From the Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Received for publication, November 21, 2002, and in revised form, December 31, 2002
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ABSTRACT |
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Drosophila 14-3-3 14-3-3 proteins comprise a ubiquitous family of highly conserved
proteins. They bind specifically to a variety of target proteins containing phosphoserine motifs and function as intracellular regulator
or adaptor proteins in diverse cellular functions (1, 2). The crystal
structures of human 14-3-3 There are only two isoforms of 14-3-3 in Drosophila,
D14-3-3 To investigate the molecular mechanism of this regulatory protein
complex, we explored the role of 14-3-3 dimerization in regulating dSlo
channel function. We report here that D14-3-3 Antibodies--
Rabbit polyclonal anti-dSlo, anti-Slob, and
anti-14-3-3 antibodies were generated and purified as described
previously (13, 14). Monoclonal anti-FLAG antibody (M2) was purchased
from Sigma. Anti-1D4 monoclonal antibody was kindly provided by Dr. D. Oprian (Brandeis University).
cDNA Constructs and Mutagenesis--
The 14-3-3 dimerization-deficient mutants were created using site-directed
mutagenesis of the following D14-3-3 Transfection and Immunoprecipitation--
tsA201 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum. A calcium phosphate transfection protocol was
used to introduce cDNAs into the cells. Forty-eight hours after
transfection, cells were lysed in lysis buffer containing 1% CHAPS, 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 120 mM NaCl, 50 mM KCl, 2 mM
dithiothreitol, and protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml each of aprotinin, leupeptin, and pepstatin A (Sigma)). After a centrifugation to remove
insoluble debris from the lysate, the supernatant was precleared with
50 µl/ml protein A/G plus-agarose (Santa Cruz Biotechnology, Santa
Cruz, CA). 14-3-3 proteins were immunoprecipitated by incubation with
anti-1D4 or anti-FLAG antibodies (4 µg/ml) for 2 h at 4 °C, followed by overnight incubation at 4 °C with 50 µl/ml protein A/G
plus-agarose. The immunoprecipitates were then washed with lysis buffer
five times.
Chemical Cross-linking--
Transfected cells were lysed in a
HEPES-based lysis buffer containing 1% Triton, 50 mM HEPES
(pH 7.5), 10% glycerol, 150 mM NaCl, 1 mM
NaNO3, 1 mM phenylmethylsulfonyl fluoride, and
protease inhibitor mixture. Disuccinimidyl suberate (DSS) was added to the lysates to a final concentration of 0.5 mM, and they
were incubated at 4 °C with slow rotation for 1 h. The reaction
was quenched with 1× SDS gel-loading buffer.
Western Blot--
Proteins in the cell lysates or
immunoprecipitates were separated on polyacrylamide gels and
transferred to nitrocellulose membranes. After blocking with 5% nonfat
milk in TBST (0.1% Tween 20 in TBS), the blots were probed with
appropriate primary antibodies in blocking buffer at 4 °C overnight.
The membranes were then washed with TBST and incubated with a 1:3000
dilution of horseradish peroxidase-conjugated donkey anti-rabbit or
sheep anti-mouse IgG (Amersham Biosciences, Arlington Heights, IL) for
1 h at room temperature. After three washes of the membrane with
TBST and one wash with TBS, protein complexes were visualized using the enhanced chemiluminescence (ECL) system (Amersham Biosciences).
Whole-cell Recording--
dSlo channel activity was recorded in
the whole-cell configuration from tsA201 cells expressing dSlo-EGFP and
Slob, together with pEBFP vector, or wild-type or
dimerization-deficient mutant D14-3-3 Dimerization of D14-3-3
The ability of D14-3-3
All three D14-3-3 Dimerization-deficient Mutants of D14-3-3
Wild-type D14-3-3 Monomeric Form of D14-3-3
To determine if the D14-3-3 Modulation of dSlo Channel Activity by D14-3-3
We first asked whether R59A/R63A D14-3-3
Moreover, when an excess of R59A/R63A D14-3-3
We next investigated whether R59A/R63A D14-3-3 Since their rediscovery in the late 1980s, 14-3-3 proteins have
been recognized as a family of multifunctional proteins that bind to
and regulate the function of a variety of cellular proteins (1, 7, 17).
