G protein-coupled receptor kinase 2 positively regulates epithelial cell migration
2008; Springer Nature; Volume: 27; Issue: 8 Linguagem: Inglês
10.1038/emboj.2008.55
ISSN1460-2075
AutoresPetronila Penela, Catalina Ribas, Ivette Aymerich, Niels Eijkelkamp, Olga Barreiro, Cobi J. Heijnen, Annemieke Kavelaars, Francisco Sánchez‐Madrid, Federico Mayor,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoArticle27 March 2008free access G protein-coupled receptor kinase 2 positively regulates epithelial cell migration Petronila Penela Corresponding Author Petronila Penela Departamento de Biología Molecular and Centro de Biología Molecular 'Severo Ochoa', Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Catalina Ribas Catalina Ribas Departamento de Biología Molecular and Centro de Biología Molecular 'Severo Ochoa', Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Ivette Aymerich Ivette Aymerich Departamento de Biología Molecular and Centro de Biología Molecular 'Severo Ochoa', Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Niels Eijkelkamp Niels Eijkelkamp Laboratory of Psychoneuroimmunology, University Medical Center, Utrecht, The Netherlands Search for more papers by this author Olga Barreiro Olga Barreiro Servicio de Inmunología, Hospital Universitario La Princesa, Madrid, Spain Search for more papers by this author Cobi J Heijnen Cobi J Heijnen Laboratory of Psychoneuroimmunology, University Medical Center, Utrecht, The Netherlands Search for more papers by this author Annemieke Kavelaars Annemieke Kavelaars Laboratory of Psychoneuroimmunology, University Medical Center, Utrecht, The Netherlands Search for more papers by this author Francisco Sánchez-Madrid Francisco Sánchez-Madrid Servicio de Inmunología, Hospital Universitario La Princesa, Madrid, Spain Search for more papers by this author Federico Mayor Jr Corresponding Author Federico Mayor Jr Departamento de Biología Molecular and Centro de Biología Molecular 'Severo Ochoa', Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Petronila Penela Corresponding Author Petronila Penela Departamento de Biología Molecular and Centro de Biología Molecular 'Severo Ochoa', Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Catalina Ribas Catalina Ribas Departamento de Biología Molecular and Centro de Biología Molecular 'Severo Ochoa', Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Ivette Aymerich Ivette Aymerich Departamento de Biología Molecular and Centro de Biología Molecular 'Severo Ochoa', Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Niels Eijkelkamp Niels Eijkelkamp Laboratory of Psychoneuroimmunology, University Medical Center, Utrecht, The Netherlands Search for more papers by this author Olga Barreiro Olga Barreiro Servicio de Inmunología, Hospital Universitario La Princesa, Madrid, Spain Search for more papers by this author Cobi J Heijnen Cobi J Heijnen Laboratory of Psychoneuroimmunology, University Medical Center, Utrecht, The Netherlands Search for more papers by this author Annemieke Kavelaars Annemieke Kavelaars Laboratory of Psychoneuroimmunology, University Medical Center, Utrecht, The Netherlands Search for more papers by this author Francisco Sánchez-Madrid Francisco Sánchez-Madrid Servicio de Inmunología, Hospital Universitario La Princesa, Madrid, Spain Search for more papers by this author Federico Mayor Jr Corresponding Author Federico Mayor Jr Departamento de Biología Molecular and Centro de Biología Molecular 'Severo Ochoa', Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Author Information Petronila Penela 1, Catalina Ribas1, Ivette Aymerich1, Niels Eijkelkamp2, Olga Barreiro3, Cobi J Heijnen2, Annemieke Kavelaars2, Francisco Sánchez-Madrid3 and Federico Mayor 1 1Departamento de Biología Molecular and Centro de Biología Molecular 'Severo Ochoa', Universidad Autónoma de Madrid, Madrid, Spain 2Laboratory of Psychoneuroimmunology, University Medical Center, Utrecht, The Netherlands 3Servicio de Inmunología, Hospital Universitario La Princesa, Madrid, Spain *Corresponding authors: Departamento de Biología Molecular y Centro de Biología