Identified and potential internalization signals involved in trafficking and regulation of Na+/K+ ATPase activity

The sodium–potassium pump (NKA) or Na+/K+ ATPase consumes around 30–40% of the total energy expenditure of the animal cell on the generation of the sodium and potassium electrochemical gradients that regulate various electrolyte and nutrient transport processes. The vital role of this protein entails proper spatial and temporal regulation of its activity through modulatory mechanisms involving its expression, localization, enzymatic activity, and protein–protein interactions. The residence of the NKA at the plasma membrane is compulsory for its action as an antiporter. Despite the huge body of literature reporting on its trafficking between the cell membrane and intracellular compartments, the mechanisms controlling the trafficking process are by far the least understood. Among the molecular determinants of the plasma membrane proteins trafficking are intrinsic sequence-based endocytic motifs. In this review, we (i) summarize previous reports linking the regulation of Na+/K+ ATPase trafficking and/or plasma membrane residence to its activity, with particular emphasis on the endocytic signals in the Na+/K+ ATPase alpha-subunit, (ii) map additional potential internalization signals within Na+/K+ ATPase catalytic alpha-subunit, based on canonical and noncanonical endocytic motifs reported in the literature, (iii) pinpoint known and potential phosphorylation sites associated with NKA trafficking, (iv) highlight our recent studies on Na+/K+ ATPase trafficking and PGE2-mediated Na+/K+ ATPase modulation in intestine, liver, and kidney cells.


Introduction
The Na + /K + ATPase (NKA) or sodium-potassium (Na + / K + ) pump is ubiquitous in all animal cells but has different levels of expression [1].It belongs to the family of P-type (phospho-intermediate type) ATPases, a family of proteins that have been evolutionarily conserved and present in both prokaryotes and eukaryotes [1][2][3].Members of this family have a regulatory beta-subunit and a catalytic alpha-subunit with homologous catalytic sites.The alpha-subunit is membrane-bound and contains the binding sites for ATP, selected cation (s), and specific Na + /K + ATPase inhibitors [1,2].The Na + /K + ATPase consumes around 25% of the energy spent by the cell.It uses the energy derived from the hydrolysis of one ATP molecule to exchange two extracellular K + ions for three cytoplasmic sodium ions [4].The pump thus establishes and maintains a transmembrane sodium gradient used in the regulation of cell volume, pH, and in driving secondary active transport processes [5].The gradient plays also a key role in the generation of action potentials in excitable tissues [6].
The central role of the Na + /K + ATPase as an antiporter renders it essential for various cellular activities [7], and its presence in the membrane is mandatory for the accomplishment of the transport processes.The Na + /K + ATPase is known to traffic between the cell membrane and intracellular stores, thus the regulation of its spatial and temporal distribution in the cell is crucial for the maintenance of homeostasis.Although previous studies reported trafficking as a mechanism of NKA regulation in several cell types [8][9][10][11][12][13], the molecular determinants of this process are still unclear.The pump residence in the plasma membrane depends on the rate of endocytosis and exocytosis.Internalization of the Na + /K + ATPase was shown to be induced by various stimuli including hormones [14,15], cardiac glycosides [16,17], and anticancer agents [18,19], and was shown to occur under pathological conditions such as hypoxia [10,20], hypercapnia [21], and sepsis [22].
The NKA translocation appears to involve post-translational modifications and is directed by intrinsic sequencebased signal motifs.In this review, we discuss current knowledge on NKA trafficking.We also present a new analysis of the pump endocytic regulation, through screening of potential endocytic motifs and phosphorylation motifs in its catalytic alpha-subunit.

