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INTRODUCTION |
14-3-3 are a family of highly conserved proteins widespread in
eukaryotic organisms. A number of biological activities have been
attributed to 14-3-3, including the control of cell cycle, differentiation, apoptosis, targeting of proteins to different cellular
locations, and the coordination of multiple signal-transduction pathways (1-7). The common feature of 14-3-3 proteins is the ability
to bind target proteins, and this accounts for their diverse regulatory
functions (2, 3, 6). Most 14-3-3-interacting proteins bind their
targets in a phosphorylation-dependent manner. Muslin
et al. (8) suggested a consensus motif
RSXSpXP (Sp indicates a phosphoserine) found in
several proteins known to associate with 14-3-3 proteins. This sequence
has been defined in more detail by peptide library screening: two
different phosphoserine motifs, R[S/Ar][+/Ar]Sp[L/E/A/M]P and
RX[S/Ar][+]Sp[L/E/A/M]P (where Ar indicates an
aromatic residue and + a basic residue), defined as mode-1 and mode-2
sequences respectively, were proposed (9). However these motifs cannot
account for all 14-3-3 interactions and, even though phosphorylation
seems to be a general prerequisite for 14-3-3 binding, proteins
interacting in a phosphorylation-independent manner have also been
identified (10-12).
The 14-3-3 dimers have a characteristic cup-like shape with a highly
conserved inner surface and a more variable outer surface (13, 14). In
the concave surface, each monomer presents an amphipathic groove formed
by a cluster of basic residues (Lys49, Arg56,
Lys120, Arg127) on one side, and hydrophobic
residues (Val176, Leu216, Leu220,
Leu227) on the other side. Crystallization studies of
14-3-3
with phosphorylated peptides reproducing mode-1 consensus
sequence demonstrated that phosphoserine interacts by salt bridges with
the side chains of basic residues of the 14-3-3 groove (9, 15).
Accordingly, mutations of Lys49, Arg56, and
Arg60 hamper the association of 14-3-3
with Raf-1 and
exoenzyme S (16). Substitution of residues in the hydrophobic cluster
also affects binding properties (17).
In plants, much less information about the mechanism of interaction
between 14-3-3 proteins and their targets is available. Most of the
binding sequences so far identified are ascribable to the mode-1 motif
(2, 7). A remarkable exception is represented by the plasma membrane
H+-ATPase. In fact, it contains, at the extreme end of its
C-terminal domain, the 14-3-3 binding site YTV (18), which is totally
unrelated to those previously identified. Phosphorylation of the
threonine residue in the YTV sequence, which has been demonstrated to
occur in vivo (19), is required for the interaction with
14-3-3 proteins (18, 20). Association of 14-3-3 proteins with the
H+-ATPase is stabilized by the toxin fusicoccin
(FC)1, known to strongly
activate the H+-ATPase (21). Moreover, FC is able to
trigger the interaction of 14-3-3 with the H+-ATPase also
in the absence of phosphorylation (22).
Here we report on mutational studies carried out to elucidate the
molecular basis of the interaction between 14-3-3 proteins and the
plasma membrane H+-ATPase of plant cells. Different mutants
of the maize 14-3-3 GF14-6 isoform (K56E, K56Q, K56R, and V185E) were
produced and used in interaction studies with the plasma membrane
H+-ATPase and with a peptide reproducing the 14-3-3 binding
site of the enzyme. The ability of 14-3-3 mutants to stimulate the phosphohydrolytic activity of the H+-ATPase was also tested
and correlated to the interaction data. Finally, to investigate the
mechanism of the FC-dependent interaction, binding
experiments between 14-3-3 proteins and mutants of the extreme portion
of the H+-ATPase C terminus were carried out.
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EXPERIMENTAL PROCEDURES |
Chemicals--
FC was prepared according to Ballio et
al. (23); tritiated dihydroFC ([3H]FC) with specific
activity 0.77 TBq/mmol was obtained according to Ballio et
al. (24).
[
-32P]ATP (specific activity 110 TBq/mmol) and
thrombin were from Amersham Biosciences (Upssala, Sweden). Protein
kinase A, catalytic subunit, was from Sigma. bL15Vp peptide
biotynil-LKGLDIDTIQQNYTpV (Tp, phosphothreonine) and MHA2-776p peptide
biotynil-ALIFVTRSRSpWSFVE (Sp, phosphoserine) were synthesized by
Neosystem (Strasbourg, France). Chemicals for gel electrophoresis were
from Bio-Rad (Hercules, California, USA). All other reagents were of
analytical grade.
