Tyrosine phosphorylation inhibits the interaction of 14-3-3 proteins with the plant plasma membrane H+-ATPase.

Interaction of 14-3-3 proteins with their targets depends not only on the phosphorylation status of the target but also on that of 14-3-3 (Fu et al., 2000). In this work we demonstrated that the maize 14-3-3 isoform GF14-6 is a substrate of the tyrosine kinase insulin growth factor receptor 1. By means of site-directed mutants of GF14-6, we identified Tyr-137 as the specific tyrosine residue phosphorylated by the insulin growth factor receptor 1. Phosphorylation of GF14-6 on Tyr-137 lowered its affinity for a peptide mimicking the 14-3-3 binding site of the plant plasma membrane H+-ATPase. Moreover, phosphorylation in planta of 14-3-3 tyrosine residues, resulting from incubation with the tyrosine phosphatase inhibitor, phenylarsine oxide, decreased their association to the H+-ATPase.

Adenosine 5'-monophosphate inhibits the association of 14-3-3 proteins with the plant plasma membrane H(+)-ATPase.

Although a well ascertained evidence proves that the activity of the plant plasma membrane H(+)-ATPase is regulated by 14-3-3 proteins, information about physiological factors modulating the phosphorylation-dependent association between 14-3-3 proteins and the proton pump is largely incomplete. In this paper we show that the 5'-AMP-mimetic, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), inhibits the fusicoccin-promoted proton extrusion in maize roots. We also demonstrate that 5'-AMP inhibits the association of 14-3-3 proteins with the C-terminal domain of the H(+)-ATPase in an overlay assay as well as the 14-3-3-dependent stimulation of the Arabidopsis thaliana H(+)-ATPase AHA1 isoform expressed in yeast membranes. Finally, by means of affinity chromatography with immobilized 5'-AMP and trinitrophenyl-AMP fluorescence analysis, we demonstrate that the 14-3-3 isoform GF14-6 from maize is able to bind 5'-AMP. The possible role of 5'-AMP as a general regulator of 14-3-3 functions in the plant cell is discussed.

Phosphorylation-dependent interaction between plant plasma membrane H(+)-ATPase and 14-3-3 proteins.

The H(+)-ATPase is a key enzyme for the establishment and maintenance of plasma membrane potential and energization of secondary active transport in the plant cell. The phytotoxin fusicoccin induces H(+)-ATPase activation by promoting the association of 14-3-3 proteins. It is still unclear whether 14-3-3 proteins can represent natural regulators of the proton pump, and factors regulating 14-3-3 binding to the H(+)-ATPase under physiological conditions are unknown as well. In the present study in vivo and in vitro evidence is provided that 14-3-3 proteins can associate with the H(+)-ATPase from maize roots also in a fusicoccin-independent manner and that the interaction depends on the phosphorylation status of the proton pump. Furthermore, results indicate that phosphorylation of H(+)-ATPase influences also the fusicoccin-dependent interaction of 14-3-3 proteins. Finally, a protein phosphatase 2A able to impair the interaction between H(+)-ATPase and 14-3-3 proteins was identified and partially purified from maize root.

Binding of 14-3-3 protein to the plasma membrane H(+)-ATPase AHA2 involves the three C-terminal residues Tyr(946)-Thr-Val and requires phosphorylation of Thr(947).

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 (Tyr(946)-Thr-Val). Binding of 14-3-3 protein to this target was induced by phosphorylation of Thr(947) (K(D) = 88 nM) and was in practice irreversible in the presence of fusicoccin (K(D) = 7 nM). Mass spectrometry analysis demonstrated that AHA2 expressed in yeast was phosphorylated at Thr(947). We conclude that the extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein.

Fusicoccin effect on the in vitro interaction between plant 14-3-3 proteins and plasma membrane H+-ATPase.

A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced. The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study. The cDNA of GF14-6 was expressed in Escherichia coli as a 32P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H+-ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H+-ATPase. The binding was dependent on Mg2+ and was strongly increased by fusicoccin. Controlled trypsin digestion of H+-ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme. To confirm this result, the C-terminal domain of H+-ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments. GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.

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 the 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.

Pseudomonas syringae pv. syringae phytotoxins reversibly inhibit the plasma membrane H(+)-ATPase and disrupt unilamellar liposomes.

The Pseudomonas syringae pv. syringae phytotoxins syringomycin-E and syringopeptins 22-A and 25-A reversibly and noncompetitively inhibit purified H(+)-ATPase solubilized from plasma membrane of maize roots. Moreover, they increase the passive permeability to protons in phosphatidylcholine/phosphatidylethanolamine liposomes. Both effects are more pronounced with syringopeptins than with syringomycin-E. Activity on phospholipid bilayers is detectable at phytotoxin concentrations not affecting H(+)-ATPase activity.

