Báo cáo khoa học: The guanine nucleotide exchange factor RasGRF1 directly binds microtubules via DHPH2-mediated interaction docx

The guanine nucleotide exchange factor RasGRF1 directly
binds microtubules via DHPH2-mediated interaction
Greta Forlani, Simona Baldassa, Paola Lavagni, Emmapaola Sturani and Renata Zippel
Department of Biomolecular Sciences and Biotechnology, University of Milan, Italy
Microtubules are crucial elements in the generation
and maintenance of neuronal morphology. They play
a role not only in the establishment of neuronal out-
growth during brain development but are also involved
in the remodeling of mature neurons [1].
The dynamics of microtubules as well as their inter-
actions with other cytoskeletal elements are regulated
by microtubule-binding proteins [2]. Some of them,
such as microtubule-associated proteins (MAPs), pro-
mote the assembly of microtubules, whereas others,
such as stathmin, increase microtubule instability.
Other microtubule-binding proteins are involved in the
transport of organelles and cargos along the micro-
tubule network. Microtubule dynamics is modulated by
different extracellular signaling molecules [3], and the
monomeric GTP-binding proteins Rho and Rac are
implicated in these processes [4]. In fibroblasts, Rho
activity induces microtubule stabilization independ-
ently from its effect on actin filaments [5,6]. p21-activa-
ted kinase, one the effectors of Rac, phosphorylates
stathmin, thus inhibiting its destabilizing effect on
microtubules [7].
RasGRF is a family of guanine nucleotide exchange
factors consisting of two members: RasGRF1 [8–10]
exclusively expressed in neurons of the central nervous
system [11] and in b cells of the pancreas [12]; Ras-
GRF2, highly expressed in the brain but also present

in other tissues [13]. RasGRF proteins show an overall
homology close to 80% and share a common modular
structure: the C-terminal region contains the catalytic
Keywords
DHPH2 module; microtubule; Ras; RasGRF;
sodium arsenate
Correspondence
R. Zippel, Department of Biomolecular
Sciences and Biotechnology, University of
Milan, via Celoria 26, 20133 Milan, Italy
Fax: +39 025031 4912
Tel: +39 025031 4914
E-mail:
(Received 16 January 2006, revised 7 March
2006, accepted 13 March 2006)
doi:10.1111/j.1742-4658.2006.05226.x
RasGRF is a family of guanine nucleotide exchange factors with dual spe-
cificity for both Ras and Rac GTPases. In this study, using mouse brain
extracts, we show that both RasGRF1 and RasGRF2 interact with micro-
tubules in an in vitro microtubule assembly system and this binding is very
tight. To characterize this association, recombinant purified proteins con-
taining different regions of RasGRF1 were tested for their ability to bind
microtubules preassembled from pure tubulin. Only the DHPH2 tandem
directly associates with microtubules, whereas the isolated DH or PH2
domains do not, indicating that the entire DHPH2 region is required for
this association. The interaction occurs with high affinity (K
d
 2 lm) and
with a stoichiometry, at saturating conditions, of one DHPH2 molecule for
two tubulin dimers. Competition experiments support the hypothesis that

the DHPH2 module is largely responsible for RasGRF1–microtubule inter-
action. In vivo colocalization of RasGRF1 and microtubules was also
observed by fluorescence confocal microscopy in nonneuronal cells after
stimulation with an oxidative stress agent and in highly differentiated
neuron-like cells. Identification of microtubules as new binding partners of
RasGRF1 may help to elucidate the signaling network in which RasGRF1
is involved.
Abbreviations
DH, Dbl-homology domain; ERK, extracellular regulated kinase; GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase;
LPA, lysophosphatidic acid; MAP, microtubule-associated protein; MAPK, mitogen-activated protein kinase; MBP, maltose-binding protein;
PH, Pleckstrin homology domain.
FEBS Journal 273 (2006) 2127–2138 ª 2006 The Authors Journal compilation ª 2006 FEBS 2127
guanine nucleotide exchange domain for Ras, and the
Dbl-homology domain (DH) and the Pleckstrin-
homology domain (PH) which are present in the cen-
tral part of the molecule are responsible for Rac
exchange activity [14–17]. The N-terminal region con-
tains a PH domain, a coiled-coil region, and an IQ
domain which binds calmodulin in a calcium-depend-
ent manner [18]. Very recently, a ‘neuronal domain’
located in the central part of RasGRF1, but absent
from RasGRF2, has been identified [19]. This region
has been found to be responsible for the physical inter-
action of RasGRF1 with the NR2B subunit of the
NMDA subtype of glutamate receptor.
RasGRF1 is activated by G-protein-coupled recep-
tors [20,21] and requires both calcium and calmodulin
to exert its activity on Ras [18,21]. The protein is phos-
phorylated on serine, threonine and tyrosine residues
in vivo and is a substrate for different kinases in vitro

