Thermodynamic and Kinetic Characterization of the Interaction
between the Ras Binding Domain of AF6 and Members of the Ras
Subfamily*
Thomas
Linnemann,
Matthias
Geyer
,
Birgit K.
Jaitner,
Christoph
Block,
Hans Robert
Kalbitzer
,
Alfred
Wittinghofer, and
Christian
Herrmann§
From the Abteilung Strukturelle Biologie, Max-Planck-Institut
für Molekulare Physiologie, Postfach 102664, 44026 Dortmund and
the
Institut für Biophysik und Physikalische
Biochemie, Universität Regensburg,
Postfach, 93040 Regensburg, Germany
 |
ABSTRACT |
Cellular signaling downstream of Ras is highly
diversified and may involve many different effector molecules. A
potential candidate is AF6 which was originally identified as a fusion
to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and compared with other
effectors. The binding characteristics are quite similar
to Raf and RalGEF, i.e. nucleotide dissociation as
well as GTPase-activating protein activity are inhibited,
whereas the intrinsic GTPase activity of Ras is unperturbed by AF6
binding. Particularly, the dynamics of interaction are similar to Raf
and RalGEF with a lifetime of the Ras·AF6 complex in the millisecond
range. As probed by 31P NMR spectroscopy one of two major
conformational states of Ras is stabilized by the interaction with AF6.
Looking at the affinities of AF6 to a number of Ras mutants in the
effector region, a specificity profile emerges distinct from that of
other effector molecules. This finding may be useful in defining the
biological function of AF6 by selectively switching off other pathways
downstream of Ras using the appropriate effector mutant. Notably, among
the Ras-related proteins AF6 binds most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation.
 |
INTRODUCTION |
Cellular functions like growth and differentiation are regulated
by a complex network of interacting proteins with either enzymatic or
adaptor functions. Ras plays a central role in this signaling network.
It is bound to GDP in its resting state and bound to GTP after
activation (1). Only this activated form can interact with effector
molecules like Raf kinase, RalGEF, and
PI(3)kinase1 (2). A variety
of incoming signals is directed to Ras activation, and this in turn
triggers the activation of different signal pathways that lead to
diverse consequences such as induction of DNA transcription, inhibition
of apoptosis, and/or cytoskeletal rearrangements (3). Synergism with
other signaling events or inhibition by competing pathways may
supplement or modulate the signal amplitude resulting in a distinct
effect. When Ras binds to the effectors they become activated by
different and not fully understood mechanisms. This leads to the
activation of other molecules located further downstream in the signal
cascade. There are other small GTP-binding proteins closely related to
Ras like Rap1A/2A, TC21, and R-Ras which also bind to the Ras
effectors. So far little is known about their biological functions.
The effectors c-Raf-1 (4), RalGEF (5), and PI(3)kinase (6, 7) have been
shown to become activated upon Ras binding. For other possible Ras
targets like Rin1/2 (8), PKC
(9), Krit (10), or Nore1 (11) the
requirements for a Ras effector have been fulfilled in that they bind
in a GTP-dependent manner and do not bind to the effector
mutant Ras(D38A), but functional data are missing for these proteins.
Recently, AF6 was discovered as another putative effector of Ras (12,
13). This protein was originally described as a fusion partner of ALL-1
in acute myeloid leukemia (14). AF6 is a homolog of Canoe in
Drosophila which is genetically linked to the Notch cascade
and other signaling pathways (15). Canoe and AF6 have the PDZ domain
which in the case of AF6 was shown to bind to the Eph3B receptor (16).
The amino terminus of AF6 was identified as the Ras-binding site (12, 13). ZO-1, a protein involved in the formation of tight junctions, also
binds to AF6 close to the amino terminus thereby competing with Ras
binding (17). These data suggest a participation of AF6 in the
regulation of cell-cell contacts via a Ras-modulated interaction with
ZO-1. Further evidence for this role of AF6 comes from the discovery of
l-Afadin, a larger AF6 splicing variant in rat which
additionally carries an actin-binding site at the carboxyl terminus
(18). l-Afadin appears to serve as a linker between the
actin cytoskeleton and the plasma membrane in adherens junctions. A
recent finding that the de-ubiquitinating enzyme Fam (fat
facets in mouse) interacts with AF6 suggests that the AF6
action is based on the turnover of participating proteins (19).
