(Received for publication, December 12, 1996, and in revised form, March 12, 1997)
From the Department of Pharmacology and the
¶ Graduate Program of Cell and Developmental Biology, Emory
University School of Medicine, Atlanta, Georgia 30322, the
Dana
Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
02115, and the ** Department of Biochemistry, University of Leicester,
Leicester LE1 7RH, United Kingdom
14-3-3 proteins are a family of conserved dimeric
molecules that bind to a range of cellular proteins involved in signal
transduction and oncogenesis. Our solution of the crystal structure of
14-3-3 revealed a conserved amphipathic groove that may allow the
association of 14-3-3 with diverse ligands (Liu, D., Bienkowska, J.,
Petosa, C., Collier, R. J., Fu, H., and Liddington, R. (1995)
Nature 376, 191-194). Here, the contributions of three
positively charged residues (Lys-49, Arg-56, and Arg-60) that lie in
this Raf-binding groove were investigated. Two of the charge-reversal
mutations greatly (K49E) or partially (R56E) decreased the interaction
of 14-3-3
with Raf-1 kinase, whereas R60E showed only subtle effects on the binding. Interestingly, these mutations exhibited similar effects on the functional interaction of 14-3-3
with another target
protein, exoenzyme S (ExoS), an ADP-ribosyltransferase from
Pseudomonas aeruginosa. The EC50 values of
14-3-3
required for ExoS activation increased by ~110-, 5-, and
2-fold for the K49E, R56E, and R60E mutants, respectively. The drastic
reduction of 14-3-3
/ligand affinity by the K49E mutation is due to a
local electrostatic effect, rather than the result of a gross
structural alteration, as evidenced by partial proteolysis and circular
dichroism analysis. This work identifies the first point mutation
(K49E) that dramatically disrupts 14-3-3
/ligand interactions. The
parallel effects of this single point mutation on both Raf-1 binding
and ExoS activation strongly suggest that diverse associated proteins share a common structural binding determinant on 14-3-3
.
14-3-3 proteins are a family of dimeric eukaryotic molecules involved in diverse cellular processes (see Ref. 1 for review). They were originally identified as brain-specific proteins (2) and later found to be present in most mammalian tissues as well as in other eukaryotic organisms, including yeast and plants. Members of the 14-3-3 family have been highly conserved throughout evolution (>60% identity between yeast and mammalian isoforms), reflecting the fundamental importance of 14-3-3 proteins in cellular physiology. Indeed, deletion of the two isoforms of yeast 14-3-3 is lethal to both Saccharomyces cerevisiae and Schizosaccharomyces pombe (3-5).
Multiple biochemical activities have been ascribed to the 14-3-3 family of proteins, although the precise function of 14-3-3 in cellular regulation is unclear. They have been shown to activate tyrosine and tryptophan hydroxylases (6, 7) and are involved in the regulation of protein kinase C activity (8-10). In vitro, 14-3-3 directly activates a bacterial ADP-ribosyltransferase, exoenzyme S (ExoS),1 from Pseudomonas aeruginosa (11). Because of its specificity and sensitivity, activation of ExoS has been used as a simple functional assay for the presence of 14-3-3 proteins (12). Recently, members of the 14-3-3 protein family were found to associate with a number of proto-oncogene and oncogene products (13) as well as with proteins involved in apoptosis. These 14-3-3-associated proteins include Raf kinases (12, 14-16), Bcr and Bcr-Abl (17), Cdc25 phosphatases (18), phosphatidylinositol 3-kinase (19), Cbl (20), the middle tumor antigen of polyoma virus (21), A20 (22), and Bad (23). Through protein/protein interactions, 14-3-3 may regulate diverse cellular processes.
The 14-3-3/Raf interaction has been extensively studied because of the central importance of Raf kinases in mitogenic signal transduction. Both in vivo and in vitro experimental data strongly support a biologically relevant interaction of 14-3-3 with Raf kinases. For instance, overexpression of either the yeast homologue, BMH1, or mammalian 14-3-3 stimulates the biological activity of mammalian Raf in budding yeast (14, 15). Additionally, microinjection of 14-3-3 mRNA into Xenopus oocytes promotes Raf-dependent oocyte maturation (16). Raf immunoprecipitates from yeast or oocytes in the above experiments have increased kinase activity. However, when Raf kinase activity is measured directly with purified 14-3-3 in vitro, no activation of Raf can be demonstrated (12, 24, 25). Consistent with this, Dent et al. (26) found that 14-3-3 proteins can block the dephosphorylation and inactivation of Raf kinase by phosphatases in vitro. Recent demonstration of oligomerization-induced Raf activation suggests an intriguing role of 14-3-3 in promoting Raf dimerization/oligomerization during the activation process (27, 28) because it is possible for one dimeric 14-3-3 molecule to bind two Raf kinase molecules (29, 30). A negative role of 14-3-3 in Raf regulation has also been postulated based on the observation that Raf-1 mutants unable to stably interact with 14-3-3 exhibit enhanced kinase activity in mammalian cells and Xenopus oocytes and are biologically activated (31). However, it remains elusive how 14-3-3 proteins contribute to Raf signaling pathways.