While efforts continue to identify novel 14-3-3 binding partners,
recent attention has focused on investigating the molecular mechanism
of 14-3-3 function, especially the role of 14-3-3 dimerization (2, 18).
Two modes of action have been attributed to the dimeric nature of
14-3-3 proteins. First, because one 14-3-3 dimer allows simultaneous
binding of two ligands, 14-3-3 proteins can serve as a scaffold to
bridge the interaction between two different proteins. Such
14-3-3-mediated association has been reported for several proteins
(19-21). On the other hand, 14-3-3 proteins can cause conformational
changes in their binding partners via interaction of the 14-3-3 dimers
with multiple sites in the target protein (8, 22).
We demonstrated previously that D14-3-3 Using a combination of molecular biological and biochemical methods, we
demonstrate here that D14-3-3 It is not uncommon to find examples in which dimerization of 14-3-3 is
not essential for its function (25, 26). Our studies provide evidence
that binding of monomeric D14-3-3 It is well-established that ion channels are subject to modulation by
intimately associated proteins, including auxiliary channel subunits,
protein kinases, phosphatases, and other regulatory proteins. Although
14-3-3 proteins are abundant in brain, exploration of the function of
14-3-3 proteins in the nervous system has just begun. Several groups
have reported that 14-3-3 proteins interact with various types of ion
channels and membrane receptors, including Ca2+-activated
Cl (D14-3-3
)
modulates the activity of the Slowpoke calcium-dependent
potassium channel (dSlo) by interacting with the dSlo binding protein,
Slob. We show here that D14-3-3
forms dimers in vitro.
Site-directed mutations in its putative dimerization interface result
in a dimerization-deficient form of D14-3-3
. Both the wild-type and
dimerization-deficient forms of D14-3-3
bind to Slob with similar
affinity and form complexes with dSlo. When dSlo and Slob are expressed
in mammalian cells, the dSlo channel activity is similarly modulated by
co-expression of either the wild-type or the dimerization-deficient
form of D14-3-3
. In addition, dSlo is still modulated by wild-type
D14-3-3
in the presence of a 14-3-3 mutant, which does not itself
bind to Slob but forms heterodimers with the wild-type 14-3-3. These data, taken together, suggest that monomeric D14-3-3
is capable of
modulating dSlo channel activity in this regulatory complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and 14-3-3
reveal that 14-3-3 proteins
share a similar dimeric structure, with each monomer consisting of nine
-helices organized in an antiparallel array (3, 4).
Co-crystallization of several ligand-bound 14-3-3 complexes further
demonstrates that the 14-3-3 dimer is arranged in such a way that the
ligand binding groove runs in opposite directions in each monomer,
allowing simultaneous binding of two ligands to one 14-3-3 dimer (5).
The ability of 14-3-3 proteins to form homodimers or heterodimers in
cells has also been demonstrated using biochemical approaches (6, 7).
However, the significance of 14-3-3 dimerization for target protein
regulation has only been shown in a few instances. For example,
although Raf binds equally well to both the dimeric and monomeric forms of 14-3-3, only the dimeric form of 14-3-3 is able to maintain Raf in
an inactive state in the absence of GTP-bound Ras and to stabilize an
active conformation of Raf in vivo (8). Other evidence
supporting the importance of 14-3-3 dimerization comes from studies
with a mutant form of 14-3-3 that sequesters endogenous 14-3-3 into
heterodimers, in which only the wild-type subunit is capable of binding
to target proteins (9, 10).
and
. D14-3-3
is highly enriched in brain and
presynaptic termini of motor axons and plays important roles in
regulating olfactory learning and synaptic functions (11, 12). In
previous studies, we have shown that D14-3-3
forms a regulatory
protein complex with the Drosophila
calcium-dependent potassium channel (dSlo),1 mediated by its
binding to a novel channel binding protein, Slob (13, 14). The
interaction between D14-3-3
and Slob is dependent upon the
phosphorylations of two serine residues located at the N-terminal
domain of Slob. Co-expression of dSlo with D14-3-3
and Slob in
transfected cells results in a dramatic decrease in dSlo channel
activity (14). These results provide evidence that D14-3-3
may
participate in the regulation of ion channel activity and synaptic
transmission in the nervous system.
