title: Na,K-ATPase alpha1-subunit dephosphorylation by protein phosphatase 2A is necessary for its recruitment to the plasma membrane.
abstract: In alveolar epithelial cells, G-protein coupled-receptors agonists (GPCR) induce the recruitment of the Na,K-ATPase to the plasma membrane. Here we report that for the recruitment of the Na,K-ATPase to occur, dephosphorylation of its alpha1-subunit at serine 18 is necessary, as demonstrated by in vitro phosphorylation, mutation of the serine 18 to alanine, and use of a specific phospho-antibody. Several approaches strongly suggest dephosphorylation to be mediated by protein phosphatase 2A (PP2A): 1) Na,K-ATPase dephosphorylation and recruitment were prevented by okadaic acid (OA); 2) the Na,K-ATPase alpha1-subunit is an in vitro substrate for PP2A; and 3) glutathione S-transferase (GST)-fusion proteins binding assays demonstrate a direct interaction between the catalytic subunit of PP2A and the first 90 amino acids of the Na,K-ATPase alpha1-subunit. Finally, GPCR agonists induced a rapid translocation of PP2A from the cytosol to the membrane fraction, which corresponded with increased coimmunoprecipitation and colocalization of PP2A and the Na,K-ATPase. Accordingly, we provide evidence that GPCR agonists promote PP2A translocation to the membrane fraction, leading to the dephosphorylation of the Na,K-ATPase alpha1-subunit at the serine 18 residue and its recruitment to the cell plasma membrane, which is of biological and physiological importance.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Feinberg School of Medicine, Northwestern University, 240 E. Huron, McGaw M410, Chicago, IL 60611, USA. e-lecuona@northwestern.edu
authors.forename: Emilia
authors.initials: E
authors.lastname: Lecuona
authors.forename: Laura A
authors.initials: LA
authors.lastname: Dada
authors.forename: Haiying
authors.initials: H
authors.lastname: Sun
authors.forename: Maria L
authors.initials: ML
authors.lastname: Butti
authors.forename: Guofei
authors.initials: G
authors.lastname: Zhou
authors.forename: Teng-Leong
authors.initials: TL
authors.lastname: Chew
authors.forename: Jacob I
authors.initials: JI
authors.lastname: Sznajder
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D008297:Male; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D054648:Protein Phosphatase 2; D021122:Protein Subunits; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000819:agonists; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D043562:Receptors, G-Protein-Coupled; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D000254:Sodium-Potassium-Exchanging ATPase