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

Abstract

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.

Keywords: Endocytic motifs; Internalization; K+ ATPase; Na+; PGE2; Phosphorylation; Trafficking.

Fig. 1

Topological representation of human sodium–potassium ATPase alpha, beta, and γ (FXYD)-subunits, using Protter software [24]. Topology model of human Na⁺/K⁺ ATPase alpha-subunit (A), beta-subunit (B), and γ-subunit (C). A: Human Na⁺/K⁺ ATPase alpha-subunit arranged in 10 transmembrane domains. The orange diamonds indicate glutamic acid (E223) and arginine (R551) forming the salt bridge which allows ATP binding. The yellow square indicates aspartate residue (D376) that is phosphorylated and dephosphorylated during each cycle. UniProt protein accession: P05023. Gene: ATP1A1. B: Topology of the human Na⁺/K⁺ ATPase beta-subunit, composed of 303 amino acids. The green squares indicate glycosylation sites, N158, N193, and N265. UniProt protein accession: P05026. Gene: ATP1B1. C: Topology of γ-subunit FXYD motif in the N-terminal extracellular end. UniProt protein accession: P54710. Gene: FXYD2

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 non-classical tyrosine-based motifs. The green squares indicate classical tyrosine-based motifs including YxxL/I and YxxxL/I. Tyrosine-like based motifs such as FxxL/I and FxxxL/I are presented in blue circles (⁷⁶⁹FDNL and ⁷⁵⁵FASI) and orange squares (⁹²FCRQL and ³⁹³FDNQI), respectively. The previously identified motifs at position 537 (⁵³⁷YLEL) is indicated in green squares. Non-classical tyrosine-based motifs are indicated by brick-colored squares (Y778TLTSN and Y824EQ). Dileucine-based motifs with similarity to [DE]xxxL[L/I] (ESALL⁴⁵⁷, EPQHLL⁵⁰⁵, DMILL⁷⁴⁷, EGRLI⁷⁶⁸) and L[L/I] (ENLPIL⁴³⁸, DIL⁶⁸⁰, SSILL⁵¹⁹, ILL⁷⁴⁹, IL⁸⁹⁷) are indicted by red squares

Fig. 3

The homologous sequences of the CCT binding sites and human Na⁺/K⁺ ATPase alpha-subunit are shown. Similarities at three positions within N-terminus (32–37) and intracellular loops of Na + /K + ATPase alpha-subunit at positions 32–37, 525–530, and 607–612. A fourth similarity at position 998–1003 falls within a transmembrane domain

Fig. 4

Reported and potential phosphorylation sites on human Na + /K + ATPase alpha-subunit. Minimal consensus motifs for serine/threonine kinases consist of having serine of threonine at position 0 and sequence similarity at positions − 1, − 2, and + 1(Rust and Thompson, 2011). Previously detected PKC phosphorylation site at serine18, PKC(det), is indicated in orange circles. Another PKC potential site, PKC(pot), is found at position and indicated with brown circles. Potential phosphorylation sites are shown—dark peach color for calcium/calmodulin dependent protein kinase I (CAMK1), violet for cyclin-dependent kinase 2/4/5 (cdk2/4/5), yellow for cyclin-dependent kinase 1 (cdk1), green for extracellular signal-regulated kinase (ERK), blue for protein kinase A (PKA), and red for tyrosine kinase (TK) site