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: 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 (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. References 1 Diaz-Nido J (1997) The Cytoskeleton. BIOS Scientific Publisher, Oxford. 2 Avila J, Dominguez J & Diaz-Nido J (1994) Regulation of microtubule dynamics by microtubule-associated pro- tein expression and phosphorylation during neuronal development. Int J Dev Biol 38, 13–25. 3 Gundersen GG & Cook TA (1999) Microtubules and signal transduction. Curr Opin Cell Biol 11, 81–94. 4 Etienne-Manneville S & Hall A (2002) Rho GTPases in cell biology. Nature 420, 629–635. 5 Cook TA, Nagasaki T & Gundersen GG (1998) Rho guanosine triphosphatase mediates the selective stabili- zation of microtubules induced by lysophosphatidic acid. J Cell Biol 141, 175–185. 6 Gundersen GG, Kim I & Chapin CJ (1994) Induction of stable microtubules in 3T3 fibroblasts by TGF-beta and serum. J Cell Sci 107, 645–659. 7 Daub H, Gevaert K, Vandekerckhove J, Sobel A & Hall A (2001) Rac ⁄ Cdc42 and p65PAK regulate the microtu- bule-destabilizing protein stathmin through phosphoryla- tion at serine 16. J Biol Chem 276, 1677–1680. 8 Cen H, Papageorge AG, Zippel R, Lowy DR & Zhang K (1992) Isolation of multiple mouse cDNAs with cod- ing homology to Saccharomyces cerevisiae CDC25: identification of a region related to Bcr, Vav, Dbl and CDC24. EMBO J 11, 4007–4015. 9 Martegani E, Vanoni M, Zippel R, Coccetti P, Brambil- la R, Ferrari C, Sturani E & Alberghina L (1992) Clon- ing by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cer- evisiae RAS activator. EMBO J 11, 2151–2157. 10 Wei W, Das B, Park W & Broek D (1994) Cloning and analysis of human cDNAs encoding a 140-kDa brain guanine nucleotide-exchange factor, Cdc25GEF, which regulates the function of Ras. Gene 151, 279–284. 11 Zippel R, Orecchia S, Sturani E & Martegani E (1996) The brain specific Ras exchange factor CDC25 Mm: modulation of its activity through Gi-protein-mediated signals. Oncogene 12, 2697–2703. 12 Font de Mora J, Esteban LM, Burks DJ, Nunez A, Garces C, Garcia-Barrado MJ, Iglesias-Osma MC, Moratinos J, Ward JM & Santos E (2003) Ras-GRF1 signaling is required for normal beta-cell development and glucose homeostasis. EMBO J 22, 3039–3049. 13 Fam NP, Fan WT, Wang Z, Zhang LJ, Chen H & Moran MF (1997) Cloning and characterization of Ras- GRF2, a novel guanine nucleotide exchange factor for Ras. Mol Cell Biol 17, 1396–1406. 14 Kiyono M, Satoh T & Kaziro Y (1999) G protein beta gamma subunit-dependent Rac-guanine nucleotide exchange activity of Ras-GRF1 ⁄ CDC25 (Mm). Proc Natl Acad Sci USA 96, 4826–4831. 15 Kiyono M, Kaziro Y & Satoh T (2000) Induction of rac-guanine nucleotide exchange activity of Ras- GRF1 ⁄ CDC25 (Mm) following phosphorylation by the nonreceptor tyrosine kinase Src. J Biol Chem. 275, 5441–5446. 16 Innocenti M, Zippel R, Brambilla R & Sturani E (1999) CDC25 (Mm) ⁄ Ras-GRF1 regulates both Ras and Rac signaling pathways. FEBS Lett 460, 357–362. 17 Fan WT, Koch CA, de Hoog CL, Fam NP & Moran MF (1998) The exchange factor Ras-GRF2 activates Ras-dependent and Rac-dependent mitogen-activated protein kinase pathways. Curr Biol 8, 935–938. 18 Farnsworth CL, Freshney NW, Rosen LB, Ghosh A, Greenberg ME & Feig LA (1995) Calcium activation of Ras mediated by neuronal exchange factor Ras-GRF. Nature 376, 524–527. 19 Krapivinsky G, Krapivinsky L, Manasian Y, Ivanov A, Tyzio R, Pellegrino C, Ben-Ari Y, Clapham DE & Medina I (2003) The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRF1. Neuron 40, 775–784. 20 Mattingly RR & Macara IG (1996) Phosphorylation- dependent activation of the Ras-GRF ⁄ CDC25Mm exchange factor by muscarinic receptors and G-protein beta gamma subunits. Nature 382, 268–272. 21 Zippel R, Balestrini M, Lomazzi M & Sturani E (2000) Calcium and calmodulin are essential for Ras-GRF1- mediated activation of the Ras pathway by lysopho- sphatidic acid. Exp Cell Res 258, 403–408. 