To understand the biological consequence of the 14-3-3/Raf interaction
in vivo, it is important to determine the key residues of
14-3-3 required for Raf binding. Defined point mutants will provide
essential tools for dissecting the role of 14-3-3 proteins in Raf
activation and in cellular regulation in general. Toward this goal, we
have solved the three-dimensional crystal structure of the -isoform
of the 14-3-3 proteins (30). In the structure, each monomer of the
dimeric protein consists of a bundle of nine
-helices organized in
an antiparallel fashion. The four N-terminal
-helices participate in
dimer formation. When viewed along the mean helix axis, the bundle of
helices,
-helices 3-9, form a palisade. Residues that are conserved
among members of the 14-3-3 family line the inside of this palisade and
define a conserved groove. Not surprisingly, 14-3-3 proteins are also
conserved at the tertiary structural level. The structure of 14-3-3
(29) exhibits a very similar folded conformation to the
-isoform, and this should facilitate the identification of common residues of
14-3-3 required for ligand binding. Inspection of the surface property
of the conserved groove of the 14-3-3
monomer reveals an amphipathic
structure that is lined with charged residues on one side and
hydrophobic residues on the other (30). Based on the fact that 14-3-3 proteins bind to a wide range of cellular partners, it is possible that
this conserved groove of 14-3-3
is involved in ligand binding.
To test the crystal structure model, we set about to systematically
analyze the contribution of individual residues in the amphipathic
groove to ligand binding. Here, we report the identification of the
first point mutation (K49E) of 14-3-3 proteins that disrupts the
association of 14-3-3 with Raf-1 kinase and diminishes its ability
to activate ExoS. The impaired binding is not due to gross alteration
of the mutant 14-3-3
protein based on our partial proteolysis and CD
analysis. The concomitant effects of a single point mutation on both
Raf binding and ExoS activation strongly suggest that diverse ligands
may share a common site on 14-3-3 proteins and that Lys-49 may
represent a general site for ligand binding.
Cell Growth Conditions and DNA Manipulations
Escherichia coli strains were grown routinely at 37 °C in LB broth or on LB agar plates (15 g/l). Ampicillin was used as required at a concentration of 100 µg/ml. Strain XL1-Blue (Stratagene, La Jolla, CA) was used for the propagation of plasmids, and strain BL21(DE3) was used for expression of proteins using bacteriophage T7 promoter-based overexpression vectors (32). S. cerevisiae strains were grown at 30 °C either in rich medium or in synthetic medium supplemented with relevant amino acids and 2% glucose or galactose as a carbon source (33). Minipreparation of plasmids, restriction enzyme digestion, subcloning procedures, cell transformation, and other molecular biology procedures were performed essentially as described (33) or as indicated. Double-stranded DNA sequencing was performed using Sequenase Version 2.0 (Amersham Corp.). Restriction enzymes and other molecular biology reagents were obtained from New England Biolabs Inc. (Beverly, MA), Promega (Madison, WI), and Life Technologies, Inc.
Plasmids
General VectorspHAF625 is a pUC19-based shuttle vector for
14-3-3 subcloning. The DNA sequence encoding 14-3-3
was amplified
by polymerase chain reaction using pHAF612 (11) as a template. The
following primers were used to introduce required restriction sites for the subsequent in-frame fusion constructions:
5
-GTCAAGAGGGAATTCCATATGGATAAAAACGAGCTG-3
(EcoRI and NdeI sites underlined) and
5
-CGTATAGAATTCGTCGACTCATTAATTTTCCCCTCC-3
(EcoRI and SalI sites underlined). The amplified
DNA fragments were digested with EcoRI and subcloned into
pUC19 at the EcoRI site with the 14-3-3
gene in the
opposite orientation of lacZ. To introduce appropriate
restriction sites for in-frame fusion of various versions of Raf-1
kinase, we constructed three basic Raf vectors. For this purpose, Raf
sequences were amplified by polymerase chain reaction with added
restriction sites using a full-length raf gene as a template
(33). The following primers were used: 1)
5
-GGATCCGAATTCCATATGGAGCACATACAG-3
(EcoRI and NdeI sites underlined), 2)
5
-CAGGGTACCATCGATCTATGGTGCCCGCTCTCT-3
(ClaI
site underlined), 3) 5
-CATATGGAATTCGTATCTGGGACCCAG-3
(EcoRI site underlined), and 4)
5
-CAGGGTACCATCGATCTAGAAGACAGGCAG-3
(ClaI site
underlined). Primer pairs 1/4, 1/2, and 3/4 were used to amplify the
full-length sequence of the Raf-1 kinase and the N-terminal (Raf-N,
residues 1-320) and C-terminal (Raf-C, residues 321-648) fragments,
respectively. These polymerase chain reaction fragments were digested
with EcoRI and ClaI and subcloned into pKS(+)
(Stratagene), generating pHW102 (Raf-1), pHW103 (Raf-N), and pHW105
(Raf-C). The integrity of the polymerase chain reaction sequences was
verified by sequencing.
