title: A phosphorylation in the c-terminal auto-inhibitory domain of the plant plasma membrane H+-ATPase activates the enzyme with no requirement for regulatory 14-3-3 proteins.
abstract: The plant plasma membrane H(+)-ATPase is regulated by an auto-inhibitory C-terminal domain that can be displaced by phosphorylation of the penultimate residue, a Thr, and the subsequent binding of 14-3-3 proteins. By mass spectrometric analysis of plasma membrane H(+)-ATPase isoform 2 (PMA2) isolated from Nicotiana tabacum plants and suspension cells, we identified a new phosphorylation site, Thr-889, in a region of the C-terminal domain upstream of the 14-3-3 protein binding site. This residue was mutated into aspartate or alanine, and the mutated H(+)-ATPases expressed in the yeast Saccharomyces cerevisiae. Unlike wild-type PMA2, which could replace the yeast H(+)-ATPases, the PMA2-Thr889Ala mutant did not allow yeast growth, whereas the PMA2-Thr889Asp mutant resulted in improved growth and increased H(+)-ATPase activity despite reduced phosphorylation of the PMA2 penultimate residue and reduced 14-3-3 protein binding. To determine whether the regulation taking place at Thr-889 was independent of phosphorylation of the penultimate residue and 14-3-3 protein binding, we examined the effect of combining the PMA2-Thr889Asp mutation with mutations of other residues that impair phosphorylation of the penultimate residue and/or binding of 14-3-3 proteins. The results showed that in yeast, PMA2 Thr-889 phosphorylation could activate H(+)-ATPase if PMA2 was also phosphorylated at its penultimate residue. However, binding of 14-3-3 proteins was not required, although 14-3-3 binding resulted in further activation. These results were confirmed in N. tabacum suspension cells. These data define a new H(+)-ATPase activation mechanism that can take place without 14-3-3 proteins.
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 4-15, 1348 Louvain-la-Neuve, Belgium.
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Piette
authors.forename: Rita
authors.initials: R
authors.lastname: Derua
authors.forename: Etienne
authors.initials: E
authors.lastname: Waelkens
authors.forename: Marc
authors.initials: M
authors.lastname: Boutry
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Duby
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000502:physiology; D020125:Mutation, Missense; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
chemical_list: D048948:14-3-3 Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases