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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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Effect of Src inhibitor PP2 on the cellular distribution of Cav1. TCN23-19 cells were treated with 2 μM of PP2 for 5 h; afterward, both control and PP2-treated cells were stained for Cav1. The plasma membrane signals were labeled by solid arrows in both treated and untreated cells to illustrate the recovery of Cav1 signal in the plasma membrane of PP2-treated cells. A set of representative images from three experiments is shown. Bar, 5 μm.
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fig10: Effect of Src inhibitor PP2 on the cellular distribution of Cav1. TCN23-19 cells were treated with 2 μM of PP2 for 5 h; afterward, both control and PP2-treated cells were stained for Cav1. The plasma membrane signals were labeled by solid arrows in both treated and untreated cells to illustrate the recovery of Cav1 signal in the plasma membrane of PP2-treated cells. A set of representative images from three experiments is shown. Bar, 5 μm.

Mentions: It is known that Src plays an important role in controlling Cav1 endocytosis (Sharma et al., 2004; Pelkmans and Zerial, 2005; Le Lay et al., 2006). We have reported that there is a pool of Na/K-ATPase–interacting Src in LLC-PK1 cells (Liang et al., 2006; Tian et al., 2006). Binding of Src to this pool of Na/K-ATPase keeps Src in an inactive state and knockdown of Na/K-ATPase releases this pool of Src, resulting in an increase in basal Src activity. Thus, the Na/K-ATPase knockdown-induced Src activation could be responsible for the observed increase in the mobility of Cav1 vesicles in TCN23-19 cells. To test this postulation, TCN23-19 cells were treated with PP2, a Src family kinase inhibitor, for 5 h and then immunostained for Cav1. Cell imaging showed that PP2 treatment caused a significant increase in plasma membrane Cav1 signal (42.6 ± 1.5% v. 26 ± 1%; n = 28; P < 0.01) (Fig. 10). The above experiment was repeated in Cav1-YFP transfected cells, and showed similar result (44.2 ± 2% in PP2 treated cells vs. 32 ± 1% in nontreated TCN23-19 cells; n = 30; P < 0.01). In accordance, we found that PP2 significantly reduced the mobility of Cav1-YFP–positive vesicles in TCN23-19 cells (Fig. 9, B and C and Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200712022/DC1).


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)

Effect of Src inhibitor PP2 on the cellular distribution of Cav1. TCN23-19 cells were treated with 2 μM of PP2 for 5 h; afterward, both control and PP2-treated cells were stained for Cav1. The plasma membrane signals were labeled by solid arrows in both treated and untreated cells to illustrate the recovery of Cav1 signal in the plasma membrane of PP2-treated cells. A set of representative images from three experiments is shown. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: Effect of Src inhibitor PP2 on the cellular distribution of Cav1. TCN23-19 cells were treated with 2 μM of PP2 for 5 h; afterward, both control and PP2-treated cells were stained for Cav1. The plasma membrane signals were labeled by solid arrows in both treated and untreated cells to illustrate the recovery of Cav1 signal in the plasma membrane of PP2-treated cells. A set of representative images from three experiments is shown. Bar, 5 μm.
Mentions: It is known that Src plays an important role in controlling Cav1 endocytosis (Sharma et al., 2004; Pelkmans and Zerial, 2005; Le Lay et al., 2006). We have reported that there is a pool of Na/K-ATPase–interacting Src in LLC-PK1 cells (Liang et al., 2006; Tian et al., 2006). Binding of Src to this pool of Na/K-ATPase keeps Src in an inactive state and knockdown of Na/K-ATPase releases this pool of Src, resulting in an increase in basal Src activity. Thus, the Na/K-ATPase knockdown-induced Src activation could be responsible for the observed increase in the mobility of Cav1 vesicles in TCN23-19 cells. To test this postulation, TCN23-19 cells were treated with PP2, a Src family kinase inhibitor, for 5 h and then immunostained for Cav1. Cell imaging showed that PP2 treatment caused a significant increase in plasma membrane Cav1 signal (42.6 ± 1.5% v. 26 ± 1%; n = 28; P < 0.01) (Fig. 10). The above experiment was repeated in Cav1-YFP transfected cells, and showed similar result (44.2 ± 2% in PP2 treated cells vs. 32 ± 1% in nontreated TCN23-19 cells; n = 30; P < 0.01). In accordance, we found that PP2 significantly reduced the mobility of Cav1-YFP–positive vesicles in TCN23-19 cells (Fig. 9, B and C and Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200712022/DC1).

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