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PIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor
1 Department of Internal Medicine and Clinical Immunology and 2 Department of Molecular Biology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura Kanazawa-ku, Yokohama 236-0004 Japan
3 Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Ibaraki 305-8575, Japan
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Abstract |
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PIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor (GEF), and they co-localized at the tips of lamellipodia in motile cells. The involvement of PIX in the RP1-induced Cdc42 activation was demonstrated by the significant dominant negative effect of a point mutant of PIX, PIX (L383R, L384S), lacking GEF activity. Our data strongly support that ILK and affixin provide a novel signalling pathway that links integrin signalling to Cdc42/Rac1 activation. |
Introduction |
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The stimulation of Rho GTPases is of special importance in this respect. These molecules are essential for the organization of actin cytoskeleton and promote the actin structures such as filopodia (Cdc42), lamellipodia (Rac1) and stress fibre (RhoA) (Etienne-Manneville & Hall 2002; Nobes & Hall 1995). Integrins have seemed to stimulate Rho GTPases (Price et al. 1998; Ren et al. 1999) via some of integrin binding proteins such as focal adhesion kinase (FAK) and paxillin (Parsons et al. 2000). FAK binding partners provide important informations about how FAK serves to mediate signalling from integrin adhesion complexes. First, a p130Cas-Crk-DOCK180 complex can activate Rac1 (Kiyokawa et al. 1998). Second, PI3-kinase can stimulate the guanine nucleotide exchanging factor (GEF) activity of Vav and Sos-1 (Das et al. 2000). Third, paxillin-paxillin kinase linker (PKL) recruit a p21-activated serine-threonine kinase (PAK)-PAK-interacting exchange factor (PIX) complex into focal complexes and play an important role in the regulation of Rac1 activity (Brown et al. 2002; Turner et al. 1999; West et al. 2001). Since each of these complex is included in one of GEF molecules, that can stimulate the exchange GDP-bound form (inactive state) of Rho GTPases for GTP-bound form (active state), and their complex formation are induced by integrins, they have been considered to be involved in integrin-induced Rho GTPases activation. However, the precise molecular mechanism how the integrins activate these GEF proteins is largely unknown.
Recently, integrin linked kinase (ILK), a ubiquitously expressed serine-threonine kinase, which is capable of interacting with integrin ß1 and ß3 cytoplasmic domains (Hannigan et al. 1996), has been suggested to stimulate Rho GTPases (Rosenberger et al. 2003). Several studies showed that ILK is acutely activated upon cell-substrate adhesion and is involved in the integrin-dependent cell adhesion, spreading and cell-shape change of cultured cells (Brakebusch & Fassler 2003; Wu & Dedhar 2001). Much progress has been made in identifying various ILK-interacting proteins such as paxillin and PINCH and each of them can modulate actin cytoskeleton in a direct or an indirect manner (Brakebusch & Fassler 2003). Among them, the kinase domain of ILK can recruit a new family of proteins which has a tandem of calponin homology (CH) domains. This family has three members and was identified independently in several laboratories including us and has multiple names. We have found affixin (Yamaji et al. 2001), which is identical to ß-parvin identified by Olski et al. (2001). Tu et al. (2001) identified the calponin homology-ILK binding protein (CH-ILKBP), which is identical to actopaxin described by Nikolopoulos & Turner (2000) or -parvin (Olski et al. 2001), and 
-parvin (Olski et al. 2001).
Affixin and ILK co-localize in focal complexes, focal adhesions and the leading edge of lamellipodia of well-spread CHO fibroblasts and, in reseeded cells, they are recruited into nascent cell-substrate adhesion structures on much earlier stage of cell spreading than FAK. Importantly, the over-expression of the CH2 domain of affixin in CHO cells blocked cell spreading after reseeding (Yamaji et al. 2001). Moreover, the ILK–parvin interaction was investigated in a myoblast cell line, in which disruption of the ILK--parvin complex retarded formation of stress fibers as well as focal adhesions and delayed cell spreading (Tu et al. 2001). These reports have suggested that the ILK–affixin complex can modulate actin cytoskeleton on the earlier stage than FAK. However, the downstream targets of affixin to which it transmits integrin-ILK signalling still have remained unclear.