The fungal H(+)-ATPase from Neurospora crassa reconstituted with fusicoccin receptors senses fusicoccin signal.

Fusicoccin affects several physiological processes regulated by the plasma membrane H(+)-ATPase in higher plants while other organisms having P-type H(+)-ATPases (e.g., fungi) are fusicoccin-insensitive. We have previously shown that fusicoccin binding to its receptor is necessary for H(+)-ATPase stimulation and have achieved the functional reconstitution into liposomes of fusicoccin receptors and the H(+)-ATPase from maize. In this paper we show that fusicoccin sensitivity can be conferred on the H(+)-ATPase from Neurospora crassa, a fungus insensitive to fusicoccin. In fact, H+ pumping by purified H(+)-ATPase from Neurospora crassa reconstituted into liposomes containing crude or partially purified fusicoccin receptors from maize was markedly enhanced by fusicoccin. The stimulation of H+ pumping by fusicoccin is dependent upon pH, fusicoccin, and protein concentration, as was reported for the system reconstituted with both proteins from maize.

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 14-3-3-like 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.

Purification and photoaffinity labeling of fusicoccin receptors from maize.

Crude soluble proteins from plasma membranes of maize shoots were purified (following the increase of fusicoccin-binding specificity) by using an original multi-step HPLC procedure. The method, based on a combination of adsorption, ion-exchange and gel-filtration chromatographies, is quick, efficient and does not damage the binding activity. It allows a 5000-fold increase of specific activity; SDS/PAGE of purified fractions shows two doublets that correspond to proteins with apparent molecular masses of 90 kDa and 30 kDa. Crude or partially purified material was irradiated for various periods in the presence of a tritiated azido analogue of fusicoccin. The electrophoretic analysis of the irradiated material shows that with a short irradiation time only the 90-kDa band is radiolabeled, whereas, as the irradiation time increases, a 30-kDa band becomes radiolabeled and less radioactivity is detected in the 90-kDa band. Irradiation of the crude material in the absence of the analogue results in a decrease of the binding capability of fusicoccin. The irradiated preparation also shows a decrease of photolabeling of the 90-kDa band. Our data suggest that the 90-kDa protein is the functional fusicoccin receptor. This conclusion is at variance with results of other authors who suggest the 30-kDa protein as the true receptor.

Phospholipase A2 affects the activity of fusicoccin receptors.

Biochemical properties of fusicoccin receptors are strongly influenced by the phospholipid environment. In this report we have studied the effect of different exogenous phospholipases on fusicoccin binding ability of both plasma membrane and solubilised receptors. Among the phospholipases tested only phospholipase A2 showed an inhibitory effect on fusicoccin binding. In particular, the influence of this enzyme on the time course and reversibility of the fusicoccin binding reaction was studied. The inhibitory effect of phospholipase A2 was the consequence of fatty acid release. The usual fatty acids of plasma membrane phospholipids were active in inhibiting the interaction of fusicoccin with its receptors. It is concluded that a phospholipid associated to the fusicoccin receptor might play a significant role in the modulation of binding.

Binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds to serine proteinases: a comparative study.

The effect of pH and temperature on kinetic and thermodynamic parameters (i.e., k(on),k(off),Ka,delta G0, delta H0 and delta S0 values) for the binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds (ETI) to bovine beta-trypsin, bovine alpha-chymotrypsin, the human tissue plasminogen activator, human alpha-, beta- and gamma-thrombin, as well as the M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator (also named urokinase) has been investigated. At pH 8.0 and 21.0 degrees C: (i) values of the second-order rate constant (K(on)) for the proteinase:ETI complex formation vary between 8.7 x 10(5) and 1.4 x 10(7)/M/s; (ii) values of the dissociation rate constant (k(off)) for the proteinase: ETI complex destabilization range from 3.7 x 10(-5) to 1.4 x 10(-1)/s; and (iii) values of the association equilibrium constant (Ka) for the proteinase:ETI complexation change from < 1.0 x 10(4) to 3.8 x 10(11)/M. Thus, differences in k(off) values account mostly for the large changes in Ka values for ETI binding. The affinity of ETI for the serine proteinases considered can be arranged as follows: bovine beta-trypsin > human tissue plasminogen activator > bovine alpha-chymotrypsin >> human alpha-, beta- and gamma-thrombin approximately M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator. Moreover, the serine proteinase:ETI complex formation is an endothermic, entropy-driven, process.(ABSTRACT TRUNCATED AT 250 WORDS)

Some Properties of a Functional Reconstituted Plasmalemma H-ATPase Activated by Fusicoccin.

Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H(+)-translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V(max) of the enzyme. Fusicoccin stimulation of maize H(+)-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.).

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to serine (pro)enzymes: a comparative thermodynamic study.

The binding of the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis to serine (pro)enzymes belonging to the chymotrypsin and subtilisin families has been investigated from the thermodynamic viewpoint, between pH 4.5 and 9.5 and from 10 degrees C to 40 degrees C. The affinity of eglin c for the serine (pro)enzymes considered shows the following trend: Leu-proteinase [the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves] greater than human leucocyte elastase congruent to human cathepsin G congruent to subtilisin Carlsberg congruent to bovine alpha-chymotrypsin greater than bovine alpha-chymotrypsinogen A congruent to porcine pancreatic elastase congruent to bovine beta-trypsin. The serine (pro)enzyme-inhibitor complex formation is an entropy-driven process. On increasing the pH from 4.5 to 9.5, the affinity of eglin c for the serine (pro)enzymes considered increases thus reflecting the acid pK shift of the invariant hystidyl catalytic residue from approximately to 6.9 in the free serine proteinases and bovine alpha-chymotrypsinogen A to congruent to 5.1 in the serine (pro)enzyme-inhibitor complexes. Considering the known molecular models, the observed binding behaviour of eglin c was related to the inferred stereochemistry of the serine (pro)enzyme-inhibitor contact regions.

Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature-independent over the range explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

IP3 levels and their modulation FY fusicoccin measured by a novel [3H] IP3 binding assay.

A recently developed sensitive assay based on the binding reaction of IP3 to bovine adrenal preparations has been utilized for determining the level of endogenous inositol-1,4,5 trisphosphate (IP3) in maize roots and coleoptiles. The amount of IP3 found in these tissues ranges from 0.1 to 1.0 nmol g-1 fresh weight. Reproducible results were obtained with extracts of tissues from a same harvest, while they showed a 2-3 fold variation when different batches of plantlets were compared. The fungal phytotoxin fusicoccin (FC) known to affect several physiological processes in higher plants, increases the level of IP3 in coleoptiles. This observation suggests that IP3 might be involved in the transduction of the FC encoded signal from its receptors at the plasmalemma level to the cell machinery.

Fusicoccin: receptors and endogenous ligands.

The first event in the mode of action of the fungal phytotoxin fusicoccin is the binding to specific receptors located at the plasma membrane. They are very likely related to the presence of endogenous compounds able to compete with FC for binding to its receptors. Some biochemical properties of these proteins conform to criteria commonly used for receptors. A functional relationship between fusicoccin receptors and the plasma membrane H(+)-ATPase which is assumed to be the final physiological target of fusicoccin, has been recently suggested. New support in favour of this relationship has been sought by studying a model system prepared by insertion into liposomes of preparations from maize tissues of the two proteins. The model system is capable of H(+)-translocation on ATP addition and the translocation is markedly stimulated by fusicoccin. Thus, it represents a promising tool for the understanding of fusicoccin signal transduction at the molecular level, since it can assist in the detection and assay of the transduction chain components contained in the impure protein preparations.

Properties of proteoliposomes containing fusicoccin receptors from maize.

We have recently described a fusicoccin (FC)-sensitive system reconstituted by inserting into liposomes FC-receptors and H(+)-ATPase-enriched preparations from maize tissues. While the proteoliposomes of maize H(+)-ATPase had been already investigated, those of FC-receptors required a careful characterization before use in the dual system. In particular, the influence of the phospholipid environment on time-course, reversibility, and pH-dependence of the FC-binding reaction has been studied by comparing these properties in microsome-bound, solubilized, and liposome-entrapped receptors. Similarities and differences between the results of this investigation and those previously obtained with FC-receptors from spinach leaves suggest that functionally similar binding proteins from monocot and dicot plants have distinct structural features.

Functional reconstitution of a proton-translocating system responsive to fusicoccin.

Crude fusicoccin binding proteins and a partially purified plasma membrane H+-transporting ATPase (EC 3.6.1.34), both solubilized from maize tissues, were simultaneously inserted into liposomes by the freeze-thaw method. ATP-driven intravesicular acidification in the proteoliposomes, measured by the fluorescence quenching of the dye 9-amino-6-chloro-2-methoxyacridine, markedly increased upon addition of fusicoccin to the reconstituted system. This effect could not be observed when binding sites and ATPase preparations were separately reconstituted into the proteoliposomes, thus demonstrating that fusicoccin binding to its receptor is a prerequisite for ATPase stimulation.