Novel role for Na,K-ATPase in phosphatidylinositol 3-kinase signaling and suppression of cell motility

The Na,K-ATPase, consisting of alpha- and beta-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase beta1-subunit (Na,K-beta) in highly motile Moloney sarcoma virus-transformed Madin-Darby canine kidney (MSV-MDCK) cells suppressed their motility. However, until now, the mechanism by which Na,K-beta reduces cell motility remained elusive. Here, we demonstrate that Na,K-beta localizes to lamellipodia and suppresses cell motility by a novel signaling mechanism involving a cross-talk between Na,K-ATPase alpha1-subunit (Na,K-alpha) and Na,K-beta with proteins involved in phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway. We show that Na,K-alpha associates with the regulatory subunit of PI3-kinase and Na,K-beta binds to annexin II. These molecular interactions locally activate PI3-kinase at the lamellipodia and suppress cell motility in MSV-MDCK cells, independent of Na,K-ATPase ion transport activity. Thus, these results demonstrate a new role for Na,K-ATPase in regulating carcinoma cell motility.     

A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

The heterodimeric Na,K-ATPase has been implicated in vertebrate and invertebrate epithelial cell junctions, morphogenesis and oncogenesis, but the mechanisms involved are unclear. We previously showed that the Drosophila Na,K-ATPase is required for septate junction (SJ) formation and that of the three beta-subunit loci, only Nrv2 isoforms support epithelial SJ barrier function and tracheal tube-size control. Here we show that Nrv1 is endogenously co-expressed with Nrv2 in the epidermis and tracheal system, but Nrv1 has a basolateral localization and appears to be excluded from the Nrv2-containing SJs. When the normally neuronal Nrv3 is expressed in epithelial cells, it does not associate with SJs. Thus, the beta-subunit is a key determinant of Na,K-ATPase subcellular localization as well as function. However, localization of the Na,K-ATPase to SJs is not sufficient for junctional activity because although several Nrv2/Nrv3 chimeric beta-subunits localize to SJs, only those containing the extracellular domain of Nrv2 have junctional activity. Junctional activity is also specific to different alpha-subunit isoforms, with only some isoforms from the major alpha-subunit locus being able to provide full barrier function and produce normal tracheal tubes. Importantly, mutations predicted to inactivate ATPalpha catalytic function do not compromise junctional activity, demonstrating that the Drosophila Na,K-ATPase has an ion-pump-independent role in junction formation and tracheal morphogenesis. These results define new functions for the intensively studied Na,K-ATPase. Strikingly, the rat alpha1 isoform has full junctional activity and can rescue Atpalpha-null mutants to viability, suggesting that the Na,K-ATPase has an evolutionarily conserved role in junction formation and function.     

The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K-pumps.

Na,K-ATPase and H,K-ATPase are the only members of the P-type ATPases in which a glycosylated beta-subunit is part of the purified active enzyme. In this study, we have followed the synthesis and the posttranslational processing of the beta-subunit of H,K-ATPase (beta HK) in Xenopus oocytes injected with beta HK cRNA and have tested whether it can act as a surrogate for the beta-subunit of Na,K-ATPase (beta NaK) to support the functional expression of Na,K-pumps. In Xenopus oocytes, beta HK is processed from an Endo H-sensitive 51-kDa coreglycosylated form to an Endo H-resistant 71-kDa fully glycosylated form. Similar to beta NaK, beta HK can stabilize and increase the trypsin resistance of alpha-subunits of Na,K-ATPase (alpha NaK). Finally, expression of beta HK together with alpha NaK leads to an increased number of ouabain binding sites at the plasma membrane accompanied by an increased Rb+ uptake and Na,K-pump current. Our data suggest that beta HK, similar to beta NaK, can assemble to alpha NaK, support the structural maturation and the intracellular transport of catalytic alpha NaK, and ultimately form active alpha NaK-beta HK complexes with Na,K-pump transport properties.     

Molecular cloning and sequence analysis of human Na,K-ATPase beta-subunit.

We have isolated a cDNA clone for the beta-subunit of HeLa cell Na,K-ATPase, containing a 2208-base-pair cDNA insert covering the whole coding region of the beta-subunit. Nucleotide sequence analysis revealed that the amino acid sequence of human Na,K-ATPase exhibited 61% homology with that of Torpedo counterpart (Noguchi et al. (1986) FEBS Lett. in press). A remarkable conservation in the nucleotide sequence of the 3' non-coding region was detected between the human and Torpedo cDNAs. RNA blot hybridization analysis revealed the presence of two mRNA species in HeLa cells. S1 nuclease mapping indicated that they were derived from utilization of two distinct polyadenylation signals in vivo. Total genomic Southern hybridization indicated the existence of only a few, possibly one set of gene encoding the Na,K-ATPase beta-subunit in the human genome.     