In the present study, we found that the over-expression of RP1 corresponding to the first CH domain (CH1) of affixin severely affects F-actin organization and induces prominent membrane protrusions in MDCK cells. The effect of RP1 over-expression was shown to depend on its ability to activate Cdc42/Rac1. We also found that PIX interacts with RP1 and co-localize at the tips of lamellipodia of migrating cells. Furthermore, the over-expression of a deficient mutant of PIX that lacks GEF-activity significantly suppressed the effect of the over-expression of RP1. These results suggest that affixin is involved in the integrin-induced Rho GTPase activation through the interaction between its CH1 domain and PIX.
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Results |
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In our previous study, we found that the over-expression of a deletion mutant of affixin (RP1) corresponding to CH1 domain (Fig. 4), in CHO cells tended to facilitate cell spreading after replating: cells spread completely 1 h after replating, at which control cells still showed round morphology, and exhibited significantly developed lamellipodia 4 h after replating (Yamaji et al. 2001). This is in sharp contrast with the effect of the over-expression of RP2, corresponding to the second CH (CH2) domain, which completely inhibited cell spreading after replating and kept cells round morphology. Since RP2 associates with and is phosphorylated by ILK, the over-expression of RP2 was assumed to exert a dominant negative effect on the integrin-ILK signalling pathway, which is important for the initial phase of cell spreading. On the other hand, the completely opposite effect of RP1 over-expression on cell spreading raised a possibility that the CH1 domain is an effector domain that transmits integrin-ILK signalling to downstream targets that facilitate cell spreading (Yamaji et al. 2001).
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It is well known that the Rho GTPases, Cdc42 and Rac1, play an important role in F-actin organization, and thereby critically contribute to cell migration as well as the establishment of epithelial cell polarity (Etienne-Manneville & Hall 2002; Hall 1998). To examine whether the phenotypes induced by RP1 over-expression in MDCK cells are mediated by Cdc42 and/or Rac1 activation, we examined the effect of RP1 over-expression on MDCK I cell lines that stably expressed the dominant negative mutants of Cdc42 or Rac1 (Cdc42N17 or Rac1N17, respectively), under the control of tetracycline-repressive transactivation (Jou & Nelson 1998; Jou et al. 1998). When the RP1-carrying adenovirus vector was transfected to cells cultured in the presence of doxycycline (DC: 20 ng/ml) and not expressing Cdc42N17 or Rac1N17, the cells showed fibroblastic morphology as observed in wild-type MDCK II cells (Fig. 2 DC +). However, when cells were precultured in the absence of doxycycline, which induces the expression of Cdc42N17 or Rac1N17, the effect of RP1 over-expression was significantly suppressed in both cases (Fig. 2 DC –). These results suggest that the over-expression of RP1 induced morphological changes of MDCK cells through the activation of Cdc42 and Rac1.
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PIX
Recently, Rosenberger et al. (2003) have reported that PIX/ARHGEF6, a guanine nucleotide exchange factor (GEF) specific to Cdc42 and Rac1, binds to affixin. Although the binding site on affixin and the physiological meaning of the interaction was not clarified, this indicates that PIX is a strong candidate molecule that mediates Cdc42/Rac1 activation induced by RP1-over-expression. Therefore, we first examined whether PIX binds to RP1 using a yeast two-hybrid system. Unfortunately, because the full-length ss-affixin (53–364), which is an affixin isoform lacking 52 N-terminal amino acids, as well as deletion mutants lacking a large part of the C-terminal region showed self-activation activity, the data were extremely limited. However, the results clearly indicated that the binding site with PIX resides in the N-terminal region (53–271) that covers the CH1, but not the CH2 domain. The data were supplemented with co-immunoprecipitation assays performed using CHO cells. As shown in Fig. 4B,C, PIX co-immunoprecipitated with the full-length of ss-affixin and RP1, but not with RP2. This similar interaction was also confirmed by reciprocal immunoprecipitation (data not shown). Figure 4B also suggests that RP1 showed greatly augmented activity for interaction with PIX compared with the full-length ss-affixin. In Fig. 5, we further confirmed that when T7-tagged RP1 or the full-length affixin and HA-tagged PIX were over-expressed, RP1 and PIX co-localized at the tips of leading edges of cells, as observed for full-length affixin and PIX (Rosenberger et al. 2003). Taken together, these results indicate that the PIX-binding site of affixin is located in RP1 corresponding to the CH1 domain.