forms dimers when
expressed in cell lines. Similar to mammalian 14-3-3, substitution of
key amino acids in the N-terminal domain of D14-3-3
abolishes its
dimerization and produces a monomeric form of D14-3-3
. We found that
the D14-3-3
monomer is capable of forming a complex with dSlo, via
binding to Slob. Moreover, the dSlo channel activity, measured using
the whole-cell voltage clamp technique, is similarly modulated when
dSlo is co-expressed with Slob, together with either the wild-type or
the dimerization-deficient mutant form of D14-3-3
. We also show that
modulation of dSlo channel activity by wild-type D14-3-3
is not
affected by overexpression of another 14-3-3
mutant, which can
heterodimerize with wild-type 14-3-3 but does not itself bind to Slob.
Taken together, these results suggest that monomeric D14-3-3
is
capable of modulating dSlo channel activity in this regulatory complex.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
amino acids: L15AE to Q15QR and
R88VE to N88VQ. A PCR strategy with appropriate primers was used to
introduce the pertinent mutations and to generate 1D4 or FLAG epitope
tags on the C terminus of D14-3-3
cDNA. The PCR products were
then cloned into the mammalian expression vector, pcDNA3. Wild-type
and dimerization-deficient mutant 14-3-3
-EBFP cDNAs were
constructed by cloning the respective D14-3-3
cDNA into the
vector pEBFP-N1 (Clontech). QuikChange strategy
(Stratagene) was used to generate mutations in D14-3-3
at amino acid
residues Arg-59 and Arg-63 (R59A/R63A). Construction of
dSlo-pcDNA3, dSlo-EGFP, and Slob-pcDNA3 was as described
previously (13, 14).
-EBFP. For experiments with
R59A/R63A 14-3-3-1D4, cells were transfected with dSlo-EGFP, Slob and
WT D14-3-3
-EBFP together with pcDNA3 vector, or R59A/R63A
14-3-3-1D4, in a 1:1:1:2 molar ratio. The FuGENE 6 transfection reagent
(Roche Molecular Biochemicals, Indianapolis, IN) was used to transfect
cells with these cDNAs. Recordings were done 1-2 days after
transfection on an Axiovert 25 inverted fluorescence microscope
(Zeiss). Transfected cells bearing both GFP and BFP fluorescence were
identified with the fluorescein isothiocyanate and BFP filter sets.
Patch electrodes with resistances of 1-3 M
were pulled from
borosilicate glass and fire-polished. The bath solution contained (in
millimolar): 30 KCl, 120 NaCl, 2 MgCl2, 1 EGTA, and 10 HEPES (pH 7.2). The pipette solution contained (in millimolar): 150 KCl, 2 MgCl2, 0.5 BAPTA, and 10 HEPES (pH 7.2). Free
calcium concentrations in the pipette solution were determined as
described previously (15). Whole-cell currents were filtered at 1 kHz
and digitized at 20 kHz with an Axopatch 200A amplifier. Data
acquisition and analysis were performed with pCLAMP8 software. All
results are shown as mean ± S.E. Statistical significance was
assessed by one-way analysis of variance.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
The crystal structure of human
14-3-3
shows that the dimerization interface is formed
by interaction of helix A with helices C and D (3, 4). Substitutions of
several key amino acids in both helices A and D result in a
dimer-deficient form of human 14-3-3
(8). Because D14-3-3
is
highly homologous to its mammalian counterparts, with an 81.2% amino
acid sequence identity between human 14-3-3
and D14-3-3
(Fig.
1A), we used site-directed
mutagenesis to assess the role of these key amino acids in dimer
formation of D14-3-3
. Mutations of D14-3-3
were made in helix A
(L15AE to Q15QR, MW-14-3-3), or helix D (R88VE to N88VQ, WM-14-3-3), or
both (MM-14-3-3) (Fig. 1, A and B). For
biochemical studies, cDNA constructs for both the wild-type and
mutant forms of D14-3-3
were tagged with either a 1D4 or FLAG
epitope (Fig. 1B).
