title: Identification of a mutant α1 Na/K-ATPase that pumps but is defective in signal transduction.
abstract: BACKGROUND
  It has not been possible to study the pumping and signaling functions of Na/K-ATPase independently in live cells.
  
  
  RESULTS
  Both cell-free and cell-based assays indicate that the A420P mutation abolishes the Src regulatory function of Na/K-ATPase.
  
  
  CONCLUSIONS
  A420P mutant has normal pumping but not signaling function.
  
  
  CONCLUSIONS
  Identification of Src regulation-null mutants is crucial for addressing physiological role of Na/K-ATPase. The α1 Na/K-ATPase possesses both pumping and signaling functions. However, it has not been possible to study these functions independently in live cells. We have identified a 20-amino acid peptide (Ser-415 to Gln-434) (NaKtide) from the nucleotide binding domain of α1 Na/K-ATPase that binds and inhibits Src in vitro. The N terminus of NaKtide adapts a helical structure. In vitro kinase assays showed that replacement of residues that contain a bulky side chain in the helical structure of NaKtide by alanine abolished the inhibitory effect of the peptide on Src. Similarly, disruption of helical structure by proline replacement, either single or in combination, reduced the inhibitory potency of NaKtide on Src. To identify mutant α1 that retains normal pumping function but is defective in Src regulation, we transfected Na/K-ATPase α1 knockdown PY-17 cells with expression vectors of wild type or mutant α1 carrying Ala to Pro mutations in the region of NaKtide helical structure and generated several stable cell lines. We found that expression of either A416P or A420P or A425P mutant fully restored the α1 content and consequently the pumping capacity of cells. However, in contrast to A416P, either A420P or A425P mutant was incapable of interacting and regulating cellular Src. Consequently, expression of these two mutants caused significant inhibition of ouabain-activated signal transduction and cell growth. Thus we have identified α1 mutant that has normal pumping function but is defective in signal transduction.
authors.affiliation: Department of Physiology, Pharmacology, and Medicine, University of Toledo College of Medicine, Toledo, Ohio 43614, USA.
authors.forename: Fangfang
authors.initials: F
authors.lastname: Lai
authors.forename: Namrata
authors.initials: N
authors.lastname: Madan
authors.forename: Qiqi
authors.initials: Q
authors.lastname: Ye
authors.forename: Qiming
authors.initials: Q
authors.lastname: Duan
authors.forename: Zhichuan
authors.initials: Z
authors.lastname: Li
authors.forename: Shaomeng
authors.initials: S
authors.lastname: Wang
authors.forename: Shuyi
authors.initials: S
authors.lastname: Si
authors.forename: Zijian
authors.initials: Z
authors.lastname: Xie
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D051242:Caveolin 1 / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D051381:Rats; D012414:Rubidium Radioisotopes / Q000378:metabolism; D015398:Signal Transduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D051242:Caveolin 1; D010446:Peptide Fragments; D012414:Rubidium Radioisotopes; D010042:Ouabain; D019061:src-Family Kinases; C499075:Atp1a1 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Cardiotonic Steroids; Cell Signaling; Membrane Proteins; Mutant; Na,K-ATPase; Src