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PIX GEF activity is required for RP1-induced effects observed in MDCK cells
To confirm that the effect of RP1 over-expression is dependent on GEF activity of PIX, we established MDCK II cells that stably expressed wild-type PIX (PIXwt) or a GEF activity-deficient mutant, PIX (L383R, L384S) (Daniels et al. 1999; Manser et al. 1998), under the control of tetracycline-inducible transactivation (so called ‘tet-on system’). In these cells, the addition of doxycycline (> 20 ng/mL) significantly induced the expression of PIXwt or PIX (L383R, L384S) within 24 h (Fig. 6A,B). To examine the dominant negative effect of PIX (L383R, L384S) on the effect of RP1 over-expression, the cells were preincubated in the presence or absence of doxycycline (100 ng/mL) for 24 h, and then transfected with adenovirus expression vector of RP1. As shown in Fig. 6B, the over-expression of RP1 induced significant spreading and scattering of both cells precultured in the absence of doxycycline as observed for wild-type MDCK II cells (Fig. 1). In contrast, the induction of PIX (L383R, L384S), but not PIXwt, significantly suppressed the effect of RP1 over-expression: even cells highly expressing RP1 showed a compact cobblelike morphology with few membrane protrusions and remained clustered in tightly packed-cell islands. Rhodamine-phalloidin staining revealed that cortical F-actin bundles were restored in these cells (Fig. 6C). Figure 7 further demonstrates that the expression of PIX (L383R, L384S) suppressed Cdc42 activation induced by RP1 over-expression. In the cells coexpressing PIX (L383R, L384S) and RP1, the Thr402 phosphorylation of PAK, that is correlated with PAK activation (Yu et al. 1998), is also reduced to about 30% compared with the cells expressing only RP1. Unfortunately, we could not obtained reproducible results on the effect of PIX (L383R, L384S) on the RP1-induced Rac1 activation. However, these results clearly indicate that the GEF activity of PIX mediates the RP1-induced activation of Cdc42, which results in PAK activation and F-actin reorganization.
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Discussion |
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PIX
In this study, we analysed the positive effect of the CH1 domain of affixin on cell spreading that was preliminarily observed in CHO cells (Yamaji et al. 2001). For this purpose, we used an epithelial cell line, MDCK cells, which show a cobblelike morphology and few membrane protrusions. By over-expressing a deletion mutant of affixin, corresponding to the CH1 domain, we found that this domain has a potential to transform static epithelial cells into depolarized and flat cells that frequently separated from cell islands. This activity of the CH1 domain was found to depend on its ability to activate Cdc42/Rac1 which have been shown to play pivotal roles in actin reorganization and cell migration. Then, the CH1 domain-induced morphological change and Cdc42 activation in MDCK cells were found to depend on the GEF activity of PIX, which specifically binds to this domain. Taken together, the present work suggests that the interaction between the CH1 domain of affxin and PIX directly or indirectly induces the GEF activity of PIX, which leads to the activation of Cdc42 and PAK, F-actin reorganization and enhanced cell spreading.
