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Epac signaling pathway involves STEF, a guanine nucleotide exchange factor for Rac, to regulate APP processing
FEBS Letters 581 (2007) 5814–5818
Epac signaling pathway involves STEF, a guanine nucleotide exchange factor for Rac, to regulate APP processing Natalia Zalduaa, Monique Gastineaub,c, Mikio Hoshinod,e, Frank Lezoualc’hb,c,*, Jose´ L. Zugazaa,* a
d
Department of Proteomic, CIC bioGUNE, Parque Tecnolo´gico de Bizkaia, Edificio 801A, 48160 Derio, Bizkaia, Spain b Inserm, U769, Signalisation et Physiopathologie Cardiaque, Chaˆtenay-Malabry F-92296, France c Univ Paris-Sud, Faculte´ de Pharmacie, IFR141, UMR-S769, Chaˆtenay-Malabry F-92296, France Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan e Precursory Research for Embryonic Science and Technology, Science and Technology Agency, Kawaguchi 332-0012, Japan Received 21 August 2007; revised 6 November 2007; accepted 16 November 2007 Available online 3 December 2007 Edited by Irmgard Sinning
Abstract The amyloid precursor protein (APP) is a key protein involved in the development of Alzheimer’s disease. We previously identified a signal transduction secretory pathway in which the small G protein Rac sets downstream of the cAMP/Epac/ Rap1 signalling cascade regulating the a cleavage of APP [Maillet, M. et al. (2003) Crosstalk between Rap and Rac regulates secretion of sAPPa. Nat. Cell Biol. 5, 633–639]. We now report that Rap1 can physically and specifically associate with the guanine nucleotide exchange factor (GEF) STEF through its TSS region. A deleted TSS domain of STEF cells fails to activate Rac1 and dramatically decreases secretion of the nonamyloidogenic soluble form of APP (sAPPa) induced by the cAMP-binding protein Epac. Altogether, our data show that upon Epac activation, Rap1 recruits STEF through its TSS region and activates Rac1, which mediates APP processing. Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Serotonin; Alzheimer’s disease; Amyloid; Small G protein; cAMP; Guanine nucleotide exchange factor
1. Introduction GTPases of the Ras superfamily are of special interest since they have been implicated in almost all aspects of cell biology, including proliferation, differentiation and vesicle trafficking [2]. They act as molecular switches to control cellular signalling pathways and are present in two conformations, an inactive form loaded with GDP and an active
* Corresponding authors. Fax: +33 1 46 83 54 75 (F. Lezoualc’h); fax: +34 94 406 1301(J.L. Zugaza). E-mail addresses: [email protected] (F. Lezoualc’h), jlzugaza@ cicbiogune.es (J.L. Zugaza).
Abbreviations: 5-HT, 5-Hydroxytryptamine; 8-CPT, 8-(4-Chlorophenylthio)-2 0 -O-methyladenosine-3 0 -5 0 -cyclicmonophosphate; APP, amyloid precursor protein; CHO, Chinese hamster ovary cells; FBS, foetal bovine serum; GEF, guanine nucleotide exchange factor; PHn; N-terminal pleckstrin homology; sAPPa, soluble form of the amyloid precursor protein; STEF, SIF and Tiam-1 like exchange factor; TSS, Tiam 1 STEF-SIF homologous
form loaded with GTP [3]. The exchange of GDP for GTP is enhanced and regulated by guanine exchange factors (GEFs). In their active state, small GTPases of the Rho family interact with various specific effector molecules producing a cellular response such as actin cytoskeleton reorganization [4]. Small GTPases not only can act independently, but also they can cooperate in a particular pathway to coordinate specific cellular responses. It has been shown that the serine/threonin kinase PAK, a Rac1/Cdc42 effector molecule, can phosphorylate MEK-1 and Raf and upregulate Ras pathway [5,6]. Another example of these relationships between small GTPases of different families has been elucidated recently. In B and T cells the activation of the MAPKinase cascade is produced through a Vav/Rac pathway that cooperates with a calciumand diacylglycerol-dependent Ras guanosine nucleotide releasing proteins (RasGRPs) leading to the activation of the Ras pathway [7,8]. With respect to cyclic AMP (cAMP) signalling, we have recently shown that Rac activation results from activation of Rap1, via the cAMP-sensitive GEF Epac1 [1]. At the functional level, we found that this connection between two families of small GTPases, Ras and Rho can influence the processing of the amyloid precursor protein (APP). Indeed, activation of Epac/Rap1/Rac1 signalling cascade increases the cleavage of APP via the a-secretase pathway leading to the release of sAPPa This large soluble N-terminal ectodomain subproduct of APP is secreted to the extracellular space [1,9]. Furthermore, it has been postulated that this sAPPa is a potent memory-enhancer and has neuroprotective properties [10]. Interestingly, activation of the serotonin 5-HT4 receptor, a canonical Gs-coupled-receptor, increases sAPPa through Epac1/Rap1/Rac in neuronal cells [1,9]. The identification of Rap1 and Rac1 as two elements in a signalling pathway controlling a cAMP-dependent secretory process raises the question of the identity of the molecular links between Ras and Rho family members of GTPases. Tiam-1 and STEF are GEFs which belong to the Dbl family of GEFs and are considered specific GEFs for Rac1 [11,12]. Other Rac-specific GEFs is the Vav family of proteins, which are involved in lymphocyte development and signalling [13,14]. Seeking for the molecular links between the Rap1 and Rac1 signalling pathways we investigated the role of STEF in this signalling pathway.