In contrast to the well characterized effectors Raf, RalGEF, and
PI(3)kinase, AF6 is a protein with no enzymatic function. It seems to
serve as a scaffolding component for protein complexes. This would
represent a new type of Ras effector because Ras binding in this case
does not lead to the activation of the enzymatic activity of the
effector. In contrast, for the regulation of Raf only transient Ras
binding is necessary. Therefore in this work the Ras binding domain
(RBD) of AF6 is defined, and its interaction with Ras is characterized
by thermodynamic and kinetic parameters and compared with the binding
behavior of the RBDs of Raf and RalGEF. Since for other effectors
highly specific binding to different Ras proteins was
demonstrated, all members of the Ras subfamily are included in
this investigation.
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MATERIALS AND METHODS |
Cloning--
Rat and human AF6 fragments were amplified via
standard polymerase chain reaction methods, ligated into pGEX and yeast
plasmids, respectively, and transformed into Bl21 and DH5
. Mutations
were introduced in Ras by standard polymerase chain reaction protocols (20). All clones were verified by sequencing (ABI-Prism,
Perkin-Elmer).
Yeast Two-hybrid Assay--
Yeast two-hybrid studies were
performed in Saccharomyces cerevisiae Y190 according to the
system of Chevray and Nathans (21). Briefly, competent Y190 cells were
prepared as described (22) and cotransformed with 1 µg of each of the
two-hybrid vectors. Efficient transformation was checked on synthetic
medium lacking leucine and tryptophan (
Leu/Trp). Transformed yeast
cells were grown on synthetic medium lacking leucine, tryptophan, and
histidine containing 25 mM 3-amino-1,2,3-triazole
(
Leu/Trp/His, Sigma) for the indicated time in order to detect
interaction between Ras and the AF6 constructs (Fig.
1). 2% glucose was used in all media as
the carbon source.
-Galactosidase activity was measured by
filter-lifting the yeast cells grown on
Leu/Trp/His medium and
staining the yeast cells with 1 mg/ml X-Gal at 37 °C for the indicated time.

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Fig. 1.
Interaction of amino-terminal AF6 fragments
with Ras in the yeast two-hybrid assay. Following AF6 constructs
cloned into pPC86 were probed against RasG12V-(1-166) in pPC97:
a, aa 1-141, b, aa 1-149, c, aa
1-170, d, aa 30-141, e, aa 36-206
(a-d are from the rat sequence and e is from
human). Panel A, 3 days' growth on Leu/Trp
agar plates. Panel B, 3 days' growth on
Leu/Trp/His agar plates after the colonies were replated from the
Leu/Trp agar plates. Panel C, X-Gal test with
the grown yeasts from panel B. The yeasts were incubated for
70 min with X-Gal after filter lifting them from the Leu/Trp/His agar
plates.
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Proteins--
The Escherichia coli strain Bl21 was
grown in standard I medium (Merck), and the expression of the pGEX
plasmid encoding the glutathione S-transferase/AF6-RBD
fusion protein was induced by the addition of 0.1 mM
isopropyl-
-D-thiogalactoside after OD = 0.6 was
reached. The culture was incubated at 30 °C for 4 h and then
centrifuged. The resuspended cell pellet was sonicated, and the lysate
was cleared by centrifugation (32,000 × g). The insoluble and soluble fractions were analyzed by SDS-polyacrylamide gel
electrophoresis indicating that the glutathione
S-transferase/AF6-RBD fusion protein (42 kDa) is readily
soluble (Fig. 2, lanes 1 and 2). The glutathione S-transferase fusion was
purified according to the standard procedure by means of a
glutathione-Sepharose column (Amersham Pharmacia Biotech). After
protein cleavage by thrombin (10 units/ml, overnight) and elution of
AF6-RBD from the column (Fig. 2, lanes 3-6), this protein
fragment was further purified by size exclusion chromatography
(Superdex 75, Amersham Pharmacia Biotech). The protein fractions were
analyzed for purity by SDS-polyacrylamide gel electrophoresis, pooled,
and concentrated to 20 mg/ml by ultrafiltration (Vivaspin 15, Vivascience), shock-frozen in liquid nitrogen, and stored at
80 °C. The preparation of the Ras proteins and the nucleotide
exchange procedure are described (23). The catalytic fragment of
p120-GAP comprising residues 714-1047 was kindly provided by R. Ahmadian (see Ref. 24).

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Fig. 2.