Structure of the Na + /K + ATPase
The Na + /K + ATPase is a heteromeric protein composed of an alpha-and beta (β)-subunit in a 1:1 stoichiometry.A third gamma (γ)-subunit may be present [23] in some cells (Fig. 1).
The alpha (α)-subunit through its ATPase activity provides the energy needed for the uphill transport of sodium and potassium.The movement of ions from one side of the membrane to the other occurs through a conformational change of the enzyme, which cycles between two conformations: E1 and E2.In the E1 state, three sodium ions bind and the pump is phosphorylated.The resulting conformational change causes a change into the E2 state, in which sodium is released and two potassium ions attach.The phosphate group is then liberated and the pump reverts to the E1 state [7,25].
The alpha-subunit, which performs the catalytic functions, has a molecular weight of around 110 kDa.It consists of approximately 1000 amino acid residues [26], arranged in 10 transmembrane domains (M1-M10), with 5 extracellular loops and 4 intracellular ones.Both the Nand C-termini reside in the cytosol [27].This subunit has three cytoplasmic functional domains, the A domain (actuator), the N domain (nucleotide-binding), which contains the ATP binding site, and the P domain (phosphorylation domain) a substrate of the ATPase.The A domain acts as a phosphatase and consists of the segment (Gly 1 -Gln 88 ) at the N-terminal end together with the loop between M2 and M3 [28].It has been demonstrated that ATP binds at the salt bridge that spans between glutamic acid (E223) of the A domain and arginine (R 551 ) on the N domain [29].
An aspartate residue (D 376 ) in the P domain is phosphorylated and dephosphorylated during each cycle.
So far, four distinct isoforms of the alpha-subunits have been identified (alpha1, alpha2, alpha3, and alpha4).Although distinct subpopulations of alpha (alpha1 and alpha2) can exist within different regions of a single cell, the different isoforms generally show tissue or cell specificity [30].
Alpha1 is ubiquitously expressed and is the major housekeeping isoform that exists in most tissues.It is largely predominant in kidneys and most epithelia [26].The isoforms alpha2 and alpha3 are found in neuronal tissue, skeletal muscle, and cardiac myocytes; alpha4 is expressed in the testis and modulates sperm motility [31].
Being a type II membrane protein, the beta-subunit of the NKA crosses the membrane only once and has a short cytosolic amino terminus and a large extracellular C -terminal end containing 6 cysteine residues forming three S-S bridges.Three sites (N 158 , N 193 , N 265 ) of attachments for N-glycosylated oligosaccharide structures are also present in the extracellular domain [26,32].The human beta-subunit is composed of 303 amino acids with an overall molecular mass of ~35-55 kDa, depending on the glycosylation status [33].Four isoforms have been detected so far: beta1, beta2, beta3, and beta(m).Beta1 is the most common isoform, beta2 was found in the nervous tissue while beta3 was identified in the testis and brain, and beta(m) in the heart and skeletal muscle [3].Extracellular parts of the beta-subunit interact with the alpha-subunit at the level of the fourth extracellular loop.This interaction is crucial for accomplishing a normal ion transport, and alpha-subunit expressed alone showed almost no significant ATPase activity [34][35][36].The betasubunit plays an important role in the trafficking of the alpha-subunit, especially in its transport from the ER to the plasma membrane as well as in its correct insertion into the membrane [37,38].It helps also in maintaining the polarization of epithelial cells and prevents motility by dimerizing to a neighboring beta-subunit forming beta-beta bridges that enhance adhesion between cells [39].Some evidence suggests that the beta-subunit plays also a role in ATP hydrolysis, as well as in ion transport and ouabain binding [40,41].
In addition to the alpha-and beta-subunits, a gamma (γ)-subunit, a small-membrane protein of the FXYD family, associates specifically with the NKA in a tissue-specific manner [42,43].
The FXYD protein families contains 7 members (FXYD1-7) that share all an FXYD motif (F: phenylalanine, X: any amino acid, Y: tyrosine, and D: aspartate) in the amino terminal end and two conserved glycine residues and one serine residue in the transmembrane domain.They are single span transmembrane domain proteins with the FXYD motif present on the extracellular amino terminal end [44].The subunit is a 7-11 kDa type I protein that spans the membrane once and possesses an FXYD motif in the N-terminal extracellular end.Although it interacts with the NKA, it is not an essential component of the enzyme complex [43].It does not play a role in the expression of the ATPase but can modify its transport properties by changing its affinity for its ion substrates and even for ATP, playing thus a fine-tuning role in the modulation of the pump's activity [30,44,45].
The NKA subunits are synthesized independently in the ER where they are subsequently assembled [26].Having different isoforms of each subunit allows for different combinations and variations in the pump between different tissues [39].

Overview of trafficking pathways
The plasma membrane is a highly dynamic structure that undergoes continuous remodeling through multiple exocytosis and retrieval models [46].These processes determine the spatiotemporal orchestration of membrane proteins and thus contribute to the regulation of many crucial biological functions associated with these proteins.In this section, we will present a brief summary of the exocytic and endocytic mechanisms and highlight the role of specific signals in these pathways.

