Regulation of Na,K-ATPase subunit abundance by translational repression

Abstract

The Na,K-ATPase is an alphabeta heterodimer responsible for maintaining fluid and electrolyte homeostasis in mammalian cells. We engineered Madin-Darby canine kidney cell lines expressing alpha(1)FLAG, beta(1)FLAG, or beta(2)MYC subunits via a tetracycline-regulated promoter and a line expressing both stable beta(1)MYC and tetracycline-regulated beta(1)FLAG to examine regulatory mechanisms of sodium pump subunit expression. When overexpression of exogenous beta(1)FLAG increased total beta subunit levels by >200% without changes in alpha subunit abundance, endogenous beta(1) subunit (beta(1)E) abundance decreased. beta(1)E down-regulation did not occur during beta(2)MYC overexpression, indicating isoform specificity of the repression mechanism. Measurements of RNA stability and content indicated that decreased beta subunit expression was not accompanied by any change in mRNA levels. In addition, the degradation rate of beta subunits was not altered by beta(1)FLAG overexpression. Cells stably expressing beta(1)MYC, when induced to express beta(1)FLAG subunits, showed reduced beta(1)MYC and beta(1)E subunit abundance, indicating that these effects occur via the coding sequences of the down-regulated polypeptides. In a similar way, Madin-Darby canine kidney cells overexpressing exogenous alpha(1)FLAG subunits exhibited a reduction of endogenous alpha(1) subunits (alpha(1)E) with no change in alpha mRNA levels or beta subunits. The reduction in alpha(1)E compensated for alpha(1)FLAG subunit expression, resulting in unchanged total alpha subunit abundance. Thus, regulation of alpha subunit expression maintained its native level, whereas beta subunit was not as tightly regulated and its abundance could increase substantially over native levels. These effects also occurred in human embryonic kidney cells. These data are the first indication that cellular sodium pump subunit abundance is modulated by translational repression. This mechanism represents a novel, potentially important mechanism for regulation of Na,K-ATPase expression.

FIGURE 1.

Isoform-specific subunit overexpression causes reduction in β₁ subunit abundance. MDCK/β₁FLAG or MDCK/β₂MYC cells were grown in medium containing 1 μg/ml tet for 5 days (+) or grown in tet-free medium (−). Cells were harvested and fractionated, and protein concentrations were determined. 5 μg of ER, PM, or TM protein was treated with PNGase F followed by SDS-PAGE and Western blot detection. Membranes were probed with anti-actin, anti-β-catenin, anti-α₁, anti-β₁, anti-MYC, or anti-FLAG antibodies. A, relative abundance of β₁E subunits from the ER- or PM-enriched fractions of MDCK/β₁FLAG cells were quantified and are displayed in C. B, total membranes from MDCK/β₂MYC cells were analyzed by Western blot, and β₁ subunit abundance was quantified and is shown in D. Quantification of β₁E subunit expression from +tet samples (light bars) was determined and corrected for β-catenin or β-actin loading controls and is shown as a percentage from −tet samples (dark bars).

FIGURE 2.

Reduced protein synthesis of β₁E when β₁FLAG expression is induced. MDCK/β₁FLAG cells were grown in the presence or absence of tet for 5 days. Cells were then radiolabeled with ³⁵S-labeled methionine/cysteine for 24 h with or without tet present in the growth medium. Cells were harvested and fractionated, and protein concentrations were determined. 20 μg of PM-enriched protein was subjected to IP by anti-α₁ loop antibody, treated with PNGase F, resolved by SDS-PAGE, and analyzed by autoradiogram (A) or Western blot detection using anti-α₁ and anti-β₁ antibodies (B). Endogenous β₁ signal intensities from −tet (dark bars) or +tet (light bars) samples from autoradiogram or Western blot were quantified and are displayed in C.

FIGURE 3.

β₁E subunit abundance varies with overexpression level of β₁FLAG. MDCK/β₁FLAG cells were grown in tet for varying amounts of time (A) or at varying concentrations (C). A, 10 μg of TM from MDCK/β₁FLAG cells grown in 1 μg/ml tet for 0, 4, 72, 96, or 264 h was subjected to PNGase F treatment, SDS-PAGE, and Western blot analysis using anti-actin, anti-β₁, or anti-α₁ antibodies. In the anti-β₁ antibody immunoblot, the upper band signifying β₁FLAG (dashed line) and the lower band corresponding to β₁E (solid line) were quantified, corrected for actin loading, and plotted as a percentage of β₁E at 0-h tet induction and are displayed in B. C, 5 μg of PM protein from MDCK/β₁FLAG cells treated with 0, 0.005, or 0.05 μg/ml tet for 3 days was treated with PNGase F and subjected to SDS-PAGE, and immunoblots were probed with anti-α₁, anti-β₁, anti-β-catenin, or anti-FLAG antibodies. Quantitation of β₁E expression (lower band in anti-β₁ immunoblot) was corrected for actin loading and determined as a percentage of expression in no-tet samples (D).