Sperm motility is dependent on a unique isoform of the Na,K-ATPase

The Na,K-ATPase, a member of the P-type ATPases, is composed of two subunits, alpha and beta, and is responsible for translocating Na(+) out of the cell and K(+) into the cell using the energy of hydrolysis of one molecule of ATP. The electrochemical gradient it generates is necessary for many cellular functions, including establishment of the plasma membrane potential and transport of sugars and ions in and out of the cell. Families of isoforms for both the alpha and beta subunits have been identified, and specific functional roles for individual isoforms are just beginning to emerge. The alpha4 isoform is the most recently identified Na, K-ATPase alpha isoform, and its expression has been found only in testis. Here we show that expression of the alpha4 isoform in testis is localized to spermatozoa and that inhibition of this isoform alone eliminates sperm motility. These data describe for the first time a biological function for the alpha4 isoform of the Na,K-ATPase, revealing a critical role for this isoform in sperm motility.     

p120 Catenin is required for growth factor-dependent cell motility and scattering in epithelial cells

Cadherin-mediated cell-cell adhesion is dynamically modulated during epithelial-mesenchymal transition triggered by activation of receptor tyrosine kinases (RTK) in epithelial cells. Several cadherin-binding proteins have been identified that control cell-cell adhesion. However, the mechanisms by which intercellular adhesion and cell motility are coregulated are still unknown. Here, we delineate a hitherto uncharted cooperation between RTKs, RhoA GTPase, and p120 catenin in instructing a motile behavior to epithelial cells. We found that expression of an N-terminus-deleted p120 catenin in a variety of epithelial cell types, including primary keratinocytes, effectively competes for endogenous p120 at cadherin binding sites and abrogates EGF-stimulated cell motility as well as HGF-induced cell scattering. The deleted mutant also inhibits the PI3K-dependent RhoA activation ensuing receptor activation. Conversely, we also show that the ectopic expression of full-length p120 in epithelial cells promotes cytoskeletal changes, stimulates cell motility, and activates RhoA. Both motogenic response to p120 and RhoA activation require coactivation of signaling downstream of RTKs as they are suppressed by ablation of the Ras/PI3K pathway. These studies demonstrate that p120 catenin is a necessary target of RTKs in regulating cell motility and help define a novel pathway leading to RhoA activation, which may contribute to the early steps of metastatic invasion.     

Characterization of Na,K-ATPase and H,K-ATPase enzymes with glycosylation-deficient beta-subunit variants by voltage-clamp fluorometry in Xenopus oocytes

The role of N-linked glycosylation of beta-subunits in the functional properties of the oligomeric P-type ATPases Na,K- and H,K-ATPase has been examined by expressing glycosylation-deficient Asn-to-Gln beta-variants in Xenopus oocytes. For both ATPases, the absence of the huge N-linked oligosaccharide moiety on the beta-subunit does not affect alpha/beta coassembly, plasma membrane delivery or functional activity of the holoenzyme. Whereas this is in line with several previous glycosylation studies on Na,K-ATPase, this is the first report showing that the cell surface delivery and enzymatic activity of the gastric H,K-ATPase is unaffected by the lack of N-linked glycosylation. Sulfhydryl-specific labeling of introduced cysteine reporter sites with the environmentally sensitive fluorophore tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes enabled us to further investigate potential effects of the N-glycans on more subtle enzymatic properties, like the distribution between E 1P/E 2P states of the catalytic cycle and the kinetics of the E 1P/E 2P conformational transition under presteady state conditions. For both Na,K-ATPase and H,K-ATPase, we observed differences in neither the voltage-dependent E 1P/E 2P ratio nor the kinetics of the E 1P/E 2P transition between holoenzymes comprising glycosylated and glycosylation-deficient beta-subunits. We conclude that the N-linked glycans on these essential accessory subunits of oligomeric P-type ATPases are dispensable for proper folding, membrane stabilization of the alpha-subunit and transport function itself. Glycosylation is rather important for other cellular functions not relevant in the oocyte expression system, such as intercellular interactions or basolateral versus apical targeting in polarized cells, as demonstrated in other expression systems.     