0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.11.053
N. Zaldua et al. / FEBS Letters 581 (2007) 5814–5818
2. Materials and methods 2.1. Materials Culture media, sera and antibiotics were from Invitrogen (Barcelona, Spain). Serotonin and 8-(4-chloro-phenylthio)-2 0 -O-methyladenosine-3 0 -5 0 -cyclicmonophosphate (8-CPT) were from Sigma–Aldrich (Madrid, Spain). Anti-HA, anti-Rac and anti-sAPPa antibody (6E10 clone) were from Cell Signalling (Beverly, USA), UBI (New York, USA) and Signet (Pathology Systems, Dedham, USA) respectively. The rabbit polyclonal anti-Rap1 antibody was purchased from Santa Cruz Biotech (USA). 2.2. Cell culture and DNA transfections Chinese hamster ovary cells (CHO) cells coexpressing the human 5HT4(e) receptor isoform and APP695 in a stable manner were cultured as previously described [15]. Transient transfection experiments were performed using jetPEIä (Polyplus-transfection, Illkirch, France) according to the manufacturer instructions. COS-1 cells were cultured at 37 °C and a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum plus antibiotics. COS-1 cells were transfected with DEAE-Dextrane method. Briefly, a solution containing 4 ml of DMEM plus 160 ll of DEAE-dextran/chloroquine solution and 5 lg of cDNA was prepared.
5815 Cells were washed twice, the DNA mix was added and they were incubated for 4 h at 37 °C. Then the DNA mix was aspirated and 4 ml 10% DMSO in DPBS was added for 2 min and aspirated, finally the cells were left with complete medium for 24 h. 2.3. Generation and purification of proteins The STEF and Tiam-1 N-terminal pleckstrin homology (PHn) and Tiam 1 STEF-SIF homologous (TSS) domains were generated by PCR and cloned into the pGEX4T-3 vector (General Electric Healthcare). All GST proteins were purified by chromatography onto glutathione beads following standard procedures [16]. 2.4. Rap and Rac activation assays Rap1, Rap1Q63E, and RapS17N pull down experiments were performed using a GST fusion protein containing the Rap1-binding domain of Ral-GDS. For Rac1 pull down experiments we used a GST fusion protein containing the Rac1 interacting binding domain of PAK. The activation state of these small GTPases was determined as previously described [1]. 2.5. Measurements of sAPPa by Western blot Secreted sAPPa was detected by Western blot as previously described [9] using the 6E10 antibody. We have previously shown that 5-HT4 receptor activation and its associated signalling cascade do not influence APP gene expression [17]. The double band observed in our immunoblot may result from a different glycosylation state of secreted sAPPa. Such a migration profile for sAPPa was observed in other works [9,18].
A
STEF Tiam-1 STEF Tiam-1
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Fig. 1. Analysis of the interaction between Rap1Q63L and Vav, Tiam1 and STEF. COS-1 cells were transfected with a plasmid encoding the constitutively active Rap1 (HA-Rap1Q63L). Total cell lysates were subjected to pull down experiments with (A), GST-CH-Ac (1), GSTCH-Ac-DH (2), GST-PH (3), GST-SH2 (4) fusion proteins of differents Vav domains, or GST–PHn (1), or GST–TSS (2) fusion proteins of Tiam-1 (B), and STEF (C). Immunoreactive bands were visualised using anti-HA antibody. The top panels show the Rap1Q63L association or not with the different fusion proteins used in the experiment, the lower panels show the total amount of HA-Rap1 Q63L. Data are representative of three independent experiments.
Fig. 2. (A) TSS domain of STEF binds specifically Rap1 active form. COS-1 cells were transfected with plasmids encoding either HARap1S17N, or HA-Rap1Q63L (5 lg), as indicated. Total cell lysates were subjected to pull down experiments with GST–PHn, and GST– TSS fusion proteins of Tiam-1 or STEF. Immunoreactive bands were visualised using anti-HA antibody. The top panel shows the Rap1 Q63L association with GST–TSS of STEF, the lower panel shows the total amount of HA-Rap1 S17N and HA-Rap1 Q63L. (B) FBS induces the binding of Rap1-GTP to the TSS domain of STEF. COS-1 cells were transfected with the tagged HA wild type form of Rap1. One day after transfection, cells were deprived of serum for 24 h and stimulated or not with 10% FBS for 10 min. Then, HA-Rap1-GTP was pulled down using GST–PHn, and GST–TSS fusion proteins of Tiam1 and STEF, as indicated. Immunoreactive bands were visualised using anti-HA antibody. The top panel shows the accumulated Rap1 in the GTP-bound form that associates with GST–TSS of STEF, the lower panel shows the total amount of HA–Rap1. The results shown are representative of two independent experiments.
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N. Zaldua et al. / FEBS Letters 581 (2007) 5814–5818
B
A 1 µ M 5-HT
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Fig. 3. 5-HT4 receptor- (A) or Epac- (B) induced Rac1 activation is inhibited by STEF lacking its TSS domain. CHO cells stably expressing the 5HT4 receptor were transfected either with HA-STEF wt or HA-STEF-DTSS. One day after transfection, cells were stimulated or not with either 1 lM 5-HT (A) or 10 lM 8-CPT (B) for 10 min and then Rac1–GTP was pulled down with GST-CRIB of PAK and analyzed by Western blot. In (A) and (B), the top panels show the accumulated Rac1 in the GTP-bound form that associates with GST-CRIB, the middle panels show the total amount of endogenous Rac, and the lower panels show the total amount of HA-STEF and HA-STEF-DTSS. The graphs indicate the amounts of Rac1-GTP which were expressed as fold activation over control. Rac1-GTP in unstimulated cells was defined arbitrarily as 1. Inset, STEF-DTSS does not block Rap1 activation induced by 5-HT. CHO cells expressing the 5-HT4 receptor were transfected either with empty vector or HA-STEF-DTSS. One day after transfection, cells were stimulated or not with 1 lM 5-HT for 10 min and then Rap1-GTP was pulled down and analyzed by Western blot. The top panel shows the accumulated Rap1 in the GTP-bound form that associates with fusion protein, the middle panels show the total amount of endogenous Rap1 and the lower panels show the total amount of HA-STEF-DTSS. Results are means ± S.E.M for three independent experiments, P < 0.01, P < 0.001 compared with untreated control cells (Student’s t-test).