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Fig. 1.
Site-directed mutagenesis for dimerization of
D14-3-3 . A, amino acid
alignment for human and Drosophila 14-3-3
isoforms. The
conserved amino acids are shown as capital letters. The
regions corresponding to helices
A-
I of human (H)
14-3-3
are indicated with shaded bars. The amino acids
mutated for dimerization are surrounded with solid boxes for
D14-3-3
or dotted boxes for H14-3-3
. B,
schematic representation of the wild-type (WT) and mutant
forms of D14-3-3
constructed in this study. Mutations are introduced
in two separate regions within helix A and D. The mutations are
indicated above their respective positions. Constructs with mutations
only in helix A are denoted as MW (for mutant-wild-type),
only in helix D as WM (wild-type-mutant), and in both
helices as MM (mutant-mutant). Each construct is fused with
either a 1D4 or FLAG epitope tag.
to dimerize was first tested using a
co-immunoprecipitation approach. Wild-type D14-3-3
s with the 1D4 and
FLAG epitope tags were co-expressed in tsA201 cells. D14-3-3
-FLAG was immunoprecipitated from cell lysates with anti-FLAG antibody, and
the immunoprecipitate was probed with anti-1D4 antibody on a Western
blot. As shown in Fig. 2A,
lane 1, D14-3-3
-1D4 co-immunoprecipitates with
D14-3-3
-FLAG. In addition, we used a chemical cross-linking method
to verify the dimerization of D14-3-3
. When cell lysates from
wild-type D14-3-3
-transfected cells were incubated with DSS, two
bands corresponding to the molecular weights of the monomeric and
dimeric D14-3-3
were detected on the Western blot (Fig.
2B, lane 1). Taken together, these results show
that wild-type D14-3-3
is capable of forming dimers in
vitro.
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Fig. 2.
Dimerization of D14-3-3 .
A, tsA201 cells were co-transfected with WT-FLAG and WT-1D4
(lane 1), WM-FLAG and WM-1D4 (lane 2), MW-FLAG
and MW-1D4 (lane 3), and MM-FLAG and MM-1D4 (lane
4) forms of D14-3-3
. Cell lysates were probed for the
expression of wild-type and mutant forms of D14-3-3
with either the
anti-1D4 (middle panel) or anti-FLAG (bottom
panel) antibodies. FLAG immunoprecipitates were probed with
anti-1D4 antibody to detect the presence of 1D4-tagged D14-3-3
. A
band corresponding to the molecular weight of D14-3-3
is detected in
immunoprecipitates of WT- and WM-D14-3-3
but not MW- and
MM-D14-3-3
transfected cells (top panel). B,
cell lysates from tsA201 cells co-transfected with WT-FLAG and WT-1D4
(lane 1), WM-FLAG and WM-1D4 (lane 2), MW-FLAG
and MW-1D4 (lane 3), MM-FLAG and MM-1D4 (lane 4),
and WT-FLAG and vector (lane 5) were chemically cross-linked
with DSS, and probed on Western blot with a polyclonal antibody that
recognizes D14-3-3
. Bands corresponding to the dimer form of
D14-3-3
are present in WT and WM (lanes 1 and
2) but not MW and MM (lanes 3 and 4)
D14-3-3
-transfected cell lysates. The dimer band of D14-3-3
is
also seen in lysates from WT-FLAG transfected cells (lane
5).
mutants are readily expressed in tsA201
cells. On a Western blot, they migrate at positions similar to that of
wild-type D14-3-3
(Fig. 2A), suggesting that they are metabolically stable. We then examined the effect of these mutations on
D14-3-3
's ability to dimerize. As shown in Fig. 2 (A and
B), although substitutions of the two amino acids in helix D
(WM-D14-3-3
) have little effect on the dimerization of D14-3-3
(lane 2), mutations of the three amino acids in helix A
(MW-D14-3-3
) completely abolish the formation of a D14-3-3
dimer
in vitro, as determined by both co-immunoprecipitation and
cross-linking assays (lane 3). In addition, substitution of
all five amino acids in both helices A and D (MM-D14-3-3
) also
results in a total loss of D14-3-3
dimerization (Fig. 2, A and B, lane 4). These data indicate
that the key amino acids identified in human 14-3-3
are conserved
and critical for dimerization of D14-3-3
as well (8).