Molecular Severo Ochoa, CSIC-Universidad Autónoma Madrid, Universidad Autónoma de Madrid, Madrid, Madrid 28049, Spain. Tel.: +34 91 196 4626; Fax: +34 91 196 4420; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2008)27:1206-1218https://doi.org/10.1038/emboj.2008.55 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration. Introduction Signal-directed migration requires a spatiotemporal integration of information from mechanical cues and from diffusible molecules such as chemokines, bioactive lipids and growth factors. Failures in this process might result in aberrant migration, leading to chronic inflammatory disorders, tumour metastasis, impaired wound healing or vascular diseases (Ridley et al, 2003). Cellular motility demands dynamic regulation of cell anchorage. Adhesion structures are nucleated by integrins, membrane receptors that orchestrate multi-protein complexes, namely focal adhesions and focal contacts that modulate cell growth, survival and cytoskeleton remodelling, linking the actin cellular cytoskeleton to components of the extracellular matrix (Brunton et al, 2004). Focal adhesion turnover is under the control of multiple signalling inputs. Activation of members of the FAK and Src families of tyrosine kinases decreases adhesive interactions and disorganizes the associated cytoskeleton by means of phosphorylation of numerous proteins (Brunton et al, 2004). In addition, extracellular signal-regulated protein kinase/mitogen-activated protein kinase (ERK/MAPK) activity can also promote focal adhesion assembly/disassembly by modifying paxillin, calpain or MLCK activities (Carragher and Frame, 2004; Webb et al, 2004), thereby enhancing cell migration. For migration to progress efficiently, pro-migratory signals from a variety of G protein-coupled receptors (GPCRs) must be precisely coordinated with those arising from integrins (Li et al, 2003; Luttrell and Luttrell, 2004; DeFea, 2007). Most chemokines and lipid messengers, such as sphingosine-1-phosphate (S1P), emit signals to the actin cytoskeleton and to adhesion structures by binding to plasma membrane receptors of the GPCR family (Spiegel et al, 2002; DeFea, 2007). Concomitantly, GPCR occupancy activates desensitization mechanisms that are also required for proper migration. Agonist-occupied receptors are phosphorylated by a family of serine/threonine kinases known as G protein-coupled receptor kinases (GRKs), an event that triggers binding of arrestins, uncoupling from G proteins and receptor internalization (Penela et al, 2003). The GRK family of kinases comprises seven isoforms, of which G protein-coupled receptor kinase 2 (GRK2) is the most ubiquitously expressed (Penela et al, 2003). Consistent with its role in GPCR desensitization, GRK2 has been shown to attenuate chemokine-induced migration in T cells and monocytes (revised by Vroon et al, 2006), thus emerging as a relevant modulator of inflammatory responses. However, pro-migratory effects of β-arrestins and of a member of a distinct GRK subfamily, GRK6, have also been reported in response to different GPCRs in several cell types (Vroon et al, 2006; DeFea, 2007), thereby suggesting other unknown functional roles different from those related to GPCR desensitization. Interestingly, GRK2 has been shown to interact with a variety of proteins involved in migration (MEK, Akt, ezrin, PI3Kγ or GIT; reviewed by Ribas et al, 2007). However, the functional role of such interactions in cell migration and whether GRK2 influences motility in cellular types other than immune cells and towards different chemotactic stimuli have not been investigated. In this report, we have studied the impact of altering GRK2 protein levels on integrin-dependent chemotaxis. We show for the first time that GRK2 positively regulates epithelial cell migration by mechanisms involving coordinated fibronectin- and S1P-mediated signalling and the modulation of the Rac/PAK/MEK/ERK1/2 pathway in response to S1P and adhesion, based on the dynamic regulation of the interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Results Increased GRK2 expression promotes profound changes in epithelial cell morphology and enhances motility towards fibronectin To explore the potential impact of GRK2 on cell migration, wild-type (wt) GRK2 was stably overexpressed in two different model epithelial-derived cell lines, COS7 and HEK-293 cells. Increased kinase expression resulted in marked morphological changes characteristic of motile cells (Figure 1A and Supplementary Figure S1A). Increased GRK2 levels trigger cortical actin rearrangements at the cell periphery, accompanied by a reduction in stress fibres. Interestingly, expression of a kinase-dead mutant of GRK2 (GRK2-K220R) at comparable levels promoted changes similar to those observed with wt GRK2 (Figure 1B). In both cases, increased GRK2 expression promoted a diffuse cytosolic localization of paxillin, in marked contrast to its dot-like distribution at peripheral and central focal adhesions observed in control COS7 cells (Figure 1B). Such paxillin redistribution suggested a role for GRK2 in focal adhesion turnover independent of kinase activity. Figure 1.GRK2 expression levels influence cell morphology and migration to fibronectin. (A, B) COS7 cells stably overexpressing wt GRK2 or the inactive GRK2-K220R mutant were plated on coverslips coated with 10 μg/ml fibronectin (FN) in the presence of 10% serum and their morphology was compared to that of parental cells by phase-contrast microscopy (objective × 20) (A) or by confocal fluorescence microscopy as described in Materials and methods. Scale bars, 50 μm (B). (C–E) GRK2 expression enhances fibronectin-directed cell migration. Serum-starved COS7 (C) or HeLa cells (D) expressing or not increased wt GRK2 or GRK2-K220R levels (3- to 10-fold over endogenous protein levels as determined by immunoblot analysis; see insets) were seeded on Transwell filters precoated with 20 μg/ml FN or 10 μg/ml collagen fraction IV and cell migration was assessed as detailed in Materials and methods. Data are the mean±s.e.m. of 4–6 independent experiments performed in duplicate. **P<0.01, compared to control cells. Number of control HeLa cells migrating to FN and collagen was 32±10 and 22±9 cells/field, respectively, whereas that of COS7 cells migrating to FN was 73±19 cells/field (means±s.d.). In (E), directed migration of COS7 cells expressing wt GRK2 was analysed upon addition of FN (20 μg/ml) to either the bottom chamber alone (chemotactic gradient) or to both upper and bottom chambers (uniform concentration). (F, G) Cellular migration to FN was significantly decreased upon reduction of GRK2 expression (see inset blots) in HeLa cells infected with an adenoviral-GRK2 shRNA construct (F) and in MEFs (G) derived from hemizygous GRK2+/− mice as compared to parental HeLa cells and MEFs from wt GRK2 mice, respectively. Data are the mean±s.e.m. of 3–4 independent experiments performed in duplicate. *P<0.05, **P<0.01, compared to control cells. Control values for HeLa and wt MEFs migrating to FN were 37±7 and 201±33 cells/field (means±s.d.), respectively. Download figure Download PowerPoint To address whether such GRK2-induced changes result in altered cell motility, we analysed migration towards fibronectin in different epithelial cell lines stably expressing GRK2 or the GRK2-K220R mutant. Increased GRK2 expression markedly enhanced migration in COS7, HeLa (Figure 1C and D) or HEK-293 cells (Supplementary Figure S1B), in a kinase activity-independent manner. Such response did not depend on the interaction of GRK2 with Gαq subunits (Supplementary Figure S1C). Expression of a C-terminal deletion mutant no longer capable of binding to Gβγ subunits did not significantly enhance cell migration, whereas a membrane-targeted GRK2 mutant strongly enhanced cell motility, suggesting the involvement of a scaffolding function for GRK2 when recruited to specific membrane locations. This pro-migratory effect was clearly reduced when the fibronectin gradient was abolished, indicating that GRK2 impacts preferentially the cell chemotactic responses (Figure 1E). Furthermore, the effect of GRK2 is specific and does