3. Results The most specific GEFs for Rac GTPase which belong to the Dbl family are Vav proteins [19], Tiam-1 [11] and STEF which is directly related with Tiam-1 and the Drosophila SIF protein [12]. One possible explanation which could clarify the Rap1/ Rac1 connection was that Rap1 could interact with one of these exchange factors of the Dbl family to mediate Rac activation. Based on this hypothesis, we examined whether the active form of Rap1 could bind to one of the Rac specific GEFs. More precisely, we first examined the potential association of Rap1 with Vav1. To do so, we expressed Rap1Q63E (constitutively active form of Rap1) in COS-1 cells and conducted pull-down experiments using fusion proteins constituted by different domains of Vav tagged with GST, as indicated in Fig. 1A. We found that Rap1Q63E did not bind to any domain of Vav Fig. 1A ((1)– CH + Ac, (2)–CH + Ac + DH, (3)–PH, (4)–SH2). Then, we examined by pull down assay whether Tiam-1 was able to bind to Rap1 or not. In order to do so, we developed fusion proteins tagged with GST for the amino terminal PH domain (PHn) and TSS region of Tiam1. Both domains were unable to bind Rap1Q63E (Fig. 1B). Nevertheless when we used the fusion proteins GST–PHn and GST–TSS from STEF, we demonstrated that TSS region of STEF bind specifically Rap1Q63E as shown in Fig. 1C. We then examined the specificity of this interaction between STEF and the active form of Rap1. We expressed either Rap1Q63E or a dominant negative form of Rap1, Rap1S17N
in COS-1 cells and performed pull-down experiments using GST–PHn and GST–TSS from STEF and Tiam-1. As shown in Fig. 2A, only the constitutively active form of Rap1, Rap1Q63E was able to bind to TSS region of STEF. Consequently, we examined whether foetal bovine serum (FBS)induced Rap1 activation could bind to STEF. Cells were transfected with the wild type form of Rap1 and then stimulated or not with 10% FBS (Fig. 2B). As expected, FBS-induced Rap1 activation but neither PH domain nor TSS region of Tiam-1 were able to pull-down Rap1-GTP (Fig. 2B). However, the TSS region of STEF bound specifically to the active form of Rap1 (Fig. 2B). Altogether, these data demonstrated that Rap1-GTP bind specifically to STEF by its TSS region. We had previously shown that the serotonin 5-HT4 receptor increases Rac activation in a cAMP-dependent manner through the GEF, Epac and its effector, Rap1 [1]. Therefore, we next tested whether the stimulating effects of the 5-HT4 receptor and Epac on Rac activation were STEF dependent. CHO cells expressing the 5-HT4 receptor were transiently transfected with STEF wild type (STEF wt) or STEF mutant which lacks in the TSS region (STEF-DTSS). As shown in Fig. 3A, 1 lM 5-HT induced a solid activation of endogenous Rac1 in cells transfected with STEF wt (Fig. 3A). In contrast, overexpression of STEFDTSS blocked Rac1 activation induced by the 5-HT4 receptor without affecting Rap1 activation (Fig. 3A, inset). Altogether, these results show that Rac1 activation induced by the 5-HT4 receptor involves STEF. Next, we tested the involvement of
N. Zaldua et al. / FEBS Letters 581 (2007) 5814–5818
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A -
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Rac1 since it regulates Rac1 activation upon 5-HT4 receptor and Epac/Rap1 activation. In order to validate this new signalling pathway in a more physiological way, we analysed its possible involvement in sAPPa secretion. In order to do so, we examined whether STEF is involved in APP processing upon 5-HT4 receptor activation. CHO cell line stably coexpressing the h5-HT4(e) receptor and human APP695 [15] were transfected with STEF wt or STEF-DTSS. Over-expression of STEF-wt significantly potentiated basal level of sAPPa secretion as compared to mock transfected cells (Fig. 4A). Furthermore, sAPPa secretion was further increased in CHO cells expressing STEF wt and treated with 1 lM 5-HT (Fig. 4A). In contrast, STEF-DTSS significantly inhibited 5-HT-induced sAPPa secretion (Fig. 4A). Similar findings were found when these experiments were performed in the presence or absence of Epac selective activator, 8-CPT. Indeed, this compound at 10 lM increased sAPPa secretion in cells transfected with STEF wt (Fig. 4B). On the contrary, STEF-DTSS blocked the stimulating effect of 8-CPT (10 lM) on sAPPa secretion in response to 8-CPT (Fig. 4B). Altogether, our results indicate that this new signalling pathway involving Epac1 and STEF is implicated in cAMP-dependent APP processing.