Exocytosis mechanisms
The exocytosis pathways are regulated by vesicular formation, transport through ER-Golgi, membrane targeting, docking, and vesicle fusion with the target membrane [47].Coat proteins that are recruited to donor membranes are crucial players in this process.In the exocytotic pathway, coat protein complex I (COPI) and II(COPII), and clathrin, are the major coat proteins that facilitate the formation of most of the intracellular trafficking vesicles [48].COPII is known to mediate anterograde transport from ER to Golgi through assembly of COPII complex containing five cytosolic proteins: the small GTPase Sar1p, the Sec23p/24p complex, and the Sec13p/Sec31p complex [49,50].Sec24p is crucial for coat assembly as well as for cargo selection, mainly binding proteins with specific signals such as the diphenylalanine motif [51].After passing from ER to Golgi, cargo proteins are sorted at the trans-Golgi network (TGN) to be directed spatially to target membranes including apical and basolateral membranes and different endosomal compartments [52].
Clathrin-coated vesicles (CCV) mediate the transport of cargo molecules from the trans-Golgi network (TGN) to the membrane.Formation of CCV is mediated through the recruitment of several adaptor proteins and cargos proteins containing (D/E)XXXL(L/I) motif and LL motifs in the presence of Arf1-GTP and phosphatidylinositol-4-monophosphate (PI4P) [53,54].

Endocytosis pathways
For most integral membrane proteins, the availability at the plasma membrane is an output of regulation of the exocytosis process and retrieval process.Several endocytic pathways have been identified with clathrin-mediated endocytosis (CME) being the most understood.Accordingly, we will briefly summarize these processes of clathrin-dependent and -independent endocytosis.Clathrin-dependent endocytosis is initiated through the recruitment of AP-2 adapter proteins to plasma membrane-enriched phosphatidylinositol lipid, PI(4,5)P2, followed by binding of additional scaffolding proteins including FCH domain only (FCHO) proteins, EGFR pathway substrate 15 (EPS15), and intersectins [55].The binding of AP-2 is mediated through endocytic motifs present in cytoplasmic tails of cargo proteins such as YXXΦ, LL motifs, or (D/E)XXXL(L/I) motif [48].Another crucial step in the detachment of the clathrin-coated vesicles from the plasma membrane is mediated by the GTPase protein dynamin [56].
Caveolin-dependent endocytosis is another well-studied pathway in which caveolins are the main structural proteins in the formation of invaginations, known as caveolae, at the plasma membrane [57].In this pathway, cholesterol-rich domain functions as a scaffolding domain for binding caveolins.In addition to caveolin complexes, several proteins were identified to be important in caveolae formation such as ATPase EHD2 [58].Similar to CME, vesicular fission is dependent on dynamin.Like clathrin endocytic motifs, caveolin binding motifs have been reported.They are hydrophobic and rich in aromatic residues [59].

Identification of potential trafficking motifs in the alpha-subunit of the Na + /K + ATPase
Na + /K + ATPase plasma membrane residence and activity are modulated by molecular mechanisms regulating exocytosis and endocytosis of the pump.Na + /K + ATPase transition from ER to Golgi is in the form of Na + /K + ATPase alpha and beta-assembled complexes packaged through COPII vesicles with assistance of spectrin and ankyrin complexes [60].In the ER, both alpha-and beta-subunits undergo cotranslation and post-translation maturation through interaction with ER chaperones [61][62][63].During ER maturation, all isoforms of beta-subunit are N-glycosylated.The role of these glycosylations in the assembly of alpha and beta-complex and exit from ER is dependent on the type of isoform.While N-glycans were shown not to be involved in Na + /K + ATPase alpha1: beta1 assembly, they were found to play an important role in the assembly of the alpha-subunit with the beta2-subunit [64].Moreover, three disulfide bonds in the beta-subunit were demonstrated to differentially affect the assembly of alpha-beta complexes [33,65].Upon exit from Golgi, vesicles containing membrane proteins, including Na + /K + ATPase, are targeted directly to plasma membrane through constitutive exocytosis, while other vesicles are targeted to plasma membrane only upon receiving a specific signal via regulated exocytosis.
Regulated exocytosis of Na + /K + ATPase to the basolateral membrane has been shown to be initiated by dopamine in alveolar epithelial cells as well as upon activation of β-adrenergic receptors [66] and involvement of actin cytoskeleton [67,68].Hundal et al. (1992) demonstrated regulated translocation of isoform-specific Na + /K + ATPase subunits from different intracellular compartments to the cell surface [69].
The same group followed up with an interesting study depicting insulin-dependent translocation in kidney cells using an exofacially epitope-tagged Na + /K + ATPase alpha1subunit [70].
On the other hand, several studies shed light on the different endocytotic processes that regulate the abundance of the pump at plasma membrane.In the Na + /K + ATPase alpha-subunit, the caveolin binding motif (CBM) was identified in the proximity of the first transmembrane helix.Interestingly, the study by Wang et al. (2020) utilizing a point mutant of the CBM alpha1-subunit suggested that this CBM is not essential for the ATPase activity or plasma membrane residence [71].Several other clathrin-independent pathways have been described [72] that depend on small GTPases such as dynamin, RhoA, Rac1, and Arf6.An additional endocytosis mechanism was dependent on flotillins, a group of proteins that are tightly associated with the inner leaflet of the plasma membrane [73].Interestingly, studies demonstrated an association of flotillins with dynamin-dependent and -independent endocytosis [73,74].Reported also was the binding of flotillins with dileucine sorting signal in the trafficking of some proteins [75].The association of different isoforms of the alpha-subunit with specific trafficking mechanisms is summarized in Table 1.
Previous works revealed a connection between NKA activity and its plasma membrane abundance [76][77][78][79][80][81][82][83][84].Chibalin et al. [76] using sucrose density gradient fractionation showed a decrease in the expression of the Na + /K + ATPase on the surface of proximal convoluted tubular cells after dopamine treatment.The authors elegantly showed enhanced localization of NKA alpha-subunits in clathrincoated vesicles, early, and late endosomes isolated from proximal convoluted tubular cells exposed to dopamine, as compared with untreated cells.The specificity of NKA recycling was supported by their data showing no change in the cellular distribution of glucose transporter type 4 and mannose 6-phosphate receptor, corroborating the assumption that changes in Na + /K + ATPase are specific and not due to dopamine-induced bulk transport.Dopamine-mediated endocytosis was blocked in the presence of nocodazole, a microtubule depolymerizing drug, without disrupting the incorporation of Na + /K + ATPase into clathrin-coated vesicles, suggesting a differential regulation of endocytosis and vesicular incorporation.
The specific recycling of plasma membrane proteins is known to be due to the presence of short-sequence motifs within their cytoplasmic regions allowing interaction with intracellular trafficking machinery [85].Several endocytic motifs in clathrin-mediated internalization have been identified in a large number of plasma membrane proteins, including the canonical tyrosine-based motif, dileucine-based motif, NPxY (x is any amino acid), and several other noncanonical motifs [85,86].