not affect the overall cell motility, as migration of COS7 or HeLa cells towards collagen fraction IV was unaltered by overexpression of wt GRK2 or GRK2-K220R (Figure 1C and D). Interestingly, migration towards fibronectin was significantly reduced upon downregulation of endogenous GRK2 expression in HeLa cells using either a specific GRK2-siRNA construct (not shown) or adenoviral-mediated shRNA delivery (Figure 1F), and in mouse embryonic fibroblasts (MEFs) derived from hemizygous GRK2+/− mice, which display a 40–50% reduction in kinase levels compared to wt animals (Figure 1G). These results indicate that GRK2 expression levels positively correlate with cell migration to specific matrix proteins in primary cells and in different epithelial-derived cell lines. GRK2-dependent increased migration to fibronectin is mediated by a PTX-sensitive GPCR Regarding the mechanisms underlying the effect of GRK2 on cell migration, initial experiments excluded the participation of a Rho-dependent pathway (Supplementary Figure S2A). Given the key role of GRK2 in GPCR modulation and signalling, we hypothesized that a GPCR activity might be involved. Addition of pertussis toxin (PTX), an inhibitor of Gi-coupled receptor signalling, completely suppressed the increase in GRK2-dependent migration (Figure 2A). As our assays were performed in the absence of soluble stimuli and using cells deprived of serum, we hypothesized that an autocrine signal acting through GPCRs might be implicated. In this regard, S1P is endogenously generated by the activity of sphingosine kinase (SK) and is able to stimulate S1P1 (Gi-coupled) and S1P3 GPCRs, present in fibroblasts and epithelial cell lines, in a paracrine/autocrine manner (Spiegel et al, 2002; El-Shewy et al, 2006; Hait et al, 2006). Figure 2.The effect of GRK2 on cell migration to FN involves a PTX-sensitive S1P GPCR. Migration to FN of HeLa cells overexpressing or not wt GRK2 (A, B, D) or MEFs derived from wt or hemizygous GRK2 mice (C) was determined as in Figure 1D in the presence of vehicle or upon cell treatment with pertussis toxin (PTX) (D), SKI (B, C) or VPC23019 (D) as detailed in Materials and methods. In control conditions, the number of HeLa cells migrating to FN was 15±4 (A), 26±4 (B) and 44±2 (D), whereas that of wt MEFs was 123±22 (C) cells/field (means±s.d.). (E) Increased GRK2 expression enhances the migratory response of HeLa cells upon direct challenge of Gi-coupled S1P receptors. Migration of cells treated or not with PTX was assessed towards 1 μM S1P as above. In all panels, data are the mean±s.e.m. of four independent experiments performed in duplicate. *P<0.05, **P<0.01, compared to untreated control cells; †P<0.05, comparison between vehicle and inhibitor-treated HeLa cells stably expressing wt GRK2. (F, G) COS7 cells with or without extra GRK2 were kept in suspension for 1 h in the presence of serum and allowed to adhere on FN-coated (10 μg/ml) or collagen IV-coated (10 μg/ml) coverslips for the indicated times. Adhered cells were fixed, permeabilized and stained with an anti-S1P1 polyclonal antibody to analyse receptor subcellular localization by confocal fluorescence microscopy (G). Despite showing an unspecific nuclear staining, anti-S1P1 antibody clearly labels plasma membrane and intracellular vesicles. Cells positive for membrane receptor presence were quantified as detailed in Supplementary data (F). Data are the mean±s.e.m. of three independent experiments. *P<0.05, compared to collagen-adhered cells. Scale bars, 10 μm. Download figure Download PowerPoint Interestingly, pharmacological inhibition of SK abolished the effect of increased GRK2 expression on migration to fibronectin in HeLa cells (Figure 2B) and COS7 cells (Supplementary Figure S3) as well as impaired motility in wt and GRK2+/− MEFs (Figure 2C). The presence of a selective S1P1/S1P3 receptor antagonist decreased the migration rate of HeLa cells stably expressing GRK2 (Figure 2D), thus ruling out receptor-independent actions of S1P. Treatment with PTX decreased migration in both GRK2-expressing and control