Parental vectors for the interaction trap system were kind
gifts from Dr. Roger Brent (Harvard Medical School) and included pEG202
and pJG4-5 (34). pEG202 carries the sequence encoding the DNA-binding
domain of the bacterial LexA protein, the expression of which is under
the control of the ADH promoter. pJG4-5 carries the B42
transcriptional activation sequence fused to a nuclear localization
sequence under the control of a GAL1 promoter. An epitope
tag (influenza virus hemagglutinin) was inserted between the nuclear
localization sequence and the sequence of interest to enable
surveillance of the expressed fusion protein by immunoblotting. For
studying 14-3-3/Raf interaction, the wild-type 14-3-3
sequence and its mutant derivatives (EcoRI-SalI fragments;
see description below) were obtained from pHAF625 and subcloned into
either pEG202 (EcoRI-SalI sites) or pJG4-5
(EcoRI-XhoI sites), generating in-frame fusion
constructs. Similarly, the EcoRI-SalI fragments
of pHW102, pHW103, and pHW105 were ligated to the LexA sequence in
pEG202, generating pHW107 (LexA-Raf-1), pHW108 (LexA-Raf-N), and pHW110 (LexA-Raf-C), and to the B42 sequence in pJG4-5, generating pHW112 (B42-Raf-1), pHW113 (B42-Raf-N), and pHW115 (B42-Raf-C),
respectively.
For protein expression in E. coli, the NdeI-EcoRI fragments of pHAF625
encoding the mutant 14-3-3 proteins (see below) were subcloned into
NdeI-EcoRI-cut pET-15b (Novagen, Madison, WI), generating pLZ121 (K49E), pHW167 (K49L), pHW168 (K49Q), pHW169 (K49R),
pLZ125 (R56E), and pLZ126 (R60E).
Site-directed Mutagenesis
Oligonucleotide-directed mutagenesis was performed with
double-stranded plasmid as the starting material according to the unique site elimination method (35). Two primers were used in this
process, a mutagenic primer containing a desired mutation and a
selection primer containing a mutation that eliminated a unique
SspI site in pHAF625
(5-AAATGCTTCAATGATATCGAAAAAGGAAG-3
; a new
EcoRV site was introduced (underlined)). To facilitate the screening of the desired mutations and based on the degeneracy of the
codons, restriction sites were purposely introduced into the mutagenic
primers. The incorporation of the mutagenic primers was screened by
restriction enzyme digestion, and the introduced mutations were
verified by sequencing. The mutagenic primers used are listed below
(mutations generated with the corresponding oligonucleotides are
underlined, and the SacI sites introduced for screening are in lower-case letters): 1)
5
-GCTTATGAAAATGTTGTAGgagctcGTAGGTC-3
(Lys-49 to Glu); 2)
5
-GTTGCTTATCTAAATGTTGTAGgagctcGTAGG-3
(Lys-49 to Leu); 3)
5
-GCTTATCAAAATGTTGTAGgagctcGTAGG-3
(Lys-49 to Gln); 4)
5
-GCTTATAGAAATGTTGTAGgagctcGTAGG-3
(Lys-49 to Arg); 5)
5
-GTAGgagctcGTGAGTCATCTTGG-3
(Arg-56 to Glu); and 6)
5
-GTAGgagctcGTAGGTCATCTTGGGAGGTCGTCTC-3
(Arg-60 to
Glu).
Yeast Two-hybrid/Interaction Trap Assay
A modified version of the yeast two-hybrid system (36), the
interaction trap (34), was used for studying 14-3-3/Raf interactions in vivo (33). S. cerevisiae EGY48
(MATa trp1 ura3 his3 LEU2::pLexAop6LEU2) was used as a host for all
interaction experiments. Both pSH18-34 and pJK103 were used as
reporters. pSH18-34 directs expression of a GAL1-lacZ gene
from eight high affinity ColE1 LexA operators, whereas pJK103 uses two
ColE1 LexA operators. For the protein/protein interaction analysis,
EGY48 harboring a lacZ reporter was cotransformed with
pEG202 derivatives and pJG4-5 derivatives by the lithium acetate method
(33). Transformants were maintained in synthetic medium with glucose
(2%) under selection for the URA3, HIS3, and
TRP1 markers. Colonies were patched onto synthetic medium
plates containing galactose (2%), raffinose (1%), and X-gal (Gold
Biotechnology, Inc., St. Louis, MO) to induce and detect the expression
of the lacZ reporter gene. The time required for color
development of positive interactions on X-gal selection plates ranged
from 8 to 24 h.
Assay of -Galactosidase Activity
For the quantitative liquid assay, yeast transformants were
grown overnight in the appropriate synthetic selecting medium with
glucose to A600 ~ 1.0. Cells were washed with
and transferred to the galactose induction medium with 1% raffinose.
After 12 h of induction, cells were prepared in fresh Z buffer and
permeabilized using three drops of chloroform and two drops of 0.1%
SDS as described (33). For quantitation,
chlorophenyl-red--D-galactopyranoside (Boehringer
Mannheim) was added as a chromogenic substrate. The amount of liberated
chlorophenol red was determined by A574, and the
specific activity in Miller units was calculated based on the following
equation: unit = (A574/(A600 × V × T)) × 1000, where A600 is the cell density used, V is
the volume of the culture in ml, and T is the reaction time
in min. Three independent colonies were used for each activity
determination.
Protein Expression and Purification
Single colonies from LB/ampicillin plates, freshly streaked with
E. coli BL21(DE3) harboring pET-15b-derived plasmids
expressing 14-3-3 or its mutant derivatives, were transferred into
3-ml cultures of LB/ampicillin. These cultures were fermented overnight at 37 °C on a rotary wheel. On the second day, 2-liter flasks, each
containing 250 ml of LB/ampicillin, were inoculated with the 3-ml
overnight culture. The flasks were aerated on a rotary shaker (300 rpm;
Innova 4000, New Brunswick Scientific, Edison, NJ) at 37 °C. The
cultures were induced with
isopropyl-
-D-thiogalactopyranoside (1 mM)
when A600 reached ~0.5. The cells were
harvested after 4 h, immediately chilled, and centrifuged at
4000 × g for 15 min at 4 °C. The pellets were
washed once with Tris/EDTA buffer (pH 8.0).