Plant Material--
Maize seeds (Zea mays L. cv.
Santos) from Dekalb (Mestre, Italy) were germinated and grown in the
dark for 6 days, as already described (25).
Escherichia Coli Strains--
E. coli
strains were grown at 37 °C in LB liquid medium or agar plates (15 g/l). Ampicillin was used at concentration of 100 µg/ml. Strain
DH5
was used for plasmid propagation and strain BL21(DE3) was used
for protein expression.
Site-directed Mutagenesis of 14-3-3 Protein--
pGEX-2TK
carrying the GF14-6 cDNA (pX18 DNA clone) was used as a template
for mutagenesis of GF14-6 14-3-3 protein. Site-directed mutagenesis of
Lys56 was obtained by PCR amplification of the 108-787
fragment using a 5' oligonucleotide primer containing the desired
mutations and SacI restriction site and a 3' oligonucleotide
primer containing EcoRI restriction site complementary to
the 3' end of GF14-6 cDNA. PCR product was directly cloned in
pGEM-T vector (Promega, Madison, Wisconsin, USA) and, after sequencing,
the mutated SacI-EcoRI fragment was recovered and
subcloned in SacI-EcoRI cut pX18.
Mutagenic primers used were (mutations generated are underlined and
restriction sites are in lowercase): Lys56
Glu,
5'-gagctcACTGTGGAGGAGCGAAACCTCTTGTCTGTTGCATACGAGAAC-3'; Lys56
Gln,
5'-gagctcACTGTGGAGGAGCGAAACCTCTTGTCTGTTGCATAC
CAGAAC-3'; Lys56
Arg,
5'-gagctcACTGTGGAGGAGCGAAAC
CTCTTGTCTGTTGCATACAGGAAC-3'; 3' primer,
5'-gaattcCTTACTGCCCCTCGCTCGAGTCCCGCTTG-3'.
Site-directed mutagenesis of Val185 was performed by using
the QuikChange site-directed mutagenesis method (Stratagene, La Jolla, CA). The mutagenic primers used were (mutation generated is
underlined): coding primer
5'-TTAACTTCTCAGAGTTCTACTATGAGATTCTG-3'; complementary primer 5'-CATAGTAGAACTCTGAGAAGTTAAGTGCCA-3'. After 18 cycles of PCR (30 s at 95 °C, 1 min at 53 °C, 11 min 40 s at
68 °C), 10 units of DpnI were added to the mixture to
digest the pX18 DNA template, and reaction was carried out at 37 °C
for 2 h. 20 µl of the mixture were used to transform E. coli DH5
competent cells. Incorporation of mutation was
controlled by DNA sequencing.
Site-directed Mutagenesis of the H+-ATPase C-terminal
Domain--
pGEX-2T carrying the cDNA fragment encoding the last
103 amino acid residues of the H+-ATPase MHA2 isoform
(Maize H+-ATPase isoform
2) (pGEX-E DNA clone) (22) was used as a template. Mutagenesis was obtained by PCR using mutagenic oligonucleotide primers
complementary to the 3' end of the cDNA fragment, containing the
desired mutations and EcoRI restriction site, and a primer based on the 5' end of the cDNA fragment corresponding to
2738-2752 region of the MHA2 gene (26) containing BamHI
restriction site.
Mutagenic primers were (mutations generated are underlined and
restriction sites are in lowercase): Glu938
Ala,
5'-gaattcTCACACCGTGTAGTTCTGCTGGATGGTGTCGA-TGGCCAG-3'; Glu940
Ala,
5'-gaattcTCACACCGTGTAGTTCTGCTGGATGGTGGCGATGTCCAG-3';
Glu938 and Glu940
Ala,
5'-gaattcTCACACCGTGTAGTTCTGCTGGATGGTGGCGATGGCCAG-3';
Thr947
Glu,
5'-gaattcTCACACCTCGTAGTTCTGCTGG-3'; 5' primer,
5'-GCGggattcAGCGGCAGG GCATGG-3'.
Expression in E. coli of Recombinant
Proteins--
Wild-type and mutated GF14-6 proteins were expressed in
E. coli as fusion proteins with the
glutathione-S-transferase (GST) using pGEX-2TK vector,
whereas wild-type and mutant C-terminal domain of MHA2
H+-ATPase were expressed as GST-fusion proteins using
pGEX-2T vector, following the procedure described by Fullone et
al. (22).