[22–24].
This exchange factor is expressed only after birth in
parallel with the maturation of synaptic connections. It
is localized at synaptic junctions and is enriched in
postsynaptic densities [25,26].
Mice lacking RasGRF1 show defects in synaptic
plasticity and memory consolidation, although some
controversy exists about the molecular nature of these
impairments [27,28]. In addition, these mice exhibit a
higher intrinsic neuronal excitability, are more suscept-
ible to convulsant drugs [29], and do not develop toler-
ance to chronic exposure to cannabinoids [30,31].
Up to now, little is known about the protein net-
work connected with RasGRF1 in neurons. In this
paper, we provide evidence that RasGRF1 binds
microtubules, and the entire DHPH2 module appears
to be largely responsible for this association. Scatchard
analysis reveals strong binding, with an estimated dis-
sociation constant of  2 lm. The stoichiometry of the
association is up to one molecule of DHPH2 per two
tubulin dimers. RasGRF1 does not appear to modu-
late microtubule dynamics. Moreover in vivo interac-
tion has been demonstrated. Thus, although further
investigation is required to elucidate the functional sig-
nificance of this association, identification of micro-
tubules as new binding partners for RasGRF1 may
help to us gain information on how the activity of this
bifunctional guanine nucleotide exchange factor is
modulated.
Results

Interaction of RasGRFs with microtubules
RasGRF1 is expressed in adult brain and, as previ-
ously shown, it is enriched in postsynaptic densities
[26]. However, RasGRF1 is also present in the cytoso-
lic fraction (Fig. 1A; S100). As well as the expected
140-kDa band, antibodies to RasGRF1 detected in
brain extracts another band of slightly lower molecular
mass which is also present in RasGRF1 knockout
mice. This band was isolated, analyzed by MALDI
TOF, and found to correspond to RasGRF2
(Fig. 1B,C).
In the course of a more general study on proteins
that interact with RasGRF1, we investigated its poss-
ible association with tubulin. Unpolymerized tubulin
did not significantly associate with RasGRF1, as indi-
cated by coimmunoprecipitation experiments on cyto-
solic brain extracts (data not shown). Further
Fig. 1. Distribution of RasGRF1 and Ras-
GRF2 in mouse brain extracts. (A) Equal
amounts of protein from total brain extracts,
particulate fraction (P100) and soluble frac-
tion (S100) were analyzed with antibodies to
RasGRF1. (B) Brain extracts obtained from
wild-type and RasGRF1 knockout mice [28]
were immunoprecipitated with antibodies to
RasGRF1 (lane 2, 4) or with nonrelated IgG
(lanes 1 and 3). Immunoprecipitates were
analyzed by SDS ⁄ PAGE and silver staining.
(C) The lower band present in knockout
mice was isolated and analyzed by MALDI

TOF.
RasGRF1 directly binds microtubules G. Forlani et al.
2128 FEBS Journal 273 (2006) 2127–2138 ª 2006 The Authors Journal compilation ª 2006 FEBS
investigation was carried out on polymerized tubulin.
Microtubules were assembled when mouse brain high-
speed supernatant was incubated at 30 °C for 30 min
[32]. Microtubules and MAPs were then isolated by
sedimentation over a sucrose cushion. Soluble and
pellet fractions were analyzed by immunoblotting.
Figure 2A shows that most of both RasGRF1 and
RasGRF2 present in the cytosol (input) cosedimented
with microtubules, although slightly different distribu-
tions between pellet and supernatant were found in
various experiments (see also Fig. 3A). Other proteins
involved in Ras signaling showed different behavior:
Erk2 was mainly found in the supernatant and both
p21-Ras and mSos1 were only found in the soluble
fraction (Fig. 2A).
Figure 2B shows that sedimentation of RasGRFs in
the pellet only occurred when conditions that allow
tubulin polymerization (37 °C) were used. Conversely,
when extracts were incubated at 4 °C (a temperature
that does not allow microtubule assembly), RasGRFs
were mainly present in the supernatant, together with
the soluble unpolymerized tubulin (compare lanes 2
and 3 with lanes 5 and 6). Moreover the amount of
RasGRFs that cosedimented with microtubules was
higher when microtubules were prepared in the pres-
ence of the polymerizing agent taxol, in parallel with
the higher efficiency of tubulin assembly (compare