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Fig. 4. Secretion of sAPPa induced by the activation of the 5-HT4 receptor (A) or Epac (B) is mediated by STEF. CHO cells stably expressing the 5-HT4 receptor and APP695 were transfected either with an empty vector or a plasmid encoding HA-STEF wt or HASTEF-DTSS. One day after transfection, cells were stimulated or not with either 1 lM 5-HT (A) or 10 lM 8-CPT (B) for 30 min. Then, sAPPa was detected in the culture medium by immunoblot using the 6E10 antibody (upper panels in A and B). Lower panels in A and B, immunoblots showing the expression of HA-STEF wt and HA-DTSS. The graphs represent the amounts of extracellular sAPPa which were expressed as fold activation over control. sAPPa expression in unstimulated cells was defined arbitrarily as 1. Results are means ± S.E.M for three independent experiments *P < 0.05; **P < 0.01 compared with untreated control cells (Student’s t-test).
the cAMP-GEF, Epac1 in STEF-dependent Rac activation. To do this, we transfected CHO cells with STEF wild type or STEF-DTSS and we treated cells with the Epac-specific cAMP analogue, 8-pCPT-2 0 -O-Me-cAMP (8-CPT) [20]. As shown in Fig. 3B, 8-CPT (10 lM) strongly induced Rac1 activation but failed to do so in cells transfected with STEF-DTSS. These data suggest that STEF is the molecular link between Rap1 and
4. Discussion In the present study, we provide evidence that STEF is involved in the regulation of Rac1 GTPase activity via Epac/ Rap1 after 5-HT4 receptor activation in intact cells. We have found that the constitutively active form of Rap1, Rap1Q63L, binds in an efficient- and specific-manner to STEF. This interaction occurs through the TSS domain of STEF which is localized just behind the PHn domain [12]. This interaction was not only established with Rap1Q63L. Indeed, TSS region of STEF pulled-down specifically Rap1-GTP upon cell treatment with FBS. This TSS region of STEF has been described to bind in vitro to the PDZ1-2 domains of PAR-3. It also hangs on the PAR-6-PAR-3 complex formation to facilitate Rac activation in neurons [21]. We show for the first time that TSS region of STEF can function as a Rap1 interacting binding domain. The STEF domains required for Rac1 activation are DH, PHn and TSS domains [22]. The DH domain is the catalytic domain and is therefore essential to facilitate the exchange between GDP and GTP of the small GTPase. Furthermore, we have previously shown that mutation in DH domain of Vav1 prevents Rac1 activation [23]. PHn and TSS domains are necessary for the membrane localization of STEF and Rac1 activation [22]. In this study, we show that only the TSS domain of STEF binds in vitro to Rap1 whereas PHn domain does not. This is probably because PHn is only required for membrane localization and TSS for binding to upstream regulating molecules (this work and [21]). Regarding the effect of STEF on Rac1 in living cells upon activation of the 5-HT4 receptor, we found that deletion of the TSS domain impairs the ability of STEF to activate Rac1. Thus, we hypothesize that Rac1 activation mediated by STEF might require two steps: firstly, STEF would be localized in the cell membrane by the PHn domain and secondly Rap1-GTP would bind to the TSS domain of STEF allowing this GEF to activate Rac1. In absence of the TSS domain of STEF, Rap1-GTP cannot bind to STEF and
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consequently Rac1 is not activated, even upon 5-HT4 receptor activation. To check the results obtained with 5-HT/STEF on Rac1 activation, we also examined whether STEF regulated Rac1 activation after Epac engagement. The cAMP analogue, 8-CPT binds to Epac which rapidly catalyzes the passage from the inactive to the active state of Rap [24,25]. In the present study, we demonstrate that 8-CPT/Epac- induced Rac1 activation is regulated by STEF. Indeed, we show that STEF-DTSS form failed to activate Rac1. On the basis of the different experimental approaches presented here, we can conclude that 5-HT4 receptor mediates Rac1 activation on living cells through Epac/Rap1/STEF signal transduction pathway. STEF has been described as a molecule which takes part in neuronal plasticity. Nishimura and colleagues have proposed that the complex PAR-6-PAR-3 mediates the crosstalk between Cdc42 and Rac through STEF and that the STEF domain required to establish the neuronal polarity is TSS [21]. Here in, we describe that a crosstalk between Epac/Rap1 and Rac1 induced by the 5-HT4 receptor is involved in the cAMP-dependent processing of APP. Consistent with the critical role of STEF in Epac/Rap1/Rac1 signalling pathway, transfection of STEF wt increased basal secretion of sAPPa. Accordingly, 5HT and 8-CPT enhanced STEF- induced sAPPa secretion and these effects were strongly blocked by STEFF-DTSS. To conclude, our findings show that STEF is the molecular link between the cAMP/Epac/Rap1 and Rac1 pathways. STEF activation is produced by a physical interaction between its TSS domain and Rap1–GTP which mediates Rac1 activation and this active form of Rac regulates the a cleavage of APP, generating the sAPPa. Acknowledgements: We would like to thank Drs X.R. Bustelo and M.E. Greenberg for the generous gift of Vav fusion proteins and the cDNA encoding for Tiam-1 respectively. Drs V. Lang and L. Parada for helpful comments on the manuscript. FL is supported by INSERM and a Grant from Fondation pour la Recherche Me´dicale (e´quipe FRM). This work was supported by the Department of Industry of the Basque Government ETORTEK and SAIOTEK Programs to J.L.Z. and by the Spanish Ministry of Education and Science (MES) Biomedicine Program to J.L.Z. (SAF2005-07930-C02-02). J.L.Z. is a Ramo´n y Cajal Investigator (MES) associated to CIC bioGUNE.
