Sabina Visconti

Binding of 14-3-3 Protein to the Plasma Membrane H+-ATPase AHA2 Involves the Three C-terminal Residues Tyr946-Thr-Val and Requires Phosphorylation of Thr947

14-3-3 proteins play a regulatory role in a diverse array of cellular functions such as apoptosis, regulation of the cell cycle, and regulation of gene transcription. The phytotoxin fusicoccin specifically induces association of virtually any 14-3-3 protein to plant plasma membrane H+-ATPase. The 14-3-3 binding site in the Arabidopsis plasma membrane H+-ATPase AHA2 was localized to the three C-terminal residues of the enzyme (Tyr946-Thr-Val). Binding of 14-3-3 protein to this target was induced by phosphorylation of Thr947 (KD = 88 nm) and was in practice irreversible in the presence of fusicoccin (KD = 7 nm). Mass spectrometry analysis demonstrated that AHA2 expressed in yeast was phosphorylated at Thr947. We conclude that the extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein.

The Potassium Channel KAT1 Is Activated by Plant and Animal 14-3-3 Proteins

14-3-3 proteins modulate the plant inward rectifier K+ channel KAT1 heterologously expressed in Xenopus oocytes. Injection of recombinant plant 14-3-3 proteins into oocytes shifted the activation curve of KAT1 by +11 mV and increased the τon. KAT1 was also modulated by 14-3-3 proteins of Xenopus oocytes. Titration of the endogenous 14-3-3 proteins by injection of the peptide Raf 621p resulted in a strong decrease in KAT1 current (∼70% at –150 mV). The mutation K56E performed on plant protein 14-3-3 in a highly conserved recognition site prevented channel activation. Because the maximal conductance of KAT1 was unaffected by 14-3-3, we can exclude that they act by increasing the number of channels, thus ruling out any effect of these proteins on channel trafficking and/or insertion into the oocyte membrane. 14-3-3 proteins also increased KAT1 current in inside-out patches, suggesting a direct interaction with the channel. Direct interaction was confirmed by overlay experiments with radioactive 14-3-3 on oocyte membranes expressing KAT1.

From cytosol to organelles: 14-3-3 proteins as multifunctional regulators of plant cell.

14‐3‐3 proteins are a class of highly conserved proteins widespread in eukaryotes. They regulate several cellular processes through phosphorylation‐dependent interaction with their targets. Since their discovery in plants, a number of peculiar functions have been ascertained, such as regulation of primary metabolism, ion transport, cellular trafficking, chloroplast and mitochondrial enzyme activities and gene transcription. The still increasing body of evidence suggests that 14‐3‐3s may function as versatile proteins able to move from cytosol to different cellular organelles. This review will focus on the broad range of regulatory tasks carried out by 14‐3‐3s in the different compartments.

ZmMPK6, a novel maize MAP kinase that interacts with 14-3-3 proteins.

Although an increasing body of evidence indicates that plant MAP kinases are involved in a number of cellular processes, such as cell cycle regulation and cellular response to abiotic stresses, hormones and pathogen attack, very little is known about their biochemical properties and regulation mechanism. In this paper we report on the identification and characterization of a novel member of the MAP kinase family from maize, ZmMPK6. The amino acid sequence reveals a high degree of identity with group D plant MAP kinases. Recombinant ZmMPK6, expressed in Escherichia coli, is an active enzyme able to autophosphorylate. Remarkably, ZmMPK6 interacts in vitro with GF14-6, a maize 14-3-3 protein and the interaction is dependent on autophosphorylation. The interacting domain of ZmMPK6 is on the C-terminus and is comprised between amino acid 337 and amino acid 467. Our results represent the first evidence of an interaction between a plant MAP kinase and a 14-3-3 protein. Possible functional roles of this association in vivo are discussed.

Binding of phosphatidic acid to 14-3-3 proteins hampers their ability to activate the plant plasma membrane H+-ATPase.

Phosphatidic acid is a phospholipid second messenger implicated in various cellular processes in eukaryotes. In plants, production of phosphatidic acid is triggered in response to a number of biotic and abiotic stresses. Here, we show that phosphatidic acid binds to 14‐3‐3 proteins, a family of regulatory proteins which bind client proteins in a phosphorylation‐dependent manner. Binding of phosphatidic acid involves the same 14‐3‐3 region engaged in protein target binding. Consequently, micromolar phosphatidic acid concentrations significantly hamper the interaction of 14‐3‐3 proteins with the plasma membrane H+‐ATPase, a well characterized plant 14‐3‐3 target, thus inhibiting the phosphohydrolitic enzyme activity. Moreover, the proton pump is inhibited when endogenous PA production is triggered by phospholipase D and the G protein agonist mastoparan‐7. Hence, our data propose a possible mechanism involving PA that regulates 14‐3‐3‐mediated cellular processes in response to stress.