Bind Slob and Form
Complexes with dSlo--
To determine if the dimerization-deficient
mutants of D14-3-3
are capable of binding Slob, we again utilized
the co-immunoprecipitation strategy. Wild-type or
dimerization-deficient mutants of D14-3-3
were co-expressed with
Slob in tsA201 cells, and lysates were immunoprecipitated with
antibodies specific for 14-3-3. The immunoprecipitates were probed with
anti-Slob antibody for 14-3-3-bound Slob. As shown in Fig.
3, all three mutants of D14-3-3
bind
Slob as well as does wild-type D14-3-3
, suggesting that these
mutations in D14-3-3
do not affect its ability to interact with
Slob. These results suggest that the D14-3-3
monomer is sufficient
to bind Slob.
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Fig. 3.
Dimerization of D14-3-3
is not required for Slob binding. tsA201 cells were
transfected with Slob together with one of the FLAG-tagged D14-3-3
constructs: WT (lane 1), WM (lane 2), MW
(lane 3), MM (lane 4), or a control vector
(lane 5). D14-3-3
was immunoprecipitated with anti-FLAG
antibody and probed on Western blot with anti-Slob antibody (top
panel). Slob is present in immunoprecipitates from the WT
(lane 1) and all three variants of mutant D14-3-3
(lanes 2-4) but not in that from vector-transfected cells
(lane 5). Expression of both Slob (bottom panel)
and all forms of D14-3-3
(middle panel) is similar under
all transfection conditions.
interacts with the dSlo channel, via its binding
to Slob (14). We asked whether the dSlo·Slob·D14-3-3
protein complex persists with monomeric D14-3-3
. Either the
wild-type or dimerization-deficient mutant D14-3-3
(MM-D14-3-3
)
was co-expressed with both Slob and dSlo in tsA201 cells, and the
14-3-3 immunoprecipitate was probed on a Western blot with anti-dSlo
antibody for dSlo protein present in the complex. As shown in Fig.
4, dSlo was co-immunoprecipitated equally
well with both the wild-type (lane 1) and
dimerization-deficient mutant MM-D14-3-3
(lane 2). These
results show that, in the presence of Slob, the monomeric D14-3-3
is
able to enter into a protein complex with the dSlo channel.
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Fig. 4.
The dimerization-deficient mutant
D14-3-3 forms a complex with dSlo
channels. tsA201 cells were transfected with dSlo and Slob
together with FLAG-tagged WT (lane 1), MM-D14-3-3
(lane 2), or a control vector (lane 3).
D14-3-3
was immunoprecipitated with anti-FLAG antibody and probed on
Western blot with anti-dSlo antibody (top panel). dSlo is
present in immunoprecipitates from both WT (lane 1) and the
MM-D14-3-3
(lane 2) but not in that from
vector-transfected cells (lane 3). Expression of dSlo
(middle panel) and the WT and MM-D14-3-3
(bottom
panel) is similar under all transfection conditions.
Modulates dSlo Channel
Activity--
We have shown previously that co-transfection of
D14-3-3
, together with Slob, dramatically decreases the activity of
dSlo channels (14). To investigate whether dimerization of D14-3-3
is necessary for modulation of the dSlo channel by D14-3-3
, we measured dSlo currents using the whole-cell voltage-clamp
configuration. dSlo-EGFP and D14-3-3
-EBFP were used in these
experiments to identify co-transfected cells by fluorescence. In tsA201
cells transfected with cDNAs for both dSlo and Slob, whole-cell
K+ currents were elicited by depolarizing voltage steps
from a holding potential of
80 mV, with 30 µM
intracellular free Ca2+ (Fig.
5A). When wild-type D14-3-3
was co-expressed with Slob and dSlo, dSlo channel activity was
decreased dramatically (Fig. 5B), as evidenced by a slowdown
of activation kinetics (Fig. 5D) and a reduction of peak
current amplitude (Fig. 5E) evoked by the same
depolarizations. This is consistent with our previous study in
detached membrane patches (14).