22 Baouz S, Jacquet E, Accorsi K, Hountondji C, Bales- trini M, Zippel R, Sturani E & Parmeggiani A (2001) Sites of phosphorylation by protein kinase A in CDC25Mm ⁄ GRF1, a guanine nucleotide exchange factor for Ras. J Biol Chem 276, 1742–1749. RasGRF1 directly binds microtubules G. Forlani et al. 2136 FEBS Journal 273 (2006) 2127–2138 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... a RhoA-specific guanine nucleotide exchange factor that interacts with microtubules J Biol Chem 276, 4948–4956 FEBS Journal 273 (2006) 2127–2138 ª 2006 The Authors Journal compilation ª 2006 FEBS 2137 RasGRF1 directly binds microtubules G Forlani et al 48 Ren Y, Li R, Zheng Y & Busch H (1998) Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for Rac... taxol Methods Enzymol 134, 104–115 RasGRF1 directly binds microtubules 36 Buchsbaum R, Telliez JB, Goonesekera S & Feig LA (1996) The N-terminal pleckstrin, coiled-coil, and IQ domains of the exchange factor Ras-GRF act cooperatively to facilitate activation by calcium Mol Cell Biol 16, 4888–4896 37 Freshney NW, Goonesekera SD & Feig LA (1997) Activation of the exchange factor Ras-GRF by calcium requires... 34954–34960 49 Glaven JA, Whitehead I, Bagrodia S, Kay R & Cerione RA (1999) The Dbl-related protein, Lfc, localizes to microtubules and mediates the activation of Rac signaling pathways in cells J Biol Chem 274, 2279– 2285 50 Krendel M, Zenke FT & Bokoch GM (2002) Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton Nat Cell Biol 4, 294–301 2138 51 Huang C, Bode... (1997) Ras-GRF, the activator of Ras, is expressed preferentially in mature neurons of the central nervous system Brain Res Mol Brain Res 48, 140–144 26 Sturani E, Abbondio A, Branduardi P, Ferrari C, Zippel R, Martegani E, Vanoni M & Denis-Donini S (1997) The Ras Guanine nucleotide Exchange Factor CDC25Mm is present at the synaptic junction Exp Cell Res 235, 117–123 27 Giese KP, Friedman E, Telliez JB,... to the activation of exchange factor activity by muscarinic receptors J Biol Chem 274, 37379–37384 24 Shou C, Wurmser A, Suen KL, Barbacid M, Feig LA & Ling K (1995) Differential response of the Ras exchange factor, Ras-GRF to tyrosine kinase and G protein mediated signals Oncogene 10, 1887–1893 25 Zippel R, Gnesutta N, Matus-Leibovitch N, Mancinelli E, Saya D, Vogel Z & Sturani E (1997) Ras-GRF, the. .. (2002) Interaction of Rac exchange factors Tiam1 and Ras-GRF1 with a scaffold for the p38 mitogen-activated protein kinase cascade Mol Cell Biol 22, 4073–4085 34 Verhey KJ, Meyer D, Deehan R, Blenis J, Schnapp BJ, Rapoport TA & Margolis B (2001) Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules J Cell Biol 152, 959–970 35 Vallee RB (1986) Purification of brain microtubules. .. Silva AJ (2001) Hippocampus-dependent learning and memory is impaired in mice lacking the Ras -guanine- nucleotide releasing factor 1 (Ras-GRF1) Neuropharmacology 41, 791–800 28 Brambilla R, Gnesutta N, Minichiello L, White G, Roylance AJ, Herron CE, Ramsey M, Wolfer DP, Cestari V, Rossi-Arnaud C, et al (1997) A role for the Ras signalling pathway in synaptic transmission and long-term memory Nature 390,... initiation factor 2 inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure J Biol Chem 280, 16925–16933 46 Tonini R, Mancinelli E, Balestrini M, Mazzanti M, Martegani E, Ferroni A, Sturani E & Zippel R (1999) Expression of Ras-GRF in the SK-N-BE neuroblastoma accelerates retinoic-acid-induced neuronal differentiation and increases the functional expression of the. .. 2 by an epidermal growth factor receptor-mediated pathway in normal human keratinocytes Br J Dermatol 149, 1116–1127 40 Ivanov PA, Chudinova EM & Nadezhdina ES (2003) Disruption of microtubules inhibits cytoplasmic ribonucleoprotein stress granule formation Exp Cell Res 290, 227–233 41 Kranenburg O & Moolenaar WH (2001) Ras-MAP kinase signaling by lysophosphatidic acid and other G protein-coupled receptor... lysophosphatidic acid and other G protein-coupled receptor agonists Oncogene 20, 1540– 1546 42 Agell N, Bachs O, Rocamora N & Villalonga P (2002) Modulation of the Ras ⁄ Raf ⁄ MEK ⁄ ERK pathway by Ca(2+), and calmodulin Cell Signal 14, 649–654 43 Seger R & Krebs EG (1995) The MAPK signaling cascade FASEB J 9, 726–735 44 Huang C, Li J, Ding M, Wang L, Shi X, Castranova V, Vallyathan V, Ju G & Costa M (2001) Arsenic-induced . The guanine nucleotide exchange factor RasGRF1 directly binds microtubules via DHPH2-mediated interaction Greta Forlani, Simona. performed to test whether the DHPH2 module is the only region responsible for the interaction of the entire RasGRF1 molecule with microtubules. Pure microtubules