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.
Trang 1Microtubules 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 Micromicro-tubule 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: renata.zippel@unimi.it
(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 (Kd 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.
Trang 2guanine 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 reccalcium-depend-ently, 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.
Trang 3investigation was carried out on polymerized tubulin.
Microtubules were assembled when mouse brain
high-speed supernatant was incubated at 30C 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 4C (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-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.
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 m M 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 ) or 8 M urea and centrifuged at
30 C The microtubule pellets and supernatants were analyzed by immunoblotting.
Trang 4hydrolyzable 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 24C 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.
Trang 5centrifugation, 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 A350 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
A350 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
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.
Trang 6addition 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 reprimag-esent 20 lm Similar rimag-esults were obtained with arsenite.
Trang 7compound 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).
Trang 8Using 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 microtubmod-ule
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 interacinhibi-tion 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
Trang 9For 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 MgCl2, 1 mm 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, 4C, before use
Microtubule cosedimentation assays
In vitromicrotubule 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
MgSO4, 1 mm 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, 4C) followed by a high-speed (100 000 g,
60 min, 4C) step The supernatant (Input), was then
incu-bated at 30C 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 30C
Superna-tant, usually 200 lL total volume, was collected and
sup-plemented with 4· SDS sample buffer, and the pellet was
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
24C in a final volume of 50 lL of G-PEM buffer (80 mm Pipes, pH 6.9, 1 mm EGTA, 0.5 mm MgCl2, 1 mm GTP) in the presence of 20 lm taxol Pellets and supernatant, collec-ted after centrifugation (30 000 g, 30 min, 24C) 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;
5 mgÆmL )1in G-PEM buffer) was monitored spectrophoto-metrically (A350) at 37C 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
Trang 10anti-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 Ricercato R.Z (COFIN
2003) and by FIRST 2003-4 to R.Z
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