AP-2
Several works focused on the identification of intrinsic domains within the Na+/K+ ATPase alpha-subunit allowing its binding to proteins of the endocytic machinery like adaptor protein-2 (AP-2).Through a series of site-directed mutagenesis using rat GFP-tagged Na+/K+ ATPase, Doné et al. [87] tested several potential endocytic motifs that were known in literature to bind AP-2 and revealed, through sequence analysis, the presence of several intracellular sites in the NKA alpha-subunit sequence for possible interaction with AP-2, including Y 50 KRH at the N-terminus, IVVY 255 in the loop between TM 2 and TM3, and Y 469 IKT, Y 537 LEL, and ILRY 679 in the loop between TM4 and TM5.The approach of the authors was based on the evidence for known binding motifs for two clathrin-associated protein complexes, specifically AP-2 -chain binding to a consensus NPXY or YppØ motif [81,88], where Y designates tyrosine, X stands for any amino acid, Ø stands for hydrophobic amino acid, and p stands for positively charged residues.Testing the role of some identified potential endocytic motifs in dopamine-induced inhibition of the Na + /K + ATPase in OK cells showed partial reversion of the inhibitory response only in cells with Na + /K + ATPase-Y 537 F mutant [78], suggesting that other additional mechanisms for pump internalization may be involved since the reversion was partial and not total.This partial reversion was not associated with changes in protein expression levels and probably occurred via a mechanism that does not affect the protein expression of the pump.The authors confirmed the essential role of this motif through a second mutation of tyrosine 737 to alanine, which completely blocked the inhibitory effect of dopamine on Na+/K+ ATPase activity.Interestingly, this study did not report the changes in the abundance of Na+/ K+ ATPase molecules present in clathrin-coated vesicles isolated from the Y537A mutants despite the absence of immunoprecipitation with AP-2.The authors suggested that these resident NKA molecules in clathrin-coated vesicles originated from recycling endosomes in their shuttling to the Alpha1 and alpha2 Ventricular myocytes Short-term α-adrenoreceptor (α-AR) activation

Reduced expression of alpha1
Increased α 1 -isoform expression in early endosomes Alpha1 and alpha2 Cell lines derived from LLC-PK1 cells

Membrane cholesterol reduction
Reduced expression Cholesterol, Src, and Caveolin-1 dependent [84] plasma membrane.It also remains possible that an AP-2-independent recycling is involved in this process.In addition, Bonifacino and Traub [88] investigated the potential role of additional two dileucine-based motifs that were reported to bind to adaptor complexes of endocytic machinery including AP and other modulatory complexes [89].This study did not present the evidence of changes in Na+/K+ ATPase at the plasma membrane in cells expressing L 499 A or L 554 A mutants but showed that these mutations did not alter the inhibitory action of dopamine or the stimulatory effect of phorbol esters, suggesting no major role for the two motifs in dopamine-mediated decrease in Na+/K+ ATPase activity.