Isolation of Maize Plasma Membranes--
Two-phase partitioned
plasma membranes from maize roots were obtained as previously
described (25).
Purification of ER from Yeast Expressing AHA1--
Plasma
membrane H+-ATPase AHA1 isoform (Arabidopsis
thaliana H+-ATPase isoform
1) was expressed in Saccharomyces cerevisiae as previously described (27). After cell homogenization, membranes were
purified by differential centrifugation and ER, containing most of the
AHA1, was isolated by sucrose gradient centrifugation (28).
SDS-PAGE and Overlay Assay--
SDS-PAGE was performed as
described by Laemmli (29), in a Mini Protean apparatus (Bio-Rad).
The system used for expression of wild-type and mutated GF14-6 proteins
produces a GST-fused 14-3-3 containing a cAMP-dependent protein kinase phosphorylation site and a thrombin site between the two
polypeptides. The 32P-labeled wild-type and mutated GF14-6
were obtained as described (22). Specific activities of all the labeled
proteins were similar (about 3 MBq/mg).
The overlay assay was carried out according to Fullone et
al. (22), with minor modifications. Briefly, two-phase partitioned maize plasma membranes (10 µg protein) containing the
H+-ATPase, wild-type and mutated GST-C-terminal domain of
the MHA2 (1 µg) or wild-type and mutated GF14-6 proteins (0.5 µg)
were subjected to SDS-PAGE and blotted onto nitrocellulose membrane, using a semidry LKB apparatus (2 h, 0.8 mA cm
2). The
membrane was blocked with 5% fatty acid-free milk in 25 mM
Hepes-OH, 75 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, 0.05% Tween
20, pH 7.5 (buffer H) and then incubated overnight at 4 °C in the
same buffer containing 2% fatty acid-free milk, 3 µg of
32P-labeled wild-type or mutated GF14-6 proteins
(corresponding to 9 kBq/ml) and, where indicated, 10 µM
FC. After incubation, the membrane was washed three times with buffer
H, dried, and subjected to autoradiography at
80 °C. Each overlay
experiment was performed at least three times, and similar results were obtained.
Binding of GF14-6 to Resin-bound Phosphopeptides--
0.5 nmol
of bL15Vp or MHA2-776p peptides were immobilized onto 40 µl of
streptavidin-agarose resin (Sigma) and incubated in 50 µl of buffer H
along with 3 µg of 32P-labeled wild-type or mutated
GF14-6 (1.4 kBq/µg) for 60 min at room temperature in the absence or
in the presence of 10 µM FC. Resin was then centrifuged
at 2000 × g for 5 min and washed three times with 1 ml
of buffer H containing, where indicated, 10 µM FC.
Resin-bound radioactivity was measured in a liquid scintillation
counter (LKB Wallac 1410).
[3H]FC Binding Assay to the 14-3-3/Phosphopeptide
Complex--
bL15Vp and MHA2-776p peptides were immobilized onto the
streptavidin-agarose resin, in the conditions already described, and incubated for 60 min at room temperature with 10 µg of wild-type or
mutated GST-GF14-6 proteins in the presence of 30 nM
[3H]FC, in 100 µl of buffer H. After three washes with
1 ml of buffer H, resin was added to 10 ml of scintillation liquid and
radioactivity was measured in the
counter (LKB Wallac 1410).
Specific binding was calculated subtracting to the total
[3H]FC-binding the value measured in the presence of
10 µM FC.
ATPase Activity--
AHA1 phosphohydrolitic activity of yeast ER
membranes was assayed according to Marra et al. (28) with
minor modifications: 10 µg of sucrose gradient purified yeast ER were
preincubated with different concentrations of wild-type or GF14-6
mutants (ranging from 0 to 1 µM) in 500 µl of 50 mM Tris-Mes, 5 mM MgCl2, 50 mM KNO3, 5 mM NaN3, 0.2 mM ammonium molybdate and 10 µM FC, pH 7.2 (buffer A). After 20 min incubation, 2 mM ATP was added.
ATP hydrolysis was measured according to Serrano (30).
Analytical Methods--
Protein concentration was determined by
the method of Bradford (31) using bovine serum albumin as the standard.