For the in vitro binding assay, the pellets were resuspended
in ice-cold sonication buffer composed of 50 mM
Na2HPO4 (pH 8.0), 100 mM KCl, 0.1%
Tween 20, 1.0 mM phenylmethylsulfonyl fluoride, and 1.0 mM EDTA. The resuspended pellets were sonicated four times on ice for 20 s each using a Branson 450 Sonifier cell disruptor, with the power control at 3 and the duty cycle at 50%. Cellular debris
was pelleted by centrifugation at 13,000 × g for 10 min at 4 °C. The supernatants were used as the source of
hexahistidine-tagged 14-3-3. For the purpose of protein
purification, crude extracts were made as described above, except that
the sonication buffer contained 20 mM Tris-HCl (pH 7.9),
500 mM NaCl, 5 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride. Subsequently, wild-type 14-3-3
and its mutant derivatives were purified using
Ni2+ chelating chromatography essentially as described
(11). Hexahistidine tags were removed by thrombin digestion (0.5 unit/mg of proteins). The 14-3-3 proteins used for the quantitative
assays were further purified by gel filtration chromatography (Superdex
200) in a Pharmacia FPLC system.
Solid-phase Binding Experiments
For the in vitro binding assays, hexahistidine-tagged
14-3-3 proteins in the crude extracts were incubated with
Ni2+-charged Sepharose 6B beads (Novagen) for 1 h at
4 °C. Radiolabeled Raf-1 proteins were generated using the TNT
in vitro transcription-translation system (Promega). The DNA
template (pKS(+)-Raf-1) was incubated with TNT rabbit reticulocyte
lysates in the presence of [35S]methionine (final
concentration, 0.8 µCi/ml) for 90 min at 30 °C according to the
manufacturer's specifications. For the binding assays, the immobilized
14-3-3
proteins (~5 µg each) were mixed with
35S-labeled Raf-1 in Nonidet P-40 buffer (1% Nonidet P-40,
137 mM NaCl, 1 mM MgCl2, and 40 mM Tris-HCl (pH 8.0)) for 1 h at 4 °C with
rotation. The 14-3-3
complexes were extensively washed with Nonidet
P-40 buffer three times, followed by radioimmune precipitation assay
wash (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 137 mM NaCl, and 20 mM Tris-HCl (pH 8.0)) three
times, and boiled for 3 min in SDS sample buffer before resolution by
SDS-PAGE (12.5%). The presence of the radiolabeled products in the
14-3-3
complexes was visualized using a PhosphorImager (Molecular
Dynamics, Inc.).
ExoS Activation Assay
The NAD+:SBTI ADP-ribosyltransferase assay measures
the rate of incorporation of the [32P]ADP-ribose moiety
of NAD+ into the trichloroacetic acid-precipitable SBTI
fraction of the reaction mixture. SBTI is used as an artificial
substrate, and the assays were performed essentially as described
previously (11, 37). The reaction mixtures contained (in a final volume of 25 µl) 0.2 M sodium acetate (pH 6.0), 20 nM purified ExoS, 30 µM SBTI, 30 µM
[adenylate-32P]NAD+, 1.5 µM bovine serum albumin, and varying concentrations of
14-3-3 or its mutant proteins. After 30 min at 25 °C, reactions
were stopped by removing 15 µl of the reaction mixture, which was
directly pipetted onto trichloroacetic acid-saturated filter paper. The filter pads were placed immediately into ice-cold trichloroacetic acid
solution (5%) and washed three to five times by gentle agitation on a
platform rocker until no counts could be detected in the discarded wash
solutions. The filter pads were then washed twice for 5 min each in
ice-cold methanol, dried, and counted with 3 ml of liquid scintillation
mixture in a Beckman LS6500 scintillation counter. Incorporation of
ADP-ribose into SBTI was calculated based on the increase in counts
(minus background) with <20% of the reactants having been utilized.
Activities were obtained from at least three separate experiments, each
performed in duplicate. Data are expressed as pmol of ADP-ribose
incorporated per min/pmol of ExoS. To verify the specific incorporation
of the ADP-ribose into SBTI, 5 µl of the reaction mixtures were
loaded onto an SDS-polyacrylamide (12.5%) gel and visualized using
autoradiography. The amount of 32P incorporated
specifically into SBTI was quantified using a PhosphorImager with Image
Quant software (Molecular Dynamics, Inc.) and was found to be
comparable to that obtained with the liquid assay described above. ExoS
activation data were fit to the following equation using SigmaPlot
(Jandel Scientific): v =
Vmax[A]/(EC50 + [A]), where Vmax is the maximal
activation expressed as pmol of ADP-ribose incorporated per min/pmol of
ExoS, v is the observed enzyme activity, EC50 is
the concentration of 14-3-3 proteins giving half-maximal activation,
and [A] is the 14-3-3 concentration.