lanes 1 and 2).
RasGRF association with microtubules is
mediated by neither motor proteins nor MAPs
It has been demonstrated that RasGRF1 binds
IB2 ⁄ JIP2 [33], a scaffold protein for the Jun N-ter-
minal kinase signaling pathway. JIP proteins also link
the motor proteins kinesins with the cargo complex to
be transported along the microtubules [34]. In vitro
microtubule-binding motor proteins can be released
from microtubules by treatment with high concentra-
tion of ATP [32]. To verify that motor proteins medi-
ate RasGRF–microtubule interaction, mouse brain
high-speed supernatant was subjected to microtubule
assembly in the presence of either ATP or the non-
A
B
Fig. 2. RasGRFs cosediment with in vitro assembled microtubules.
(A) Cytosolic high-speed supernatant of a mouse brain homogenate
(Input) was used for the microtubule cosedimentation assay (see
Experimental procedures). Microtubules were polymerized at
37 °C, then loaded on a sucrose cushion and sedimented by cen-
trifugation. The microtubule pellet (MT) was resuspended in the
same volume as the supernatant (SN), and an equal volume of
each fraction and the Input were resolved by electrophoresis, and
analyzed by western blotting. (B) Mouse brain high-speed supernat-
ant was used in a microtubule cosedimentation assay, following
different procedures: microtubules were induced to polymerize at
37 °C with (lanes 1, 4) or without (lanes 2, 5) 10 l
M taxol. To avoid
tubulin polymerization, the extracts were maintained on ice (lanes

3, 6). Supernatants and pellets were isolated by centrifugation, and
equal volumes were used for SDS ⁄ PAGE and analyzed by immuno-
blotting using monoclonal antibodies to a-tubulin and RasGRF1.
Lane 7 represents a low exposure of lane 1.
A
B
Fig. 3. Motor proteins or MAPs do not mediate RasGRF1 associ-
ation with microtubules. (A) Mouse brain high-speed supernatant
was divided into three aliquots and incubated with 10 l
M taxol in
the presence of 10 m
M ATP or 10 mM p[NH]ppA or left untreated
(control). After centrifugation both the supernatant and microtubule
fractions were analyzed by western blotting with antibodies to kine-
sin heavy chain, a-tubulin and RasGRF1. (B) Taxol-stabilized micro-
tubules were resuspended in PME buffer containing either different
concentrations of NaCl (0.5, 1.0
M)or8M urea and centrifuged at
30 °C. The microtubule pellets and supernatants were analyzed by
immunoblotting.
G. Forlani et al. RasGRF1 directly binds microtubules
FEBS Journal 273 (2006) 2127–2138 ª 2006 The Authors Journal compilation ª 2006 FEBS 2129
hydrolyzable ATP analog, adenylyl imidodiphosphate
(p[NH]ppA). The collected pellets and supernatants
were then analyzed for tubulin, RasGRFs and kinesin
heavy chain. The data in Fig. 3A show that neither
treatments modified the amount of RasGRFs bound
to microtubules. Conversely, kinesin heavy chain only
remained associated with the microtubules in the pres-
ence of p[NH]ppA (Fig. 3A).

Also MAPs seem not to be involved in the in vitro
association of RasGRFs with microtubules. In fact
treatment of isolated taxol-stabilized microtubules with
high salt concentration, a condition reported to disso-
ciate MAPs [35], did not affect the amount of Ras-
GRFs in the pellet (Fig. 3B). Urea also did not
dissociate RasGRFs from microtubules.
These results indicate that neither motor proteins
nor MAPs mediate RasGRF interaction with micro-
tubules and suggest that this association is very tight.
The DHPH2 module directly interacts with
microtubules
To verify whether pure microtubules are also able to
bind RasGRF1, microtubules preassembled from pure
commercial tubulin were incubated with a small
aliquot of extracts (5 lg) of Hek293 cells expressing
RasGRF1. Microtubules were then recovered by
centrifugation and analyzed by western blotting for
RasGRF1. Figure 4 shows that most of the RasGRF1
cosedimented with pure microtubules. As expected in
the absence of preassembled microtubules, RasGRF1
was found in the supernatant.
The latter experiment shows that RasGRF1 interacts
with pure microtubules but its direct association is not
yet proven, as proteins present in Ras-GRF1 extracts
could mediate this interaction. To investigate this
point, purified tagged recombinant proteins coding for
different regions of RasGRF1 were prepared (Fig. 5A).
Microtubules preassembled from pure commercial tub-
ulin were incubated with purified proteins, and both

pellets and supernatants were then analyzed. Figure 5B
shows that the DHPH2 module [maltose-binding
protein (MBP)-DHPH2] associated with microtubules,
whereas neither the N-terminal region (MBP-
PHCCIQ) nor the C-terminal catalytic domain (GST-
Cat) did. Moreover, neither the isolated DH nor the
PH2 domain bound microtubules. In the absence of
microtubules, none of the recombinant proteins was
found in the pellet fraction. These findings indicate
that the DHPH2 module, but not the DH or PH2
domain separately, directly interacts with microtu-
bules.
To better characterize DHPH2–microtubule associ-
ation, a constant concentration of microtubules pre-
assembled from pure tubulin (5 lm tubulin dimer)
was incubated with increasing concentrations of
DHPH2 (from 1 to 5 lm) for 20 min at 24 °C. After
Fig. 4. RasGRF1 binds to pure microtubules. (A) HEK293 cells were
transfected with RasGRF1. High-speed cell extract (5 lg) was
incubated with taxol-stabilized microtubules (25 lg pure tubulin
dimers; + MT) or without (– MT). Microtubules and associated
proteins were isolated and Input, supernatant and microtubule
pellet (P) fractions were analyzed by western blotting using anti-
bodies to RasGRF1 and a-tubulin.
A
B
Fig. 5. DHPH2 domain of RasGRF1 binds directly to microtubules.
In vitro binding of recombinant regions of RasGRF1 to pure micro-
tubules. (A) The diagram illustrates the different RasGRF1 fusion
proteins used for in vitro microtubule-binding assay. The table on