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Fig. 5.
dSlo channel activity is modulated by both
dimeric and monomeric D14-3-3 . dSlo
currents were evoked by 350-ms depolarizing voltage steps from
30 mV
in 10-mV increments, in the whole-cell voltage clamp configuration.
Representative current traces recorded from tsA201 cells transfected
with dSlo and Slob together with a control vector (A), the
WT-D14-3-3
(B), or the MM-D14-3-3
(C).
D, the activation time constant (
) (mean ± S.E.),
from single-exponential fits of dSlo currents evoked at +150 mV, is
significantly increased with co-transfection of either the WT or
MM-D14-3-3
(p < 0.05). E, the peak dSlo
current density at +150 mV (mean ± S.E.), normalized to membrane
capacitance, is significantly lower in cells co-transfected with either
the WT or MM D14-3-3
(p < 0.05).
dimer is required to modulate the dSlo
channel activity in this protein complex, we fused EBFP to one of the
dimerization-deficient mutants of D14-3-3
, MM-D14-3-3
, which does
not dimerize but interacts with dSlo via Slob (Figs. 2-4). Fusion of
EBFP to wild-type and mutant D14-3-3
does not interfere with their
binding to Slob (data not shown). In addition, we used chemical
cross-linking to confirm that EBFP-tagged wild-type 14-3-3 proteins
still form dimers, but MM-D14-3-3-EBFP does not dimerize (data not
shown). As shown in Fig. 5C, co-transfection of
MM-D14-3-3
, together with Slob and dSlo, also decreases
the whole-cell current of dSlo channels. The extent of reduction of
current amplitude and the slowdown of activation by co-transfection of
D14-3-3
is not significantly different between wild-type and
dimerization-deficient forms of D14-3-3
(Fig. 5, D and
E). At +150 mV, the time constant for activation (
)
of dSlo current is 7.8 ± 4.3 ms in the absence of D14-3-3
and
120.9 ± 43.2 ms or 134.9 ± 71.5 ms in the presence of
wild-type or mutant D14-3-3
, respectively (Fig. 5D). The
dSlo current density at +150 mV is 805 ± 397 pA/picofarad (pF) in
control and 353 ± 80 pA/pF in WT co-transfected and 334 ± 126 pA/pF in MM-D14-3-3
co-transfected cells (Fig. 5E).
Both parameters are significantly different between control and either
the wild-type or mutant D14-3-3
co-transfected cells but not between
wild-type and mutant D14-3-3
groups. This suggests that the
monomeric D14-3-3
is sufficient to modulate dSlo channel activity.
Is Not Affected
by Overexpression of a D14-3-3
Mutant That Does Not Bind
Slob--
To explore further the role of dimerization of D14-3-3
in
the modulation of dSlo channel activity, we constructed another mutant,
R59A/R63A D14-3-3
, which no longer binds Slob (Fig
6A) but still forms a dimer
with wild-type D14-3-3
(Fig. 6B, lane 1). It
has been reported that similar mutants of mammalian 14-3-3 sequester
wild-type 14-3-3 proteins into a heterodimer form, in which only the
wild-type subunit is able to bind the target protein. This mutant thus
can function as a dominant negative when dimer binding is required for
the targeting of 14-3-3 proteins (9, 16).
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Fig. 6.
R59A/R63A D14-3-3
does not bind Slob but forms a heterodimer with 14-3-3. A, tsA201 cells were transfected with Slob together with
either WT or R59A/R63A D14-3-3
-1D4. D14-3-3
was
immunoprecipitated with anti-1D4 antibody and probed on a Western blot
with anti-Slob antibody (top panel). Slob is only present in
immunoprecipitates from WT (lane 1) but not in those from
R59A/R63A D14-3-3
-1D4 (lane 2) co-transfected cells.