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[7]
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[13] [14] [15] [16] [17]
[18]
[19] [20]
[21]
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Epac signaling pathway involves STEF, a guanine nucleotide exchange factor for Rac, to regulate APP processing Natalia Zalduaa, Monique Gastineaub,c, Mikio Hoshinod,e, Frank Lezoualc’hb,c,*, Jose´ L. Zugazaa,* a
d
Department of Proteomic, CIC bioGUNE, Parque Tecnolo´gico de Bizkaia, Edificio 801A, 48160 Derio, Bizkaia, Spain b Inserm, U769, Signalisation et Physiopathologie Cardiaque, Chaˆtenay-Malabry F-92296, France c Univ Paris-Sud, Faculte´ de Pharmacie, IFR141, UMR-S769, Chaˆtenay-Malabry F-92296, France Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan e Precursory Research for Embryonic Science and Technology, Science and Technology Agency, Kawaguchi 332-0012, Japan Received 21 August 2007; revised 6 November 2007; accepted 16 November 2007 Available online 3 December 2007 Edited by Irmgard Sinning
Abstract The amyloid precursor protein (APP) is a key protein involved in the development of Alzheimer’s disease. We previously identified a signal transduction secretory pathway in which the small G protein Rac sets downstream of the cAMP/Epac/ Rap1 signalling cascade regulating the a cleavage of APP [Maillet, M. et al. (2003) Crosstalk between Rap and Rac regulates secretion of sAPPa. Nat. Cell Biol. 5, 633–639]. We now report that Rap1 can physically and specifically associate with the guanine nucleotide exchange factor (GEF) STEF through its TSS region. A deleted TSS domain of STEF cells fails to activate Rac1 and dramatically decreases secretion of the nonamyloidogenic soluble form of APP (sAPPa) induced by the cAMP-binding protein Epac. Altogether, our data show that upon Epac activation, Rap1 recruits STEF through its TSS region and activates Rac1, which mediates APP processing. Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Serotonin; Alzheimer’s disease; Amyloid; Small G protein; cAMP; Guanine nucleotide exchange factor
1. Introduction GTPases of the Ras superfamily are of special interest since they have been implicated in almost all aspects of cell biology, including proliferation, differentiation and vesicle trafficking [2]. They act as molecular switches to control cellular signalling pathways and are present in two conformations, an inactive form loaded with GDP and an active
* Corresponding authors. Fax: +33 1 46 83 54 75 (F. Lezoualc’h); fax: +34 94 406 1301(J.L. Zugaza). E-mail addresses: [email protected] (F. Lezoualc’h), jlzugaza@ cicbiogune.es (J.L. Zugaza).
Abbreviations: 5-HT, 5-Hydroxytryptamine; 8-CPT, 8-(4-Chlorophenylthio)-2 0 -O-methyladenosine-3 0 -5 0 -cyclicmonophosphate; APP, amyloid precursor protein; CHO, Chinese hamster ovary cells; FBS, foetal bovine serum; GEF, guanine nucleotide exchange factor; PHn; N-terminal pleckstrin homology; sAPPa, soluble form of the amyloid precursor protein; STEF, SIF and Tiam-1 like exchange factor; TSS, Tiam 1 STEF-SIF homologous
form loaded with GTP [3]. The exchange of GDP for GTP is enhanced and regulated by guanine exchange factors (GEFs). In their active state, small GTPases of the Rho family interact with various specific effector molecules producing a cellular response such as actin cytoskeleton reorganization [4]. Small GTPases not only can act independently, but also they can cooperate in a particular pathway to coordinate specific cellular responses. It has been shown that the serine/threonin kinase PAK, a Rac1/Cdc42 effector molecule, can phosphorylate MEK-1 and Raf and upregulate Ras pathway [5,6]. Another example of these relationships between small GTPases of different families has been elucidated recently. In B and T cells the activation of the MAPKinase cascade is produced through a Vav/Rac pathway that cooperates with a calciumand diacylglycerol-dependent Ras guanosine nucleotide releasing proteins (RasGRPs) leading to the activation of the Ras pathway [7,8]. With respect to cyclic AMP (cAMP) signalling, we have recently shown that Rac activation results from activation of Rap1, via the cAMP-sensitive GEF Epac1 [1]. At the functional level, we found that this connection between two families of small GTPases, Ras and Rho can influence the processing of the amyloid precursor protein (APP). Indeed, activation of Epac/Rap1/Rac1 signalling cascade increases the cleavage of APP via the a-secretase pathway leading to the release of sAPPa This large soluble N-terminal ectodomain subproduct of APP is secreted to the extracellular space [1,9]. Furthermore, it has been postulated that this sAPPa is a potent memory-enhancer and has neuroprotective properties [10]. Interestingly, activation of the serotonin 5-HT4 receptor, a canonical Gs-coupled-receptor, increases sAPPa through Epac1/Rap1/Rac in neuronal cells [1,9]. The identification of Rap1 and Rac1 as two elements in a signalling pathway controlling a cAMP-dependent secretory process raises the question of the identity of the molecular links between Ras and Rho family members of GTPases. Tiam-1 and STEF are GEFs which belong to the Dbl family of GEFs and are considered specific GEFs for Rac1 [11,12]. Other Rac-specific GEFs is the Vav family of proteins, which are involved in lymphocyte development and signalling [13,14]. Seeking for the molecular links between the Rap1 and Rac1 signalling pathways we investigated the role of STEF in this signalling pathway.