the right summarizes the results of binding assays shown in (B).
(B) Purified proteins MBP-PHCCIQ, MBP-DHPH2, MBP-DH, GST-
PH2, GST-Cat, MBP and GST (0.2 l
M) were incubated with (+)
or without (–) pure preassembled microtubules (10 l
M, relative to
tubulin dimers) in a microtubule binding assay (see Experimental
procedures). Input, pellet (P) and supernatant (SN), were analyzed
by western blotting with antibodies to MBP, GST or a-tubulin.
RasGRF1 directly binds microtubules G. Forlani et al.
2130 FEBS Journal 273 (2006) 2127–2138 ª 2006 The Authors Journal compilation ª 2006 FEBS
centrifugation, both supernatants and microtubule pel-
lets were analyzed by SDS ⁄ PAGE and Coomassie Brilli-
ant Blue staining (Fig. 6A). The intensity of the bands
was then determined by densitometry. Plots of the con-
centration of MBP-DHPH2 in the pellets versus total
MBP-DHPH2 protein added to the reaction mixture
are reported in Fig. 6B, and Scatchard analysis is shown
in Fig. 6C. The data reveal that DHPH2 binds microtu-
bules with high affinity, showing an estimated dissoci-
ation constant of  2 lm. The stoichiometry of the
interaction at saturating conditions of MBP-DHPH2 is
one MBP-DHPH2 molecule per two tubulin dimers.
An in vitro competition experiment was performed
to test whether the DHPH2 module is the only region
responsible for the interaction of the entire RasGRF1
molecule with microtubules. Pure microtubules were
incubated for 20 min with RasGRF1-containing cell
extracts (as in Fig. 4) and with increasing concentra-
tions of purified DHPH2 protein. As shown in Fig. 7,

the addition of DHPH2 increasingly reduced the
amount of RasGRF1 bound to microtubules, although
it did not prevent this association completely. These
data support the hypothesis that the DHPH2 domain
is largely responsible for the interaction between full-
length RasGRF1 and microtubules.
The DHPH2 module does not affect in vitro
microtubule dynamics
To gain information on a possible role for RasGRF1
in microtubule dynamics, the effect of the DHPH2
fusion protein on the kinetics of tubulin polymeriza-
tion was investigated by monitoring A
350
with a ther-
mostatically controlled spectrophotometer. As shown
in Fig. 8, the same kinetics was observed in the pres-
ence of 10 lm MBP alone or 10 lm MBP-DHPH2
(Fig. 8, polymerization, compare a with b), suggesting
that the DHPH2 module has no specific effects on
tubulin polymerization.
We then investigated whether DHPH2 protects
microtubules from the disassembling activity of stath-
min, a protein involved in the control of microtubule
dynamics, preventing tubulin polymerization and ⁄ or
promoting microtubule depolymerization. Tubulin, in
the presence of MBP-DHPH2 or MBP, was allowed to
polymerize until a plateau was reached. Then recombin-
ant stathmin (20 lm) was added to the solution, and
A
350

was monitored for further 30 min. As shown in
Fig. 8 (depolymerization), stathmin caused a large
decrease in the steady-state level of polymerized tubulin,
and DHPH2 did not prevent stathmin-induced micro-
tubule depolymerization. Moreover the simultaneous
A
B
C
Fig. 6. Kinetics of DHPH2 binding to microtubules. (A) Constant
amounts of pure taxol-stabilized microtubules (5 l
M, relative to tub-
ulin dimer) were incubated with increasing concentrations of MBP-
DHPH2 (1–5 l
M). Input, microtubule pellet (MT) and supernatant
(SN) were resolved by SDS ⁄ PAGE and stained with Coomassie Bril-
liant Blue. (B) Plots of the amounts of MBP-DHPH2 in the pellets
(bound MBP-DHPH2) (l
M) as a function of total MBP-DHPH2 pro-
tein added to the binding assays (total MBP-DHPH2) (l
M) shown in
(A). The amounts of MBP-DHPH2 were quantitated by densitom-
etry. The intensity of single bands was compared with that calcul-
ated for known amounts of BSA used as standard control and was
expressed as micromolar. Results of the Scatchard analysis are
reported in (C).
Fig. 7. DHPH2 domain competes with RasGRF1 for microtubule
binding. Constant amounts of pure taxol-stabilized microtubules
(5 l
M, relative to tubulin dimer) were incubated with extracts (5 lg)
of HEK293 cells expressing RasGRF1 and increasing concentrations