The CH1 domain may be an effector domain of affixin that transmits integrin-ILK signalling to Cdc42/Rac1
Previous study showed that affixin binds to ILK through its C-terminal CH2 domain, which is phosphorylated by ILK acutely activated upon initial cell–substrate interaction (Yamaji et al. 2001). Moreover, the over-expression of RP2, corresponding to the CH2 domain, suppresses cell spreading, suggesting that affixin–ILK interaction is important for lamellipodia development and focal adhesion formation (Yamaji et al. 2001). In this context, the present results strongly support a notion that the CH1 domain is an effector domain of affixin that transmits integrin-ILK signals that activate Cdc42/Rac1 through its interaction with PIX. It is very interesting in this respect that the binding activity of the CH1 domain with PIX is higher than that of the full-length affixin (Fig. 4B). This suggests that affixin is conformationally constrained in the resting state and requires some modification such as the phosphorylation of the CH2 domain by ILK to expose a functional PIX-binding site in the CH1 domain.
To examine whether ILK, affixin, and PIX present in the same complex, we performed co-immunoprecipitation assays in 293T cells. When all proteins were coexpressed, affixin, but not PIX, was co-immunoprecipitated with ILK. Consistently, affixin, but not ILK, was co-immunoprecipitated with PIX (data not shown). These results seemed to indicate that ILK and PIX were not able to interact with affixin simultaneously in spite of their discrete binding sites on affixin. On the other hand, we could not also observe any enhancement of the affixin/PIX interaction induced by ILKwt coexpression (data not shown). This result seemed to be inconsistent with our hypothesis that the phosphorylation of CH2 domain of affixin by ILK changes affixin into an ‘activated form’ to bind PIX. However, we could not rule out a possibility that something was missing in these experiments performed under the non-physiological condition, and the three proteins may present in the same complex in a much limited situation such as very early stage of cell spreading and/or in an insoluble cytoskeletal structures. Actually, it has been recently reported by Attwell et al. (2003) that the kinase activity of ILK is elevated in the cytoskeletal fraction, and that the interaction of CH-ILKBP with ILK within the cytoskeleton stimulates ILK activity. Hence, further studies, including the determination of ILK-phosphorylation sites on affixin, will be required to completely verify our hypothesis about the role of ILK in affixin–PIX interaction.
ILK-affixin provides a novel signalling pathway that links integrin signalling to Cdc42/Rac1 activation
It was demonstrated that integrin-mediated cell–substrate interaction induces the activation of Cdc42 and Rac1 (Price et al. 1998; Ren et al. 1999). Since some integrin binding proteins such as paxillin and FAK interact with the various GEF molecules via integrin-induced protein complexes, they have been considered to be involved in integrin-induced Cdc42/Rac1 activation (Parsons et al. 2000; Turner 2000). However, the precise molecular mechanism how integrins activate these GEF proteins remains to be clarified.
PAK, one of the most important downstream effectors of Cdc42/Rac1, is considered to mediate some of the effects of these Rho GTPases on actin organization, cell motility and adhesion through the phosphorylation of cofilin, resulting in the regulation of actin polymerization (Bagrodia & Cerione 1999; Bagrodia et al. 1995). PIX is a PAK-binding partner that localizes at focal complexes with PAK (Manser et al. 1998) and has been shown to stimulate PAK kinase activity dependent as well as independent on its GEF activity toward Cdc42 and Rac1 (Daniels et al. 1999). These data suggest that PIX is one of the most important candidates of the Cdc42/Rac1 activators in integrin signalling. Importantly, recent works by Brown et al. (2002), Turner et al. (1999), and West et al. (2001) have demonstrated that the PAK-PIX complex is recruited into the focal complex by interacting with paxillin, a multidomain focal contact adaptor protein, and paxillin kinase linker (PKL), ARF-GTPase-activating protein (ARF-GAP) interacting with paxillin and PIX. Furthermore, this interaction plays an important role in the regulation of Rac1 activity. Nevertheless, it has been still uncleared how integrin signalling activates PIX, which in turn activates Cdc42/Rac1.
