Biomedical subjects

Man Liang

Publications and source records attributed to Man Liang.

3 recordsLinked to original sources

PubMed2006

Functional characterization of Src-interacting Na/K-ATPase using RNA interference assay.

We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several alpha1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing alpha1-specific small interference RNA. Although the alpha1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabain-induced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat alpha1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat alpha1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat alpha1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases.

Animals↗

PubMed2005

Ouabain-induced endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells requires caveolin-1.

BACKGROUND: We have demonstrated that ouabain causes dose- and time-dependent decreases in (86)Rb uptake in pig renal proximal tubule cell line (LLC-PK1) cells; and ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells in a clathrin-dependent pathway. Our data also suggest a role of endocytosis in both ouabain-induced signal transduction and proximal tubule sodium handling. The present study addresses the molecular mechanisms involved in this process. METHODS: Studies were performed with cultured LLC-PK1 and a stable-expressed caveolin-1 knockdown LLC-PK1 cell line by SiRNA method. RESULTS: In wild-type LLC-PK1 cells, depletion of cholesterol by methyl beta-cyclodextrin reduced ouabain-induced accumulation of Na/K-ATPase alpha-1 subunit, EGFR, Src, and MAPKs in clathrin-coated vesicles, as well as in endosomes. Depletion of cholesterol also significantly reduced the protein-protein interaction among alpha-1 subunit, AP2, PI-3K, and clathrin heavy chain. In LLC-PK1 cells expressing mock-vehicle and caveolin-1 siRNA, depletion of caveolin-1 abolished ouabain-induced decrease in Rb uptake and decrease in the plasmalemmal Na/K-ATPase content. Depletion of caveolin-1 also significantly reduced the ouabain-induced accumulation of Na/K-ATPase alpha-1 subunit, EGFR, Src, and MAPKs in clathrin-coat vesicles, as well as early and late endosomes. In addition, depletion of caveolin-1 also significantly reduced the protein-protein interaction among alpha-1 subunit, AP2, PI-3K, and clathrin heavy chain. These data suggest that caveolae are involved in ouabain-induced endocytosis and signal transduction by initiating assembly of signaling cascades through the caveolar Na/K-ATPase and/or the interaction with clathrin-mediated endocytosis of the Na/K-ATPase.

Animals↗

PubMed2004

Ouabain assembles signaling cascades through the caveolar Na+/K+-ATPase.

Based on the observation that the Na(+)/K(+)-ATPase alpha subunit contains two conserved caveolin-binding motifs, we hypothesized that clustering of the Na(+)/K(+)-ATPase and its partners in caveolae facilitates ouabain-activated signal transduction. Glutathione S-transferase pull-down assay showed that the Na(+)/K(+)-ATPase bound to the N terminus of caveolin-1. Significantly, ouabain regulated the interaction in a time- and dose-dependent manner and stimulated tyrosine phosphorylation of caveolin-1 in LLC-PK1 cells. When added to the isolated membrane fractions, ouabain increased tyrosine phosphorylation of proteins from the isolated caveolae but not other membrane fractions. Consistently, ouabain induced the formation of a Na(+)/K(+)-ATPase-Src-caveolin complex in the isolated caveolae preparations as it did in live cells. Finally, depletion of either cholesterol by methyl beta-cyclodextrin or caveolin-1 by siRNA significantly reduced the caveolar Na(+)/K(+)-ATPase and Src. Concomitantly, cholesterol depletion abolished ouabain-induced recruitment of Src to the Na(+)/K(+)-ATPase signaling complex. Like depletion of caveolin-1, it also blocked the effect of ouabain on ERKs, which was restored after cholesterol repletion. Clearly, the caveolar Na(+)/K(+)-ATPase represents the signaling pool of the pump that interacts with Src and transmits the ouabain signals.

Amino Acid Sequence↗