of MBP-DHPH2 (1, 2, 4 l
M) for 20 min at 24 °C. Input, pellet (P)
and supernatant (SN) were analyzed by western blotting with anti-
bodies to RasGRF1 and a-tubulin. Results representative of three
independent experiments are shown.
G. Forlani et al. RasGRF1 directly binds microtubules
FEBS Journal 273 (2006) 2127–2138 ª 2006 The Authors Journal compilation ª 2006 FEBS 2131
addition of DHPH2 protein and stathmin in the poly-
merization assay did not prevent the inhibitory effect of
stathmin on microtubule assembly (data not shown).
Taken together, these results suggest that the DHPH2
module does not affect in vitro microtubule dynamics.
Colocalization of Ras-GRF1 with microtubules
in intact cells
Prompted by the data reported above and in an
attempt to determine the function of microtubule–Ras-
GRF1 association, we investigated whether this inter-
action occurs in intact cells and whether it is altered
after stimulation with different agents. COS7 cells were
transfected with either full-length RasGRF1 or the
region coding for the first 625 amino acids, which con-
tains all the regions important for the responsiveness
of the protein to calcium signaling [36,37] but devoid
of the CDC25 domain active on Ras.
Serum-deprived cells were either left untreated or sti-
mulated with the calcium ionophore A23187, lysophos-
phatidic acid (LPA), which are known to activate
RasGRF1 [18,20,21], or with sodium arsenate, an
agent that induces oxidative stress [38,39] and stress
granule formation (data not shown and [40]). These

stimuli have been reported to activate different kinases
of the MAPK family (reviewed in e.g. [41–45]). After
treatment with LPA and A23187 (30 min) or with
sodium arsenate (1 h), cells were fixed and immuno-
stained using monoclonal antibodies to tubulin and
polyclonal antibodies to RasGRF1 or the N-terminal
region of RasGRF1 [28]. Immunofluorescence was
then analyzed by confocal microscopy. In unstimulated
cells, we were unable to detect a significant colocaliza-
tion of microtubules with either full-length RasGRF1
or its N-terminal region. Moreover, treatment with the
calcium ionophore or LPA also did not have any dis-
cernible effect (data not shown). Conversely, when
cells were treated with sodium arsenate, the N-terminal
region of RasGRF1 clearly associated with microtu-
bules in a large proportion of the transfected cells
(compare Fig. 9C with Fig. 9F). Identical results
were obtained with sodium arsenite, another arsenic
Fig. 8. DHPH2 tandem does not affect in vitro microtubule dynam-
ics. In vitro tubulin polymerization ⁄ depolymerization assay. Tubulin
(40 l
M) polymerization was performed at 37 °C for 30 min, and
microtubule assembly was monitored at A
350
(polymerization).
Time-course of tubulin assembly in the presence of 10 l
M MBP-
DHPH2 (A) or 10 l
M MBP recombinant proteins (B). The effect of
stathmin (20 l

M) on microtubule depolymerization was also exam-
ined in the second part of the curve (depolymerization). At the time
indicated by the arrow, 20 l
M stathmin was added to the solution
and a slow decrease in the curve was observed, indicating partial
microtubule depolymerization.
Fig. 9. Sodium arsenate induces association of the N-terminal region of RasGRF1 with microtubules in COS7 cells. COS7 cells transfected
with the N-terminal region of RasGRF1 (amino acids 1–625) also containing the DHPH2 tandem were left untreated (A,B,C) or treated with
sodium arsenate (0.5 m
M) for 1 h (D,E,F). Cells were then stained with antibodies to the N-terminal region of Ras-GRF1 (green) (A, D) and
tubulin (red) (B, E) and processed for fluorescence confocal microscopy. Yellow areas indicate red and green signal overlap in merged imag-
es (C, F). Scale bars represent 20 lm. Similar results were obtained with arsenite.
RasGRF1 directly binds microtubules G. Forlani et al.
2132 FEBS Journal 273 (2006) 2127–2138 ª 2006 The Authors Journal compilation ª 2006 FEBS
compound. This effect could not be detected when
full-length RasGRF1 was used in place of the N-ter-
minal region (data not shown). The interaction
observed above did not cause any detectable modifica-
tion of the microtubule network, as also indicated by
the comparison with untransfected cells in the same
preparation (not shown). The very well defined net-
work of microtubules remained almost unchanged
on treatment with arsenate (Fig. 9E) compared with
unstimulated cells (Fig. 9B).
To further investigate RasGRF1–microtubule inter-
action, we used the SK-N-BE neuroblastoma cell line,
stably expressing Ras-GRF1 (SO5 clone) [46]. When
induced to differentiate with retinoic acid, these cells
acquired neuronal morphological characteristics and
expressed a repertoire of proteins similar to those

