1 The Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208, USA
2 Department of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
* Author for correspondence (e-mail: laflams{at}mail.amc.edu)
Accepted 21 August 2002
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Summary |
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Key words: Rac, Integrin, ß cytoplasmic domain
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Introduction |
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Integrins regulate the activities of RhoA, Rac1 and Cdc42 (Ren et al., 1999; Arthur et al., 2000
; Danen et al., 2000
; del Pozo et al., 2000
; Etienne-Manneville and Hall, 2001
; Price et al., 1998
). These small GTP-binding proteins in turn regulate cell adhesion and changes in cell morphology by triggering dynamic changes in the actin cytoskeleton (Kjoller and Hall, 1999
). Integrin activation of Rac1 and Cdc42 signaling induces the formation of lamellapodia and filopodia, which are necessary for cell spreading (Kjoller and Hall, 1999
). The disruption of Cdc42 or Rac1 signaling by the expression of the dominant negative proteins N17Cdc42 or N17 Rac1 inhibits the spreading process (Berrier et al., 2000
; Clark et al., 1998
; Price et al., 1998
). Interestingly, the regulation of RhoA by integrins during cell adhesion is biphasic (Ren et al., 1999
). Initially, integrin signaling triggers a transient inhibition of RhoA activity upon cell attachment to promote cell spreading (Arthur and Burridge, 2001
; Ren et al., 1999
). RhoA is subsequently re-activated (Ren et al., 1999
), inducing actinomyosin contractility, the formation of stress fibers, and focal adhesions, which are important steps in the adhesion process following cell spreading (Chrzanowska-Wodnicka and Burridge, 1996
).
The mechanisms linking integrins to the regulation of Rho family GTPases are not fully defined. However, integrin-mediated activation of tyrosine kinases may play a central role. For many integrins, intact integrin ß tails are required to trigger increases in tyrosine phosphorylation of FAK, Cas and paxillin following cell adhesion (Guan et al., 1991; Schaffner-Reckinger et al., 1998
; Wennerberg et al., 1998
; Wennerberg et al., 2000
), suggesting important roles for integrin ß tails in this process. Additionally, clustering isolated integrin ß1 or ß3 tails induces the tyrosine phosphorylation of these same proteins indicating that interactions with ß tails are sufficient to trigger increases in tyrosine phosphorylation of FAK, Cas and paxillin (Akiyama et al., 1994
; Bodeau et al., 2001
; Tahiliani et al., 1997
). Since the activation of tyrosine phosphorylation provides a pathway linking integrin engagement to many downstream signaling events (Cary and Guan, 1999
; Parsons et al., 2000
; Pawson and Scott, 1997
), clustering integrin ß tail-associated proteins may also be sufficient to initiate other signaling events, such as the regulation of Rho family GTPases.
In addition to playing an important role in the activation of tyrosine kinase signaling, integrin ß tails also play a central role in regulating cell spreading. Previous studies by others demonstrated that integrin ß tails are required for integrins to promote cell spreading (Ylanne et al., 1993). We showed that the expression of tac-ß tail chimeras can inhibit cell spreading, indicating that protein interactions mediated by ß tails can regulate the spreading process (Bodeau et al., 2001
; LaFlamme et al., 1994
). More recently, we demonstrated that integrin ß tails are required downstream of Rac1 signaling in cell spreading, and that constitutively active Rac1 can restore cell spreading inhibited by tac ß tail chimeras, suggesting that ß tails may also be involved in Rac1 activation (Berrier et al., 2000
). In our current study, we examine the role of integrin ß tails in regulating Rac1 activation, and demonstrate that integrin ß tails are both required and sufficient for integrins to signal increases in Rac1 GTP-loading.