cells, indicating that specificity of receptor coupling is not altered by GRK2 overexpression (Figure 2E). Moreover, exogenous S1P reversed the effects of the SK inhibitor SKI on cell migration towards fibronectin in HeLa and COS7 cells with or without extra GRK2 (Supplementary Figure S3A and B), further supporting the paracrine/autocrine actions of S1P. We next used endogenous S1P1 receptor internalization as a read-out of physiologically relevant S1P production leading to S1P receptor transactivation. We found that in the presence of fibronectin (but not collagen), a clear S1P1 receptor internalization takes place in wt or GRK2-overexpressing HeLa cells, being slightly increased in the latter (Figure 2F and G). Direct determination of S1P levels indicated increased autocrine production of this messenger in cells stably expressing GRK2 (0.96±0.13 μM in conditioned medium and 9.61±2.2 μM in the cytosolic fraction) compared to wt HeLa cells (0.56±0.21 and 5.46±1.3 μM, respectively). Overall, these results suggested that the pro-migratory effects of GRK2 in epithelial cells involved SK activation and paracrine/autocrine actions of S1P signalling through Gi-coupled receptors. Confirming such functional interplay, cells stably expressing GRK2 or the GRK2-K220R mutant displayed higher migration rates in response to exogenously added S1P than control cells (Figure 2E and Supplementary Figure S3C), whereas S1P-induced migration was significantly reduced upon knockdown of endogenous GRK2 in HeLa cells (Supplementary Figure S3D) or in MEFs from GRK2+/− mice compared to wt cells (data not shown). Overall, our data suggested that fibronectin triggers an autocrine loop involving S1P receptors to potentiate epithelial cell migration, and that this component is positively modulated by GRK2 protein levels. GRK2 facilitates activation of the ERK pathway upon direct or integrin-mediated S1P receptor stimulation To define the role of GRK2 downstream S1P receptor signalling, we analysed the impact of altering GRK2 levels on S1P-dependent ERK1/2 activation. Increased GRK2 expression in HeLa cells promoted an enhanced (≈12-fold versus ≈8-fold) ERK1/2 peak stimulation, followed by a similar decay to baseline levels (Figure 3A). A similar effect was noted in COS7 cells with extra GRK2 or GRK2-K220R levels (Supplementary Figure S4). ERK1/2 stimulation involved Gi-coupled S1P receptors (Figure 3A, bottom panel) as in S1P-mediated migration. Consistent with the notion that GRK2 levels positively modulate S1P receptor-dependent ERK1/2 activation, decreased stimulation of this pathway by S1P was observed in MEFs from GRK2+/− mice compared to wt animals (Figure 3B). Figure 3.GRK2 expression levels modulate S1P-triggered or adhesion-dependent stimulation of ERK1/2. (A) Increased GRK2 expression promotes a more rapid and potent activation of ERK in response to S1P. The indicated cells were serum-starved for 6 h and challenged with 1 μM S1P for the indicated times. ERK1/2 activation was determined in cell lysates by using an anti-phospho-ERK1/2 antibody (P-ERK). The immunoblot was then stripped and the total cellular ERK1/2 was detected with specific antibodies. Phospho-ERK2 band densities were normalized to cognate total ERK2 densities. Preincubation with PTX markedly inhibits ERK activation. A representative blot is shown. (B) ERK1/2 activation is reduced in cells displaying lower levels of GRK2 protein. MEFs derived from GRK2+/+ or GRK2+/− mice were starved for 12 h in 0.1% serum, stimulated with 1 μM S1P for the indicated times and ERK1/2 activation was assessed as in (A). (C–E) GRK2 levels modulate ERK1/2 activity upon adhesion in a Gi protein-dependent and S1P-mediated manner. HeLa cells stably overexpressing or not wt GRK2 (C, E) or MEFs from GRK2+/+ or GRK2+/− mice (D) with or without PTX or SKI (E) were serum-starved and kept in suspension (S) for 2 h before adhesion on 10 μg/ml FN-coated plates for different periods of time. ERK stimulation was assessed as in (A). In all panels, data are the mean±s.e.m. of 3–4 independent experiments. *P<0.05, **P<0.01, ***P<0.001, compared