The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

Imagawa, Toshiaki; Kaya, Shunji; Taniguchi, Koki

doi:10.1074/jbc.m309833200

Cited by 7 publications

(7 citation statements)

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“…It had been suggested that coupling to ATPase activity would exact an insupportable cost to cells for CFTR channel activity [26]. This present study confirms that CFTR possesses a very low specific activity (approximately three orders of magnitude lower than that of the Na + /K + -ATPase) [50]. Therefore it is unlikely that the ATPase activity of CFTR will be costly to the cell.…”

Section: Discussionsupporting

confidence: 76%

The intact CFTR protein mediates ATPase rather than adenylate kinase activity

Ramjeesingh

Ugwu

Stratford

et al. 2008

Biochemical Journal

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The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

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Section: Discussionsupporting

confidence: 76%

The intact CFTR protein mediates ATPase rather than adenylate kinase activity

Ramjeesingh

Ugwu

Stratford

et al. 2008

Biochemical Journal

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“…This distortion in the V920E in the transmembrane segment might be related to the decrease in the affinity for Na ϩ and influence the conformational state of the N-domain such as to decrease the affinity for high affinity ATP effect and increase the affinity for low affinity ATP effects differently (Table I, e, f, and g). If these two apparent low affinity ATP effects structurally reflect the same single low affinity ATP binding conformation, the ratio K i,0.5 pNPPase /K 0.5 ATPase would be the same as the ratios in the data obtained from the mutagenesis of the ATP binding pocket as has already been reported (53,57). The ratio for the wild type and V920E was, respectively, 0.86 (0.43/0.50) and 0.42 (0.21/0.5).…”

Section: Fig 8 Atp Concentration Dependence Of Nak-atpase Activitysupporting

confidence: 54%

“…The relative turnover number was obtained by dividing rat Na,K-ATPase activity by the relative amount of EP max , estimated as described above. The value of the wild type was taken as 240 s Ϫ1 , which was estimated in a previous paper (57). To investigate the effect of ATP and K ϩ on Na,K-ATPase activity, 4 mM ATP and 16 mM K ϩ were replaced with 0 -4 mM ATP and 0 -16 mM K ϩ , respectively.…”

Section: Methodsmentioning

confidence: 99%

Thr-774 (Transmembrane Segment M5), Val-920 (M8), and Glu-954 (M9) Are Involved in Na+ Transport, and Gln-923 (M8) Is Essential for Na,K-ATPase Activity

Imagawa

Yamamoto

Kaya

et al. 2005

Journal of Biological Chemistry

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The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/ E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K 0.5 values for Na ؉ , as estimated by the Na ؉ -dependent phosphoenzyme formation (K 0.5 Na,EP ) or Na,K-ATPase activity (K 0.5 Na,ATPase ), were increased by around 2ϳ8-fold in the case of T774A, V920E, and E954A. The apparent K 0.5 values for K ؉ , as estimated by the Na,K-ATPase (K 0.5 K,ATPase ) or p-nitrophenylphosphatase activity (K 0.5 K,pNPPase ), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K 0.5 K,ATPase . The apparent K 0.5 values for ATP (K 0.5 EP ), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6ϳ2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K 0.5 ATPase ) and ATP-induced inhibition (K i,0.5 pNPPase ) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2-and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na ؉ and/or K ؉ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na ؉ and also in the high and low affinity ATP effects rather than the transport of K ؉ .

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“…1, D and E). Further structural analysis of the ND2 reveals that the N terminus of ND2 is highly exposed and does not contribute to ATP binding (30,31). Moreover, the corresponding domain in SR Ca 2ϩ -ATPase is known to bind phospholamban (32).…”

Section: Resultsmentioning

confidence: 99%

NaKtide, a Na/K-ATPase-derived Peptide Src Inhibitor, Antagonizes Ouabain-activated Signal Transduction in Cultured Cells

Cai

Tian

et al. 2009

Journal of Biological Chemistry

125

171

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We have previously shown that the Na/K-ATPase binds and inhibits Src. Here, we report the molecular mechanism of Na/KATPase-mediated Src regulation and the generation of a novel peptide Src inhibitor that targets the Na/K-ATPase/Src receptor complex and antagonizes ouabain-induced protein kinase cascades. First, the Na/K-ATPase inhibits Src kinase through the N terminus of the nucleotide-binding domain of the ␣1 subunit. Second, detailed mapping leads to the identification of a 20-amino acid peptide (NaKtide) that inhibits Src (IC 50 ‫؍‬ 70 nM) in an ATP concentration-independent manner. Moreover, NaKtide does not directly affect the ERK and protein kinase C family of kinases. It inhibits Lyn with a much lower potency (IC 50 ‫؍‬ 2.5 M). Third, highly positively charged leader peptide conjugates including HIV-Tat-NaKtide (pNaKtide) readily enter cultured cells. Finally, the following functional studies of pNaKtide demonstrate that this conjugate can specifically target the Na/K-ATPase-interacting pool of Src and act as a potent ouabain antagonist in cultured cells: 1) pNaKtide, unlike PP2, resides in the membranes. Consistently, it affects the basal Src activity much less than that of PP2. 2) pNaKtide is effective in disrupting the formation of the Na/K-ATPase/Src receptor complex in a dose-dependent manner. Consequently, it blocks ouabain-induced activation of Src, ERK, and hypertrophic growth in cardiac myocytes. 3) Unlike PP2, pNaKtide does not affect IGF-induced ERK activation in cardiac myocytes. Taken together, we suggest that pNaKtide may be used as a novel antagonist of ouabain for probing the physiological and pathological significance of the newly appreciated signaling function of Na/K-ATPase and cardiotonic steroids.The Na/K-ATPase is expressed in most eukaryotic cells and is essential for maintaining the transmembrane ion gradient by pumping Na ϩ out of and K ϩ into cells (1). Structurally, the enzyme consists of two non-covalently linked ␣ and ␤ subunits. Similar to other P-ATPases, the Na/K-ATPase ␣ subunit has 10 transmembrane domains with both the N and C termini located in the cytoplasm (2, 3). Based on the published crystal structures of Na/K-ATPase (4), the ␣ subunit consists of several well-characterized domains. The actuator (A) 2 domain consists of the N terminus and the second cytosolic domain (CD2) connected to transmembrane helices M2 and M3, and the highly conserved discontinuous phosphorylation (P) domain is close to the plasma membrane, while the nucleotide-binding (N) domain is relatively isolated (2). There is a significant amount of movement of both the A and N domains during the ion-pumping cycle as in the SR Ca 2ϩ -ATPase (4 -6). It appears that the A domain rotates, while the N domain closes during the transport cycle. Interestingly, these domains have also been implicated in interacting with many protein partners, including inositol 1,4,5-trisphosphate receptors, phosphoinositide 3-kinase, phospholipase C-␥ (PLC-␥), ankyrin, and cofilin (7-12).Src, a member of the Src family non...

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The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

Cited by 7 publications

References 51 publications

The intact CFTR protein mediates ATPase rather than adenylate kinase activity

The intact CFTR protein mediates ATPase rather than adenylate kinase activity

Thr-774 (Transmembrane Segment M5), Val-920 (M8), and Glu-954 (M9) Are Involved in Na+ Transport, and Gln-923 (M8) Is Essential for Na,K-ATPase Activity

NaKtide, a Na/K-ATPase-derived Peptide Src Inhibitor, Antagonizes Ouabain-activated Signal Transduction in Cultured Cells

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