Expression of Slob (middle panel) and the WT and R59A/R63A
D14-3-3
-1D4 (bottom panel) are similar under both
transfection conditions. B, R59A/R63A D14-3-3
-1D4 was
co-transfected with either WT or the dimerization-deficient mutant
MM-14-3-3-EBFP. D14-3-3
-1D4 was immunoprecipitated with anti-1D4
antibody and probed on a Western blot with anti-14-3-3 antibody
(top panel). 14-3-3-EBFP is present in immunoprecipitates
from WT (lane 1) but not in those from MM-D14-3-3
-EBFP
(lane 2) co-transfected cells. Expression of WT and
MM-14-3-3-EBFP (middle panel) and R59A/R63A D14-3-3
-1D4
(bottom panel) are similar under both transfection
conditions.
can influence the binding
of WT-D14-3-3
homodimers to Slob, via forming heterodimers with
WT-D14-3-3
. To demonstrate that Slob is able to bind 14-3-3 homodimers, we used a different approach to detect the 14-3-3 proteins
in this protein complex. Slob was co-expressed with either wild-type or
the dimerization-deficient mutant MM-D14-3-3
, and lysates were
immunoprecipitated with antibody specific for Slob. The
immunoprecipitates were then probed with anti-14-3-3 antibody for
Slob-bound 14-3-3. As shown in Fig. 7,
there is more 14-3-3 protein in Slob immunoprecipitates from cells
co-transfected with WT- D14-3-3
than those with MM-D14-3-3
(compare lanes 2 and 4 in the top
panel). Because both WT- and MM-D14-3-3
s have similar affinity
for Slob (Fig. 3), this result suggests that Slob can bind to either
14-3-3 homodimers or monomers.
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Fig. 7.
The binding of D14-3-3
homodimer to Slob is decreased by R59A/R63A
D14-3-3
. tsA201 cells were transfected
with Slob together with EBFP-tagged WT (lanes 1 and
2) or MM (lanes 3 and 4) D14-3-3
,
in the presence (lanes 1 and 3) or absence
(lanes 2 and 4) of R59A/R63A D14-3-3
-1D4. Cell
lysates were probed for the expression of either the D14-3-3
-EBFPs
with anti-14-3-3 antibody or Slob with anti-Slob antibody (middle
panels). Anti-1D4 antibody was used to detect the expression of
the R59A/R63A D14-3-3
-1D4 (bottom panel). Slob
immunoprecipitates were probed on Western blot with anti-14-3-3
antibody to detect the presence of D14-3-3
-EBFP (top
panel). Overexpression of R59A/R63A D14-3-3
decreases the
Slob-associated WT-D14-3-3
-EBFP (compare lanes 1 and
2) but not MM-D14-3-3
(compare lanes 3 and
4).
is co-expressed with
either wild-type or the dimerization-deficient mutant D14-3-3
(Fig.
7), the amount of D14-3-3
detected in Slob immunoprecipitates is
reduced only in WT (compare lanes 1 and 2) but
not in MM-D14-3-3
(compare lanes 3 and 4)
co-transfected cells. Because R59A/R63A D14-3-3
does not directly
bind to Slob and only dimerizes with WT-D14-3-3
but not with
MM-D14-3-3
(Fig. 6), the reduction of WT-D14-3-3
in the
Slob·14-3-3 complex suggests that R59A/R63A D14-3-3
heterodimers
with WT 14-3-3 compete with WT 14-3-3 homodimers for binding to Slob.
has an effect on the
modulation of dSlo channel activity by D14-3-3
. Whole-cell dSlo
currents were recorded from cells transfected with dSlo, Slob, and WT
D14-3-3
in the absence or presence of R59A/R63A D14-3-3
. We found
no significant difference in either current amplitude or
activation kinetics between cells with (Fig.
8B) or without (Fig.
8A) co-transfection of R59A/R63A D14-3-3
. At +150 mV, the
time constant for activation of dSlo current is 122.3 ± 104.2 ms
in the absence and 111.2 ± 17.9 ms in the presence of R59A/R63A
D14-3-3
(Fig. 8C), and the dSlo current density is
392 ± 201 pA/pF in control and 343 ± 118 pA/pF in R59A/R63A D14-3-3
co-transfected cells (Fig. 8D). These data show
that, although R59A/R63A D14-3-3
can heterodimerize with wild-type D14-3-3
, it does not interfere with the ability of D14-3-3
to modulate dSlo channel activity via Slob. That is, R59A/R63A D14-3-3
does not act as a dominant negative for this response. Taken together with the results in Fig. 6, these data provide further evidence that
dimer binding of D14-3-3
to Slob is not required for the modulation.