0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.11.053
N. Zaldua et al. / FEBS Letters 581 (2007) 5814–5818
2. Materials and methods 2.1. Materials Culture media, sera and antibiotics were from Invitrogen (Barcelona, Spain). Serotonin and 8-(4-chloro-phenylthio)-2 0 -O-methyladenosine-3 0 -5 0 -cyclicmonophosphate (8-CPT) were from Sigma–Aldrich (Madrid, Spain). Anti-HA, anti-Rac and anti-sAPPa antibody (6E10 clone) were from Cell Signalling (Beverly, USA), UBI (New York, USA) and Signet (Pathology Systems, Dedham, USA) respectively. The rabbit polyclonal anti-Rap1 antibody was purchased from Santa Cruz Biotech (USA). 2.2. Cell culture and DNA transfections Chinese hamster ovary cells (CHO) cells coexpressing the human 5HT4(e) receptor isoform and APP695 in a stable manner were cultured as previously described [15]. Transient transfection experiments were performed using jetPEIä (Polyplus-transfection, Illkirch, France) according to the manufacturer instructions. COS-1 cells were cultured at 37 °C and a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum plus antibiotics. COS-1 cells were transfected with DEAE-Dextrane method. Briefly, a solution containing 4 ml of DMEM plus 160 ll of DEAE-dextran/chloroquine solution and 5 lg of cDNA was prepared.
5815 Cells were washed twice, the DNA mix was added and they were incubated for 4 h at 37 °C. Then the DNA mix was aspirated and 4 ml 10% DMSO in DPBS was added for 2 min and aspirated, finally the cells were left with complete medium for 24 h. 2.3. Generation and purification of proteins The STEF and Tiam-1 N-terminal pleckstrin homology (PHn) and Tiam 1 STEF-SIF homologous (TSS) domains were generated by PCR and cloned into the pGEX4T-3 vector (General Electric Healthcare). All GST proteins were purified by chromatography onto glutathione beads following standard procedures [16]. 2.4. Rap and Rac activation assays Rap1, Rap1Q63E, and RapS17N pull down experiments were performed using a GST fusion protein containing the Rap1-binding domain of Ral-GDS. For Rac1 pull down experiments we used a GST fusion protein containing the Rac1 interacting binding domain of PAK. The activation state of these small GTPases was determined as previously described [1]. 2.5. Measurements of sAPPa by Western blot Secreted sAPPa was detected by Western blot as previously described [9] using the 6E10 antibody. We have previously shown that 5-HT4 receptor activation and its associated signalling cascade do not influence APP gene expression [17]. The double band observed in our immunoblot may result from a different glycosylation state of secreted sAPPa. Such a migration profile for sAPPa was observed in other works [9,18].
A
STEF Tiam-1 STEF Tiam-1
HA-Rap1Q63L
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HA-Rap1S17N α-HA HA-Rap1Q63L HA-Rap1S17N
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B 10% FBS
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Fig. 1. Analysis of the interaction between Rap1Q63L and Vav, Tiam1 and STEF. COS-1 cells were transfected with a plasmid encoding the constitutively active Rap1 (HA-Rap1Q63L). Total cell lysates were subjected to pull down experiments with (A), GST-CH-Ac (1), GSTCH-Ac-DH (2), GST-PH (3), GST-SH2 (4) fusion proteins of differents Vav domains, or GST–PHn (1), or GST–TSS (2) fusion proteins of Tiam-1 (B), and STEF (C). Immunoreactive bands were visualised using anti-HA antibody. The top panels show the Rap1Q63L association or not with the different fusion proteins used in the experiment, the lower panels show the total amount of HA-Rap1 Q63L. Data are representative of three independent experiments.
Fig. 2. (A) TSS domain of STEF binds specifically Rap1 active form. COS-1 cells were transfected with plasmids encoding either HARap1S17N, or HA-Rap1Q63L (5 lg), as indicated. Total cell lysates were subjected to pull down experiments with GST–PHn, and GST– TSS fusion proteins of Tiam-1 or STEF. Immunoreactive bands were visualised using anti-HA antibody. The top panel shows the Rap1 Q63L association with GST–TSS of STEF, the lower panel shows the total amount of HA-Rap1 S17N and HA-Rap1 Q63L. (B) FBS induces the binding of Rap1-GTP to the TSS domain of STEF. COS-1 cells were transfected with the tagged HA wild type form of Rap1. One day after transfection, cells were deprived of serum for 24 h and stimulated or not with 10% FBS for 10 min. Then, HA-Rap1-GTP was pulled down using GST–PHn, and GST–TSS fusion proteins of Tiam1 and STEF, as indicated. Immunoreactive bands were visualised using anti-HA antibody. The top panel shows the accumulated Rap1 in the GTP-bound form that associates with GST–TSS of STEF, the lower panel shows the total amount of HA–Rap1. The results shown are representative of two independent experiments.
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B
A 1 µ M 5-HT
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Rac 1 GTP α-Rac 1 Rac 1 α-HA
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1 µ M 5-HT - + Rap1 GTP
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STEF-ΔTSS
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4 2 0 1 µM 5-HT
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Fig. 3. 5-HT4 receptor- (A) or Epac- (B) induced Rac1 activation is inhibited by STEF lacking its TSS domain. CHO cells stably expressing the 5HT4 receptor were transfected either with HA-STEF wt or HA-STEF-DTSS. One day after transfection, cells were stimulated or not with either 1 lM 5-HT (A) or 10 lM 8-CPT (B) for 10 min and then Rac1–GTP was pulled down with GST-CRIB of PAK and analyzed by Western blot. In (A) and (B), the top panels show the accumulated Rac1 in the GTP-bound form that associates with GST-CRIB, the middle panels show the total amount of endogenous Rac, and the lower panels show the total amount of HA-STEF and HA-STEF-DTSS. The graphs indicate the amounts of Rac1-GTP which were expressed as fold activation over control. Rac1-GTP in unstimulated cells was defined arbitrarily as 1. Inset, STEF-DTSS does not block Rap1 activation induced by 5-HT. CHO cells expressing the 5-HT4 receptor were transfected either with empty vector or HA-STEF-DTSS. One day after transfection, cells were stimulated or not with 1 lM 5-HT for 10 min and then Rap1-GTP was pulled down and analyzed by Western blot. The top panel shows the accumulated Rap1 in the GTP-bound form that associates with fusion protein, the middle panels show the total amount of endogenous Rap1 and the lower panels show the total amount of HA-STEF-DTSS. Results are means ± S.E.M for three independent experiments, P < 0.01, P < 0.001 compared with untreated control cells (Student’s t-test).
