The NA/K-ATPase and its isozymes: what we have learned using the baculovirus expression system

Blanco, Gustavo

doi:10.2741/1705

Cited by 41 publications

(33 citation statements)

References 141 publications

(183 reference statements)

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“…The large intracellular region of the Na,K-ATPase between transmembrane domains 4 and 5 has been shown to interact with the C-terminus of polycystin 1 (40,41). It is interesting that we found that, in insect cells, exogenous expression of a construct that contains the transmembrane and C-terminal domains of polycystin 1 increases the sensitivity of the Na,K-ATPase to ouabain, with the enzyme exhibiting a K i for the steroid that is similar to that found in ADPKD cells (42). Therefore, Na,K-ATPase-protein interaction represents another potential mechanism that is involved in the response of the cells to ouabain.…”

Section: Discussionsupporting

confidence: 48%

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

Nguyen¹,

Wallace²,

Blanco³

2007

Journal of the American Society of Nephrology

108

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In autosomal dominant polycystic kidney disease (ADPKD), cyst formation and enlargement require proliferation of mural renal epithelial cells and the transepithelial secretion of fluid into the cyst cavity. Na,K-ATPase is essential for solute and water transport in ADPKD cells, and ouabain blocks fluid secretion in these cells. By binding to the Na,K-ATPase, ouabain also induces proliferation in some cell types. Surprisingly, it was found that nanomolar concentrations of ouabain, similar to those circulating in blood, induced ADPKD cell proliferation but had no statistically significant effect on normal human kidney (NHK) cells. Ouabain, acting from the basolateral side of the cells, also caused an increase in the level of phosphorylated extracellular signal-regulated kinases (ERK). Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocked ouabain-induced ERK activation and cell proliferation, suggesting that ouabain effect is mediated through the MEK-ERK pathway. In contrast to NHK cells, the dose-response curve for ouabain inhibition of Na,K-ATPase activity indicated that approximately 20% of the enzyme in ADPKD cells exhibits a higher affinity for ouabain. The increased ouabain affinity of ADPKD cells was not due to differences in Na,K-ATPase isoform expression because these cells, like NHK cells, possess only the ␣1 and ␤1 subunits. The ␥ variants of the Na,K-ATPase also are expressed in the cells but are elevated in ADPKD cells. Currently, the basis for the differences in ouabain sensitivity of NHK and ADPKD cells is unknown. It is concluded that ouabain stimulates proliferation in ADPKD cells by binding to the Na,K-ATPase with high affinity and via activation of the MEK-ERK pathway.

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Section: Discussionsupporting

confidence: 48%

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

Nguyen¹,

Wallace²,

Blanco³

2007

Journal of the American Society of Nephrology

108

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“…To date, four a-and b-subunit isoforms have been identified in mammals, which are expressed in a tissue-specific manner (17). A g-subunit can provide further tissue-specific regulation (18,19).…”

Section: Regulation Of the Nak-atpasementioning

confidence: 99%

“…It has been proposed that the Na,K-ATPase has other evolutionarily conserved physiological functions beyond ion transport. Particularly interesting is the requirement of the Na,K-ATPase in the formation and maintenance of intercellular junctions, where it can act as a signaling and scaffolding center (16)(17)(18).…”

Section: Regulation Of the Nak-atpasementioning

confidence: 99%

Role of Ubiquitination in Na,K-ATPase Regulation during Lung Injury

Helenius

Dada

Sznajder

2010

Proceedings of the American Thoracic Society

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During acute lung injury edema accumulates in the alveolar space, resulting in hypoxemia due to intrapulmonary shunt. The alveolar Na,K-ATPase, by effecting active Na 1 transport, is essential for removing edema from the alveolar spaces. However, during hypoxia it is endocytosed and degraded, which results in decreased Na,KATPase function and impaired lung edema clearance. Na,K-ATPase endocytosis and degradation require the phosphorylation and subsequent ubiquitination of the Na,K-ATPase. These events are the results of cross-talk between post-translational modifications, and how ubiquitination of a specific protein can result from injurious extracellular stimuli. Here, we review current knowledge on the regulation of Na,K-ATPase activity during lung injury, focusing on the role of Na,K-ATPase ubiquitination during hypoxia. A better understanding of these signaling pathways can be of relevance for the design of novel treatments to ameliorate the deleterious effects of acute lung injury.

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“…This problem can be circumvented by using heterologous expression systems. In the past, we have successfully used the baculovirus expression system to study different aspects of the enzymatic and regulatory properties of the Na-K-ATPase isoforms in insect cells (4). Sf-9 insect cells provide several advantages for the study of the Na-K-ATPase, including the production of high amounts of the ␣-and ␤-subunits and the proper delivery of catalytically competent Na-K-ATPase isozymes to the plasma membrane (4).…”

mentioning

confidence: 99%

Isoform specificity of Na-K-ATPase-mediated ouabain signaling

Pierre

Sottejeau²,

Gourbeau³

et al. 2008

American Journal of Physiology-Renal Physiology

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The ion transporter Na-K-ATPase functions as a cell signal transducer that mediates ouabain-induced activation of protein kinases, such as ERK. While Na-K-ATPase composed of the α1-polypeptide is involved in cell signaling, the role of other α-isoforms (α2, α3, and α4) in transmitting ouabain effects is unknown. We have explored this using baculovirus-directed expression of Na-K-ATPase polypeptides in insect cells and ERK phosphorylation as an indicator of ouabain-induced signaling. Ouabain addition to Sf-9 cells coexpressing Na-K-ATPase α1- and β1-isoforms stimulated ERK phosphorylation. In contrast, expression of the α1- and β1-polypeptides alone resulted in no effect, indicating that the αβ-complex is necessary for Na-K-ATPase signaling. Moreover, the ouabain effect was sensitive to genistein, suggesting that Na-K-ATPase-mediated tyrosine kinase activation is a critical event in the intracellular cascade leading to ERK phosphorylation. In addition, the Na-K-ATPases α3β1- and α4β1-isozymes, but not α2β1, responded to ouabain treatment. In agreement with the differences in ouabain affinity of the α-polypeptides, α1β1 required 100- to 1,000-fold more ouabain to signal than did α4β1 and α3β1, respectively. These results confirm the role of the Na-K-ATPase in ouabain signal transduction, show that there are important isoform-specific differences in Na-K-ATPase signaling, and demonstrate the suitability of the baculovirus expression system for studying Na-K-ATPase-mediated ouabain effects.

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The NA/K-ATPase and its isozymes: what we have learned using the baculovirus expression system

Cited by 41 publications

References 141 publications

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

Role of Ubiquitination in Na,K-ATPase Regulation during Lung Injury

Isoform specificity of Na-K-ATPase-mediated ouabain signaling

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