ILK is an ubiquitously expressed serine-threonine kinase capable of interacting with integrin ß1 and ß3 cytoplasmic domain (Hannigan et al. 1996). Several studies showed that ILK is acutely activated upon cell-substrate adhesion and is involved in the integrin-dependent cell adhesion, spreading and cell-shape change of cultured cells (Brakebusch & Fassler 2003; Wu & Dedhar 2001). Moreover, various ILK-interacting proteins such as paxillin and PINCH can modulate actin in a direct or an indirect manner (Brakebusch & Fassler 2003). Recently, Rosenberger et al. (2003) have reported that PIX/ARHGEF6, specific GEF to Cdc42 and Rac1, binds to affixin. Although the binding site on affixin and the physiological meaning of the interaction was not clarified, they suggested that the ILK-affixin complex is included in the integrin-induced Cdc42/Rac1 activation pathways. Here, we confirmed Cdc42/Rac1 activation through affixin and PIX. Our data strongly supported that ILK-affixin provides a novel signalling pathway that links integrin signalling to Cdc42/Rac1 activation.
The present work based on over-expression experiments did no more than suggest a possible involvement of affixin in adhesion-dependent PIX activation. However, the critical importance of affixin in cell spreading and locomotion has been already demonstrated by the dominant negative effect of the CH2 domain of affixin on cell spreading. It was further supported by the recent finding that siRNA knock down of affixin in human fibroblasts severely inhibited lamellipodia development and cell movement (Yamaji et al. unpublished observation). Together with the recent report on the ILK-deficient fibroblasts that cannot develop normal focal adhesions (Sakai et al. 2003), these results establish the essential role of ILK-affixin signalling in the establishment of nascent cell-substrate adhesions and the subsequent development of actin-based membrane protrusions.
The role of ILK-affixin in integrin signalling must be evolutionarily conserved, because defects in C. elegans orthologues of ILK (PAT-4) or affixin (PAT-6) result in embryonic lethality showing inability to form a mature dense body (a corresponding structure of focal adhesion) in body wall muscle similarly to integrin mutants (PAT-3). PAT-4 and PAT-6 are both required in the same steps during attachment assembly in vivo, consistent with the notion that they work together in the same multiprotein complex (Lee et al. 2001; Lin et al. 2003; Mackinnon et al. 2002). The present results should provide one of the molecular bases of this evolutionarily conserved role of ILK-affixin signalling.
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Experimental procedures |
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Plasmid expression vectors for T7/His-tagged human ss-affixin, RP1 and RP2, were constructed using an SR His vector (Yamaji et al. 2001). The plasmid encoding human PIX cDNA (KIAA 0006) was a gift from Dr T. Nagase. The seven nucleotide sequence (ATGAATC) of 5'-terminus lacking in KIAA0006 that encodes the full-length PIX was supplemented by PCR using appropriate primers designed based on the reported sequence (Nomura et al. 1994). A point mutant of PIX, PIX (L383R, L384S), was generated using primers containing appropriate nucleotide substitutions. cDNAs of PIX and PIX (L383R, L384S) were subcloned into SRD4-HA for transient transfection. For generating cells stably expressing PIX and PIX (L383R, L384S) under the control of tetracycline-inducible transactivation, cDNA fragments with the HA-tag sequence were subcloned into the pOS-Tet14MCS vector (Y. Miwa manuscript in preparation). This plasmid vector was constructed using pEB6-CAGMCS (Tanaka et al. 1999) carrying an Epstein-Barr virus’ replicational origin, oriP, and a replication initiation factor (EBNA-1) that are sufficient for autonomous replication. pOS-Tet14MCS also encodes the tetracycline-dependent transactivator (rtTA2-M2 (Urlinger et al. 2000)). For yeast two–hybrid interaction, PIX was subcloned into pGAD424 (Clontech), while the deletion mutants of affixin were subcloned into pAS2–1C (Clontech).