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Materials and Methods |
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Assay for Rac1-GTP loading
In general, cells were harvested with trypsin and allowed to recover for 2.5 hours at 37°C in serum-free medium. Cells were then kept in suspension or either allowed to adhere to fibrinogen (Enzyme Research Laboratories), collagen I (Vitrogen 100, Cohesion), or fibronectin (kindly provided by Jane Sottile). Non-adherent cells were removed from the coated dishes with two washes with cold PBS. The amount of Rac1-GTP was assayed in cell lysates as described previously (Benard et al., 1999). In brief, a fraction of the lysate was incubated with glutathione-agarose beads coated with bacterially expressed GST-PAK [PAK amino acid residues 67-150 (Benard et al., 1999
)]. The levels of Rac1 in the GST pull-down assay were analyzed by western blot with monoclonal antibody, clone 102 to Rac1 (BD transduction laboratories) and sheep anti-mouse horseradish peroxidase conjugated secondary antibody (Amersham Pharmacia Biotech). The bands were visualized with ECL enhanced chemiluminescence (Amersham Pharmacia Biotech). Quantitation was performed by densitometric scanning of the autoradiogram (Molecular Dynamics Scanner and ImageQuant software, Amersham Pharmacia Biotech). Several different autoradiogram exposures were used to quantitate each experiment. In each case, the levels of Rac1 GTP-loading in cells kept in suspension were normalized to 1. Western blot analysis of one-fiftieth of the cell lysates was performed to correct for the lysate levels of Rac1. Statistical analysis of the data was performed using the Kruskall Wallis one way analysis of variance on rank. Differences between time points were determined by Dunnett's method using SigmaStat software (Jandel Corp.). Significance was set at P<0.05.
Clustering the integrin ß tail
Transiently transfected fibroblasts were harvested with trypsin and allowed to recover for 2.5 hours at 37°C in serum-free medium. Purified 7G7B6 mouse monoclonal antibody to the tac epitope (Upstate Biotechnology) was used to coat magnetic beads conjugated with goat anti-mouse IgG (Polysciences). Chimeric receptors were clustered on the cell surface by incubating the transfected cells with antibody-coated magnetic beads in serum-free medium (Akiyama et al., 1994; Tahiliani et al., 1997
). Prior to the clustering assay, flow cytometry of a fraction of the transfected cells was performed in order to ensure that similar numbers of tac-, tac-ß1-, or tac-ß3-expressing cells were incubated with the magnetic beads. Tac-expressing cells were magnetically sorted and the levels of Rac1-GTP were assayed in lysates of the positively sorted cells as described previously (Benard et al., 1999
). The levels of Rac1-GTP in the control tac-receptor-expressing cells were normalized to 1. Since measuring endogenous Rac1 activity is unmanageable for more than one tac construct per trial due to the large numbers of transiently transfected cells needed for the analysis of Rac1 GTP-loading, we measured the activity of cotransfected Rac1.
Flow cytometry
Cells were harvested, immunostained and fluorescence intensity was analyzed on individual cells using a FACScan flow cytometer (Becton Dickinson) and Cellquest software (Becton Dickinson) as described previously (Mastrangelo et al., 1999). Surface expression of the
llbß3 integrin receptor was detected using the FITC-conjugated anti-CD41 monoclonal antibody, clone P2 (Immunotech). The surface expression of the transfected tac and tac-ß tail chimeras was detected using a FITC-conjugated anti-CD25 monoclonal antibody (Becton Dickinson). The expression of myc-tagged Rac1 was detected in cells permeabilized with Cytofix/Cytoperm (Pharmingen) using a rabbit polyclonal anti-myc antibody (Santa Cruz Biotechnology) and a goat anti-rabbit phycoerythrin conjugated secondary antibody (Molecular Probes).