3. Results The most specific GEFs for Rac GTPase which belong to the Dbl family are Vav proteins [19], Tiam-1 [11] and STEF which is directly related with Tiam-1 and the Drosophila SIF protein [12]. One possible explanation which could clarify the Rap1/ Rac1 connection was that Rap1 could interact with one of these exchange factors of the Dbl family to mediate Rac activation. Based on this hypothesis, we examined whether the active form of Rap1 could bind to one of the Rac specific GEFs. More precisely, we first examined the potential association of Rap1 with Vav1. To do so, we expressed Rap1Q63E (constitutively active form of Rap1) in COS-1 cells and conducted pull-down experiments using fusion proteins constituted by different domains of Vav tagged with GST, as indicated in Fig. 1A. We found that Rap1Q63E did not bind to any domain of Vav Fig. 1A ((1)– CH + Ac, (2)–CH + Ac + DH, (3)–PH, (4)–SH2). Then, we examined by pull down assay whether Tiam-1 was able to bind to Rap1 or not. In order to do so, we developed fusion proteins tagged with GST for the amino terminal PH domain (PHn) and TSS region of Tiam1. Both domains were unable to bind Rap1Q63E (Fig. 1B). Nevertheless when we used the fusion proteins GST–PHn and GST–TSS from STEF, we demonstrated that TSS region of STEF bind specifically Rap1Q63E as shown in Fig. 1C. We then examined the specificity of this interaction between STEF and the active form of Rap1. We expressed either Rap1Q63E or a dominant negative form of Rap1, Rap1S17N
in COS-1 cells and performed pull-down experiments using GST–PHn and GST–TSS from STEF and Tiam-1. As shown in Fig. 2A, only the constitutively active form of Rap1, Rap1Q63E was able to bind to TSS region of STEF. Consequently, we examined whether foetal bovine serum (FBS)induced Rap1 activation could bind to STEF. Cells were transfected with the wild type form of Rap1 and then stimulated or not with 10% FBS (Fig. 2B). As expected, FBS-induced Rap1 activation but neither PH domain nor TSS region of Tiam-1 were able to pull-down Rap1-GTP (Fig. 2B). However, the TSS region of STEF bound specifically to the active form of Rap1 (Fig. 2B). Altogether, these data demonstrated that Rap1-GTP bind specifically to STEF by its TSS region. We had previously shown that the serotonin 5-HT4 receptor increases Rac activation in a cAMP-dependent manner through the GEF, Epac and its effector, Rap1 [1]. Therefore, we next tested whether the stimulating effects of the 5-HT4 receptor and Epac on Rac activation were STEF dependent. CHO cells expressing the 5-HT4 receptor were transiently transfected with STEF wild type (STEF wt) or STEF mutant which lacks in the TSS region (STEF-DTSS). As shown in Fig. 3A, 1 lM 5-HT induced a solid activation of endogenous Rac1 in cells transfected with STEF wt (Fig. 3A). In contrast, overexpression of STEFDTSS blocked Rac1 activation induced by the 5-HT4 receptor without affecting Rap1 activation (Fig. 3A, inset). Altogether, these results show that Rac1 activation induced by the 5-HT4 receptor involves STEF. Next, we tested the involvement of
N. Zaldua et al. / FEBS Letters 581 (2007) 5814–5818
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A -
1 µ M 5-HT secreted sAP Pα
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secreted sAPPα
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Rac1 since it regulates Rac1 activation upon 5-HT4 receptor and Epac/Rap1 activation. In order to validate this new signalling pathway in a more physiological way, we analysed its possible involvement in sAPPa secretion. In order to do so, we examined whether STEF is involved in APP processing upon 5-HT4 receptor activation. CHO cell line stably coexpressing the h5-HT4(e) receptor and human APP695 [15] were transfected with STEF wt or STEF-DTSS. Over-expression of STEF-wt significantly potentiated basal level of sAPPa secretion as compared to mock transfected cells (Fig. 4A). Furthermore, sAPPa secretion was further increased in CHO cells expressing STEF wt and treated with 1 lM 5-HT (Fig. 4A). In contrast, STEF-DTSS significantly inhibited 5-HT-induced sAPPa secretion (Fig. 4A). Similar findings were found when these experiments were performed in the presence or absence of Epac selective activator, 8-CPT. Indeed, this compound at 10 lM increased sAPPa secretion in cells transfected with STEF wt (Fig. 4B). On the contrary, STEF-DTSS blocked the stimulating effect of 8-CPT (10 lM) on sAPPa secretion in response to 8-CPT (Fig. 4B). Altogether, our results indicate that this new signalling pathway involving Epac1 and STEF is implicated in cAMP-dependent APP processing.