Purification of AF6-RBD visualized by a
Coomassie-stained SDS-polyacrylamide gel. Bacterial lysate after
centrifugation: lane 1, pellet; lane 2, supernatant; lane 3, GSH column flow-through; lane
4, protein eluted with GSH from GSH column after 12 h
thrombin incubation; lane 5, AF6-RBD eluted with buffer;
lane 6, AF6 RBD after gel filtration. Lane M,
marker proteins with molecular masses as indicated (SDS 7;
Sigma).
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Experimental Techniques--
Protein concentrations were
measured using the dye assay described by Bradford (25). Bovine serum
albumin was used for calibration. Nucleotide concentrations in the
GTPase assay were quantified by HPLC (Beckman) using a reversed phase
C18 column (ODS Ultrasphere, Beckman). For further details see Lenzen
et al. (26). Fluorescence measurements were carried out on a
spectrofluorimeter (Fluoromax, Spex) equipped with a thermostatting
device. For stopped-flow measurements an SM-17 apparatus (Applied
Photophysics) was used. Rate constants can reliably be measured up to
500 s
1 since the dead time for the mixing of two
solutions is in the range of 1-2 ms. Ras·mant-GppNHp was mixed with
AF6-RBD in more than 5-fold molar excess in order to have pseudo-first
order conditions for the association. Accordingly, the fluorescence
traces monitored were fitted with an exponential curve yielding
kobs. Mant nucleotides were excited at 360 nm,
and the fluorescence was monitored through a 408-nm cut-off filter. If
not indicated otherwise, all experiments were carried out in a buffer
containing 20 mM Tris/HCl, pH 7.5, 100 mM NaCl,
5 mM MgCl2, and 1 mM
dithiothreitol. The proteins were transferred into other buffers using
Nap-5 columns (Amersham Pharmacia Biotech).
Differential scanning calorimetry was performed with a VP-DSC (MicroCal
Science). Evaluation of the data was done with the manufacturer's
software (Microcal Origin 4.1). Briefly, the buffer base line was
subtracted from the experimental curve. According to the two-state
model a curve was fitted to the experimental data including
Cp effects.
The NMR spectra were recorded on a Bruker DMX-500 spectrometer
operating at a phosphorous resonance frequency of 202 MHz. The signal
was referenced to 85% phosphoric acid which was sealed in a glass
sphere immersed in the sample. Further experimental conditions are
given in Fig. 8.
 |
RESULTS |
Minimization of the AF6-RBD--
For the biochemical and
structural characterization of the interaction of AF6 with Ras and
other small GTPases, bacterial synthesis of recombinant protein at
reasonable levels is required. In order to define a soluble and stable
protein fragment which comprises the minimal Ras binding domain of the
AF6 protein, different constructs were tested for their ability to bind
to Ras. In a first step this was done semi-quantitatively by the double
hybrid assay as described by Jaitner et al. (27). RasG12V
truncated by the 23 carboxyl-terminal amino acids was introduced into
pPC97 and cotransformed with different AF6 constructs ligated into
pPC86. The construct starting from the amino terminus (amino acids
1-170) as described by van Aelst et al. (12) showed a
strong signal in this system (Fig. 1) indicating a strong interaction
with Ras. These colonies grow within 3 days, and the stain seen in Fig. 1, panel C, developed after 70 min incubation with X-Gal.
Shortening this fragment by 21 and 29 amino acids at its
carboxyl-terminal end, respectively, improved binding significantly
(Fig. 1, panel B). The X-Gal response is similar to that of
Ras and Raf-RBD which are known to interact strongly (27). Under the
same conditions the sequence of the RBD of AF6 (aa 36-206) reported
earlier (13) did not support growth in the Y190 strain. A similar
result was obtained with another amino-terminal truncated sequence (aa
30-141) which contains all residues from the predicted RBD of AF6
(28). The latter showed only small colonies after 9 days of growth
which gave a poor X-Gal stain after overnight incubation (data not shown).
The DNAs encoding different AF6 constructs, namely residues 1-84,
13-133, 30-141, and 1-141, were transferred into pGEX-4T3 vectors
and expressed in the bacterial strain BL21 in order to check the
integrity and the solubility of the protein fragments. The two former
fragments were poorly soluble and were partly degraded in E. coli. From the two latter protein fragments 30-141 bound much
less strongly to Ras than 1-141 (assay see below). From these observations we concluded that the Ras binding domain (RBD) of AF6
requires residues 1-141 for full integrity and strong binding to Ras.