Protein Stability Assays
Partial Proteolytic DigestionWT 14-3-3 and its mutant
derivatives (2.5 µg) were incubated with 0.5 unit of chymotrypsin in
a final volume of 10 µl at 37 °C for 30 min. Digested fragments
were resolved by SDS-PAGE (15%), and the digestion patterns of the WT
and mutant proteins were visualized using Silver Stain Plus
(Bio-Rad).
The far-UV CD spectra were recorded on a
Jasco 600 spectropolarimeter. Results are expressed as mean residue
molar ellipticity ([],
degree·cm2·dmol
1) calculated from the
following equation: [
] = ([
]obs × MRW)/(10 × L × C), where [
]obs is
the observed ellipticity expressed in millidegree, MRW is the mean
residue molecular weight (114 for 14-3-3
), L is the
optical path length in cm (0.1 cm), and C is the final
protein concentration in mg/ml. Far-UV CD spectra were the average of
four scans obtained by collecting data at 0.2-nm intervals from 250 to
190 nm with a protein concentration of 0.4 mg/ml in phosphate-buffered
saline. For the guanidine hydrochloride denaturation experiments,
changes in the helical content of the sample were monitored at 222 nm.
Phosphate-buffered saline was used for these studies. The half-melting
concentration (Cm) is defined as the concentration
of denaturant at the midpoint of the transition. The breadth of the
transition (
C) is defined as the difference in denaturant
concentrations between the points at which the transition is one-fourth
and three-fourths complete.
SDS-PAGE and Immunoblotting
An SDS-PAGE system was used essentially as described by Laemmli (38). The enzyme-linked immunoblotting procedures of Towbin et al. (39) were followed. Proteins separated by SDS-PAGE were electrophoretically transferred to nitrocellulose membranes and incubated with antiserum (1:2000 dilutions for mouse anti-hemagglutinin monoclonal antibody (BAbCO, Richmond, CA)). Cross-reacting materials were visualized with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G using ECL (Amersham Corp.).
Protein Assay
Protein concentration was estimated by the Bio-Rad protein assay kit based on the method of Bradford (40), and bovine serum albumin was used as the standard.
In the three-dimensional
crystal structure of 14-3-3, we identified a conserved amphipathic
groove that may represent a general site for binding to associated
proteins (Fig. 1) (30). On the charged face of the
groove, exposed residues Lys-49, Arg-56, and Arg-60 are lined along
-helix 3. The side chains of these three positively charged residues
project toward the inside of the groove, which positions the charged
groups of the side chains well for potential contact with charged
groups from the interacting ligand. As a first step in the systematic
dissection of residues of 14-3-3 involved in ligand binding, we
examined the contributions of these three positively charged residues
to 14-3-3 protein/protein associations.
Mutations of Lys-49, Arg-56, and Arg-60 Decrease the Binding of 14-3-3
To test whether charged residues in the
proposed groove are involved in ligand binding, we made three
charge-reversal mutations by site-directed mutagenesis, generating
K49E, R56E, and R60E (Fig. 1). It has been established that 14-3-3
directly binds to Raf-1 kinase in vivo and in
vitro (13). Individual mutations were tested for their effect on
the interaction of 14-3-3
with Raf-1 kinase.
We used the interaction trap to determine the 14-3-3/Raf interaction
in an in vivo environment (34). Raf-1 was fused to the
DNA-binding domain of LexA protein, whereas 14-3-3
was fused to the
B42 transcriptional activation sequence as described under "Materials
and Methods." Interaction between the LexA-Raf-1 and B42-14-3-3
fusion proteins results in transcription of a lacZ reporter
and consequently
-galactosidase activity that can be visualized
using chromogenic substrates (X-gal). Initially, the X-gal plate assay
was performed to assess the relative interactions of 14-3-3
and Raf
in various combinations. Consistent with previous reports (14-16),
expression of 14-3-3
and Raf-1 induced the production of
-galactosidase activity (Table I), reflecting the
interaction of 14-3-3
with Raf-1 in yeast. No
-galactosidase
activity was detectable when LexA-Raf or B42-14-3-3
fusion protein
was expressed independently. Introduction of the charge-reversal
mutations K49E and R56E abolished
-galactosidase production,
suggesting decreased binding of 14-3-3
to Raf-1. The mutation R60E
had no significant effect on the 14-3-3
/Raf interaction in this
assay. It has been demonstrated that 14-3-3 binds to both the
N-terminal regulatory fragment of Raf-1 and the C-terminal kinase
domain (12, 14-16, 25). Consistent with these reports,
cotransformation of yeast with WT 14-3-3
and either the N- or
C-terminal sequence of Raf-1 induced blue colony formation (Table I).