Rasip1 is required for endothelial cell motility, angiogenesis and vessel formation

Ras proteins are small GTPases that regulate cellular growth and differentiation. Components of the Ras signaling pathway have been shown to be important during embryonic vasculogenesis and angiogenesis. Here, we report that Rasip1, which encodes a novel Ras-interacting protein, is strongly expressed in vascular endothelial cells throughout development, in both mouse and frog. Similar to the well-characterized vascular markers VEGFR2 and PECAM, Rasip1 is specifically expressed in angioblasts prior to vessel formation, in the initial embryonic vascular plexus, in the growing blood vessels during angiogenesis and in the endothelium of mature blood vessels into the postnatal period. Rasip1 expression is undetectable in VEGFR2 null embryos, which lack endothelial cells, suggesting that Rasip1 is endothelial specific. siRNA-mediated reduction of Rasip1 severely impairs angiogenesis and motility in endothelial cell cultures, and morpholino knockdown experiments in frog embryos demonstrate that Rasip1 is required for embryonic vessel formation in vivo. Together, these data identify Rasip1 as a novel endothelial factor that plays an essential role in vascular development.     

TRPM7 is required for ovarian cancer cell growth,migration and invasion

Our previous study demonstrated that the melastatin-related transient receptor potential channel 7 (TRPM7) was highly expressed in ovarian carcinomas and its overexpression was significantly associated with poor prognosis in ovarian cancer patients. However, the function of TRPM7 in ovarian cancer is mostly unknown. In this study, we examined the roles of TRPM7 in ovarian cancer cell proliferation, migration and invasion. We found that short hairpin RNA interference-mediated silence of TRPM7 significantly inhibited cell proliferation, colony formation, migration and invasion in multiple ovarian cancer cell lines. Mechanistic investigation revealed that silence of TRPM7 decreased phosphorylation levels of Akt, Src and p38 and increased filamentous actin and focal adhesion number in ovarian cancer cells. Thus, our results suggest that TRPM7 is required for proliferation, migration and invasion of ovarian cancer cells through regulating multiple signaling transduction pathways and the formation of focal adhesions.     

Rab11 is required for epithelial cell viability, terminal differentiation, and suppression of tumor-like growth in the Drosophila egg chamber

Background

The Drosophila egg chamber provides an excellent system in which to study the specification and differentiation of epithelial cell fates because all of the steps, starting with the division of the corresponding stem cells, called follicle stem cells, have been well described and occur many times over in a single ovary.

Methodology/Principal Findings

Here we investigate the role of the small Rab11 GTPase in follicle stem cells (FSCs) and in their differentiating daughters, which include main body epithelial cells, stalk cells and polar cells. We show that rab11-null FSCs maintain their ability to self renew, even though previous studies have shown that FSC self renewal is dependent on maintenance of E-cadherin-based intercellular junctions, which in many cell types, including Drosophila germline stem cells, requires Rab11. We also show that rab11-null FSCs give rise to normal numbers of cells that enter polar, stalk, and epithelial cell differentiation pathways, but that none of the cells complete their differentiation programs and that the epithelial cells undergo premature programmed cell death. Finally we show, through the induction of rab11-null clones at later points in the differentiation program, that Rab11 suppresses tumor-like growth of epithelial cells. Thus, rab11-null epithelial cells arrest differentiation early, assume an aberrant cell morphology, delaminate from the epithelium, and invade the neighboring germline cyst. These phenotypes are associated with defects in E-cadherin localization and a general loss of cell polarity.

Conclusions/Significance

While previous studies have revealed tumor suppressor or tumor suppressor-like activity for regulators of endocytosis, our study is the first to identify such activity for regulators of endocytic recycling. Our studies also support the recently emerging view that distinct mechanisms regulate junction stability and plasticity in different tissues.     

The functional role of the beta-subunit in the maturation and intracellular transport of Na,K-ATPase.

The minimal functional enzyme unit of Na,K-ATPase consists of an alpha-beta complex. The alpha-subunit bears all functional domains of the enzyme and so far a regulatory role for the beta-subunit in the catalytic cycle has not been established. On the other hand, increasing experimental evidence suggests that the beta-subunit is an indispensable element for the structural and functional maturation of the enzyme as well as its intracellular transport to the plasma membrane. This brief review summarizes the experimental data supporting the hypothesis that assembly of the beta-subunit is needed for the alpha-subunit to acquire the correct, stable configuration necessary for the acquisition of functional properties and its exit from the ER.     

The transmembrane segment of the human Na,K-ATPase beta-subunit acts as the membrane incorporation signal

The human Na,K-ATPase beta-subunit is anchored to the membrane by a single stretch of 28 hydrophobic amino acids; the hydrophilic amino terminus faces the cytoplasm and the carboxyl terminus is exoplasmic. Glycosylation and insertion of the Na,K-ATPase beta-subunit into the endoplasmic reticulum membrane are shown to be co-translational and SRP-dependent. The hydrophilic amino terminus is not required for the membrane insertion. The membrane-anchor domain is necessary for membrane insertion, and a 16 amino acid stretch has been identified as an element sufficient for the insertion.     

