The ‘lysine cluster’ in the N-terminal region of Na+/K+-ATPase α-subunit is not involved in ATPase activity

Ohta, Toshiko; Noguchi, Shunsuke; Nakanishi, Makoto; Mutoh, Yoshinobu; Hirata, Hajime; Kagawa, Yasuo; Kawamura, Masaru

doi:10.1016/s0005-2728(05)80200-2

Cited by 14 publications

(11 citation statements)

References 34 publications

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“…3). Although all Na,K-ATPase ␣-isoforms share more than 80% homology, the N-terminal tail shows little homology between the different isoforms (33). One exception is the lysine-rich motif (LKK) in the N-terminal tail that is conserved in almost all species.…”

Section: Discussionmentioning

confidence: 99%

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

Zhang

Malmersjö

et al. 2006

Journal of Biological Chemistry

120

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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...

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Section: Discussionmentioning

confidence: 99%

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

Zhang

Malmersjö

et al. 2006

Journal of Biological Chemistry

120

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“…Following SDS-PAGE, the proteins were transferred onto a PVDF membrane. The Coomassie blue stained bands were cut out and applied to the sequencer without Polybrene treatment, as described previously (Ohta et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

Oxidative-stress-inducible qorA encodes an NADPH-dependent quinone oxidoreductase catalysing a one-electron reduction in Staphylococcus aureus

et al. 2003

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This work characterized the putative quinone oxidoreductase gene (qorA) from Staphylococcus aureus. The deduced amino acid sequence indicated that the 333 aa protein contains an NAD(P)H-binding motif. A Northern blot analysis revealed that 2?6 kb and 1?4 kb signals were detected by using a qorA probe. Both the signals were enhanced under the presence of a redoxcycling agent, 9,10-phenanthrenequinone (PQ). It was also revealed that the expression of three genes, SA1988, SA1989 (qorA) and SA1990, was enhanced at the transcriptional level by PQ exposure. The results suggested that the 2?6 kb signal detected by the qorA probe was in two co-transcripts, i.e. SA1990-qorA and qorA-SA1988 were transcribed. Besides, primer extension analyses confirmed the enhancement of qorA and SA1990 transcripts. The GST (glutathione S-transferase)-tagged QorA protein was expressed in Escherichia coli and purified using a glutathione affinity column. In purification steps, a 36 kDa band co-purified with the GST-QorA, and it was detected even in the thrombin-cleaved fraction. N-terminal amino acid sequences for the 36 kDa protein revealed that it was an intact QorA. They showed that QorA formed a multimer under physiological conditions. The purified recombinant GST-QorA catalysed NADPH consumption in the presence of PQ as a substrate, but not NADH. To characterize the catalytic activity of QorA, superoxide anion that was generated through one-electron reduction of PQ and hydroquinone that was produced by two-electron reduction of PQ were measured. During reduction of PQ by GSTQorA, superoxide anion was generated, whereas a small amount of 9,10-dihydroxyphenanthrene (hydroquinone of PQ) was produced. These results suggest that the activity of QorA is similar to f-Crystallin, catalysing an NADPH-dependent one-electron reduction of quinone.

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“…Cleavage at this residue increases KЈ K and decreases KЈ ATP , with associated effects on the phosphorylation and dephosphorylation reactions of the enzyme (42)(43)(44), providing evidence for a shift in the E 1 /E 2 equilibrium toward E 1 forms (45,46). Several studies have demonstrated that although the lysine cluster is not essential to pump function (47)(48)(49)(50)(51), the N terminus may play a role in Na ϩ sensitivity (52), K ϩ affinity, and in the dependence on membrane potential (53,54) likely resulting from changes in rates of Na ϩ translocation (41) and K ϩ deocclusion reactions (55). 1 The abbreviation used is: SERCA, sarcoplasmic reticulum Ca 2ϩ -ATPase.…”

Section: Enzymementioning

confidence: 99%

New Insights into the Role of the N Terminus in Conformational Transitions of the Na,K-ATPase

Segall

Lane

Blostein

2002

Journal of Biological Chemistry

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The deletion of 32 residues from the N terminus of the ␣1 catalytic subunit of the rat Na,K-ATPase (mutant ␣1M32) shifts the E 1 /E 2 conformational equilibrium toward E 1 , and the combination of this deletion with mutation E233K in the M2-M3 loop acts synergistically to shift the conformation further toward E 1 (Boxenbaum, N., Daly, S. E., Javaid, Z. Z., Lane, L. K., and Blostein, R. (1998) J. Biol. Chem. 273, 23086 -23092). To delimit the region of the cytoplasmic N terminus involved in these interactions, the consequences of a series of N-terminal deletions of ␣1 beyond ⌬32 were evaluated. Criteria to assess shifts in conformational equilibrium were based on effects of perturbation of the entire catalytic cycle ((i) sensitivity to vanadate inhibition, (ii) K ؉ sensitivity of Na-ATPase measured at micromolar ATP, (iii) changes in K ATP , and (iv) catalytic turnover), as well as estimates of the rates of the conformational transitions of phospho-and dephosphoenzyme (E 1 P 3 E 2 P and E 2 (K ؉ ) 3 E 1 ؉ K ؉ ). The results show that, compared with ␣1M32, the deletion of up to 40 residues (␣1M40) further shifts the poise toward E 1 . Remarkably, further deletions (mutants ␣1M46, ␣1M49, and ␣1M56) reverse the effect, such that these mutants increasingly resemble the wild type ␣1. These results suggest novel intramolecular interactions involving domains within the N terminus that impact the manner in which the N terminus/ M2-M3 loop regulatory domain interacts with the M4-M5 catalytic loop to effect E 1 7 E 2 transitions.The Na,K-ATPase or sodium pump is a ubiquitous integral membrane protein that catalyzes the glycoside sensitive, ATPcoupled exchange of three intracellular Na ϩ for two extracellular K ϩ ions across the plasma membrane of all animal cells. The sodium pump is essential to the maintenance of the electrochemical gradients of Na ϩ and K ϩ across the cell membrane, providing the driving force for the transport of nutrients into the cell and maintaining the cellular resting membrane potential. It comprises two essential subunits: a large catalytic ␣ subunit (ϳ100 kDa), containing the ligand binding and phosphorylation sites and a smaller, highly glycosylated ␤ subunit (ϳ35-55 kDa), which acts as a chaperone for ␣ (for review see Refs. 1 and 2). A third subunit, ␥ (ϳ7 kDa), was found in the kidney where it functions as a regulator of the pump (see Refs. 3 and 4).The sodium pump is a member of a family of transporters known as P-type ATPases that are directly phosphorylated and dephosphorylated on a conserved aspartate residue within their cytoplasmic domain during the course of the reaction cycle. Both the phosphorylated and dephosphorylated forms of the enzyme can exist in at least two states that undergo conformational transitions (E 1 P 3 E 2 P and E 2 3 E 1 ) that are coupled to the ion-translocating steps. Definitive evidence for distinct E 1 and E 2 conformational states and a role of the N terminus in effecting E 1 7 E 2 transitions was first obtained in 1975 by Jorgensen (5, 6) using tryptic cleav...

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The ‘lysine cluster’ in the N-terminal region of Na+/K+-ATPase α-subunit is not involved in ATPase activity

Cited by 14 publications

References 34 publications

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

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

Oxidative-stress-inducible qorA encodes an NADPH-dependent quinone oxidoreductase catalysing a one-electron reduction in Staphylococcus aureus

New Insights into the Role of the N Terminus in Conformational Transitions of the Na,K-ATPase

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