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Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase.

Cai T, Wang H, Chen Y, Liu L, Gunning WT, Quintas LE, Xie ZJ - J. Cell Biol. (2008)

Bottom Line: Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface.These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit.Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Toledo College of Medicine, Health Science Campus, Toledo, OH 43614, USA.

ABSTRACT
Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase-depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.

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Knocking-in a wild-type, but not mCBM α1 mutant rescues Cav1 distribution in TCN23-19 cells. TCN23-19 cells were rescued by YFP-tagged wild-type α1 (wt α1) or mCBM. Stable cell lines were generated and analyzed as described in Fig. 1. (A) A set of representative Western blots from four independent experiments showing the expression of both YFP-α1/mCBM α1 (140 kD) and residual endogenous α1 (110 kD), and the distribution of Cav1 in each fraction. (B) Combined data from four different experiments shows that expression of wild-type rat α1, but not the mCBM α1, restored the distribution of Cav1 into the 4/5 fraction in TCN23-19 cells . **, P < 0.01 in comparison with control P-11. Endo α1: endogenous α1.
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fig5: Knocking-in a wild-type, but not mCBM α1 mutant rescues Cav1 distribution in TCN23-19 cells. TCN23-19 cells were rescued by YFP-tagged wild-type α1 (wt α1) or mCBM. Stable cell lines were generated and analyzed as described in Fig. 1. (A) A set of representative Western blots from four independent experiments showing the expression of both YFP-α1/mCBM α1 (140 kD) and residual endogenous α1 (110 kD), and the distribution of Cav1 in each fraction. (B) Combined data from four different experiments shows that expression of wild-type rat α1, but not the mCBM α1, restored the distribution of Cav1 into the 4/5 fraction in TCN23-19 cells . **, P < 0.01 in comparison with control P-11. Endo α1: endogenous α1.

Mentions: To functionally assess the role of the interaction between Na/K-ATPase and Cav1 in regulating cellular distribution of Cav1, we rescued TCN23-19 cells by expressing a wild-type rat α1. After the stable cell lines were established, we repeated the density fractionation assays. As shown in Fig. 5, expression of a wild-type rat α1 in TCN23-19 cells not only restored cellular Na/K-ATPase activity (Liang et al., 2006), but was also able to target Cav1 into fraction 4/5 as in the control P-11 cells (Fig. 5 B). To be sure that this restoration is not a cell-specific phenomenon, we repeated the same experiments in PY-17 cells, another Na/K-ATPase knockdown cell line (Liang et al., 2006), showing that expression of a rat α1 was indeed sufficient to restore Cav1 targeting to the fraction 4/5 in this cell line (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200712022/DC1). In contrast, rescuing TCN23-19 cells with the mCBM α1 only restored the cellular Na/K-ATPase, but failed to change Cav1 distribution because most of Cav1 remained in the heavy fractions (Fig. 5). These findings are consistent with the notion that the interaction between Na/K-ATPase and Cav1 is important for trafficking Cav1 to the plasma membrane.


Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase.

Cai T, Wang H, Chen Y, Liu L, Gunning WT, Quintas LE, Xie ZJ - J. Cell Biol. (2008)

Knocking-in a wild-type, but not mCBM α1 mutant rescues Cav1 distribution in TCN23-19 cells. TCN23-19 cells were rescued by YFP-tagged wild-type α1 (wt α1) or mCBM. Stable cell lines were generated and analyzed as described in Fig. 1. (A) A set of representative Western blots from four independent experiments showing the expression of both YFP-α1/mCBM α1 (140 kD) and residual endogenous α1 (110 kD), and the distribution of Cav1 in each fraction. (B) Combined data from four different experiments shows that expression of wild-type rat α1, but not the mCBM α1, restored the distribution of Cav1 into the 4/5 fraction in TCN23-19 cells . **, P < 0.01 in comparison with control P-11. Endo α1: endogenous α1.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2542476&req=5

fig5: Knocking-in a wild-type, but not mCBM α1 mutant rescues Cav1 distribution in TCN23-19 cells. TCN23-19 cells were rescued by YFP-tagged wild-type α1 (wt α1) or mCBM. Stable cell lines were generated and analyzed as described in Fig. 1. (A) A set of representative Western blots from four independent experiments showing the expression of both YFP-α1/mCBM α1 (140 kD) and residual endogenous α1 (110 kD), and the distribution of Cav1 in each fraction. (B) Combined data from four different experiments shows that expression of wild-type rat α1, but not the mCBM α1, restored the distribution of Cav1 into the 4/5 fraction in TCN23-19 cells . **, P < 0.01 in comparison with control P-11. Endo α1: endogenous α1.
Mentions: To functionally assess the role of the interaction between Na/K-ATPase and Cav1 in regulating cellular distribution of Cav1, we rescued TCN23-19 cells by expressing a wild-type rat α1. After the stable cell lines were established, we repeated the density fractionation assays. As shown in Fig. 5, expression of a wild-type rat α1 in TCN23-19 cells not only restored cellular Na/K-ATPase activity (Liang et al., 2006), but was also able to target Cav1 into fraction 4/5 as in the control P-11 cells (Fig. 5 B). To be sure that this restoration is not a cell-specific phenomenon, we repeated the same experiments in PY-17 cells, another Na/K-ATPase knockdown cell line (Liang et al., 2006), showing that expression of a rat α1 was indeed sufficient to restore Cav1 targeting to the fraction 4/5 in this cell line (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200712022/DC1). In contrast, rescuing TCN23-19 cells with the mCBM α1 only restored the cellular Na/K-ATPase, but failed to change Cav1 distribution because most of Cav1 remained in the heavy fractions (Fig. 5). These findings are consistent with the notion that the interaction between Na/K-ATPase and Cav1 is important for trafficking Cav1 to the plasma membrane.

Bottom Line: Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface.These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit.Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Toledo College of Medicine, Health Science Campus, Toledo, OH 43614, USA.

ABSTRACT
Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase-depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.

Show MeSH
Related in: MedlinePlus