Interestingly, the K49E and R56E mutations diminished the interaction
of 14-3-3
with both the N- and C-terminal domains. This result
suggests that the N- and C-terminal fragments of Raf-1 associate with
14-3-3
via a similar site on 14-3-3
and that 14-3-3
can
interact with the N- or C-terminal fragment of Raf-1 independently. To
rule out possible reporter effects (41), two types of lacZ
reporters were used for the above experiments: a sensitive reporter,
pSH18-34 (with eight operators for LexA binding), and a less sensitive reporter, pJK103 (with two operators). Similar results were obtained with each (data not shown).
|
To quantify the effect of mutations on 14-3-3/Raf interactions, the
above experiments were further performed using a quantitative liquid
assay. As shown in Fig. 2, 14-3-3
bound to the
full-length or truncated forms of Raf-1 with different affinities, with
Raf-N being strongest and Raf-C weakest (16). Consistent with the above
assay, the mutation K49E completely abolished the interaction of
14-3-3
with Raf-1, Raf-N, or Raf-C in a parallel fashion. The R56E
mutation greatly diminished the interaction, yielding lacZ
reporter expression reduced 50-fold compared with the WT protein. The
R60E mutation slightly decreased the
-galactosidase activity
(~20-30%). To ensure that the differences in reporter activity
among particular combinations of Raf and 14-3-3
mutants were not a
reflection of differential expression of the mutant 14-3-3 proteins, we
tested cells coexpressing Raf with various 14-3-3
proteins for the
level of each hybrid protein using immunoblotting. As shown in Fig. 2
(lower panels), relative protein levels of WT and mutant
14-3-3
proteins as fusion proteins were similar. Stripping and
reprobing the membranes with anti-Raf antibody confirmed that similar
amounts of Raf were present in all the samples (data not shown).
Therefore, it is likely that the impaired reporter gene expression is
due to the disrupted 14-3-3
/Raf interactions caused by the
charge-reversal mutations K49E and R56E.
To confirm the results obtained with the interaction trap system, an
alternative in vitro binding assay was performed. For this
purpose, WT and mutant 14-3-3 proteins were overexpressed in E. coli as hexahistidine-tagged fusion proteins and immobilized on
nickel-charged beads. The binding partner, Raf-1, was expressed and
labeled with [35S]methionine using the TNT in
vitro transcription-translation system. Equal amounts of
radiolabeled Raf-1 protein mixtures were incubated individually with
the WT or mutant 14-3-3-coated beads. After extensive washing, Raf-1
protein that was complexed with various 14-3-3 derivatives was
visualized following SDS-PAGE separation and autoradiography (Fig.
3A). Consistent with the data obtained with
the interaction trap system, K49E abolished the binding of 14-3-3
to
Raf-1, whereas R56E partially decreased the interactions. R60E bound to
Raf as strongly as did the WT protein in this assay.
Mutations of Lys-49, Arg-56, and Arg-60 Decrease the Ability of 14-3-3
Data from the above experiments
suggested the participation of Lys-49 and Arg-56, and possibly Arg-60,
in the 14-3-3/Raf interaction. However, it is unknown whether Lys-49
and Arg-56 are specific to the Raf interaction or whether they form
part of a general interaction surface for multiple ligands. To address this question, we used a defined biochemical assay, the ExoS activation reaction (11). ExoS is an ADP-ribosyltransferase that catalyzes, in a
14-3-3-dependent manner, the incorporation of the
ADP-ribose moiety of the NAD+ molecule into substrates,
including Ras, vimentin, and SBTI (11, 42).
For the kinetic studies and protein stability analysis, 14-3-3 and
its mutants were overexpressed in E. coli BL21(DE3), and individual proteins were purified by nickel chelate affinity
chromatography. Hexahistidine tags were removed by enzymatic digestion.
The proteins were further purified by gel filtration chromatography on
a Superdex 200 column and reached ~95% purity (data not shown).
The recombinant WT 14-3-3 protein activated ExoS to ADP-ribosylate
SBTI in a dose-dependent manner, with an EC50
of 3.0 nM (Table II). The specific
incorporation of ADP-ribose into the substrate SBTI was confirmed by an
SDS-PAGE-based assay (see "Materials and Methods"; data not shown).
As in the case of Raf binding, K49E drastically increased the
EC50 of 14-3-3
in the ExoS assay (334 nM;
~110-fold increase), suggesting the decreased affinity of this 14-3-3 mutant toward ExoS. The R56E mutation partially decreased the ability
of 14-3-3 to activate ExoS, as measured by the amount of 14-3-3 needed
to achieve 50% of Vmax (EC50 ~ 15.9 nM) (Table II). Again, R60E had only a slight effect
on ExoS activation. None of these mutants, however, affected the
Vmax of the reactions (Table II). Therefore,
there is a direct correlation between the effects of mutations of
Lys-49, Arg-56, and Arg-60 on interaction with Raf-1 and activation of
ExoS.
|
Among three mutants tested above, K49E
exhibited the most severe defect in 14-3-3/ligand binding. The
nature of the contribution of Lys-49 to ligand binding was further
probed by changing Lys-49 to Leu, Gln, or Arg. This panel of Lys-49
mutants was overexpressed in E. coli and purified as
described under "Materials and Methods." The effect of these Lys-49
mutations on 14-3-3
/ligand binding was investigated using the Raf
binding and ExoS activation assays described above. In comparison with
K49E, a neutral Leu substitution slightly increased the binding
affinity of 14-3-3
for Raf as measured in the interaction trap
system (Fig. 4) and the in vitro binding
assay (Fig. 3B). A polar Gln residue and a positively charged Arg residue significantly restored the binding activity (Fig.
4). The ability of this panel of 14-3-3 mutants to activate ExoS was
also examined and was found to increase in the following order in
relation to the K49E mutant: K49L < K49Q < K49R (Table II).
It appears that a positive charge is important for both Raf binding and
ExoS activation. It is interesting that the affinities of the Lys-49
mutants for Raf-1 kinase directly paralleled the efficiencies of ExoS
activation, supporting the notion that Raf-1 and ExoS bind to 14-3-3
through a common site.