View larger version (21K):
[in a new window]
Fig. 8.
Modulation of dSlo by
D14-3-3 is not affected by overexpression of
R59A/R63A D14-3-3
. Whole-cell dSlo
currents were recorded from tsA201 cells using recording conditions
identical to those described for Fig. 5. Representative current traces
recorded from tsA201 cells transfected with dSlo, Slob, and
WT-D14-3-3
together with a control vector (A) or with
R59A/R63A D14-3-3
(B) in a 1:1:1:2 molar ratio.
Co-transfection of R59A/R63A D14-3-3
does not significantly change
either the activation time constant (mean ± S.E.) (C)
(p > 0.5) or the peak dSlo current density (mean ± S.E.) (D) (p > 0.5), at +150 mV.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
modulates the activity of
the dSlo channel through its interaction with the dSlo-binding protein,
Slob (13, 14). The interaction between D14-3-3
and Slob requires
phosphorylation of Slob by calcium/calmodulin-dependent kinase II and is abolished by mutations in two phosphoserine motifs in
Slob (14). Based on these observations, we asked whether modulation of
dSlo channel activity requires dimerization of D14-3-3
. This may
involve one of the following mechanisms: 1) 14-3-3 dimers may bring
other proteins into this complex, thereby allowing these proteins to
modulate dSlo or 2) 14-3-3 may change the conformation of Slob upon
simultaneous binding of the 14-3-3 dimer to the two phosphoserine
motifs in Slob.
proteins form dimers in a heterologous
expression system, and the amino acid residues crucial for dimerization
are conserved between Drosophila and human 14-3-3 proteins,
indicating that they have similar dimerization interfaces (4). The
dimerization-deficient mutants of D14-3-3
can be stably expressed in
transfected cells. They not only bind well to Slob but also form
complexes with dSlo. This is similar to reports from other groups
showing that other proteins bind monomeric and dimeric forms of 14-3-3 with similar efficiency (23, 24). We show further that co-expression of
either the dimeric or the dimerization-deficient form of D14-3-3
,
together with Slob, decreases the activity of dSlo channels in
transfected cells. No significant difference was observed between
wild-type and the dimerization-deficient mutant of D14-3-3
in
modulating dSlo channel activity, as measured by changes in both the
activation kinetics and the peak current density of whole-cell dSlo
currents. In addition, we found that modulation of dSlo channel
activity by wild-type D14-3-3
is not affected by overexpression of a
mutant, R59A/R63A D14-3-3
, which interferes with the binding of
14-3-3 homodimers to Slob. These results, taken together, indicate that the modulation of dSlo channel activity does not require the formation of 14-3-3 dimers.
is sufficient to change dSlo
channel properties. It rules out the possibility that the modulation
observed in this overexpression system is dependent upon other
regulatory proteins that might have been bridged by a 14-3-3 dimer.
However, the precise molecular mechanism underlying the profound
modulation of dSlo channel activity by Slob and D14-3-3
remains to
be determined.
channels, growth factor receptors, and
-aminobutyric acid, type B and
2-adrenergic
receptors (27-30). Moreover, a very recent report shows that 14-3-3 proteins associate with the human ether-a-go-go-related channel and
that this protein-protein interaction amplifies and prolongs adrenergic
stimulation of human ether-a-go-go-related K+ channel
activity (16). Taken together with our finding that 14-3-3 modulates
dSlo channel activity in Drosophila, these data strongly
suggest that the 14-3-3 protein family plays important roles in
regulating diverse neuronal functions.
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ACKNOWLEDGEMENT |
---|
We are grateful to members of the Levitan laboratory for helpful discussions and critical comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by a grant from the National Institutes of Health (to I. B. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 215-898-2122; Fax: 215-573-2015; E-mail: yizhou@mail.med.upenn.edu.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M211907200
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ABBREVIATIONS |
---|
The abbreviations used are: dSlo, Drosophila calcium-dependent potassium channel; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DSS, disuccinimidyl suberate; BFP, blue fluorescent protein; EBFP, enhanced BFP; GFP, green fluorescent protein; EGFP, enhanced GFP; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.
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