Arrestin and PP2A
Kimura et al. revealed the role of arrestin in the trafficking of the Na + /K + ATPase [90].They showed through coimmunoprecipitation studies using mouse kidney tissues in situ binding of arrestins 2 and 3 and spinophilin to the Na + /K + ATPase alpha-subunit and immunolocalization of the NKA and arrestin in intracellular compartments in COS cells.There was partial redistribution of NKA to the plasma membrane when arrestin 2 was co-expressed with spinophilin, suggesting an antagonistic role for spinophilin and arrestins in Na+/K+ ATPase internalization.An earlier study showed that spinophilin binds to TGN38 which is involved in recycling between the trans-Golgi network (TGN) and the plasma membrane [91].Spinophilin is also known to bind to protein phosphatase 1 (PP-1), one of the phosphatases that regulate the activity of the Na + /K + ATPase [92].
The direct binding of arrestin 2 to the large cytoplasmic loop of the Na+/K+ ATPase was shown through the pulldown experiments of GST fused arrestin 2 and GST-NKA-large cytoplasmic loop fusion [90].Using a series of truncated mutants, the authors showed that the sequence of the first 53 amino acids of the large cytosolic loop between TM4 and TM5 is sufficient for binding to arrestin.However, constructs containing the first 155 and 175 aa of the M4-M5 cytosolic loop showed better bindings to arrestin 2, while the part containing 238 aa of the cytosolic loop showed lower binding capacity as compared to the 175, suggesting the presence within this segment of a binding site for spinophilin, another adaptor that was shown to inhibit arrestin 2 binding.Lecuona et al. [92] provided evidence for the direct interaction of PP2A with the first 90 aa of the N-terminus of the NKA ɑ-subunit.A few years later, Kimura et al. based on these findings pursued work on the regulation of Na + / K + ATPase through arrestin's binding.When truncated constructs of the NKA cytoplasmic loop 53 Δ, 155 Δ, and 175 Δ were used, PP2A binding was stronger than the binding to the full Na + /K + -ATPase cytoplasmic loop [90].It was concluded that PP2A and arrestin compete for binding to the large cytoplasmic loop of the Na + /K + -ATPase.

Clathrin and caveolin
Studies from Joseph I. Shapiro's lab reported that ouabain induced, in LLC-PK1, Na + /K + ATPase internalization through clathrin-dependent endocytosis through enhancing association between the Na + /K + ATPase, adaptor protein-2 (AP-2), and clathrin [93].In a following study using a similar model, the authors showed that ouabain-induced internalization is dependent on caveolin-1 [94].By studying plasma membrane Na + /K + ATPase through confocal imaging, and biochemically through biotinylation, Liu et al. showed that ouabain could not induce internalization of the pump [94] or the association of its alpha1-subunit with clathrin heavy chain and AP-2, when caveolin-1 was knocked down, suggesting an involvement of caveolin in the clathrin-dependent pathway [95][96][97].However, whether caveolin is involved in the clathrin-independent endocytosis was not investigated.In the same study, Liu et al. [93] also reported reduced accumulation of Na + /K + ATPase in early endosomes in the presence of methyl-beta-cyclodextrin, suggesting an important role for cholesterol in the ouabain-induced Na + /K + ATPase internalization.A different study assessing NKA internalization in MDCK cells after energy depletion showed that the process is independent of caveolin-1 but dependent on AS160, a Rab GTPase-activating protein previously shown to play a role in the translocation of glucose transporter type 4 (GLUT4) [98].Mapping the domain of interaction between AS 160 and Na + /K + ATPase alpha-subunit, identified the cytoplasmic region between transmembrane domains 4 and 5 [99].Morton et al. [100] reported that rat Na + /K + ATPase alpha binds to COP-1 through a dibasic motif at position 54.This binding promotes the retrieval of the subunit in the ER.Mutating the dibasic motif enhanced the expression of mutant subunits at the plasma membrane, as shown by biotinylation studies and immunofluorescence [100].