K49E, R56E, and R60E Mutations Do Not Result in Gross Structural Changes in 14-3-3
To explore the possibility that the reduced activity of mutant 14-3-3 proteins is a result of structural changes, we probed their structural integrity using several alternative approaches. First, the overall structural integrity of the 14-3-3 mutants was examined by assessing their ability to dimerize. Crystal structure data (29, 30) and other biochemical analyses (43, 44) suggest that 14-3-3 proteins function as a dimer. We found that dimer formation can be demonstrated in vivo using the interaction trap system (Table I). This observation provided a simple means to evaluate the relative conformational integrity of the mutant proteins in vivo because dimer formation depends on the integrity of the folded proteins (45). Despite specific defects in association with Raf-1 and activation of ExoS, each of the 14-3-3 mutants readily formed dimers with the WT (Table I) and mutant (data not shown) partners in vivo. In vitro, the purified 14-3-3 mutant proteins comigrated with the WT protein, as a dimer, on a native gel2 and on a gel filtration column (Superdex 75 HR10/30) (data not shown). These data suggest that the Lys-49 mutations, R56E, and R60E caused no drastic perturbation to the overall structure of the proteins.
Second, the relative stability of the mutant proteins compared with
that of WT 14-3-3 was evaluated by analysis of the proteins' resistance to limited proteolytic digestion with chymotrypsin. Purified
WT 14-3-3
and its mutant derivatives were incubated with
chymotrypsin for 30 min, followed by analysis of the digestion products
by SDS-PAGE and silver staining. The mutant proteins gave similar
digestion patterns compared with WT 14-3-3
(Fig. 5),
suggesting that the global structure of each mutant protein was not
altered. Similar results were obtained using additional proteases,
including Pronase and carboxypeptidase B.
Third, as a quantitative measure of the possible structural effect of
the mutations, we used CD spectroscopy to compare the overall secondary
structure of the mutant 14-3-3 proteins with that of the WT protein.
Consistent with the three-dimensional crystal structure (29, 30) and
earlier reports (46), the far-UV CD spectra of 14-3-3 proteins were
characteristic of an
-helical structure (Fig. 6).
Ellipticity minima were observed near 208 and 222 nm. As shown in Fig.
6, the Lys-49 mutations, R56E, and R60E resulted in virtually no effect
on the CD spectrum of 14-3-3
, suggesting that these mutations do not
alter the global secondary structure of 14-3-3
. Because K49E causes
the most dramatic reduction in 14-3-3
/ligand binding, we further
compared the chemical stability of the K49E mutant and the WT protein
as measured by CD at 222 nm. Strikingly, the K49E mutant, which
disrupted the 14-3-3
/Raf interaction and drastically decreased the
affinity for ExoS, was as stable as the WT protein as judged by the
guanidine hydrochloride-induced unfolding profiles. The midpoint
denaturation concentrations and the concentration intervals in which
chemical denaturation took place did not change for K49E proteins (WT: Cm = 1.31 ± 0.10 M,
C = 0.25 ± 0.03 M; K49E:
Cm = 1.35 ± 0.13 M,
C = 0.21 ± 0.05 M). Similarly, no
change in the Cm and
C values of other
Lys-49 mutants was observed. Taken together, the data presented above
strongly support the notion that the mutations K49E, K49L, K49Q, K49R,
R56E, and R60E do not induce large structural alterations of
14-3-3
.
14-3-3 proteins interact with diverse proteins involved in a wide
range of biological systems (1). Based on our crystal structure data,
we proposed that 14-3-3 binds to multiple ligands through a
conserved amphipathic groove (30). On the charged face of the groove,
three positively charged residues (Lys-49, Arg-56, and Arg-60) are
lined along
-helix 3. Here, we report that charge-reversal mutations
at these three positions disrupted the interaction of 14-3-3
with
Raf-1 kinase and ExoS to different degrees, with K49E being the most
dramatic. The disruption of the 14-3-3
/ligand interaction by
mutations of Lys-49 appears to be due to the local alteration of the
charge property at this position, rather than mutation-induced
structural change. More important, our work identifies a critical
residue, Lys-49, with general importance for ligand binding, which is
consistent with the crystal structure model. In addition, application
of a panel of Lys-49 mutations establishes a clear correlation between
the disruption of 14-3-3
/Raf association and the 14-3-3
/ExoS
interaction, suggesting that diverse associated proteins use a common
binding site on 14-3-3
. In further support of this notion, the
effective association of 14-3-3
with the N-terminal fragment of
c-Bcr kinase (encoded by the first exon) was also disrupted by the K49E
mutation (data not shown).
Lys-49 appears to be part of a binding interface in which multiple
residues are involved. Clearly, Arg-56 also contributes to ligand
binding because the charge-reversal mutation of Arg-56 partially
decreased the binding of 14-3-3 to Raf and ExoS. Only a slight
effect of the R60E mutation on ligand binding was observed. The
mechanism by which Lys-49 contributes to ligand binding was investigated by replacing the initial charge-reversal mutation residue
(Glu) with Leu, Gln, or Arg. It appears that the hydrophobic nature of
the Lys-49 side chain is not the primary determinant for 14-3-3 binding
since a hydrophobic Leu residue showed only a slight effect. The
ability of 14-3-3
to bind Raf was partially restored with the
elimination of the negative charge and the introduction of Gln or Arg,
suggesting that the positive charge of Lys-49 is involved in the
association of 14-3-3 with Raf kinase. However, at this point, we
cannot rule out the possibility that Lys-49 is indirectly involved in
ligand binding, for example, by interacting with residues in the
C-terminal loop of the 14-3-3
protein. In our crystal structure, the
C-terminal 12 amino acid residues form a poorly ordered structure and
appear lined in the proposed ligand-binding groove (30).