Oligomerization of the Na,K-ATPase in cell membranes

The higher order oligomeric state of the Na,K-ATPase alphabeta heterodimer in cell membranes is the subject of controversy. We have utilized the baculovirus-infected insect cell system to express Na,K-ATPase with alpha-subunits bearing either His(6) or FLAG epitopes at the carboxyl terminus. Each of these constructs produced functional Na,K-ATPase alphabeta heterodimers that were delivered to the plasma membrane (PM). Cells were simultaneously co-infected with viruses encoding alpha-His/beta and alpha-FLAG/beta Na,K-ATPases. Co-immunoprecipitation of the His-tagged alpha-subunit in the endoplasmic reticulum (ER) and PM fractions of co-infected cells by the anti-FLAG antibody demonstrates that protein-protein associations exist between these heterodimers. This suggests the Na,K-ATPase is present in cell membranes in an oligomeric state of at least (alphabeta)(2) composition. Deletion of 256 amino acid residues from the central cytoplasmic loop of the alpha-subunit results in the deletion alpha-4,5-loop-less (alpha-4,5LL), which associates with beta but is confined to the ER. Co-immunoprecipitation demonstrates that when this inactive alpha-4,5LL/beta heterodimer is co-expressed with wild-type alphabeta, oligomers of wild-type alphabeta and alpha-4,5LL/beta form in the ER, but the alpha-4,5LL mutant remains retained in the ER, and the wild-type protein is still delivered to the PM. We conclude that the Na,K-ATPase is present as oligomers of the monomeric alphabeta heterodimer in native cell membranes.     

Tissue-specific expression of Na,K-ATPase beta-subunit. Does beta 2 expression correlate with tumorigenesis?

We have found a substantial decrease in the level of Na,K-ATPase beta 2-subunit mRNA in xenografts of human renal, lung hepatocellular carcinomas in nude mice as compared with corresponding normal tissues, as well as in the neuroblastoma cell line as compared with the neuron primary cell culture. The level of beta 1 mRNA is decreased in kidney and lung tumor cells, but is unchanged in hepatocellular carcinoma. In the neuroblastoma cell line the level of beta 1 subunit mRNA was found to be higher then in neuron primary cell culture. The level of alpha 1 mRNA in investigated tumors was the same as in normal tissues. These results may give evidence of the involvement of beta 2-subunit in the process of tumorigenesis as was shown for some other adhesion molecules.     

Uncoupled Na+-efflux on reconstituted shark Na,K-ATPase is electrogenic

In liposomes with reconstituted shark Na,K-ATPase produced to contain sucrose addition of external Na+ and ATP induce an uncoupled Na+-efflux on inside-out oriented pumps which can be inhibited by digitoxigenin. This flux mode is found to be electrogenic and accompanied by hydrolysis of ATP. The coupling ratio of Nacyt transported per ATP split is 3:1 measured as the initial rate of rise in transmembrane potential and initial rate of liberated Pi.     

A novel Dictyostelium RasGEF is required for normal endocytosis, cell motility and multicellular development

BACKGROUND: Dictyostelium possesses a surprisingly large number of Ras proteins and little is known about their activators, the guanine nucleotide exchange factors (GEFs). It is also unclear, in Dictyostelium or in higher eukaryotes, whether Ras pathways are linear, with each Ras controlled by its own GEF, or networked, with multiple GEFs acting on multiple Ras proteins. RESULTS: We have identified the Dictyostelium gene that encodes RasGEFB, a protein with homology to known RasGEFs such as the Son-of-sevenless (Sos) protein. Dictyostelium cells in which the gene for RasGEFB was disrupted moved unusually rapidly, but lost the ability to perform macropinocytosis and therefore to grow in liquid medium. Crowns, the sites of macropinocytosis, were replaced by polarised lamellipodia. Mutant cells were also profoundly defective in early development, although they eventually formed tiny but normally proportioned fruiting bodies. This defect correlated with loss of discoidin Igamma mRNA, a starvation-induced gene, although other genes required for development were expressed normally or even precociously. RasGEFB was able to rescue a Saccharomyces CDC25 mutant, indicating that it is a genuine GEF for Ras proteins. CONCLUSIONS: RasGEFB appears to be the principal activator of the RasS protein, which regulates macropinocytosis and cell speed, but it also appears to regulate one or more other Ras proteins.