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RESULTS |
K56E and V185E Mutations Do Not Result in Significant Structural
Changes of GF14-6--
In the 14-3-3
isoform, mutations of
Lys49 and Val176 have been reported to
drastically reduce the interaction with the prototype consensus motif
of Raf-1 (16, 17). To verify whether the association between plant
14-3-3 proteins and the peculiar YTpV binding sequence of the
H+-ATPase shares the same mechanism, we generated the
corresponding mutations of the maize 14-3-3 isoform GF14-6. In Fig.
1 the alignment of 14-3-3
and GF14-6
isoforms shows that Lys49 and Val176 residues
in 14-3-3
correspond to Lys56 and Val185,
respectively, in GF14-6. K56E and V185E mutants were expressed in
E. coli as GST-fusion proteins. The overall structure
integrity of mutant proteins was examined by circular dichroism
analysis and by testing their ability to dimerize. CD spectra of
mutants overlapped that of wild-type protein (data not shown). To check the dimerization properties of mutants, overlay assay experiments were
carried out. In this system, 32P-labeled GF14-6 wild-type,
K56E, and V185E proteins were used as probes and the same unlabeled
proteins, immobilized on nitrocellulose membrane, as baits.
Autoradiography, reported in Fig. 2,
shows that both 32P-labeled mutated proteins were able to
interact each other and with the wild-type, demonstrating that the
ability of mutants to form dimers was unaffected.

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Fig. 1.
Alignment between the Zea mays
14-3-3 GF14-6 isoform and the mammalian 14-3-3
isoform. 14-3-3 residues Lys49 and
Val176, corresponding to Lys56 and
Val185 in GF14-6, are indicated in bold.
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Fig. 2.
Dimerization properties of GF14-6
mutants. Overlay assay was carried out with wild-type and mutant
proteins treated with thrombin to remove GST. 0.5 µg of K56E, V185E,
and wild-type GF14-6 were subjected to SDS-PAGE, and, after blotting,
nitrocellulose membranes were incubated with 32P-labeled
wild-type GF14-6 (a), 32P-labeled K56E mutant
(b) or 32P-labeled V185E mutant (c),
in the conditions indicated under "Experimental Procedures."
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K56E and V185E Mutations hamper the interaction of GF14-6 to the
H+-ATPase--
The effect of K56E and V185E mutations on
the association of 14-3-3 proteins with the H+-ATPase was
investigated by means of the overlay assay. 32P-labeled
K56E, V185E, and wild-type GF14-6 proteins were used as probes and the
H+-ATPase from maize roots as a bait. The results of
autoradiography are reported in Fig.
3a. K56E (2) and V185E (3)
mutations severely hampered the association of 14-3-3 protein with the
H+-ATPase. Binding of mutants, compared with wild-type, is
reduced also in the presence of FC (Fig. 3a), which is known
to strongly increase association of 14-3-3 with the
H+-ATPase (22).

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Fig. 3.
Effect of mutations K56E and V185E of GF14-6
on its interaction with the H+-ATPase. The
overlay assay was performed subjecting 10 µg of plasma membrane
proteins (a) or 1 µg of recombinant MHA2 GST-C terminus
(b) to SDS-PAGE. After blotting, nitrocellulose membranes
were incubated with 32P-labeled wild-type GF14-6
(lane 1), 32P-labeled K56E (lane 2),
or 32P-labeled V185E (lane 3) in the absence or
in the presence of 10 µM FC.
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It is known that 14-3-3 proteins can interact with the
H+-ATPase also in a phosphorylation-independent manner. In
these conditions, the association is completely dependent on FC (18,
20, 22). To verify whether K56E and V185E mutations affect also this
interaction, the ability of mutated proteins to bind the recombinant
C-terminal domain of H+-ATPase, expressed in E. coli as a GST-fusion protein, was tested. As shown in Fig.
3b, for both mutants, the interaction with the C-terminal
domain of the H+-ATPase was almost completely abolished.
Lys56 Mutations Suggest a Role for a Positive Charge in
the Interaction with the H+-ATPase--
The relevance of
the contribution of Lys56 to the H+-ATPase
binding was further studied by mutating it in Gln and Arg and
expressing the mutated GST-fusion proteins in E. coli.
The effect of mutations on the interaction was tested by overlay
experiments. As reported in Fig.