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Results |
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Clustering an isolated integrin ß1 or ß3 tail is sufficient to enhance Rac1 activation
To further our understanding of how integrin receptors regulate Rac1 activity, the mechanisms involved in the requirement for integrin ß tails were examined. Clustering integrin ß tails in normal fibroblasts is sufficient to activate protein tyrosine phosphorylation and may lead to the formation of signaling complexes through recruitment of SH2-domain containing signaling and adaptor proteins (Bodeau et al., 2001; Pawson and Scott, 1997
). We reasoned that clustering isolated integrin ß tails could trigger Rac1 activation, if integrin ß tail-associated complexes are sufficient to signal to Rac1. Alternatively, it is possible that integrin ß tails play only a permissive role, and that additional pathways are required for Rac1 activation. To distinguish between these possibilities, we tested whether clustering tac-ß-tail chimeras could trigger increases in Rac1 GTP-loading. Since we have previously clustered ß tail chimeras to induce protein tyrosine phosphorylation in transiently transfected primary human fibroblasts, we chose to perform our assay for Rac activation in this cell type. Additionally, cells were co-transfected with myc-tagged, wild-type Rac1 to facilitate detection of Rac1 by western blot in the clustering assay.
Integrin-mediated cell adhesion of several fibroblast cell lines has been shown to regulate Rac1 activity (Danen et al., 2000; del Pozo et al., 2000
; Arthur et al., 2000
; Price et al., 1998
). To confirm that integrin-mediated adhesion of primary human fibroblasts induced Rac1 activation, Rac1 GTP-loading of endogenous Rac1 was assayed following cell adhesion to collagen I. The levels of Rac1 GTP-loading increased on average 2.1-fold in primary fibroblasts adhered to collagen I for 15 minutes when compared to the levels in suspended cells (Fig. 3A,B), demonstrating that integrins can mediate the activatino of Rac1 in primary human fibroblasts. We also analyzed the effect of cell adhesion on the activity of transiently expressed recombinant wild-type Rac1. The activity of the myc-tagged wild-type Rac1 was enhanced on average 2.5-fold by adhesion to collagen I (Fig. 3C), confirming that transiently expressed myc-tagged Rac1 can also be regulated by cell adhesion. Furthermore, a comparison of the levels of GTP-loading of wild-type Rac1 or constitutively active L61Rac1-transfected cells revealed that overexpression of wild-type myc-tagged Rac1 does not induce constitutive activation of Rac1 in suspended cells (data not shown).
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To test whether integrin ß-tails are sufficient to signal Rac1 activation, human fibroblasts were transiently co-transfected with the myc-tagged wild-type Rac1 and either the control tac receptor or chimeric receptors expressing tac with the intracellular domains of integrin ß1A (tac-ß1) or ß3A (tac-ß3) (LaFlamme et al., 1994). A fraction of the tac, tac-ß1 or tac-ß3 transiently transfected cells were each stained for cell surface expression of the tac epitope and flow cytometry was performed to determine the percentage of cells that express tac, tac-ß1 or tac-ß3 (Fig. 4A). Equal numbers of tac-expressing cells were then incubated with anti-tac antibody-coated magnetic beads to cluster tac on the surface of the tac-, tac-ß1- or tac-ß3-transfected cells while in suspension. The tac-expressing cells were magnetically sorted. The enrichment of tac-epitope-expressing and Rac1 co-transfected cells by this method was confirmed by flow cytometry experiments (Fig. 4B). Lysates from the positively selected cells were prepared and analysis of Rac1 GTP-loading revealed that clustering of tac-ß1 or tac-ß3 for 15 minutes resulted in an average of 4.7 or 4.1-fold increase in Rac1 activity, respectively, in comparison to Rac1 activity induced by clustering the control tac receptor (Fig. 4C,D). These experiments indicate that clustering an isolated integrin ß1 or ß3 tail is sufficient to signal Rac1 activation in human fibroblasts.