This fragment is defined as AF6-RBD and is used for further biochemical
experiments in this study. By size exclusion chromatography and
comparison to the elution volumes of standard proteins (data not
shown), it was demonstrated that AF6-RBD is a homogeneously monomeric protein.
To check the stability of AF6-RBD and to show that it constitutes a
stable folding domain, it was further characterized by differential
scanning calorimetry. A solution of AF6-RBD was heated from 20 to
80 °C at 70 °C/h, and unfolding was monitored with the change in
the heat capacity Cp (Fig.
3). The reversibility of the unfolding
transition in a solution at pH 9.0 was reasonably high for a
thermodynamic analysis, i.e. 85% recovery of folded protein
after each scan. This analysis was carried out under the assumption of
a two-state equilibrium, i.e. the protein is either in the
native, folded state, or in the unfolded state, and no intermediates
occur (29). Such a behavior is seen for most small proteins which
constitute a single cooperativity unit. The fit to the data yielded
Hu = 62.7 kcal/mol for the enthalpy of unfolding
and Tm = 56,1 °C for the melting temperature. The
scanning rate had no influence on the melting point and the enthalpy.
At 40 and 90 °C/h 60 and 95% reversibility of unfolding was
observed, respectively, which can be explained by the different times
the unfolded protein was kept at high temperature. The quasi-reversible unfolding transition may be taken as additional evidence for the integrity of AF6-RBD.

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Fig. 3.
Differential scanning
calorimetry with AF6-RBD. 0.2 mg/ml AF6-RBD in 20 mM
glycine (pH 9.0 at 20 °C), 100 mM NaCl, and 1 mM -mercaptoethanol was scanned at a rate of 70 °C/h.
A theoretical curve (dashed line) was fitted to the
experimental temperature dependence of the heat capacity (solid
line). The calculated base line (dotted line) is also
shown. The best fit parameters for this experiment were
Tm = 56.1 °C,
Hu(Tm) = 62.7 kcal/mol, and
Cp = 0.3 kcal/mol/K.
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AF6-RBD Binding to Ras Proteins--
In our earlier studies on the
interaction of Ras proteins with effector RBDs, we made use of the
inhibition of nucleotide dissociation from Ras upon effector binding
(30). AF6-RBD also inhibits nucleotide dissociation from Ras. In this
GDI assay Ras is quantitatively loaded with the non-hydrolyzable GTP
analog with a fluorescent label attached to the ribose moiety. The
dissociation of this nucleotide mant-GppNHp is initiated by adding a
1000-fold excess of unlabeled GppNHp. The fluorescence decay due to
quasi-irreversible mant-GppNHp dissociation is monitored and fitted by
an exponential function. The obtained rate constants are plotted
versus AF6-RBD concentration and a curve fitted according to
Equation 2 yields the dissociation constant Kd as
defined in Equation 1. Equation 2 may be applied since the
equilibration of the Ras·AF6 complex is fast compared with the
nucleotide dissociation as shown below. The Ras protein, the
nucleotide, and the effector AF6-RBD are abbreviated by R,
N, and E, respectively, and k
1
and k
2 represent the nucleotide dissociation
rate constants from Ras alone and from the Ras·effector complex,
respectively. Index 0 denotes total concentrations.
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(Eq. 1)
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(Eq. 2)
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In Fig. 4 the dependence of the
observed nucleotide dissociation rate constant on the AF6-RBD
concentration is shown for Rap1A and Ras. The Kd
values obtained from these data and also for Rap2A, R-Ras, and TC21 are
listed in Table I, and it is remarkable
that Rap1A binds more than 10-fold more tightly to AF6-RBD than Ras and
Rap2A, respectively.

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Fig. 4.
GDI assay with AF6-RBD. Dissociation
rate constants of mant-GppNHp from Ras (upper panel) and
Rap1A (lower panel) in dependence of AF6-RBD concentration
at 37 °C. The rate constants were measured as described in the text,
and the Kd values obtained from the fit according to
Equation 2 are listed in Table I.
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Table I
Kd values for AF6-RBD and members of the Ras subfamily
The data were obtained with the GDI method with mantGppNHp and mantGDP
bound to the Ras protein. For some proteins Kd > 100 µM is indicated. This means that up to AF6-RBD
concentrations of 100 µM less than 50% inhibition of
nucleotide dissociation is observed.