4a, substitution of
Lys56 with the polar residue Gln only partially restored
the binding activity (3). On the other hand, the interaction with the
H+-ATPase was completely rescued by the conservative
mutation K56R (4). The results demonstrate that the positive charge is
also required in the FC-dependent interaction; in fact,
K56Q mutant showed a very weak interaction with both
H+-ATPase (Fig. 4a) and its C-terminal domain
(Fig. 4b).

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Fig. 4.
Effect of different mutations of
Lys56 on the interaction of GF14-6 with the
H+-ATPase. The overlay assay was performed
under the same conditions described in the legend of Fig. 3, using
the full-length H+-ATPase (a) or its
recombinant C-terminal domain (b) as baits in the absence or
in the presence of 10 µM FC. Lane
1, 32P-labeled wild type GF14-6; lane
2, 32P-labeled K56E; lane 3,
32P-labeled K56Q; lane 4,
32P-labeled K56R.
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Lys56 and Val185 Mutations Affect the
Interaction of GF14-6 with Peptides Reproducing 14-3-3 Binding Sites of
H+-ATPase--
A phosphorylated biotinyl-peptide (bL15Vp)
reproducing the last 15 amino acid residues of the MHA2
H+-ATPase and containing the 14-3-3-YTpV binding sequence
was used in interaction studies with GF14-6 mutants. The peptide was
immobilized onto a streptavidin-agarose resin and incubated with
32P-labeled wild-type and mutant proteins. Interestingly,
the results, reported in Fig. 5, were
only partially in accordance with those obtained by overlay assays. In
fact, although, as expected, K56E and V185E mutants did not interact
with bL15Vp peptide and K56Q interacted very weakly, the conservative
mutant K56R bound bL15Vp more efficiently than the wild-type GF14-6
did.

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Fig. 5.
Binding of wild-type and mutant GF14-6
proteins to bL15Vp and MHA2-776p phosphopeptides. Peptides were
immobilized onto streptavidin-agarose resin and incubated with
32P-labeled wild-type or mutant GF14-6 in the absence
or in the presence of 10 µM FC. After washing,
radioactivity associated with resin was measured. Data are the
mean of three independent experiments.
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The same binding experiments were carried out with a phosphorylated
peptide (MHA2-776p) reproducing a putative 14-3-3-binding site located
in the cytosolic stretch between the 8 and 9 helices of the MHA2
H+-ATPase. This peptide contains the mode-1 related
sequence RSRSpWS, and it has been reported to interact with GF14-6
protein (28); however, the physiological relevance of this interaction
has not yet been demonstrated.
The results were substantially in accordance with those obtained with
bL15Vp peptide. Yet a remarkable difference was observed when the
experiments were performed in the presence of FC; in fact, whereas FC
strongly stabilized the bL15Vp/14-3-3 complexes, it was completely
ineffective on the MHA2-776p/14-3-3 interaction (Fig. 5). These data
were confirmed by assaying the ability of peptides/14-3-3 complexes to
bind a tritiated derivative of FC. In fact, as reported in Fig.
6, only the complexes between bL15Vp peptide and wild-type GF14-6, or K56R mutant, were able to bind [3H]FC.

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Fig. 6.
[3H]FC binding to
peptides/GF14-6 proteins complexes. bL15Vp and MHA2-776p peptides
were immobilized onto streptavidin agarose beads and incubated with
wild-type or mutated GF14-6 proteins in the presence of 30 nM [3H]FC. Data are the mean of three
independent experiments.
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Lys56 and Val185 Mutations Affect
14-3-3-induced Stimulation of H+-ATPase--
The effect of
Lys56 and Val185 mutations on the in
vitro activation of the H+-ATPase induced by 14-3-3 proteins was tested by using purified ER vesicles of S. cerevisiae expressing the H+-ATPase AHA1 isoform of
Arabidopsis thaliana. In this system, a significant and
reproducible stimulation of H+-ATPase can be obtained by
exogenous 14-3-3 proteins, provided that FC is also added.
ER membranes were incubated with different amounts of wild-type or
mutated GF14-6 proteins in the presence of 10 µM FC. The results, reported in Fig. 7, demonstrate
that the ability to stimulate the H+-ATPase activity
depends on the ability to associate with it. In fact, only the
wild-type and K56R GF14-6, which efficiently bound
H+-ATPase in the interaction experiments (Fig. 4),
stimulated AHA1 phosphohydrolytic activity.