Identification of additional endocytic motifs
In an attempt to identify the additional and still unrecognized potential internalization signals within Na + /K + ATPase alpha, (Fig. 2), we searched human Na + /K + ATPase alphasubunit for canonical and noncanonical endocytic motifs based on the reported signals in the literature [78,101,102].
Interestingly, studies by Pierre et al. group demonstrated increase in basal surface expression of Na + /K + ATPase alpha1-subunits in cells expressing dileucine motif mutant L 499 V corresponding to the [D/E]XXXL[L/I] at position (500-505) identified in our screens [81].
Finally, driven by previous studies on the trafficking of human store-operated Ca2 + channel, Orai1 [103], we were curious to screen the NKA alpha-subunit for homologous sequences that can bind to chaperonin-containing TCP-1 (CCT).This chaperone is well known to be involved in the folding of several newly synthesized proteins [104] and was previously shown to regulate the trafficking of lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1 receptor) by binding to the cytoplasmic domain of the LOX-1 [105].CCT was reported as a novel regulator of Orai1 internalization through binding to its intracellular loop, and inhibition of CCT-Orai1 interaction increases Orai1 residence in the plasma membrane [103].Alignment of the LOX-1 receptor cytoplasmic domain sequence (MTFDDLKIQTVKDQPDEKSNGK-KAKGLQFLYSPGGKG) with human NKA alpha-subunit shows significant homology at one position within the Na + / K + ATPase alpha-subunit N-terminus (residues 32 to 37), and at two positions within cytoplasmic loop between transmembrane 3 and 4 (525-KEQPLDEE, 607-RSAGIKV) (Fig. 3).It remains possible that these three sequences serve as binding sites for CCT and consequently modulate the trafficking of the pump.A fourth similarity was detected at residues (998-1004, LLIFVYD.However, this sequence falls within a transmembrane domain and is unlikely to play a role in Na + /K + ATPase alpha-subunit recycling (Fig. 4).

Involvement of phosphorylation in the trafficking of the Na + /K + ATPase alpha-subunit
Phosphorylation and dephosphorylation are the main processes regulating cellular activities including protein trafficking.Phosphorylation events affecting Na + /K + ATPase structure and function have been extensively reported in the literature [106].Phosphorylation may induce changes in NKA activity, binding of the pump's subunits [107,108], interactions with signaling proteins, and the recycling of the NKA alpha-subunit between the plasma membrane and intracellular compartments [33,106].However, the link between direct phosphorylation of NKA and its trafficking has been limited to few studies.In this section, we review the current knowledge derived from these works.We also propose distinct phosphorylation sites that may orchestrate NKA activity and/or trafficking, by presenting detected (det) and potential (pot) serine/threonine and tyrosine phosphorylation sites in NKA alpha-subunit.

PKC phosphorylation sites and NKA internalization
Protein kinase C (PKC) is one of the earliest kinases known to negatively or positively modulate the Na + /K + ATPase through a direct phosphorylation of its alpha-subunit [109].Studies on rodent kidney cells have identified serine 11 and serine 18, located on the N-terminus of the alpha-subunit, as PKC phosphorylation sites [109].Interestingly, while serine 11 is conserved in almost all mammalian kidney NKA alpha-subunits, serine 18 is missing in several mammalian species [110].In proximal convoluted tubule cells, Chibalin et al. [14] demonstrated that the dopamine-induced internalization of the NKA alpha-subunit did not occur in the simultaneous presence of pharmacological inhibitors of PKC.Soon after this report, the same group used a truncated mutant of rat NKA alpha-subunit missing serine 11 and 18 and demonstrated that phosphorylation of Ser18 but not Ser-11 initiates internalization.Moreover, the mutant showed decreased abundance in clathrin-coated vesicles and early endosomes [78].
Another discovered protein kinase C phosphorylation site is serine 938 found within the cytoplasmic loop between the transmembrane regions M8 and M9 [111].This residue is the same as serine 943 in full protein, and contrary to serine18, it is conserved in all mammalian Na + /K + ATPase α-subunits.In OK cells exposed to the hormone angiotensin (ANG) II, Massey et al. [112] demonstrated that serine 938 phosphorylation stimulates the pump's activity and increased its trafficking to the plasma membrane as revealed by the amount of biotinylated wild-type Na + /K + ATPase alpha1 or S938A mutant in the presence or absence of ANG II [112].

Adaptor proteins phosphorylation and NKA internalization
Other studies demonstrated an involvement of phosphorylation events in the dopamine-mediated NKA internalization through clathrin-dependent endocytosis.However, phosphorylation was linked to adaptor proteins implicated in the pump's trafficking rather than NKA directly.On the other hand, AP-2 binding to tyrosine-based motif (Tyr-537LEL) located in the NKA alpha-subunit was shown to be vital for NKA endocytosis in response to dopamine or ROS [87].In 2006, Bertorello's laboratory reported that phosphorylation of adaptor protein AP-2 mu2 subunit mediates NKA internalization in OK and lung alveolar epithelial cells when treated with dopamine or ROS [113].

