Distinct Role of the N-terminal Tail of the Na,K-ATPase Catalytic Subunit as a Signal Transducer

Abstract

Mounting evidence suggests that the ion pump, Na,KATPase, can, in the presence of ouabain, act as a signal transducer. A prominent binding motif linking the Na,K-ATPase to intracellular signaling effectors has, however, not yet been identified. Here we report that the N-terminal tail of the Na,KATPase catalytic ␣-subunit (␣NT-t) binds directly to the N terminus of the inositol 1,4,5-trisphosphate receptor. Three amino acid residues, LKK, conserved in most species and most ␣-isoforms, are essential for the binding to occur. In wild-type cells, low concentrations of ouabain trigger low frequency calcium oscillations that activate NF-B and protect from apoptosis. All of these effects are suppressed in cells overexpressing a peptide corresponding to ␣NT-t but not in cells overexpressing a peptide corresponding to ␣NT-t⌬LKK. Thus we have identified a well conserved Na,K-ATPase motif that binds to the inositol 1,4,5-trisphosphate receptor and can trigger an anti-apoptotic calcium signal.The Na,K-ATPase is an integral plasma membrane protein that establishes the electrochemical gradient across the plasma membrane in all mammalian cells. Ouabain is a steroid derivative that binds specifically to Na,K-ATPase. Several recent studies suggest that the ouabain⅐Na,K-ATPase complex may act as a signal transducer and transcription activator (1-4) modulating cell growth (5, 6), apoptosis (7), and cell motility (8). These effects have been ascribed to the activation of a number of intracellular signaling pathways (for review see Refs. 9 and 10). Most, if not all, of these pathways involve the release of calcium (Ca 2ϩ ) from intracellular stores via the inositol 1,4,5-trisphosphate receptor (InsP 3 R), 4 and results from recent studies indicate that Na,K-ATPase tethers the InsP 3 R into a Ca 2ϩ regulatory complex (3, 4). The exact mechanisms by which Na,K-ATPase activates the InsP 3 R remains to be elucidated.Here we report that the N-terminal tail of the Na,K-ATPase catalytic ␣-subunit binds to the N terminus of the InsP 3 R. Interaction between Na,K-ATPase and InsP 3 R modulates the Ca 2ϩ oscillatory signal, which serves to protect the cell from apoptosis (11). The identification of a distinct motif in the Na,KATPase that via protein-protein interaction transmits this signal to the InsP 3 R has important implications for the many vital cell functions that are regulated by ouabain⅐Na,K-ATPase signaling. EXPERIMENTAL PROCEDURESCells and Tissue-Two types of cells were used. COS-7 cells, a cell line derived from fetal monkey kidney, were used in most protocols. Because transformed cells are not suitable for serum deprivation-induced apoptosis, rat proximal tubule (RPT) cells in primary culture were used in these protocols. COS-7 cells were purchased from the European Collection of Cell Cultures and were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum and 2 mM L-glutamine. RPT cells were prepared as described previously (1). Briefly, the kidneys of 20-day-old male Sprague-Dawley rats...

Regulation of Inositol 1,4,5-Trisphosphate Receptor-mediated Calcium Release by the Na/K-ATPase in Cultured Renal Epithelial Cells

“…Needless to say, other possibilities may also be in play. For example, knockdown of Na/K-ATPase reduces the interaction between the Na/K-ATPase and IP 3 R, which may simply reduce the sensitivity of IP 3 R to IP 3 or change the channel opening properties as suggested previously (10). These issues have to be addressed in future studies.…”

“…2ϩ Release-Results from recent studies indicate that the N terminus of the Na/K-ATPase ␣1 subunit binds to IP 3 R directly (9,10). To detect whether the effect of Na/K-ATPase on ER Ca 2ϩ release is because of the direct interaction between Na/K-ATPase and IP 3 R, we constructed a YFP fusion protein containing the ␣1 N terminus (amino acids 1-160) (␣NT-eYFP).…”

“…Caveolar Na/K-ATPase interacts with the ER IP 3 R through its N terminus. This interaction could be further stabilized by the cytoskeleton protein such as ankyrin (8,41) and enhanced by ouabain-activated signaling pathways (7,9,10). Functionally, this interaction between Na/K-ATPase and IP 3 R helps targeting IP 3 R to the proximity of PLC-coupled plasma membrane receptors (e.g.…”

“…The interaction between the Na/K-ATPase and Src, for example, forms a functional receptor complex for cardiotonic steroids such as ouabain to activate protein tyrosine phosphorylation (5,6). Interestingly, recent studies from several laboratories have demonstrated a direct interaction between the ␣ subunit of Na/K-ATPase and inositol 1,4,5-trisphosphate receptors (IP 3 Rs) 2 (7)(8)(9)(10). In addition, we have found that ouabain was capable of stimulating the formation of a functional Ca 2ϩ -signaling complex consisting of the Na/KATPase/Src/PLC-␥/IP 3 R in LLC-PK1 cells (9).…”

Cell shrinkage and monovalent cation fluxes: Role in apoptosis

“…The idea of ionic transport proteins such as exchangers or co-transporters as signal transducers during apoptosis has recently been demonstrated for the Na + , K + -ATPase by Zhang et al [77]. Here the authors showed the direct interaction of the N-terminal tail of the catalytic α-subunit of the Na + , K + -ATPase with the N-terminus of the inositol 1,4,5-triphosphate receptor.…”