FIGURE 4.

Degradation of β₁E is not increased by β₁FLAG expression. MDCK/β₁FLAG cells were grown in the presence of cycloheximide (A) or tetracycline (B) for 0, 2, 4, 7, 18 or 24 h and 10 μg of total membrane protein was PNGase F-treated and subjected to Western blot analysis using anti-α₁, anti-β₁, anti-β-catenin, or anti-FLAG antibodies. The lower bands in anti-β₁ antibody blots, representing β₁E subunit expression, from cells treated with cycloheximide (square data points) or tetracycline (circle data points) were quantified and are displayed as a percentage of expression at 0 h (C). Data points containing S.E. bars are representative of three independent experiments.

FIGURE 5.

β₁FLAG overexpression causes a decrease in β₁MYC and β₁E subunit abundance in MDCK/β₁FLAG/β₁MYC cells. MDCK/β₁FLAG/β₁MYC cells were grown in medium containing 1 μg/ml tet for 5 days (+) or grown in tet-free medium (−). Membrane-enriched fractions were harvested, and 5 μg of ER or PM protein was treated with PNGase F followed by SDS-PAGE and Western blot detection. A, membranes were probed with anti-β-catenin, anti-α, anti-β, anti-MYC, or anti-FLAG antibodies. Signal intensities from anti-β₁ (lower band) and anti-MYC immunoblots of the PM-enriched fraction were used to quantify β₁E and β₁MYC subunit abundance as a percentage of expression without tet (displayed in B).

FIGURE 6.

Overexpression of β₁/β₂ chimeras have different effects on endogenous β₁ subunit. A schematic representation of β₁/β₂-MYC and β₂/β₁-FLAG chimeras containing β₁ (light gray) and β₂ (dark gray) amino acid residues and FLAG or MYC epitope tags (gray box) is displayed in A. Both chimeras contain a short cytoplasmic amino terminus, single transmembrane segment, and long extracellular domain. MDCK/β₁/β₂-MYC or MDCK/β₂/β₁-FLAG cells were tet-treated for 6 days or left untreated prior to collecting ER- and PM-enriched fractions. Equal amounts of protein were treated with PNGase F, resolved by SDS-PAGE, and analyzed by Western blot. MDCK/β₁/β₂-MYC samples were probed with anti-α₁, anti-actin, anti-β₁, or anti-MYC antibodies (B). MDCK/β₂/β₁-FLAG samples were probed with anti-α₁, anti-actin, anti-β₁, or anti-FLAG antibodies (C). Quantitation of endogenous β₁ subunit abundance from the PM-enriched fraction was corrected for loading by anti-actin and is displayed in D as a percentage of β₁E from untreated samples.

FIGURE 7.

β₂/β₁-FLAG chimera assembles with α₁ subunit, but β₁/β₂-MYC does not. 30 μg of total cell lysate from MDCK cells expressing either β₁/β₂-MYC (A) or β₂/β₁-FLAG (B) chimeras was collected and subjected to immunoprecipitation with anti-α loop antibody. Precipitated proteins were treated with PNGase F to deglycosylate all N-linked glycans. 10% of the initial lysate input (IN) and anti-α loop precipitated proteins (IP) were analyzed by SDS-PAGE and Western blot probed with anti-α₁, anti-MYC, and/or anti-β₁ antibodies.

FIGURE 8.

Overexpression of α₁FLAG lowers α₁E abundance without changing total α₁ levels. MDCK/α₁FLAG cells were grown with (+) or without (−) tet for 3 days prior to harvesting TM-, ER-, Golgi (G)-, and PM-enriched fractions and analyzing by Western blot. Immunoblots were probed with anti-FLAG, anti-α, anti-α6F, anti-β, and anti-β-actin antibodies (A), and relative expression levels of total α₁ and α₁E from −tet (dark bars) or +tet (light bars) PM-enriched samples were quantified (B).

FIGURE 9.

α₁E abundance decreases as α₁FLAG expression increases. MDCK/α₁FLAG cells were grown in the presence of tet for 0, 1, 3, or 6 days prior to harvesting total membranes. Equal amounts of protein from each sample were analyzed by Western blot using anti-β-actin, anti-α6F, anti-FLAG, anti-α₁, and anti-β₁ antibodies (A). α₁E subunit abundance was quantified from the anti-α6F immunoblot and is displayed in B as a percentage of α₁E abundance from 0-h tet samples. C, 20 μg of total membrane protein from MDCK/α₁FLAG cells grown for 3 days with (+) or without (−) tet was subjected to anti-FLAG antibody IP. α₁FLAG and β₁ subunits were detected by Western blot analysis using anti-α₁ and anti-β₁ antibodies.