found in neurons. Thus SO5 differentiated cells were
stained for Ras-GRF1 and tubulin. As shown in
Fig. 10B, tubulin has the typical microtubule organiza-
tion of a neuronal cell. Ras-GRF1 staining was distri-
buted in the cell body and along the neurites and
excluded from the nucleus (Fig. 10A). In these cells,
RasGRF1 was found to partially colocalize with
microtubules mainly within the proximal region of
cellular processes (Fig. 10C), along those neurites in
which tubulin was well organized in microtubule bun-
dles (Fig. 10F), in the tips and in the varicosities. No
colocalization could be depicted in thinner neurites
with a less organized microtubular structure (Fig. 10F
left). We did not detect colocalization of RasGRF1
with the actin network (not shown).
Discussion
In this study, we provide evidence that RasGRF1
interacts both in vivo and in vitro with microtubules.
Both RasGRF1 and RasGRF2 present in the cytosolic
fraction of brain extracts bind microtubules, whereas
other proteins involved in the Ras pathway do not.
Neither high salt nor urea dissociates RasGRFs from
microtubules, indicating that both electrostatic and
hydrophobic interactions are involved in this tight
association. In particular, the lack of effect of high salt
suggests that MAPs are not involved in the interaction.
Also motor proteins, for instance kinesin, do not
appear to mediate this interaction, so that it is unlikely
that RasGRFs use microtubules as tracks for its trans-
port to the neurites.

Fig. 10. Ras-GRF1 partially colocalizes with microtubules in a neuron-like cell line. Confocal immunofluorescence analysis of Ras-GRF1 and
microtubules in SK-N-BE ⁄ SO5 cell lines. Differentiated SO5 cells were stained with antibodies to Ras-GRF1 (green) (A, D) and tubulin (red)
(B, E) and processed for fluorescence confocal microscopy. Yellow areas indicate red and green signal overlap in merged images (C, F).
Scale bars represent 20 lm in (A, B, C) and 40 lm in (D, E, F).
G. Forlani et al. RasGRF1 directly binds microtubules
FEBS Journal 273 (2006) 2127–2138 ª 2006 The Authors Journal compilation ª 2006 FEBS 2133
Using purified proteins coding for different regions
of RasGRF1, we found that neither the N-terminal
region fused to MBP (MBP-PH-CC-IQ) nor the C-ter-
minal catalytic domain bound microtubules. Con-
versely, the DHPH2 tandem interacted directly and
with high affinity with them. Neither the DH nor the
PH2 did separately. Competition experiments indicated
that the DHPH2 was responsible for a large part of
the interaction between RasGRF1 and microtubules,
although we cannot rule out that other regions of the
protein contributed. Thus the DHPH2 module has at
least two functions: not only, as reported previously, is
it responsible for Rac exchange activity [16], but it also
interacts with microtubules. Other DHPH-containing
proteins have been shown to bind microtubules, in
particular Lfc ⁄ GEF-H1 and p190 RhoGEF, but the
interaction involves other regions of the molecule.
These factors act on the dynamics of the cytoskeleton,
the former promoting the recruitment of elements of
the Rac1 signaling pathway, and the latter regulating
Rho activity [47–50]. However, investigating whether
the association of RasGRF1 affects microtubule
dynamics, we found that the DHPH2 domain neither
modified the kinetics of tubulin assembly nor protected

microtubules from depolymerization induced by stath-
min. Moreover, expression of the DH-PH tandem did
not affect in vivo microtubule reorganization following
recovery after nocodazole washout (data not shown).
Thus, we can reasonably assert that the DHPH2 mod-
ule of RasGRF1 does not directly affect microtubule
stability. However, there is the possibility that Ras-
GRF1 may act as scaffolding for other proteins that
modulate microtubule dynamics. We are now investi-
gating this aspect using both the yeast two hybrid sys-
tem and affinity-based chromatography.
In vivo studies have shown that arsenic compounds,
which are known oxidative stress agents [38,39,44,45,
51,52], induce the interaction between the N-terminal
part of RasGRF1 and microtubules in COS7 cells,
whereas other stimuli, known to activate RasGRF1,
such as LPA and a calcium ionophore [18,21], do not.
In this regard, it can be recalled that the agents used
(ionophore and LPA on one side and arsenic com-
pounds on the other) activate different pathways and
most probably lead to different modifications of either
RasGRF1 or microtubule structure.
It is, however, difficult to understand the different
behavior of the entire RasGRF1 and its N-terminal
region after arsenate treatment. One possible explan-
ation is that RasGRF1 with its catalytic region
strongly activates the Ras pathway, leading to inhibi-
tion of the interaction. Interestingly, in differentiated
neuron-like cells in which microtubular organization is
very different, colocalization of the entire RasGRF1