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Fig. 7.
Effect of mutations of Lys 56 and Val 185 on
the ability of GF14-6 to stimulate the phosphohydrolytic activity of
AHA1 H+-ATPase. Purified yeast ER vesicles
(10 µg) were incubated with different concentrations of wild-type or
mutated GF14-6 proteins in the presence of 10 µM FC. ,
wild type; , K56E; , K56Q; , K56R; , V185E. Data are the
mean of three independent experiments.
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Effect of the D938A/D940A Double Mutation and the T947E Mutation,
Both in the H+-ATPase C Terminus, on
FC-dependent Interaction with GF14-6--
R18 is a
synthetic peptide selected from a phage display library for its ability
to bind 14-3-3 proteins in a phosphorylation-independent manner (32).
It has been proposed that binding property is due to the motif WLDLE.
Interestingly, in the H+-ATPase C terminus a very similar
sequence, namely 936GLDID (numbered according to MHA2
isoform), located close to the C-terminal end is present. To verify
whether this sequence could be involved in the
phosphorylation-independent, FC-dependent interaction, single and double mutation of the two acidic residues,
Asp938 and Asp940, were obtained.
The wild-type and mutated C-terminal domains were expressed in E. coli as GST-fusion proteins and assayed for their ability to
interact with 32P-labeled GF14-6 in overlay experiments, in
the presence of 10 µM FC.
As shown in Fig. 8A, mutation
D940A (lane 3) did not affect the binding of recombinant
H+-ATPase C terminus, mutation D938A (lane 2)
reduced the binding, and double mutation (lane 4) completely
abolished it. These results demonstrated the relevance of the two
negatively charged residues for the FC-dependent
interaction with GF14-6.

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Fig. 8.
Role of negative charges in the
H+-ATPase C terminus in the
FC-dependent interaction with GF14-6. MHA2 GST-C
terminus mutants were subjected to overlay assay using
32P-labeled GF14-6 as a probe. A, wild-type
GST-C-terminal domain of MHA2 H+-ATPase (lane
1), D938A mutant (lane 2), D940A mutant (lane
3), and D938A-D940A double-mutant (lane 4) were assayed
in overlay assay in the presence of 10 µM FC.
B, comparison between wild-type GST-C-terminal domain of
MHA2 H+-ATPase (lane 1) and T947E mutant
(lane 2) in the interaction with 32P-labeled
GF14-6 in the absence or in the presence of 10 µM FC. The
minor signal intensity of MHA2 wild-type C-terminal domain, compared
with that shown in A, is due to a shorter time of exposure
of the autoradiography.
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A further indication of the importance of negative charges in the
FC-dependent interaction was obtained by means of the T947E mutant. It contains a negatively charged residue in place of the phosphorylatable threonine residue, within the YTV 14-3-3 binding sequence. This mutant had been produced to verify whether the negative
charge of the glutamic acid could mimic the phosphate group.
Autoradiography reported in Fig. 8B showed that no
interaction occurred between GF14-6 and the mutated C terminus.
Surprisingly, however, in the presence of FC, the T947E mutant
(lane 2) interacted more efficiently than did the wild-type
C-terminal domain.
 |
DISCUSSION |
The plasma membrane H+-ATPase of plant cell is
regulated by 14-3-3 proteins. It contains the 14-3-3 binding motif YTV,
located at the extreme end of the C-terminal autoinhibitory region of the enzyme. This is a novel sequence completely unrelated to the well
characterized mode-1 and mode-2 motifs, which accounts for most of
14-3-3 interactions with target proteins. Hence, we were interested in
identifying residues important for binding activity of a plant 14-3-3 protein and ascertaining whether the interaction with the
H+-ATPase underlies the same mechanism reported for the
interaction with canonical motifs (9, 15). To this purpose, single
amino acid mutations known to affect the binding properties of animal 14-3-3 proteins were reproduced in the plant 14-3-3 isoform GF14-6, and
their effects on the ability to interact with and to activate the
H+-ATPase were investigated.