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It was also possible to measure the affinity between Ras and AF6-RBD by
means of direct fluorescence titration. At 20 °C the fluorescence of
Ras·mant-GppNHp drops by 12% on saturation with AF6-RBD as shown in
Fig. 5, upper panel. In the
lower panel a curve is fitted to the titration data
according to Equation 2 where k
1 and
k
2 are replaced by the maximum and minimum
fluorescence values, respectively, and kobs is
replaced by the relative fluorescence. The Kd value
of 1.8 µM obtained from this fit is in good agreement
with the result of 3.0 µM from the GDI assay regarding
the difference in temperature. At 37 °C the titration is impossible
since there is almost no change in fluorescence detectable. On addition
of 100 µM AF6-RBD to Ras·mant-GDP no change in
fluorescence is seen (Fig. 5, upper panel). As for the GDI
data this is explained by the lack of binding at this concentration
which means that the affinity of AF6 to the GDP form of Ras is at least
33-fold lower compared with the GTP form.

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Fig. 5.
Fluorescence titration. Upper
panel, fluorescence spectra ( ex = 360 nm) of 0.5 µM mant-GppNHp loaded Ras in the absence (a)
and in the presence (b) of 20 µM AF6-RBD and
correspondingly for Ras·mant-GDP (c and d,
respectively) at 20 °C. Lower panel, Ras·mant-GppNHp
was titrated against AF6-RBD at 20 °C ( ex = 360 nm,
em = 440 nm).
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Evidence has accumulated during the last few years that Ras signaling
branches out into several distinct pathways (for review see Ref. 30)
the output of which may depend on the type of the cell or on the
synergism with other stimuli. By cell transformation studies it was
shown that certain mutations in the Ras effector region lead to
abrogation of signaling to distinct effectors. The mutants T35S, E37G,
and Y40C appear to cause discrimination between the effectors Raf,
RalGEF, and PI(3)kinase (31-35). As an example T35S is believed to
merely activate Raf, E37G only activates RalGEF, whereas Y40C is
reported to activate only PI(3)kinase. D38A is the most prominent
effector mutant of Ras which decreases the affinity to Raf- and
RalGEF-RBD, respectively, by approximately 2 orders of magnitude (23,
30). All these mutants were tested for their ability to bind AF6-RBD,
and the Kd values obtained by the GDI method are
listed in Table I. The mutations T35S and Y40C, respectively, prevent
interaction with AF6-RBD up to a concentration of 100 µM.
In contrast, the Ras mutant E37G decreases the affinity only 10-fold,
and the same small decrease of complex stability is found for D38A.
Mutation at position 31 which is known to be a strongly discriminative
amino acid of Ras and Rap1A for Raf and RalGEF binding (23, 36-38) did
not change the affinity to AF6.
Effect on GTP Hydrolysis--
As AF6-RBD has an influence on the
nucleotide binding to Ras, it was worthwhile to look at the effect on
GTP hydrolysis activity of Ras. At 37 °C 50 µM Ras
loaded with GTP was incubated in the absence and in the presence of
saturating amounts of AF6-RBD (100 µM). The time course
of GTP hydrolysis by Ras was monitored by HPLC analysis of the
nucleotides. The data obtained in Fig. 6 were fitted according to first order kinetics. For both experiments the
same rate constants were calculated (0.03 min
1)
demonstrating that AF6-RBD binding to Ras does not change its GTPase
activity.

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Fig. 6.
The influence on GTPase activity. 50 µM Ras·GTP was incubated at 37 °C, and aliquots were
analyzed for nucleotide concentrations by HPLC. Solid
symbols indicate the presence of 4 nM of the catalytic
fragment of GAP, and the triangles represent the experiments
in the presence of 100 µM AF6-RBD.
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Effector molecules and GAPs bind to overlapping sites on Ras (37, 39,
40). Accordingly, the catalytic activity of GAP is inhibited by Raf and
RalGEF (21). In order to check if AF6 binds to Ras in the same manner
as the other effectors, the inhibition of GAP was investigated. 50 µM Ras·GTP and 4 nM of the catalytic fragment of GAP were incubated at 37 °C, and the nucleotide
concentrations were probed at short time intervals (Fig. 6). The
addition of AF6-RBD reduced the catalytic activity of GAP suggesting
but not proving competitive binding of AF6 and GAP. In the presence of saturating amounts of AF6-RBD the binding of GAP is completely inhibited as the rate of GTP hydrolysis corresponds to the intrinsic activity of Ras (Fig. 6).
