Mutagenesis Disrupts Posttranslational Processing of the Na,K-ATPase Catalytic Subunit

Petrosian, Susan A.; Carr, Deborah L.; Guerrero, Georgina; Pressley, Thomas A.

doi:10.1006/abbi.1998.0816

Cited by 7 publications

(9 citation statements)

References 39 publications

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“…Indeed, the distinctive behavior of the isoforms is apparent when the reaction is carried out under micromolar ATP concentrations because the K ϩ deocclusion pathway of the reaction cycle becomes rate limiting (18). Under such conditions, the activity of exogenous ␣ 1 expressed in HeLa and COS-1 cells is inhibited by K ϩ , whereas ␣ 2 is stimulated (8,17). The present observations have confirmed these results in OK cells.…”

Section: Discussionsupporting

confidence: 89%

“…1). We and others have established the influence of the NH 2 -terminal region on isoform-specific enzyme kinetics and regulation (8,9,17,26). However, this work has also raised suspicions that another site on the catalytic subunit may contribute to these differences.…”

Section: Discussionmentioning

confidence: 99%

“…The activity of Na ϩ -K ϩ -ATPase in isolated membranes from transfected cells was determined from the hydrolysis of radiolabeled ATP as described (17). Briefly, membranes (1 mg/ml) were treated with 0.4-0.7 mg/ml deoxycholate for 30 min at room temperature to activate latent Na ϩ -K ϩ -ATPase activity.…”

Section: Preparation Of Chimerasmentioning

confidence: 99%

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Structure/function analysis of Na⁺-K⁺-ATPase central isoform-specific region: involvement in PKC regulation

Pierre

Duran

Carr

et al. 2002

American Journal of Physiology-Renal Physiology

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Specific functions served by the various Na+-K+-ATPase α-isoforms are likely to originate in regions of structural divergence within their primary structures. The isoforms are nearly identical, with the exception of the NH2 terminus and a 10-residue region near the center of each molecule (isoform-specific region; ISR). Although the NH2 terminus has been clearly identified as a source of isoform functional diversity, other regions seem to be involved. We investigated whether the central ISR could also contribute to isoform variability. We constructed chimeric molecules in which the central ISRs of rat α1- and α2-isoforms were exchanged. After stable transfection into opossum kidney cells, the chimeras were characterized for two properties known to differ dramatically among the isoforms: their K+ deocclusion pattern and their response to PKC activation. Comparisons with rat full-length α1- and α2-isoforms expressed under the same conditions suggest an involvement of the central ISR in the response to PKC but not in K+ deocclusion.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

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Structure/function analysis of Na⁺-K⁺-ATPase central isoform-specific region: involvement in PKC regulation

Pierre

Duran

Carr

et al. 2002

American Journal of Physiology-Renal Physiology

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“…Site-directed Mutagenesis-Rat ␣1 expression vector pRc/ CMV-␣1AAC was provided by Dr. Pressley (12). To make the expression of rat ␣1 insensitive to A4 siRNA, the ␣1 siRNAtargeted sequence was silently mutated from 2530 ggtcgtctgatcttt (GenBank TM accession number NM_012504) to 2530 ggcaggctaatattc using the QuikChange mutagenesis kit.…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

Liang

Cai

Tian

et al. 2006

Journal of Biological Chemistry

148

228

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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 ␣1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing ␣1-specific small interference RNA. Although the ␣1 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 ouabaininduced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat ␣1, 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 ␣1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat ␣1 (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. Na/K-ATPase was discovered by Skou (1) as the molecular machinery of the cellular sodium pump. It belongs to a family of evolutionarily ancient ATPases that couple the hydrolysis of ATP to membrane ion translocation (2, 3). A major difference between the Na/K-ATPase and other ATPases is its ability to bind cardiotonic steroids such as ouabain. Studies from many laboratories have now established that the binding of ouabain to this enzyme not only inhibits the ATPase activity but also stimulates protein tyrosine kinases such as Src (4, 5). The activated Src in turn transactivates epidermal growth factor receptor, resulting in the assembly and activation of multiple signaling cascades such as the ERK1/2 2 and phospholipase C-␥/ protein kinase C pathways (5, 6).Because several laboratories have demonstrated that the activation of Src is essential for ouabain-induced changes in many cellular activities including the regulation of intracellular calcium, gene expression, and cell growth (6 -9), we have recently examined whether the Na/K-ATPase interacts directly with Src to form a functional signaling receptor (10). Using in vitro glutathione S-transferase pulldown assays we have identified that the second and the third intracellular domains of the Na/K-ATPase ␣1 subunit interact with the Src SH2 and the kinase domains, respectively. Functionally these interactions keep Src in an inactive state, and binding of ouabain to this inactive Na/K-ATPase⅐Src complex frees and then activates the associated Src (10). These new findings suggest that the cellular Src-interacting Na/K-ATPase may play an...

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“…81 More recent studies in HeLa cells indicated differences in isoform interaction with extracellular Na ϩ and K ϩ . 82 In addition, the group of Pressley, by using Cos-1 and kidney opossum cells, 24,83 and Daly et al, 84 working with HeLa cells, showed that the N-terminal portion of the ␣ polypeptides is important in defining the K ϩ kinetic differences among isoforms. This last group also determined that the mechanistic basis for these dissimilarities depend on isozyme differences in E1/E2 conformational equilibrium, as well as on the rates of partial reactions associated with the translocation of Na ϩ and K ϩ .…”

Section: Kinetic Properties Of the Nak-atpase Isozymesmentioning

confidence: 99%

Na,K-ATPase Subunit Heterogeneity as a Mechanism for Tissue-Specific Ion Regulation

Blanco

2005

Seminars in Nephrology

259

267

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Mutagenesis Disrupts Posttranslational Processing of the Na,K-ATPase Catalytic Subunit

Cited by 7 publications

References 39 publications

Structure/function analysis of Na⁺-K⁺-ATPase central isoform-specific region: involvement in PKC regulation

Structure/function analysis of Na⁺-K⁺-ATPase central isoform-specific region: involvement in PKC regulation

Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

Na,K-ATPase Subunit Heterogeneity as a Mechanism for Tissue-Specific Ion Regulation

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Mutagenesis Disrupts Posttranslational Processing of the Na,K-ATPase Catalytic Subunit

Cited by 7 publications

References 39 publications

Structure/function analysis of Na+-K+-ATPase central isoform-specific region: involvement in PKC regulation

Structure/function analysis of Na+-K+-ATPase central isoform-specific region: involvement in PKC regulation

Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

Na,K-ATPase Subunit Heterogeneity as a Mechanism for Tissue-Specific Ion Regulation

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Structure/function analysis of Na⁺-K⁺-ATPase central isoform-specific region: involvement in PKC regulation

Structure/function analysis of Na⁺-K⁺-ATPase central isoform-specific region: involvement in PKC regulation