Patrizia Aducci

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 30-kilodalton protein present in purified fusicoccin receptor preparations is a 14-3-3-like protein.

We have recently reported on the purification of the fusicoccin (FC) receptor from corn (Zea mays L.) and its identification by photoaffinity labeling (P. Aducci, A. Ballio, V. Fogliano, M.R. Fullone, M. Marra, N. Proietti [1993] Eur J Biochem 214: 339–345). Pure preparations of FC receptors, obtained under nondenaturing conditions, showed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis two doublets of proteins with apparent molecular masses of 30 and 90 kD. In the present paper we describe the isolation and identification of the primary structure of the 30-kD doublet proteins. Sequencing studies of peptides resulting from the digestion of the 30-kD protein showed a full identity with a 14–3–3like protein from corn, named GF14. The 14–3–3 family is a class of proteins that is widely distributed in eukaryotes and is known to play various regulatory roles. The 30-kD protein has been immunologically identified by specific antibodies prepared against a synthetic peptide based on the determined amino acid sequence. A similar protein is recognized in partially purified FC receptor preparations from bean and spinach leaves.

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.

Inactivation of solubilised fusicoccin-binding sites by endogenous plant hydrolases

The poor stability of crude solutions of fusicoccin-binding sites, prepared from acetonedried microsomal fractions of spinach leaves, results from the attack by endogenous phosphatase and α-mannosidase. The addition of either of these enzymes to solubilised binding sites preincubated with [3H]fusicoccin promptly releases most of the bound radioactivity. A satisfactory stabilization of the crude preparations is obtained with fluoride added either during homogenization of the tissue, or immediately after solubilisation. The results indicate that the fusicoccin-binding sites are phosphorylated glycoproteins.

Fusicoccin receptors. Evidence for endogenous ligand.

The binding of fusicoccin to the microsomal preparations of maize roots in vitro is increased several-fold when segments of the tissue are washed for 2 h in distilled water before homogenization. Addition of freeze-dried wash solution to microsomal preparations of spinach leaves or fresh roots, washed roots, or coleoptiles of maize inhibited the binding of fusicoccin to particulate fractions. The freeze-dried material also blocked fusicoccin-promoted H+ extrusion from maize root segments. Roots may contain one or more water-soluble compounds competing with fusicoccin at the receptor level; such ligands might play a physiological role as modulators of the H+/K+ exchange system in higher plants.

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.

The H+-ATPase purified from maize root plasma membranes retains fusicoccin in vivo activation

The activity of ‘P‐type’ ATPases is modulated through the C‐terminal autoinhibitory domain. The molecular bases of this regulation are unknown. Their understanding demands functional and structural studies on the activated purified enzyme. In this paper the plasma membrane H+‐ATPase from maize roots activated in vivo by fusicoccin was solubilised and fractionated by anion‐exchange HPLC. Results showed that the H+‐ATPase separated from fusicoccin receptors retained fusicoccin activation and that it was more evident after enzyme insertion into liposomes. These data suggest that fusicoccin stimulation does not depend on a direct action of the fusicoccin receptor on the H+‐ATPase, but rather, fusicoccin brings about a permanent modification of the H+‐ATPase which very likely represents a general regulatory mechanism for ‘P‐type’ ATPases.

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.