to control cells or to vehicle-treated cells at each time point of adhesion (E). In (D), the normalized fold stimulation of ERK2 activity versus basal (suspension) conditions is indicated in the representative blot. Download figure Download PowerPoint Similarly, increased GRK2 expression results in a stronger stimulation of ERK1/2 upon integrin-mediated adhesion (Figure 3C), whereas a more rapid attenuation of ERK stimulation was detected in GRK2+/− compared to wt MEFs in these conditions (Figure 3D). Consistently, knockdown of endogenous GRK2 in HeLa cells also leads to decreased fibronectin- or S1P-mediated ERK stimulation (data not shown). Moreover, the effect of GRK2 on ERK1/2 activation during adhesion was attenuated in the presence of either PTX or an SK1 inhibitor (Figure 3E), thus indicating cooperation between S1P and integrin receptors in this signalling pathway. We next explored whether β-arrestins are involved in the positive effects of GRK2 on ERK1/2 activation and cell migration by using MEFs lacking β-arrestin-1 and -2 expression (βarr1/2-KO MEFs). S1P promoted a robust increase in ERK signalling in these cells, and migration to fibronectin was markedly increased (2.5-fold) in knockout MEFs compared to controls (Supplementary Figure S4A and B). Interestingly, we had previously reported that GRK2 levels are higher in βarr1/2-KO MEFs compared to control MEFs (Salcedo et al, 2006), which could promote enhanced migration and S1P/ERK signalling. Moreover, stably increasing GRK2 levels in β-arrestin KO MEFs further enhanced migration towards fibronectin (Supplementary Figure S4C), thus stressing a β-arrestin-independent effect of GRK2 on cell motility. In keeping with this, GRK2-K220R, a mutant that does not trigger β-arrestin recruitment to the receptor complex, potentiates S1P-induced ERK1/2 stimulation as efficiently as wt GRK2 (Supplementary Figure S4D). Phosphorylation of GRK2 on tyrosine and serine residues regulates ERK signalling and cell motility in response to S1P and fibronectin Interestingly, the effect of GRK2 on fibronectin-directed epithelial cell migration was clearly decreased by pharmacological inhibition of the MEK/ERK pathway or the activity of c-Src or PI3K, but did not involve Akt activation (Supplementary Figures S2 and S5). We have previously reported that GRK2 itself can be phosphorylated by c-Src upon GPCR activation (Ribas et al, 2007), which modulates its activity and interaction with other molecules (Mariggio et al, 2006), whereas phosphorylation of serine 670 by ERK1/2 impairs Gβγ-dependent catalytic activity (Penela et al, 2003). Interestingly, cell adhesion and S1P challenge were able to promote such phosphorylation events (Supplementary Figure S6). Remarkably, GRK2 phosphorylation at S670 was preceded by an increase in GRK2 phosphotyrosine levels and by maximal ERK1/2 pathway activity (see Figure 3) either upon S1P stimulation or cellular adhesion, consistent with a functional link between these processes. Next, we addressed whether an adequate timing of tyrosine and serine phosphorylation of GRK2 might influence cellular responses to either S1P or fibronectin, by using specific GRK2 phosphorylation mutants. Mimicking 'permanent' tyrosine phosphorylation of GRK2 (GRK2-Y2D mutant) did not cause an increase in cell migration observed with the transient expression of either wt GRK2 or a mutant with decreased phosphorylation by c-Src (Figure 4A). On the other hand, HeLa cells stably expressing a GRK2 mutant unable to be phosphorylated at S670 (GRK2-S670A) displayed a markedly decreased migration towards fibronectin (Figure 4B). Interestingly, the presence of a GRK2 mutant that would mimic permanent phosphorylation at this site, GRK2-S670D, was unable to enhance motility of HeLa cells as effectively as wt GRK2. A similar trend was observed in the migratory responses to S1P challenge (Figure 4C). The effect of GRK2 mutants on motility was not caused by an unspecific impairment in cellular locomotion, as cell migration towards collagen was unaltered in the presence of such