Binding Kinetics--
When Ras binds to Raf, RalGEF, or
PI(3)kinase, it activates the enzymatic activity of these effector
molecules by not fully understood mechanisms. Although Ras binds
tightly to Raf, it was shown that these proteins form a short-lived
complex (41, 42). This highly dynamic behavior of the Ras·Raf complex
could be important for the activation of Raf kinase activity or for the
down-regulation of Ras by GAP. For the Ras·RalGEF complex similar
interaction dynamics are
observed.2
The binding kinetics of Ras/AF6 were investigated by means of the
stopped-flow technique. The fluorescence change observed on binding of
AF6-RBD to mant-GppNHp-loaded Ras proteins as described above (Fig. 5)
was used as detection signal. For Ras as well as for Rap1A the observed
rate constant kobs showed a linear dependence on
the concentration of AF6-RBD up to 15 µM. From the linear
slope fitted to the data kon, the apparent
association rate constant, is obtained, and the intercept corresponds
to the dissociation rate constant koff. The
dissociation rate constant can be measured more accurately by
displacement experiments. Ras·mant-GppNHp is displaced from the
complex with AF6-RBD by addition of a large molar excess of non-labeled
Ras·GppNHp. In a typical experiment a solution containing 0.5 µM Ras·mant-GppNHp and 6 µM AF6-RBD was
mixed with 60 µM Ras·GppNHp. The observed rate constant
in this case is governed by the dissociation process. The dissociation rate constants obtained by this method agree with the intercepts mentioned above. In the case of Rap1A the dissociation rate constant is
smaller than the experimental error for the intercept. Here the
dissociation rate constant can only be obtained from the displacement experiments. At 26 °C lifetimes of the AF6·RBD complexes with Ras
and Rap1A of 10 and 100 ms are calculated, respectively. This is
similarly short-lived as the Ras·Raf-RBD complex (42). The results
for the temperature range between 10 and 26 °C are listed in Table
II together with the ratios
koff/kon which agree well with the Kd values reported in the upper
section.
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Table II
Stopped-flow kinetics
Rate constants are given for the interaction of AF6-RBD with
Ras.mantGppNHp and Rap.mantGppNHp, respectively, obtained by
stopped-flow experiments.
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From the temperature dependence of the rate constants the energies of
activation for complex dissociation and association Eaoff and
Eaon, respectively, can be obtained.
According to the Arrhenius equation the energies of activation
Eaoff = 16 kcal/mol and
Eaon = 14 kcal/mol for Ha-Ras and
Eaoff = 9.5 kcal/mol and
Eaon = 10 kcal/mol for Rap1A are
calculated, respectively.
At room temperature and at concentrations higher than 20 µM kobs is too large to be
measured by means of the stopped-flow technique. However, at 10 °C
saturation of Ras/AF6 binding kinetics is observed at higher
concentrations which is an indication of a two-step binding process.
The formation of an initial weak complex is followed by a
conformational rearrangement leading to tight binding. Under the
assumption that the dissociation of the initial complex is fast
compared with its conformational rearrangement, the observed rate
constant for the two-step binding process is described by Equation 3.
K1 is the association constant for the initial
weak complex, and k+2 and
k
2 represent the rate constants of the forward
and backward conformational rearrangements, respectively. Note that at
concentrations far below 1/K1 Equation 3 can be
approximated by a linear equation (see above).
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(Eq. 3)
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In Fig. 7 the observed rate
constants measured under pseudo-first order conditions are plotted
versus AF6-RBD concentration. From the fit to the data
according to Equation 3 a value of K1 = 25,000 M
1 is obtained which corresponds to a
dissociation constant of the initial encounter complex of 40 µM. The maximum k+2 + k
2 is reached at 310 s
1. For
Rap1A no sign of saturation of AF6-RBD binding kinetics could be
observed up to 50 µM where the upper limit for
stopped-flow experiments of kobs = 500 s
1 is reached (data not shown).

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Fig. 7.
Saturation of the binding kinetics. In a
thermostatted (10 °C) stopped-flow apparatus Ras·mant-GppNHp (0.5 µM) was mixed with AF6-RBD at concentrations as
indicated. Fluorescence was excited at 360 nm and monitored through a
cut-off filter of 408 nm. The data are fitted according to Equation 3.
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31P NMR Studies on the Ras·AF6-RBD Complex--
By
31P NMR experiments it was shown previously that the
GppNHp-bound form of Ras exists in two conformational states (43) which
is reflected by the band splitting of the
- and
-phosphorus resonances in the bottom panel of Fig.