Drastic deletion analysis of 14-3-3 implicates a different region of
the protein in ligand binding. Luo et al. (47) reported the
importance of the C-terminal sequence (amino acids 179-245) of
14-3-3
in the binding of Raf-1 kinase in COS cells. Interestingly, only the inactive form of Raf kinase was found associated with this
C-terminal 14-3-3 fragment, and the active Raf form was complexed only
with the full-length 14-3-3 protein. A C-terminal fragment of 14-3-3
(residues 166-208; termed BoxI) was also found to be necessary and
sufficient for association with phosphorylated tyrosine hydroxylase
(48). However, Liu et al. (20) found that deletion of the
N-terminal sequence, which abolished dimer formation, also impaired the
binding of 14-3-3
to Raf, Cbl, and p85 (phosphatidylinositol 3-kinase). It is possible that the N-terminal sequence directly or
indirectly participates in ligand binding. It is also conceivable that
an initial contact at the Lys-49 region may induce a conformational change that is required to expose the primary binding site in the
C-terminal fragment of 14-3-3.
The data presented here support the notion that 14-3-3 uses the
conserved amphipathic groove for binding to multiple ligands, which
include Raf, Bcr, and ExoS. A complementary amphipathic conformation in
the ligand may permit the specific interaction of 14-3-3
with
diverse associated proteins. For instance, the phosphorylation in
ligands may provide part of the charged interface for 14-3-3 binding
since 14-3-3 has been shown to bind only to phosphorylated ligands in a
number of cases (7, 23, 31, 49, 50). Such a mode of action is
reminiscent of the mechanism proposed for calmodulin, another highly
conserved protein that can bind a range of regulatory proteins (see
Ref. 51 for review). Calmodulin adopts an amphipathic structure that
forms a binding site for amphipathic helices. Besides the amphipathic
structure, 14-3-3 shares several additional features with calmodulin.
For example, they are both highly conserved, abundant, and ubiquitous eukaryotic proteins. Both interact with a range of diverse proteins and
enzymes, including kinases and hydroxylases. In addition, the binding
of Ca2+/calmodulin or 14-3-3 to target proteins alters
their activity, although the role of 14-3-3 binding, in many cases, is
unclear. Such similarities between 14-3-3 and calmodulin raise the
possibility that 14-3-3, like calmodulin, may regulate the activity of
the associated proteins directly. Evidence is accumulating. The first example is the stimulatory role of 14-3-3 for tyrosine and tryptophan hydroxylases after phosphorylation by
Ca2+/calmodulin-dependent kinase II (6). 14-3-3 has also been found to inhibit (8) or activate (9) protein kinase C and
serves as an obligatory activator of ExoS ADP-ribosyltransferase (11). Interestingly, 14-3-3 proteins serve as inhibitors of nitrate reductase
from spinach leaves in a phosphorylation-dependent manner (52). However, a direct role of 14-3-3 in Raf kinase activation seems
unlikely because the addition of purified 14-3-3 to the Raf protein has
no significant effect (12, 24, 25, 31).
14-3-3 proteins have been shown to interact with Raf kinases and are
implicated in Raf-mediated signal transduction. Earlier experiments
have demonstrated that 14-3-3 binds to Raf via multiple sites. In
support of this notion, two phosphorylation-dependent high
affinity binding sites in Raf have been identified, which are the
N-terminal regulatory domain sequence containing Ser-259 and the
C-terminal kinase domain sequence containing Ser-621 (49). Consistent
with these data, we show here that 14-3-3 can bind independently to
the Raf-N and Raf-C fragments (Fig. 2). Moreover, the point mutation
K49E abolished the binding of 14-3-3
to either Raf-N or Raf-C. We
conclude that Raf-N and Raf-C utilize a similar structural determinant
on 14-3-3
for binding that involves Lys-49. Such an interaction
could enable a single 14-3-3 dimer to bind to the N- and C-terminal
domains of the same Raf molecule simultaneously or to bind to two
molecules of Raf. Alternatively, it also allows one 14-3-3 dimer to
tether one molecule of Raf and another 14-3-3-associated partner
together.
Because Lys-49 is conserved among all 14-3-3 isoforms, Lys-49 equivalent residues in other isoforms may also participate in ligand binding in a similar fashion. However, isoform specificity and ligand specificity issues have not been addressed by current studies. It is possible that residues that are not conserved in 14-3-3 and/or binding sequences in the target proteins confer different levels of binding specificity.
We thank Joe Barbieri for kindly providing ExoS, Roger Brent for vectors of the interaction trap system, and Tom Roberts for anti-Raf antibody. We thank Todd Milne and Hongzhu Yang for critical reading of the manuscript; Keith Wilkinson, Dale Edmonson, Shane Masters, and Romesh Subramanian for valuable discussions; and Susan Qu for assistance in CD experiments.