was detected in particular sites of the cell.
At the moment we do not understand the functional
significance of RasGRF1–microtubule interaction. It
has been shown that arsenite causes hyperphosphoryla-
tion of tau protein at specific sites similarly to what
has been reported in Alzheimer’s disease and that this
inhibits its association with microtubules [53]. More-
over microtubules and MAPs play a role in neuro-
degenerative processes. The interaction of RasGRF1
with microtubules may be important for this aspect.
In conclusion, this identification of microtubules as
binding partners for RasGRF1 may help to clarify the
complicated signaling network and the physiological
and possible pathological processes in which RasGRF1
is involved.
Experimental procedures
Plasmids
Plasmids coding for full-length RasGRF1 and the N-ter-
minal region (amino acids 1–625; PHC21) have been pre-
viously described [16,21]. Plasmids coding for PHCCIQ
(amino acids 1–239), DHPH2 (amino acids 239–591), DH
(amino acids 239–480) fused to MBP or the catalytic domain
(amino acids 1027–1259) fused to glutathione S-transferase
(GST) were kindly provided by E. Jacquet (Ecole Polytech-
nique, Palaiseau Cedex, France) and have been previously
described [22]. Plasmid coding for PH2 (amino acids 480–
591) fused to GST was a gift from P. Crespo (Instituto de
Investigaciones Biomedicas, Consejo Superior de Investigaci-
ones Cientificas, Madrid, Spain). pET-28b vector (Novagen,
Darmstadt, Germany) coding for histidine-tagged stathmin

was kindly provided by A. Colombatti (University of Udine,
Udine, Italy).
Cell culture and transfections
Human SK-N-BE neuroblastoma cells expressing cDNA
for HA-tagged-Ras-GRF1 (SO5 clone) [46] were cultured in
RPMI 1640 medium supplemented with fetal bovine serum
(Gibco, Invitrogen Corporation, Carlsbad, CA, USA).
HEK293 cells and COS7 cells grown in Dulbecco’s modi-
fied Eagle’s medium supplemented with 10% fetal bovine
serum were transfected using the Lipofectamine (Invitrogen,
Life Technologies) method according to the manufacturer’s
instructions. All media were from Gibco, Invitrogen
Corporation.
Immunofluorescence analysis
For immunofluorescence studies, COS7 cells, plated on
glass coverslip, were transfected with different constructs
RasGRF1 directly binds microtubules G. Forlani et al.
2134 FEBS Journal 273 (2006) 2127–2138 ª 2006 The Authors Journal compilation ª 2006 FEBS
and serum deprived over night before stimulation with
sodium arsenate (0.5 mm) for 1 h. SO5 cells were treated
with 10 lm retinoic acid. Seven days later, cells were used
for immunofluorescence analysis.
For tubulin and Ras-GRF1 staining, cells were fixed and
double-stained with polyclonal antibodies to Ras-GRF1
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) or the
N-terminal region [28] and with monoclonal antibodies to
a-tubulin. Alexa Fluor 488-conjugated anti-rabbit IgG and
Alexa Fluor 594-conjugated anti-mouse IgG (Molecular
Probes, Eugene, OR, USA) were used as secondary anti-
bodies. Microscopic analysis was performed with a Leica

TCS NT laser scan microscope imaging system (Leica Micro-
system, Milan, Italy), equipped with an Ar ⁄ Kr laser and a
60 · oil immersion objective. All images were obtained
from scansions taken at 1 lm optical sections.
Expression and purification of RasGRF1
recombinant proteins
Bacterially synthesized proteins, containing different frag-
ments of RasGRF1 fused to GST or MBP, were purified as
previously described [22]. The bacterially synthesized N-ter-
minal histidine-tagged stathmin was purified on Ni ⁄ nitrilo-
triactetate beads as recommended by the manufacturer
(Invitrogen). After elution, purified MBP fusion proteins
and His fusion proteins were dialyzed against buffer con-
taining 0.1 m Pipes (sequisodium salt), pH 6.6, 1 mm
EGTA, 1 mm MgCl
2
,1mm dithiothreitol, 150 mm NaCl,
and 20% (v ⁄ v) glycerol, and GST fusion proteins were dia-
lyzed against buffer containing 50 mm Tris ⁄ HCl, pH 7.5,
50 mm NaCl, 20% (v ⁄ v) glycerol, 7 mm 2-mercaptoethanol.
All the purified proteins were concentrated with Stirred
Ultrafiltration Cells and with Centricon (Millipore, Billeri-
ca, MA, USA). Recombinant proteins were ultracentrifuged
with the TL100-A rotor (Beckman Instruments, Palo Alto,
CA, USA) at 100 000 g for 1 h, 4 °C, before use.
Microtubule cosedimentation assays
In vitro microtubule assembly was essentially performed as
described [32,35]. Brains from adult CD1 mice (Harlan, S.
Pietro al Natisone, Italy) were mechanically homogenized
in PME buffer (0.1 m Pipes, pH 6.9, 2 mm EGTA, 1 mm

