisville.edu.
authors.forename: Eleanor D
authors.initials: ED
authors.lastname: Lederer
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D019295:Computational Biology; D030541:Databases, Genetic; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism; D013058:Mass Spectrometry; D009893:Opossums; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; C105789:KHSRP protein, human; D010281:Parathyroid Hormone; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C493554:SLC34A1 protein, human; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D015534:Trans-Activators
keywords: hnRNPK-homology-type-splicing regulatory protein; messenger ribonucleic acid; messenger ribonucleic acid stability; parathyroid hormone; phosphoproteome; proximal tubule; ribonucleic acid-binding proteins; type IIa sodium-phosphate cotransporter