8. The higher populated state (61%) was
interpreted as the conformation with the effector loop (switch I) in
close proximity to the phosphate groups exhibiting a ring current shift
of the Tyr-32 side chain onto the phosphorus atoms (state 2). In the
other state Tyr-32 is pointed away from the nucleotide (state 1). From
the bottom to the top panel of Fig. 8 increasing
amounts of AF6-RBD are added to a Ras·GppNHp solution. The change of
the
- and
-phosphorus resonances reflects a shift from state 1 to
state 2. At saturation only state 2 is populated indicating a
stabilization of this conformation by AF6-RBD binding.

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Fig. 8.
31P NMR spectra of a titration
series of AF6-RBD to Ras· GppNHp. 500 MHz spectra of
Ras·GppNHp at 1 mM and AF6-RBD from bottom to
top panel at 0, 0.2, 0.5, and 1 mM,
respectively, at 5 °C. The resonances between 12 and 11 ppm
correspond to P (where P indicates phosphate), between 4 and 2
to P , and at 0.3 to P . The peak at 2.3 ppm originates from free
phosphate ions.
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DISCUSSION |
Previously, AF6 was identified as a potential Ras target (12, 13)
which would add to the list of well established effectors like Raf
kinase, RalGEF, and PI(3)kinase. It was also demonstrated that AF6
binds to tight junctions in epithelial cells via the interaction of the
AF6 amino terminus with ZO-1 which appears to be regulated by Ras (17).
This mechanism would further increase the complexity of Ras signaling
and could explain the perturbation of cell-cell contacts induced by
activated Ras. Activation of some Ras effectors by Rap, R-Ras, or TC21
was also demonstrated (44-46). Therefore, quantitative knowledge about
the interaction of AF6 with all putative partners is necessary in order
to estimate physiological consequences like inhibition or synergism in
the context of the cell where all Ras proteins can be present.
Furthermore, information about the dynamics of protein-protein
interactions could be an important criterion for the assessment of
signaling or scaffolding complexes. In this work the interactions
between AF6 and members of the Ras subfamily have been characterized by biophysical methods. A stable protein fragment constituting the RBD of
AF6 was defined, and its binding specificity and the dynamic behavior
of Ras interaction were investigated.
Apart from Ras and Rap1A (12, 13) no other Ras proteins had been
reported to interact with AF6 so far. The data presented here
demonstrate that the closely related Rap2A, TC21, and R-Ras also bind
to AF6-RBD. Moreover, like the RBDs of the effectors Raf and RalGEF,
the RBD of AF6 showed different affinities to the various Ras proteins.
Intriguingly, AF6 forms the most stable complex with Rap1A which is
more than 10 times tighter than with Ras, Rap2A, TC21, and R-Ras,
respectively, as shown in Table I. For Rap1 an influence on the
regulation of cell morphology via the cytoskeleton was shown in various
organisms like yeast (47, 48), Neurospora crassa (49),
Dictyostelium (50), and in the Swiss 3T3 cell line (51). It
is uncertain whether the affinity of the Ras·effector complex
correlates with its physiological importance, but it will be worthwhile
to investigate the biological function of the Rap1A-AF6 interaction.
When specificity is discussed it should be stressed that Rap2A and Ras
have the same affinity. This is surprising since throughout the
effector region (residues 20-45) the homology is higher between Rap1A
and
2A than between Rap1A and Ras (see Fig.
9). Residues that are different between
Rap1A and -2A are the same for Rap1A and Ras, and residues different
between Rap1A and Ras are the same for Rap1A and -2A. So, the observed
affinities could be due to subtle differences of the Rap1A and -2A
conformations that are not obvious from the primary sequence. The
involvement of switch II of Ras in AF6-RBD binding is unlikely since
two mutants in this region, E62A and E63A, do not alter significantly
the affinity that is also observed for Raf and RalGEF
binding.2

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|
Fig. 9.
Primary sequence alignment of the effector
region of Ras-like proteins. The one-letter code for
amino acids is used to compare the residues of the Ras relatives in the
effector region (residues 32-40). Dots represent conserved
amino acids.
|
|
Partial loss of function mutations of Ras were reported from various
investigators. These particular mutants have been found to bind and
activate only distinct effectors and so they are useful for reducing
the complexity of Ras signaling in biological experiments. The
affinities of these Ras mutants to AF6-RBD were quantified (Table I),
and together with the affinities to the different Ras proteins one may
say that the binding specificity of AF6 has a similar profile as
RalGEF. Exceptions though are represented by the Ras/Rap residue 31 and
by D38A. These findings may be helpful for the design of experiments
addressing the biological function of AF6. By the appropriate design of
a Ras or Rap1A mutant, AF6 binding may be preferred (e.g.