MgSO
4
,1mm dithiothreitol, 0.5 mm GTP) supplemented
with EDTA-free protease inhibitor cocktail (Roche Phar-
maceuticals, Basel, Switzerland) at a ratio of 1 mL per g
brain tissue. The homogenate was centrifuged at low speed
(1000 g, 10 min, 4 °C) followed by a high-speed (100 000 g,
60 min, 4 °C) step. The supernatant (Input), was then incu-
bated at 30 °C for 30 min in the presence of 20 lm taxol,
loaded on a 13% (w ⁄ v) sucrose cushion in the above buffer
and centrifuged at 45 000 g, for 30 min at 30 °C. Superna-
tant, usually 200 lL total volume, was collected and sup-
plemented with 4 · SDS sample buffer, and the pellet was
resuspended in SDS sample buffer in the same total volume
of supernatant. Input and equal volumes of the supernatant
and pellet were separated by SDS ⁄ PAGE and blotted on
nitrocellulose membranes (Schleicher & Schuell Bioscience,
Dassel, Germany), for western blotting. Bound antibodies
were visualized by enhanced chemiluminescence (ECL)
detection (Amersham Pharmacia Biotech, Milan, Italy)
using horseradish peroxidase-conjugated secondary antibo-
dies (Jackson Immunoresearch Laboratories, West Grove,
PA, USA).
Binding assays
For binding assays, microtubules were prepared from pure
bovine brain tubulin according to the protocol described
by the manufacturer (Cytoskeleton, Denver, CO, USA).
Extracts of RasGRF1-expressing cells or purified GST and
MBP fusion proteins were then incubated with preas-
sembled, taxol-stabilized, pure microtubules for 20 min at

24 °C in a final volume of 50 lL of G-PEM buffer (80 mm
Pipes, pH 6.9, 1 mm EGTA, 0.5 mm MgCl
2
,1mm GTP) in
the presence of 20 lm taxol. Pellets and supernatant, collec-
ted after centrifugation (30 000 g, 30 min, 24 °C) were sep-
arated by SDS ⁄ PAGE and either stained with Coomassie
Brilliant Blue (Bio-Rad Laboratories, Milan, Italy) or ana-
lyzed by western blotting.
Densitometric analysis was performed using the scion
image beta 4.02 win. Software (Scion Corporation, Fred-
erick, MD, USA), interfaced to an HP precision image
scanner (Hewlett-Packard Development Co, USA).
Assembly and disassembly of microtubules
in vitro
Assembly of pure bovine brain tubulin (cytoskeleton;
5mgÆmL
)1
in G-PEM buffer) was monitored spectrophoto-
metrically (A
350
)at37°C using a thermostatically con-
trolled Ultraspec 300 spectrophotometer (Pharmacia
Biotech, Uppsala, Sweden). To determine the effect of puri-
fied recombinant proteins on microtubule assembly, equal
volumes of MBP (10 lm) or MBP-DHPH2 (10 lm) recom-
binant proteins were added to the cuvette, and the assembly
reaction was started with the addition of 1 mm GTP.
Absorbance was monitored for 30 min.
To assess the effect of recombinant proteins on the destab-

ilizing activity of stathmin, microtubules were assembled as
described above and when the steady-state was reached
recombinant stathmin (20 lm) was added. Absorbance was
monitored for a further 30 min.
Antibodies and chemicals
Polyclonal antibodies to RasGRF1 (sc-224), Sos1 (sc-256),
Erk2 (sc-154), and K-Ras (sc-30) and monoclonal anti-
G. Forlani et al. RasGRF1 directly binds microtubules
FEBS Journal 273 (2006) 2127–2138 ª 2006 The Authors Journal compilation ª 2006 FEBS 2135
bodies to GST (sc-138) were from Santa Cruz Biothec-
nology (Santa Cruz, CA, USA). Monoclonal antibodies to
a-tubulin (B-5-1-2) and kinesin heavy chain (clone IBII)
were from Sigma (Milan, Italy). Polyclonal antibodies to
MBP were from New England Biolabs. Polyclonal anti-
bodies to the N-terminal region of RasGRF1 have been
previously described [28]. Chemicals were from Sigma
unless otherwise indicated.
Acknowledgements
We are grateful to N. Gnesutta for critical reading of
the manuscript, to U. Fascio for technical assistance
with the confocal microscope, and to G. Cappelletti
and M. V. Schiaffino for their valuable advice. This
work was supported by grants from Ministero dell’Ist-
ruzione, dell’Universita
`
e della Ricerca to R.Z. (COFIN
2003) and by FIRST 2003-4 to R.Z.
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