Identification of potential phosphorylation sites inducing trafficking
In an attempt to identify the critical residues that can be phosphorylated and consequently modulate NKA activity and/or trafficking, we highlight the reported and potential phosphorylation sites within intracellular loops of the pump which could be of interest in future studies.We searched the sequence of alpha-subunit for serine/threonine protein kinase [114] and tyrosine kinase consensus sequences [115,116] and highlighted the minimal consensus motifs for serine/ threonine kinases presented as serine or threonine at position 0 and sequence similarity at position − 1, − 2, and + 1.For tyrosine kinases, we highlight the sites with [DE]-x(3)-Y.Future studies will identify which of those potential sites are bona fide phosphorylation sites and which can be directly involved in NKA trafficking.

External signals and Na + /K. + ATPase trafficking: the case of PGE2
Prostaglandin E2 (PGE2), produced by cyclooxygenase 2 (COX2) conversion of arachidonic acid, is one of the most important biologically active lipid mediators found throughout the gastrointestinal tract [117] and kidneys [118].Works conducted on several animal models showed that PGE2 in the gastrointestinal (GI) tract acts through multiple prostaglandin E2 (EP) receptors and mediates important physiological processes such as gastric acid secretion, GI motility, and mucus secretion [117].Additionally, increased levels of PGE2 detected in the GI mucosal membranes of patients and in experimental models of inflammatory bowel disease have been associated with improved epithelial repair [118].In kidneys, PGE2 can exert protective or harmful effects through different target receptors [119].Moreover, PGE2 is also a main regulator of liver function.It induces lipid accumulation in hepatocytes [120] and improves hepatic bile acid synthesis [121].On the other hand, PGE2 exerts hepatoprotective effects against many liver injuries mainly by promoting proliferation or mitigating hepatocyte injury [122].
PGE2 is an important regulator of Na + /K + ATPase activity in numerous cell types, including intestinal, kidney, and liver cells.However, limited evidence is available on the effect of PGE2 on the Na + /K + ATPase trafficking.

PGE2 and NKA trafficking in intestine
Nepal et al. [123] showed that in the small intestine, PGE2 reduces the Na + /K + ATPase activity, In addition, the authors demonstrated by Western blot and immunofluorescent imaging of fixed cells that this inhibition is due to transcriptional repression and consequently to a decrease in the alpha1 and beta1-subunits at the plasma membrane.However, in this study, the decrease at the plasma membrane was associated with a decrease in the overall signal of Na + /K + ATPase, suggesting that PGE2 causes a reduction in total Na + /K + ATPase expression.Similarly, our group showed in Caco-2 cells that the pump's activity was reduced by PGE2 and that this reduction was associated with lower protein levels of the Na + /K + ATPase in both the cell homogenate and the membrane fraction [124], suggesting reduced localization of the pump at the plasma membrane.However, one interpretation of the results obtained could be that PGE2 regulates directly the trafficking and the translocation of the Na + /K + ATPase to intracellular compartments where it is degraded; another interpretation could be that PGE2 reduces the overall expression of the pump.Consistent with these results, we demonstrated in a separate study using Caco-2 cells that FTY720P, an analogue of sphingosine 1-phosphate, mediates the inhibition of Na + /K + ATPase activity by activating S1PR2 and inducing PGE2 release [125].Recently, we showed, using confocal imaging of live Caco-2 cells co-expressing GFP-tagged alpha Na + /K + ATPase and mcherry-tagged membrane marker, that FTY720P-mediated inhibition of the NKA pump results from a decrease in its abundance in the plasma membrane [13].In addition to PGE2, FTY720P was found to induce NO release in these cells.Previous studies have shown that the vesicle fusing ATPase, N-ethylmaleimide sensitive factor (NSF) is a target for NO.It is also a major component of the trafficking machinery regulating vesicular targeting which includes, in addition to NSF, soluble NSF attachment receptor proteins (SNAREs), the Sec/Munc family, and the Rab family members [126].Nitric oxide can also decelerate exocytosis by destabilizing the SNARE complex [127] and regulating small GTPases such as Ras and Rac that may lie upstream of the exocytic machinery [128].Finally, by nitrosylation of ryanodine receptors on the sarcoplasmic reticulum (SR), NO can modulate cytosolic calcium levels that are important regulators of exocytosis [129,130].On the other hand, NO is known to accelerate endocytosis by enhancing S-nitrosylation of the GTPase dynamin which is well known to play an important role in the scission of nascent vesicles from the plasma membrane [131].Whether FTY720P-mediated NO release is involved in the decrease in the amount of Na + /K + ATPase at plasma membrane is still not clear.

