Human express several different isoforms of Na/K-ATPase. While a1 isoform is expressed ubiquitously in all cells, a2 is predominantly found in muscles, astrocytes and adipocytes. Besides the canonic pumping function, we and others have demonstrated that the a1 Na/K-ATPase has a receptor-like function, allowing its ligands including cardiotonic steroids (CTS) to regulate cellular signal transduction via protein and lipid kinase cascades. Mechanistically, we have shown that the a1 Na/K-ATPase interacts with Src to form a functional receptor complex. We have further demonstrated that the ligand-induced changes in the conformation of Na/K-ATPase activate the associated Src kinase, which in turn transactivates EGF receptor, resulting in the assembly and activation of different protein/lipid kinase cascades in a cell-specific manner. Interestingly, a2 isoform lacks Src binding, and consequently fails to transmit CTS binding to the activation of protein/lipid kinases. The major focus of this dissertation is to understand whether this Src-interacting capacity of a1 and the lack of Src-interacting capacity of a2 confer a specific phenotype in cell physiology.
Following up an accidental observation that a2 Na/K-ATPase-expressing cells appear to acidify culture medium faster than that by a1-expressing cells, we have discovered that Na/K-ATPase regulates aerobic glycolysis and mitochondrial respiration in an isoform-specific manner in mammalian cells. Specifically, we have found that the expression of a2 isoform produces a Warburg-like phenotype, which is an increase in aerobic glycolysis with a concomitant decrease in mitochondrial respiration. Moreover, these cells show no metabolic plasticity and are prone to metabolic stress. Under the same condition, expression of a1 reduces aerobic glycolysis and increases mitochondrial respiration, thus reversing the Warburg Effect. Furthermore, a1 cells exhibited good metabolic plasticity and are resistant to metabolic stress. This difference in metabolic regulation is related to the two conserved Src-binding sites in a1 Na/K-ATPase and to the conserved lack of Src-binding sites in a2 isoform. Consistently, a loss-of-Src-binding mutation in a1 Na/K-ATPase results in a a2-like phenotype in cellular metabolism whereas gain-of-Src binding mutations in a2 produce a1-like phenotypic changes. Mechanistically, expression of a2 increased the expression and tyrosine phosphorylation of PKM2, a fetal isoform of pyruvate kinase that is known to promote the Warburg Effect in cancer cells. Interestingly, when PKM2 was analyzed in mouse skeletal muscle, we found that skeletal muscle-specific knockout of a1 increased the expression and tyrosine phosphorylation of PKM2. Physiologically, it is known that a2 isoform is highly expressed in tissues/cells such as skeletal muscle, astrocytes and adipocytes where glucose is utilized as a major fuel for ATP generation or for providing building materials as well as metabolic intermediates. Thus, our new findings could be highly relevant to the a2-specific cell physiology. Moreover, our new findings also expand our appreciation of a1 Na/K-ATPase, specifically its Src-binding sites, in cell physiology. Finally, we speculate that co-expression of a1 in a2-expressing cells could allow a dynamic regulation of cellular metabolism that meets the need of muscle, astrocytes and adipocytes.
Although the role of Src kinase in the signaling function of Na/K-ATPase has been well accepted, it remains as an outstanding question whether there is a direct interaction between the Na/K-ATPase and Src. In the process of studying the role of Src interaction, we found that Y260 site in the a1 second cytosolic domain is specifically phosphorylated by Src family kinases and the phosphorylated Y260 serves as a Src SH2 ligand, capable of interacting and recruiting Src to the a1 Na/K-ATPase. While this site is conserved in mammalian a1 Na/K-ATPase, it is completely missed in a2 Na/K-ATPase. Functionally, a Y260A mutation in a1 Na/K-ATPase is sufficient to produce a a2-like phenotype as mutant cells exhibit an increase in aerobic glycolysis. These new data not only support our claim that a1 Na/K-ATPase interacts directly with Src kinase, but also reveal the significance of such interaction in the regulation of cell metabolism by Na/K-ATPase.
Finally, because Warburg effects are well documented in cancer cells, we have explored whether a2 expression is induced or a1/Src interaction is impaired in cancer cells. Although the expression of a2 was detected in several human cancer cell lines, it appears unremarkable since control prostate and breast epithelial cells also express a2. On the other hand, we have found a significant decrease in the expression of a1 Na/K-ATPase in many types of human cancers. This decrease is most severe in metastatic tumor samples. Furthermore, in accordance with the notion that Na/K-ATPase-mediated Src regulation may be defective in human cancers, we have also found that Y260 phosphorylation is significantly reduced in several human cancer cell lines. These new data suggest that the a1 expression level together with Y260 phosphorylation could serve as a biomarker for developing individualized medication targeting Na/K-ATPase/Src signaling pathways in cancer.