title: Specificity of ε and non-ε isoforms of arabidopsis 14-3-3 proteins towards the H+-ATPase and other targets.
abstract: 14-3-3 proteins are a family of ubiquitous dimeric proteins that modulate many cellular functions in all eukaryotes by interacting with target proteins. 14-3-3s exist as a number of isoforms that in Arabidopsis identifies two major groups named ε and non-ε. Although isoform specificity has been demonstrated in many systems, the molecular basis for the selection of specific sequence contexts has not been fully clarified. In this study we have investigated isoform specificity by measuring the ability of different Arabidopsis 14-3-3 isoforms to activate the H+-ATPase. We observed that GF14 isoforms of the non-ε group were more effective than ε group isoforms in the interaction with the H+-ATPase and in the stimulation of its activity. Kinetic and thermodynamic parameters of the binding of GF14ε and GF14ω isoforms, representative of ε and non-ε groups respectively, with the H+-ATPase, have been determined by Surface Plasmon Resonance analysis demonstrating that the higher affinity of GF14ω is mainly due to slower dissociation. The role of the C-terminal region and of a Gly residue located in the loop 8 and conserved in all non-ε isoforms has also been studied by deletion and site-specific mutagenesis. The C-terminal domains, despite their high divergence, play an auto-inhibitory role in both isoforms and they, in addition to a specific residue located in the loop 8, contribute to isoform specificity. To investigate the generality of these findings, we have used the SPOT-synthesis technology to array a number of phosphopeptides matching known or predicted 14-3-3 binding sites present in a number of clients. The results of this approach confirmed isoform specificity in the recognition of several target peptides, suggesting that the isoform specificity may have an impact on the modulation of a variety of additional protein activities, as suggested by probing of a phosphopeptide array with members of the two 14-3-3 groups.
authors.affiliation: Department of Biology, University of Rome "Tor Vergata", Rome, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Pallucca
authors.affiliation: Department of Biology, University of Rome "Tor Vergata", Rome, Italy.
authors.forename: Sabina
authors.initials: S
authors.lastname: Visconti
authors.affiliation: Department of Biology, University of Rome "Tor Vergata", Rome, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Camoni
authors.affiliation: Department of Biology, University of Rome "Tor Vergata", Rome, Italy; IRCSS, Research Institute "Fondazione Santa Lucia", Rome, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Cesareni
authors.affiliation: Department of Sciences and Chemical Technologies, University of Rome "Tor Vergata", Rome, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Melino
authors.affiliation: Department DiBEST, University of Calabria, Rende, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Panni
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Torreri
authors.affiliation: Department of Biology, University of Rome "Tor Vergata", Rome, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Aducci
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry*; D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
chemical_list: D048948:14-3-3 Proteins; C556234:14-3-3chi protein, Arabidopsis; D029681:Arabidopsis Proteins; D002135:Calcium-Binding Proteins; C087620:GF14 protein, Arabidopsis; D020033:Protein Isoforms; D006180:Proton-Translocating ATPases; C544444:AHA1 protein, Arabidopsis