PGE2 and NKA trafficking in liver cells
Na + + ATPase trafficking is also implicated in PGE2 regulation of the pump's activity in liver cells.We previously showed that PGE2 enhances Na + /K + ATPase activity in HepG2 through activation of EP4, production of cAMP, and PKA activation [12].Our results also reported an important role for calcium released from intracellular stores in the PGE2 effect.Using pharmacological tools and fluorescent imaging, we uncovered the pivotal role of NKA trafficking in the PGE2-induced increase in its activity.Immunoblotting showed, in PGE2-treated cells, a higher expression of Na + /K + ATPase alpha1 in cell membrane fractions but not in the whole cell homogenate, inferring that the increased abundance at the plasma membrane (PM) is not due to the increased protein expression.Interestingly, the stimulatory effect of PGE2 was completely blocked when cells were incubated on ice, demonstrating the dynamic nature of Na + /K + ATPase vesicular transport.The PGE2 enhanced abundance of the pump at PM was also confirmed by confocal imaging of HepG2 cells expressing GFP-tagged Na + / K + ATPase alpha1.Our time-lapse imaging also revealed a PGE2-induced highly dynamic pattern of Na + /K + ATPase vesicles and a higher abundance of the pump at the cell membrane.

PGE2 and NKA trafficking in the kidney
In the kidney, several reports demonstrated modulation of NKA activity by prostaglandins.Although PGE2 is the major prostaglandin synthesized in the kidney [132].
Herman et al. [133] reported in primary cultures of rabbit renal proximal tubule (RPT) cells, stimulation of the pump not only by PGE2 but also by other prostaglandins like PGE1 and PGF2α.The increased activity was reported in both short-term and long-term prostaglandin treatments.Long exposure of RPT cells to PGE1 and PGE2 resulted in an increase in Na + /K + ATPase mRNA and total protein levels.Interestingly, under short-term treatments with PGE2, biotinylation assays of cells cultured on transwell membranes showed an increase in the level of NKA in the basolateral membrane without an increase in the overall level of cellular NKA.These results implicate a role for directional membrane trafficking in the regulation of NKA activity in these cells.
Similar findings were reported in Madin Darby Canine Kidney (MDCK) cells, where long exposures to PGE (4 h), acting via EP1 and EP2, induced an increase in mRNA and protein level of Na + /K + ATPase [134,135].Although these studies demonstrated a role for Ca2 + in the PGE2-induced stimulation of the pump, the impact of calcium on pump trafficking was not studied.Interestingly, an independent work studying the short-term effect of PGE2 in MDCK showed an inhibition of the Na + /K + ATPase, presumably due to impoverished residence at the plasma membrane rather than to changes in Na + /K + ATPase expression [136].
Collectively, prior and current studies suggest that PGE2modulation of the pump's activity in liver, intestine, and kidney cells may be due to an effect on its trafficking and gene expression.However, more studies are required to address the molecular mechanisms linking PGE2-dependent modulation to Na + /K + ATPase trafficking.

Conclusions
The Na + /K + ATPase is a vital enzyme in all types of animal cells.It maintains an essential ion gradient across the plasma membrane needed to drive normal cellular processes and sustain homeostasis.Alterations in the pump's activity have been associated with several disorders including cardiovascular, neurological, kidney, and metabolic diseases [137].Developing therapeutic strategies against these complications requires proper understanding of the processes involved in modulating directly or indirectly the activity of the pump, at both the cellular and molecular levels.Although the NKA pump has been extensively studied, the association between signal transduction pathways and its membrane trafficking has still not been fully elucidated.We tried in this review to focus on reported studies investigating the role of NKA trafficking in the modulation of the pump's activity with an emphasis on the involvement of potential short-sequence endocytic motifs and phosphorylation sites.

Fig. 2
Fig. 2 Known and predicted internalization signals in human sodium-potassium ATPase α-subunit.Motifs were identified based on canonical and noncanonical endocytic motifs reported in literature.The full sequence of the alpha-subunit was searched for tyrosine-based and dileucine-based motifs.The tyrosine-based motifs identified were either classical sequences including classical or nonclassical tyrosine-based motifs.The green squares indicate classical tyrosine-based motifs including YxxL/I and YxxxL/I.Tyrosinelike based motifs such as FxxL/I and FxxxL/I are presented in blue

Table 1
Correlation between the tissue expression of different isoforms of the alpha-subunit with specific trafficking mechanisms