Adenovirus vectors
Adenovirus vectors expressing RP1 were constructed by the homologous recombination method. Briefly, pJM17 (Microbix) and a shuttle plasmid, pCA3, carrying RP1 cDNA were co-transfected into human embryonic kidney 293 cells (HEK293, ATCC) by the calcium phosphate precipitation method. The generated virus was plaque purified twice, propagated in HEK293 cells, and concentrated by the caesium chloride-density gradient method. The virus stock was titred on the HEK 293 cells and stored at –80 °C.
Cell culture
MDCKII and 3Y1 cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco's modified essential medium (DMEM) containing 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco). CHO-K1 cells were similarly cultured except for the use of F-12 instead of DMEM. MDCK tet-off cell lines stably expressing Rac1N17 or Cdc42N17 under the control of tetracycline repressive transactivation were gifts from Dr W. James Nelson (Jou & Nelson 1998; Jou et al. 1998). They were routinely cultured under the same conditions except for the addition of 20 ng/ml doxycycline (DC). The expression of Rac1N17 or Cdc42N17 was induced after replating by removing DC from the culture medium. For the Cdc42N17-expressing cell line, 2.5 nM sodium butyrate was added to the medium to enhance the expression of the mutant gene. Transient transfection of plasmid vectors was performed using polyfect (QIAGEN) according to the manufacturer's instruction. Adenovirus infection into MDCK cells was carried out as previously described (Mishima et al. 2002). MDCK tet-on cells stably expressing PIX or PIX(L383R, L384S) were obtained by transiently transfecting cells with pOS-Tet14MCS carrying the appropriate cDNA and simply selecting geneticin-resistant cells (Gibco: 400 µg/ml) without further cloning. The expression of PIX or PIX (L383R, L384S) was induced following the addition of 100 ng/ml DC.
Antibodies
The following antibodies were used in this study: rabbit Omni-probe polyclonal antibody (M-21), which recognizes His/T7-tag sequence (Santa Cruz), mouse anti-c-myc monoclonal antibody (9E10: Santa Cruz), rat anti-HA monoclonal antibody (Roche), mouse anti-T7 monoclonal antibody (Novagen), rhodamine-phalloidin (Molecular Probes), rabbit anti-PAK polyclonal antibody (Santa Cruz), and rabbit anti-P-PAK polyclonal antibody (Santa Cruz).
Immunoprecipitation and pull down assay
T7/His-tagged ss-affixin and HA-tagged PIX were immunoprecipitated from CHO-K1 cells as previously described (Yamaji et al. 2001). Briefly, cells were transfected with appropriate expression vectors, and lysed the next day with 20 mM HEPES (pH 7.5) containing 150 mM NaCl, 1 mM EDTA, 10 µg/ml leupeptin, 1 mM PMSF, 1% Triton X-100 and 0.1% deoxycholate. The immunocomplex was precipitated from cell lysates using protein G-sepharose (Amercham Pharmacia Biotech) conjugated with 2 µg of anti-T7 or anti-HA monoclonal antibody. Pull down assay was performed using Cdc42, Rac1 and RhoA activation assay kits (Cytoskeleton) according to the manufacturer's instructions.
Immunofluorescence staining
Cells grown on collagen-coated coverslips were fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. Cells were then permeabilized with PBS containing 0.5% (v/v) Triton X-100 for 10 min and blocked with 10% calf serum in PBS for 1 h at room temperature. Then the cells were treated with appropriate primary antibodies for 45 min at 37 °C in a moist chamber. After washing with PBS containing 0.05% Tween 20, the cells were incubated with secondary antibodies. Samples were observed under a fluorescence microscope (BX50: Olympus).
Two–hybrid interaction
The Y187(a) yeast strain was transfected with the appropriate combination of plasmid vectors, and co-transformants of the bait and prey plasmids were grown for 4 days at 30 °C on minimum essential plates lacking tryptophan, leucine and uracil. ß-galactosidase filter assay was performed 4 days after transfection according to the manufacturer's (Clontech) instructions.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: ishigats{at}med.yokohama-cu.ac.jp
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Received: 3 September 2003
Accepted: 17 December 2003
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