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Discussion |
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Our result that clustering isolated ß tails can trigger Rac1 activation indicates that proteins that bind to ß tails can initiate the pathway leading to increases in Rac1 activity. Although we do not know the identity of these ß tail binding protein(s), we have recently shown that clustering isolated ß tails is sufficient to trigger the tyrosine phosphorylation of p130Cas, paxillin and FAK (Akiyama et al., 1994; Bodeau et al., 2001
). The phosphorylation of these proteins provides potential pathways linking ß tails to Rac1 activation. The tyrosine phosphorylation of p130Cas is known to trigger the formation of a Cas/Crk complex with p180Dock, which in turn can activate Rac1 (Kiyokawa et al., 1998a
; Kiyokawa et al., 1998b
). Phosphorylated paxillin can also bind CrkII, suggesting that CrkII may similarly couple paxillin to Rac1 activation (Petit et al., 2000
). Also, integrin-triggered tyrosine phosphorylation of FAK can lead to the activation of PI 3-kinase, and PI 3-kinase signaling is known to participate in Rac1 activation (Chen and Guan, 1994
; Kjoller and Hall, 1999
; Reiske et al., 1999
).
Additional studies are needed to define the molecular pathway linking integrin ß tails with Rac1 activation. If tyrosine phosphorylation is required for ß tails to trigger Rac1 activation, talin is an attractive candidate for a ß tail binding protein responsible for this linkage. Talin binds to both the ß tail and to FAK (Chen et al., 1995; Liu et al., 2000
), and thus could nucleate the assembly of signaling complexes leading to FAK activation, a cascade of tyrosine phosphorylation and the subsequent activation of Rac1. Mutations in regions of the ß tail implicated in talin binding inhibit the induction of tyrosine phosphorylation by ß tails (Bodeau et al., 2001
; Tahiliani et al., 1997
), supporting a role for talin in the activation of tyrosine phosphorylation by integrins. However, there are other potential pathways linking ß tails with the regulation of Rac1. ILK is also a ß tail binding protein that could initiate signaling to Rac1. ILK can bind to paxillin linking integrin ß tails to Rac1 through the formation of an ILK/paxillin/p95PKL/PIX complex. However, it is important to note that although PIX is a Rac1 GEF, the effect of the formation of this complex on PIX exchange activity has not yet been determined. ILK can also bind to PINCH and could link integrin ß tails with Rac1 through the formation of an ILK/PINCH/Nck2/Dock180 complex, since Dock180 can trigger Rac1 activation (Tu et al., 2001
; Turner, 2000
; Wu and Dedhar, 2001
; Kiyokawa et al., 1998a
). However, the role of integrin-mediated adhesion or tyrosine kinase signaling in the formation of these complexes is also not yet clear. Future studies will focus on identifying the components linking ß tails with Rac1 and the mechanisms of their action.
After the completion of this work and while this paper was in preparation, Hirsch and colleagues (Hirsch et al., 2002) published that adhesion-triggered Rac1 activation is inhibited in cells expressing ß1 integrins containing mutant ß tails. Our results are in agreement with these findings since we demonstrate that truncation of ß tails on the integrins mediating adhesion inhibits the integrin-activation of Rac1. We extend these results by demonstrating that integrin ß tails are sufficient to trigger increases in Rac1 GTP-loading. Our results indicating that wild-type
IIbß3 expressed in CHO cells can activate Rac1, however, differ from those published recently by Miao and colleagues (Miao et al., 2002
). These authors conclude that ß1 but not ß3 integrins can activate Rac1 upon adhesion to fibronectin. We demonstrate that
IIbß3-mediated adhesion to fibrinogen and
5ß1-mediated adhesion to fibronectin can both trigger Rac1 activation, indicating that both ß1 and ß3 integrins can regulate Rac1. Our experiments and the studies by Miao and colleagues differ in experimental design and this may account for the observed difference in the ability of ß3 integrins to activate Rac1. We assayed Rac1 activity after 15 minutes of adhesion in contrast to 4 hours used in the assay described by Miao and colleagues. Also, we assayed Rac1 activation after adhesion to fibrinogen, whereas Miao and colleagues measured Rac1 activity following adhesion to fibronectin. Thus, it is possible that integrin transmembrane signaling to Rac1 may differ depending on whether particular ß3 integrins bind to fibronectin or fibrinogen. Clearly, additional studies are needed to fully understand the mechanisms by which integrins regulate Rac1 and how these mechanisms may differ for different integrin heterodimers and different integrin ligand interactions.
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Acknowledgments |
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