P-type ATPases in Health and Disease

P-type ATPases are a large group of evolutionary related ion and lipid pumps that have in common that they catalyze a transient phosphorylated intermediate at a key conserved aspartate residue within the pump in order to function. While all the P-type ATPases perform active transport across cellular membranes, they have different transport specificities and serve diverse physiological functions. The ion pumps of the P-type ATPase family create electrochemical gradients that are essential for transepithelial transport, nutrient uptake and membrane potential. They mediate cellular signaling and provide the ligands for metalloenzymes. Phospholipid flippases, also members of the P-type ATPase superfamily, regulate the asymmetric lipid distribution across the lipid bilayer and are critical for the biogenesis of cell membranes. Since all of these ATPases serve fundamental cellular functions, malfunctioning is associated with various pathophysiological processes and dysfunctions of P-type ATPases are known to contribute to cardiovascular, neurological, renal and metabolic diseases. However, with the ever growing knowledge about the diseases associated with the malfunction of P-type ATPases, they are also promising targets for future drug development. In eukaryotes the most prominent examples of P-type ATPases are the Na+,K+-ATPase (sodium pump), the H+-ATPase (proton pump), the H+,K+-ATPase (proton-potassium pump) and the Ca2+-ATPases (calcium pumps). Mutations in the alpha2 and alpha3 subunit of Na,K-ATPase have been associated with neurological diseases, including rapid-onset dystonia-parkinsonism, familial hemiplegic migraine and alternating hemiplegia of childhood. Dysregulation and loss of expression of Na,K-ATPase and plasma membrane Ca-ATPases may be involved in cancer progression. Malfunctioning of the Ca-ATPases is also thought to contribute to hypertension and neurodegenerative diseases and mutations can cause cardiac dysfunction, deafness, hypertension and cerebellar ataxia. Mutations in the SERCA calcium pumps can cause heart failure, Brody myopathy and Darier disease and mutations in the Cu-ATPase genes cause Menkes and Wilson disease. Deficiencies in phospholipid flippases have been linked to progressive familial intrahepatic cholestasis, obesity, diabetes, hearing loss and neurological diseases

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

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+ transport, is essential for removing edema from the alveolar spaces. However, during hypoxia it is endocytosed and degraded, which results in decreased Na,K-ATPase 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

Mitochondrial Ca2+ and ROS take center stage to orchestrate TNF-α–mediated inflammatory responses

Proinflammatory stimuli induce inflammation that may progress to sepsis or chronic inflammatory disease. The cytokine TNF-α is an important endotoxin-induced inflammatory glycoprotein produced predominantly by macrophages and lymphocytes. TNF-α plays a major role in initiating signaling pathways and pathophysiological responses after engaging TNF receptors. In this issue of JCI, Rowlands et al. demonstrate that in lung microvessels, soluble TNF-α (sTNF-α) promotes the shedding of the TNF-α receptor 1 ectodomain via increased mitochondrial Ca2+ that leads to release of mitochondrial ROS. Shedding mediated by TNF-α–converting enzyme (TACE) results in an unattached TNF receptor, which participates in the scavenging of sTNF-α, thus limiting the propagation of the inflammatory response. These findings suggest that mitochondrial Ca2+, ROS, and TACE might be therapeutically targeted for treating pulmonary endothelial inflammation

Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-ζ

During ascent to high altitude and pulmonary edema, the alveolar epithelial cells (AEC) are exposed to hypoxic conditions. Hypoxia inhibits alveolar fluid reabsorption and decreases Na,K-ATPase activity in AEC. We report here that exposure of AEC to hypoxia induced a time-dependent decrease of Na,K-ATPase activity and a parallel decrease in the number of Na,K-ATPase α(1) subunits at the basolateral membrane (BLM), without changing its total cell protein abundance. These effects were reversible upon reoxygenation and specific, because the plasma membrane protein GLUT1 did not decrease in response to hypoxia. Hypoxia caused an increase in mitochondrial reactive oxygen species (ROS) levels that was inhibited by antioxidants. Antioxidants prevented the hypoxia-mediated decrease in Na,K-ATPase activity and protein abundance at the BLM. Hypoxia-treated AEC deficient in mitochondrial DNA (ρ(0) cells) did not have increased levels of ROS, nor was the Na,K-ATPase activity inhibited. Na,K-ATPase α(1) subunit was phosphorylated by PKC in hypoxia-treated AEC. In AEC treated with a PKC-ζ antagonist peptide or with the Na,K-ATPase α(1) subunit lacking the PKC phosphorylation site (Ser-18), hypoxia failed to decrease Na,K-ATPase abundance and function. Accordingly, we provide evidence that hypoxia decreases Na,K-ATPase activity in AEC by triggering its endocytosis through mitochondrial ROS and PKC-ζ–mediated phosphorylation of the Na,K-ATPase α(1) subunit

α1-AMP-Activated Protein Kinase Regulates Hypoxia-Induced Na,K-ATPase Endocytosis via Direct Phosphorylation of Protein Kinase Cζ▿

Hypoxia promotes Na,K-ATPase endocytosis via protein kinase Cζ (PKCζ)-mediated phosphorylation of the Na,K-ATPase α subunit. Here, we report that hypoxia leads to the phosphorylation of 5′-AMP-activated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK α subunit (AMPK-DN) construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. The overexpression of the reactive oxygen species (ROS) scavenger catalase prevented hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondrion-deficient ρ0-A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKCζ translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK α phosphorylates PKCζ on residue Thr410 within the PKCζ activation loop. Importantly, the activation of AMPK α was necessary for hypoxia-induced AMPK-PKCζ binding in alveolar epithelial cells. The overexpression of T410A mutant PKCζ prevented hypoxia-induced Na,K-ATPase endocytosis, confirming that PKCζ Thr410 phosphorylation is essential for this process. PKCζ activation by AMPK is isoform specific, as small interfering RNA targeting the α1 but not the α2 catalytic subunit prevented PKCζ activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK α1 isoform, which binds and directly phosphorylates PKCζ at Thr410, thereby promoting Na,K-ATPase endocytosis