E31K, D38A) or specifically blocked (e.g. T35S, Y40C), respectively.
Proteins in general are constituted of largely independent building
blocks with distinct functions like catalytic activity or docking to
other proteins (e.g. Refs. 52 and 53). Our studies were done
with only a fragment of the AF6 protein implying that only this part of
the protein is responsible for Ras binding. Support for this assumption
may be taken from our earlier findings with Raf where the binding
constants of several RBD mutants showed a good correlation with the
activation of Raf by Ras and with the binding of Ras to full-length Raf
carrying the same mutations as probed by a reporter gene assay in a
mammalian cell line and by the double hybrid method in yeast cells,
respectively (54, 55).
From kinetic studies with Raf-RBD it was concluded that the association
with Ras occurs in two steps (42). The formation of an initial complex
with low stability is followed by a conformational rearrangement of the
proteins leading to tighter binding (induced fit). This is indicated by
the saturation of the binding kinetics at high concentrations where the
rate of the second step becomes rate-limiting. This behavior is also
observed for the association of Ras and AF6-RBD. In summary, the
mechanism of Ras binding is similar for AF6-RBD and Raf-RBD, and
particularly, the complexes are similarly short-lived. The difference
in nature of an enzymatically acting effector like Raf or RalGEF and a
potential scaffolding protein like AF6 is not reflected in the dynamics
of complex formation and dissociation of their RBDs.
Further evidence for a similar interaction is taken from the
influence of the Ras-AF6-RBD interaction on the dynamics of the effector region as probed by 31P NMR spectroscopy. Notably,
no shift of the phosphorus resonances occurs when AF6-RBD binds to Ras
which indicates that AF6-RBD attaches to Ras remote from the
nucleotide-binding site like the RBDs of Raf and RalGEF. Moreover,
after addition of saturating AF6-RBD concentrations, the band splitting
disappears in favor of the state where Tyr-32 is located in closer
proximity to the nucleotide. The same stabilization of this
conformational state was reported for the Ras/Raf and Ras-RalGEF interactions.
In conclusion, the members of the Ras superfamily recognize AF6 in a
manner characteristic for all effectors, and Rap1A shows the highest
affinity. Whereas the RBDs of Raf and RalGEF have the same size and
despite their low sequence homology show the same structural fold, the
RBD of AF6 with 141 residues is significantly larger. Nevertheless, the
binding characteristics as probed by mutagenic and kinetic studies as
well as by 31P NMR spectroscopy are quite similar for all
these effector RBDs. The influence of AF6 on the nucleotide
dissociation from Ras and the competition of AF6 and GAP strongly
suggest the same type of interaction as Raf and RalGEF. Specific
differences are observed, however, such as the different contributions
to binding by residues 31 and 38 from Ras. Structural results must be
waited for in order to compare the Ras-AF6 interaction on an atomic
level to the other RBDs. It will be interesting to see if AF6 is
another effector with the same RBD topology as Raf and RalGEF, how the
additional residues are accommodated, and how ZO-1 binding to AF6 or
the interaction with other proteins is regulated by Ras or its relatives.
 |
ACKNOWLEDGEMENTS |
We thank Linda vanAelst and Kozo Kaibuchi for
kindly providing the cDNA of rat AF6 (aa 1-170) and human AF6, respectively.
 |
FOOTNOTES |
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant He 2679/1.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.
§
To whom correspondence should be addressed. Tel.: 49 231 1206 351; Fax: 49 231 1206 230; E-mail:
christian.herrmann{at}mpi-dortmund.mpg.de.
2
T. Linnemann and C. Herrmann, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
PI(3)kinase, phosphatidylinositol 3-kinase;
GDI, guanine nucleotide dissociation
inhibition;
GppNHp, guanyl-5'-yl imidodiphosphate;
mant, 2',3'-(N-methylanthraniloyl), a fluorophor attached at 2'-
or 3'-position to the nucleotide;
HPLC, high pressure liquid
chromatography;
RalGEF, Ral guanine nucleotide exchange factor;
RBD, Ras binding domain;
X-Gal, 5-bromo-4-chloro-3-indolyl-
-galactopyranoside;
aa, amino acids;
GAP, GTPase-activating protein.
 |
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