Post-transcriptional Control of Na,K-ATPase Activity and Cell Growth by a Splice Variant of FXYD2 Protein with Modified mRNA

Kathleen J Sweadner,Jennifer L Pascoa,Cynthia A Salazar,Elena Arystarkhova

doi:10.1074/jbc.m111.241901

Kathleen J Sweadner, Jennifer L Pascoa + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.241901

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Abstract

In kidney, FXYD proteins regulate Na,K-ATPase in a nephron segment-specific way. FXYD2 is the most abundant renal FXYD but is not expressed in most renal cell lines unless induced by hypertonicity. Expression by transfection of FXYD2a or FXYD2b splice variants in NRK-52E cells reduces the apparent Na(+) affinity of the Na,K-ATPase and slows the cell proliferation rate. Based on RT-PCR, mRNAs for both splice variants were expressed in wild type NRK-52E cells as low abundance species. DNA sequencing of the PCR products revealed a base alteration from C to T in FXYD2b but not FXYD2a from both untreated and hypertonicity-treated NRK-52E cells. The 172C→T sequence change exposed a cryptic KKXX endoplasmic reticulum retrieval signal via a premature stop codon. The truncation affected trafficking of FXYD2b and its association with Na,K-ATPase and blocked its effect on enzyme kinetics and cell growth. The data may be explained by altered splicing or selective RNA editing of FXYD2b, a supplementary process that would ensure that it was inactive even if transcribed and translated, in these cells that normally express only FXYD2a. 172C→T mutation was also identified after mutagenesis of FXYD2b by error-prone PCR coupled with a selection for cell proliferation. Furthermore, the error-prone PCR alone introduced the mutation with high frequency, implying a structural peculiarity. The data confirm truncation of FXYD2b as a potential mechanism to regulate the amount of FXYD2 at the cell surface to control activity of Na,K-ATPase and cell growth.

Highlights

Expression of the FXYD2b protein can be achieved in NRK-52E cells only by transfection [9], here we discovered that mRNA for both FXYD2a and FXYD2b is expressed in low abundance in control NRK-52E cells
Base Change in mRNA—The FXYD2 subunit is abundantly expressed in several nephron segments, but NRK-52E and other renal epithelial cells lack its expression as a protein in basal conditions [8, 26, 29]
We identified a novel form of the FXYD2b regulatory subunit that is retained intracellularly when expressed

Summary

EXPERIMENTAL PROCEDURES

Antibodies and Cell Lines—Polyclonal antiserum K3 was used for detection of the ␣1 and ␤1 subunits of Na,K-ATPase on blots [22]. The RNGB polyclonal antibody raised against the N-terminal peptide of the rat FXYD2b splice variant [6] was used to identify the protein on blots as well as in immunostaining of cells. The following primers contained nucleotide sequences from the 5Ј- and 3Ј-ends of the coding sequence for rat FXYD2b plus EcoRI and BamHI sites for unidirectional cloning, respectively: forward primer, 5Ј-GCGAATTCCACCATGGACAGGTGGTAC-3Ј; reverse primer, 5Ј-CGCGGATCCTCACAGCTCATCTTCATTGAC-3Ј. Construction of c⌬7—Construction of c⌬7 was performed by PCR using the plasmid containing full-length cDNA for rat FXYD2b as a template. The following primers contained nucleotide sequences from FXYD2b (including the new stop codon in the reverse primer) plus EcoRI and BamHI sites for unidirectional cloning: forward, 5Ј-GCGAATTCCACCATGGACAGGTGGTAC-3Ј; reverse, 5Ј-CGCGGATCCCTACCTATGCTTCTTACTGCCCCC-3Ј. Cells were scraped and triturated, and insoluble material was removed by centrifugation at 3,000 ϫ g for 10 min (Sorvall SS-34)

The abbreviations used are

RESULTS

DISCUSSION