The phytotoxin fusicoccin promotes platelet aggregation via 14-3-3-glycoprotein Ib-IX-V interaction.

The fungal toxin fusicoccin induces plant wilting by affecting ion transport across the plasma membrane of plant cell. The activity of this toxin is so far unknown in humans. In the present study we show that fusicoccin is able to affect the platelet aggregation process. The toxin associates with platelet intracellular binding sites and induces aggregation in platelet-rich plasma in a dose-dependent manner. We identified the adhesion receptor glycoprotein Ib-IX-V as fusicoccin target. The toxin promotes the binding of the regulatory 14-3-3 proteins to glycoprotein Ibα and hampers that to glycoprotein Ibβ subunit. As a result, platelet adhesion to von Willebrand factor is stimulated, leading to platelet spreading and integrin αIIbβ3 activation. We anticipate the present study to be a starting point for future therapeutic use of fusicoccin in genetic bleeding diseases characterized by qualitative or quantitative abnormalities of the platelet membrane-adhesion receptors. Furthermore, the present study also sets the stage for future work to determine the potential pharmacological application of fusicoccin as a drug directed to other 14-3-3-target complexes.

Specificity of ε and non-ε isoforms of arabidopsis 14-3-3 proteins towards the H+-ATPase and other targets.

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.

Role of the 14-3-3 C-terminal region in the interaction with the plasma membrane H+-ATPase.

The 14-3-3 proteins are a family of proteins present in a number of isoforms in all eukaryotes and involved in the control of many cellular functions. Regulation of different activities is achieved by binding to phosphorylated targets through a conserved mechanism. Although in many systems isoform specificity has been demonstrated, the underlying molecular basis is still unclear. The sequences of 14-3-3 isoforms are highly conserved, divergence occurring at the N- and C-terminal regions. Recently it has been suggested that the C-terminal domain of 14-3-3 may regulate protein binding to the targets. Here we study the role of the C-terminal region of maize isoform GF14-6 in the interaction with the plant plasma membrane H(+)-ATPase. Results obtained demonstrate that removal of the last 22 amino acids residues of GF14-6 increases binding to H(+)-ATPase and stimulation of its activity. C-terminal deletion, moreover, reduces 14-3-3 sensitivity to cations. We also show that a peptide reproducing the GF14-6 C-terminus is able to bind to the C-terminal domain of H(+)-ATPase and to stimulate the enzyme activity. The implications of these findings for a integrated model of 14-3-3 interaction with H(+)-ATPase are discussed.

The phytotoxin fusicoccin, a selective stabilizer of 14‐3‐3 interactions?

Summary The study of the structure and dynamics of protein–protein interaction networks has become overwhelming in all biological systems. Most of the biological events are the consequence of several protein–protein interactions finely regulated by covalent modifications and physiological effectors. Moreover, several studies have shown that the complex interactome responsible for the progress and control of vital processes is disturbed in diseases. Besides the basic information on the mechanisms involved in the processes driven by protein–protein interactions, it appears nowadays extremely challenging to study possible regulators of the lifespan of protein networks. Small molecules able to stabilize or to inhibit protein complexes could easily find applications as potential innovative drugs. In this article, we hypothesize that a natural product, the fungal phytotoxin fusicoccin, can play a role as a stabilizer of interactions between 14‐3‐3 proteins and specific natural targets. A very specific stabilizer molecule is the ideal starting point for the development of a family of structurally related drugs able to selectively tune 14‐3‐3 interaction with their targets.

The phytotoxin fusicoccin differently regulates 14‐3‐3 proteins association to mode III targets

Modulation of the interaction of regulatory 14‐3‐3 proteins to their physiological partners through small cell‐permeant molecules is a promising strategy to control cellular processes where 14‐3‐3s are engaged. Here, we show that the fungal phytotoxin fusicoccin (FC), known to stabilize 14‐3‐3 association to the plant plasma membrane H+‐ATPase, is able to stabilize 14‐3‐3 interaction to several client proteins with a mode III binding motif. Isothermal titration calorimetry analysis of the interaction between 14‐3‐3s and different peptides reproducing a mode III binding site demonstrated the FC ability to stimulate 14‐3‐3 the association. Moreover, molecular docking studies provided the structural rationale for the differential FC effect, which exclusively depends on the biochemical properties of the residue in peptide C‐terminal position. Our study proposes FC as a promising tool to control cellular processes regulated by 14‐3‐3 proteins, opening new perspectives on its potential pharmacological applications.