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Fig. 4. Secretion of sAPPa induced by the activation of the 5-HT4 receptor (A) or Epac (B) is mediated by STEF. CHO cells stably expressing the 5-HT4 receptor and APP695 were transfected either with an empty vector or a plasmid encoding HA-STEF wt or HASTEF-DTSS. One day after transfection, cells were stimulated or not with either 1 lM 5-HT (A) or 10 lM 8-CPT (B) for 30 min. Then, sAPPa was detected in the culture medium by immunoblot using the 6E10 antibody (upper panels in A and B). Lower panels in A and B, immunoblots showing the expression of HA-STEF wt and HA-DTSS. The graphs represent the amounts of extracellular sAPPa which were expressed as fold activation over control. sAPPa expression in unstimulated cells was defined arbitrarily as 1. Results are means ± S.E.M for three independent experiments *P < 0.05; **P < 0.01 compared with untreated control cells (Student’s t-test).
the cAMP-GEF, Epac1 in STEF-dependent Rac activation. To do this, we transfected CHO cells with STEF wild type or STEF-DTSS and we treated cells with the Epac-specific cAMP analogue, 8-pCPT-2 0 -O-Me-cAMP (8-CPT) [20]. As shown in Fig. 3B, 8-CPT (10 lM) strongly induced Rac1 activation but failed to do so in cells transfected with STEF-DTSS. These data suggest that STEF is the molecular link between Rap1 and
4. Discussion In the present study, we provide evidence that STEF is involved in the regulation of Rac1 GTPase activity via Epac/ Rap1 after 5-HT4 receptor activation in intact cells. We have found that the constitutively active form of Rap1, Rap1Q63L, binds in an efficient- and specific-manner to STEF. This interaction occurs through the TSS domain of STEF which is localized just behind the PHn domain [12]. This interaction was not only established with Rap1Q63L. Indeed, TSS region of STEF pulled-down specifically Rap1-GTP upon cell treatment with FBS. This TSS region of STEF has been described to bind in vitro to the PDZ1-2 domains of PAR-3. It also hangs on the PAR-6-PAR-3 complex formation to facilitate Rac activation in neurons [21]. We show for the first time that TSS region of STEF can function as a Rap1 interacting binding domain. The STEF domains required for Rac1 activation are DH, PHn and TSS domains [22]. The DH domain is the catalytic domain and is therefore essential to facilitate the exchange between GDP and GTP of the small GTPase. Furthermore, we have previously shown that mutation in DH domain of Vav1 prevents Rac1 activation [23]. PHn and TSS domains are necessary for the membrane localization of STEF and Rac1 activation [22]. In this study, we show that only the TSS domain of STEF binds in vitro to Rap1 whereas PHn domain does not. This is probably because PHn is only required for membrane localization and TSS for binding to upstream regulating molecules (this work and [21]). Regarding the effect of STEF on Rac1 in living cells upon activation of the 5-HT4 receptor, we found that deletion of the TSS domain impairs the ability of STEF to activate Rac1. Thus, we hypothesize that Rac1 activation mediated by STEF might require two steps: firstly, STEF would be localized in the cell membrane by the PHn domain and secondly Rap1-GTP would bind to the TSS domain of STEF allowing this GEF to activate Rac1. In absence of the TSS domain of STEF, Rap1-GTP cannot bind to STEF and
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consequently Rac1 is not activated, even upon 5-HT4 receptor activation. To check the results obtained with 5-HT/STEF on Rac1 activation, we also examined whether STEF regulated Rac1 activation after Epac engagement. The cAMP analogue, 8-CPT binds to Epac which rapidly catalyzes the passage from the inactive to the active state of Rap [24,25]. In the present study, we demonstrate that 8-CPT/Epac- induced Rac1 activation is regulated by STEF. Indeed, we show that STEF-DTSS form failed to activate Rac1. On the basis of the different experimental approaches presented here, we can conclude that 5-HT4 receptor mediates Rac1 activation on living cells through Epac/Rap1/STEF signal transduction pathway. STEF has been described as a molecule which takes part in neuronal plasticity. Nishimura and colleagues have proposed that the complex PAR-6-PAR-3 mediates the crosstalk between Cdc42 and Rac through STEF and that the STEF domain required to establish the neuronal polarity is TSS [21]. Here in, we describe that a crosstalk between Epac/Rap1 and Rac1 induced by the 5-HT4 receptor is involved in the cAMP-dependent processing of APP. Consistent with the critical role of STEF in Epac/Rap1/Rac1 signalling pathway, transfection of STEF wt increased basal secretion of sAPPa. Accordingly, 5HT and 8-CPT enhanced STEF- induced sAPPa secretion and these effects were strongly blocked by STEFF-DTSS. To conclude, our findings show that STEF is the molecular link between the cAMP/Epac/Rap1 and Rac1 pathways. STEF activation is produced by a physical interaction between its TSS domain and Rap1–GTP which mediates Rac1 activation and this active form of Rac regulates the a cleavage of APP, generating the sAPPa. Acknowledgements: We would like to thank Drs X.R. Bustelo and M.E. Greenberg for the generous gift of Vav fusion proteins and the cDNA encoding for Tiam-1 respectively. Drs V. Lang and L. Parada for helpful comments on the manuscript. FL is supported by INSERM and a Grant from Fondation pour la Recherche Me´dicale (e´quipe FRM). This work was supported by the Department of Industry of the Basque Government ETORTEK and SAIOTEK Programs to J.L.Z. and by the Spanish Ministry of Education and Science (MES) Biomedicine Program to J.L.Z. (SAF2005-07930-C02-02). J.L.Z. is a Ramo´n y Cajal Investigator (MES) associated to CIC bioGUNE.
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[7]
[8] [9]
[10] [11] [12]
[13] [14] [15] [16] [17]
[18]
[19] [20]
[21]
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