mutants (Figure 4B). Moreover, blockade of migration by expression of GRK2-S670A does not result from an altered cell attachment (Supplementary Figure S7). Figure 4.Phosphorylation of GRK2 at defined tyrosine or serine residues regulates migration and ERK1/2 signalling in response to S1P challenge and FN-mediated adhesion. (A–C) HeLa cells transiently transfected with either wt GRK2 or the tyrosine phoshorylation mutants GRK2-Y13,86,92F or GRK2-Y86,92D (A) or stably overexpressing similar levels (B, inset blot) of wt GRK2 or the GRK2-S670A or GRK2-S670D mutants (B, C) were subjected to migration assays towards 20 μg/ml FN (A, B), 10 μg/ml collagen fraction IV (B) or 1 μM S1P (C), as detailed in Materials and methods. Data are the mean±s.e.m. of 2–4 independent experiments performed in duplicate. *P<0.05, **P<0.01, compared to control HeLa cells; †P<0.05, compared to cells transfected with GRK2-Y13,86,92F. Control cells migrating towards fibronectin were 21±1 (A), 33±10 (B) or 23±1 (C) cells/field (mean±s.d.), and towards collagen 21±4 (B). (D, E) Expression of GRK2-S670A completely abrogates ERK1/2 activation in response to S1P and upon adhesion to fibronectin. HeLa cells or cells stably expressing wt GRK2, GRK2-S670A or GRK2-S670D were serum-starved and challenged with 1 μM S1P (D) or kept in suspension (S) before adhesion to 10 μg/ml FN (E) for the indicated times. ERK activation was determined as in Figure 3. Representative blots from four independent experiments are shown, and fold stimulation of ERK2 activity normalized by total ERK2 is included in (D). Download figure Download PowerPoint These results indicate that timely and dynamic regulation of serine and tyrosine phosphorylation of GRK2 was strictly required, in a step following integrin engagement, for proper migration in response to S1P and fibronectin. As the extent and duration of ERK1/2 activation is instrumental in cell motility (Brahmbhatt and Klemke, 2003), we explored whether GRK2 phosphorylation status could modulate agonist-mediated stimulation of the ERK1/2 pathway. Expression of GRK2-S670A completely abrogated ERK1/2 activation in response to S1P where that of GRK2-S670D led to a pattern of stimulation similar to that observed in control HeLa cells expressing endogenous levels of GRK2 (Figure 4D, see quantified blots for comparison), in line with the fact that these cells migrate similar to HeLa parental cells in response to S1P and fibronectin. Activation of ERK1/2 during adhesion to fibronectin was also severely inhibited in the presence of GRK2-S670A (Figure 4E), thus stressing the notion that GRK2 modulates cell motility by means of regulating ERK activation. Signalling integration between adhesion and the ERK pathway involves integrin-induced phosphorylation of MEK1 at S298 by PAK1, which enhances both phosphorylation of MEK1 by Raf and MEK1–ERK interaction, thus leading to efficient ERK activation (Slack-Davis et al, 2003; Edin and Juliano, 2005). Interestingly, S1P triggered a more potent increase of Raf-dependent phosphorylation of MEK1 in cells overexpressing wt GRK2 (or GRK2-S670D) as compared to control cells (Figure 5A), which correlates with the higher activation of ERK observed in HeLa-GRK2 wt cells (Figure 3). In contrast, the presence of GRK2-S670A completely abrogated MEK1 activation by S1P (Figure 5A). A similar trend in MEK1 activation was observed upon adhesion to fibronectin in cells expressing wt GRK2 or the S670A mutant (Figure 5B). Moreover, PAK-mediated MEK1 phosphorylation at S298 was markedly impaired in the presence of such GRK2 mutant in S1P-stimulated adherent cells (Figure 5C) and upon adhesion to fibronectin (Figure 5D). Figure 5.Phosphorylation of GRK2 at S670 is required for triggering Rac/PAK1/MEK1 activation in response to S1P or fibronectin. HeLa cells with or without extra wt GRK2 or GRK2-S670A were serum-starved and stimulated with 1 μM S1P (A, C) or kept in suspension for 2 h (S) and then adhered to FN-coated plates (B, D, E) for the indicated times. The phosphoryl
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