In this study, we show that K56E and V185E mutations drastically hamper
the interaction of GF14-6 with the H+-ATPase. These results
indicate that Lys56 and Val185 are both crucial
for binding to the YTpV sequence, thereby suggesting that basic and
hydrophobic clusters in the conserved amphipathic groove of 14-3-3 are
involved also in this interaction. This finding is worth noting,
because the H+-ATPase binding site lacks the main features
of classical consensus sequences, namely a basic residue at the
3/
4
position as well as a proline at the +2 position. It has been suggested
that the proline residue is important to fold the binding site,
allowing the entrance of the C-terminal portion back toward to the
binding groove (33); probably in the H+-ATPase the
localization of the phosphoamino acid residue at the extreme C-terminal
end of the protein renders the role of the proline residue unnecessary.
Interestingly, the 14-3-3 binding sequence GHSL of the glycoprotein
Ib-IX-V complex protein (34), which also lacks the proline at +2 Sp
position, is likewise located at the extreme C-terminal end of the protein.
The importance of a positively charged residue at position 56 of GF14-6
has been further probed by substituting the Lys residue with a Gln or
Arg residue. In comparison with the mutation K56E, the polar K56Q
partially, and the conservative K56R completely, restored the binding
activity. Interestingly, efficiencies of H+-ATPase
stimulation by mutants directly paralleled binding affinities.
The interaction mechanism with H+-ATPase was further
investigated by studying the binding of wild-type and mutant GF14-6 to the bL15Vp peptide, reproducing the last 15 residues of MHA2
H+-ATPase and containing the YTpV sequence. The results
were in good agreement with those observed with the whole enzyme in the overlay experiments. However, the K56R mutant, compared with the wild-type GF14-6, displays a much higher affinity toward the bL15Vp peptide than to the H+-ATPase. A possible explanation is
that the bL15Vp peptide does not exactly match the conformational
features occurring in the whole enzyme, which can be important in the
physiological interaction of the enzyme.
It is worth noting that no FC effect was detected when the interaction
studies were performed with the MHA2-776p peptide, which reproduces a
putative 14-3-3 binding site in the H+-ATPase (28) related
to a mode-1 binding motif. This strongly suggests that the ability to
bind FC is a unique feature of the complex between 14-3-3 protein and
the extreme portion of the C-terminal domain of the proton pump.
FC strongly stabilizes the interaction between 14-3-3 and the
H+-ATPase and brings about 14-3-3 binding also in the
absence of enzyme phosphorylation. The results of the overlay assay
with the recombinant unphosphorylated C-terminal domain of the
H+-ATPase demonstrated that Lys56 and
Val185 are both essential for FC-induced binding. This
indicates that the same amphipathic 14-3-3 binding groove is involved
also in the FC-dependent interaction, occurring in the
absence of phosphorylation. This result appears particularly relevant
because it has been observed that binding with unphosphorylated 14-3-3 targets does not required the same hydrophobic residues involved in the
interaction with phosphorylated proteins (35).
It has been proposed (9) that the function of the basic cluster in
14-3-3 proteins is to interact with the negatively charges of the
phosphoamino acid within the binding site; in this respect, the
question is open about the effect of Lys56 mutation, which
abolishes the binding also in the absence of phosphorylation of the
C-terminal domain of the H+-ATPase. An explanation for this
result was obtained by studying the effect of point mutation of the
H+-ATPase C terminus. In fact, substitution of the two Asp
with Ala residues, within the sequence 936GLDID (numeration
according to MHA2 isoform) located close to the YTV sequence,
completely abolished the ability of recombinant C-terminal domain of
H+-ATPase to bind GF14-6 in the presence of FC. This
demonstrates that negative charges play a role also in the
FC-dependent interaction, probably mimicking the phosphate
group function in the 14-3-3/target complex formation. This hypothesis
has also been proposed for the unphosphorylated R18 peptide, which
contains the very similar sequence WLDLE; in fact, in the 14-3-3
/R18
complex, the two Asp and Glu acidic residues of peptide are located
next to the basic cluster of the 14-3-3 protein (15). Intriguingly
exoenzyme S, which interacts with 14-3-3 proteins in a
phosphorylation-independent manner, possesses a homologous motif,
245FGADAE, in its C-terminal region (12).
The role of negative charges in the FC-dependent
interaction can also be inferred from data obtained with the T947E
mutant. In fact, the introduction of a further negatively charged
residue brought about a marked increase in the ability to bind 14-3-3 protein.
In conclusion, our data show that positively charged residues in GF14-6
binding groove and negatively charged residues in the C-terminal domain
of H+-ATPase play a crucial role in binding between these
proteins, even when, as in the FC-dependent interaction, it
occurs in the absence of H+- ATPase phosphorylation.