Department of Biology, Wesleyan University, Hall-Atwater Labs, Room 257, Lawn Ave, Middletown, CT 06459, USA
*Author for correspondence (e-mail: lgrabel{at}wesleyan.edu)
Accepted March 27, 2001
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SUMMARY |
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Key words: Phosphorylation, ß1 Integrin, Serine, GD25, F9
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INTRODUCTION |
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Two regions of the ß1 cytoplasmic domain appear to be key in mediating interactions with these focal adhesion proteins. The first is a membrane proximal region that has been implicated in FAK and paxillin associations and in stabilizing /ß interactions, presumably by creating a salt bridge between the aspartate residue (D759) and a conserved arginine in the
cytoplasmic tail. The second region contains two highly conserved NPXY domains that are present in all of the ß subunits except ß4 (Filardo et al., 1995; OToole et al., 1995; Reszka et al., 1992). This second region has been implicated in associations with a variety of proteins including
-actinin, FAK, talin and paxillin.
Many protein-protein interactions are modulated by reversible phosphorylation. The ß1 integrin cytoplasmic domain contains five potential phosphorylation sites (Y783, Y795, S785, T788, T789) within a sequence of 16 amino acids (780-795) that includes the two highly conserved NPXY motifs. Several observations suggest that these phosphorylation sites are important for regulating the function of ß1 integrins. First, tyrosine-phosphorylated ß1 in Rous Sarcoma virus-transformed fibroblast cells localizes to podosomes, whereas the unphosphorylated form is found in focal adhesions (Johansson et al., 1994). Focal adhesion targeting is also inhibited by site directed substitutions at S785, Y783 and Y795, when these residues are mutated to mimic specific phosphorylation states (Barreuther and Grabel, 1996; Reszka et al., 1992). Conservative Y to F mutations at Y783 and Y795 result in wild-type levels of adhesion to ECM substrates and these proteins localize to focal adhesion sites but fail to mediate directed cell motility effectively (Sakai et al., 1998). Conservative T to A mutations at threonine residues 788 and 789 produce a ß1 that cannot support fibronectin fibril formation or attachment to fibronectin substrates (Wennerberg et al., 1998). In addition, these T to A mutations result in an inactive, non-ligand-binding form of the extracellular domain, suggesting that phosphorylation at these threonines may regulate inside-out signaling (Wennerberg et al., 1998).
Most of these studies are limited in that they only investigate the functional outcomes of conservative mutations from a residue that can be phosphorylated to one that cannot be phosphorylated. This approach will not detect a loss of function owing to phosphorylation. In other words, if an unphosphorylated residue supports ß1 function and phosphorylation at this site does not, it would be missed. Therefore, in our investigations of the functional significance of S785, we included a site-directed substitution of an amino acid mimicking a constitutively phosphorylated form of the protein, which allows us more fully to reveal the functional significance of phosphorylation at this site.
As F9 cells differentiate into parietal endoderm in the presence of cyclic AMP plus retinoic acid, focal adhesions form and there is a concomitant dephosphorylation of the ß1 cytoplasmic domain. Levels of ß1 phosphorylation in parietal endoderm cells are increased twofold by treatment with the phosphatase inhibitor okadaic acid (Mulrooney et al., 2000). Phosphoamino acid analysis revealed that phosphorylation is on a serine residue and that there is only one serine residue, at amino acid 785 (Dahl and Grabel, 1989). These results led us to hypothesize that the dephosphorylation of serine 785 was required for focal adhesion localization. We tested this by expressing chicken ß1 with site-directed substitutions in F9 cells and determining the subcellular localization of the integrin using an antibody specific for the chicken form of the protein (Barreuther and Grabel, 1996). When transfected F9 cells expressed chicken ß1 with either a serine at position 785 (wildtype) or a methionine at this site, the integrin localized to focal adhesion sites. By contrast, ß1 containing an aspartate at this site, which mimics a constitutively phosphorylated residue, failed to localize to the focal adhesion sites (Barreuther and Grabel, 1996). In this study, we were restricted from more completely examining the role of S785 in ß1 function by the presence of wild-type endogenous ß1. We now report two approaches that allow us to examine the role of S785 in multiple ß1-supported functions. First, we have used a mouse-specific ß1-function-blocking antibody to examine a number of ß1-dependent behaviors in F9 cell lines expressing various forms of chicken ß1. Second, we have expressed these mutant chicken ß1 constructs in the ß1-deficient mouse fibroblast cell line GD25. The data suggest that phosphorylation at S785 promotes adhesion but inhibits cell spreading and directed cell migration in both F9 and GD25 cells.
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MATERIALS AND METHODS |
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Transfection of GD25 with chicken ß1
Three RSV-neo constructs encoding the sequence of chicken ß1, (WT, S790M, S790D) were obtained from A. Horwitz. The ß1 sequence was excised with EcoRI and XhoI and ligated into the same sites of the multiple cloning region of pCDNA3.1zeo (Invitrogen). The constructs were linearized and electroporated as previously described (Wennerberg et al., 1996) into GD25 fibroblast cells. Cells were selected for two weeks in the presence of 500 µg ml-1 Zeocin (Invitrogen).
FACS analysis
Zeocin resistant clones were removed from the dishes using calcium and magnesium free Hepes-Hanks Buffer (CMF-HH) supplemented with 0.02% EDTA, collected and resuspended in 500 µl CMF-HH containing 2% bovine serum albumin (BSA) (blocking buffer; BB). 20 µg of W1B10 (chicken ß1 specific antibody) was added and incubated for 30 minutes at 37°C. Cells were centrifuged, washed in BB three times, resuspended in BB containing FITC-conjugated goat anti-mouse IgG (1:100) (Cappel Laboratories) and incubated for 30 minutes at 37°C. Cells were washed twice in BB and three times in CMF-HH, and resuspended in CMF-HH. Cells were then analyzed and sorted in a Becton-Dickson FACScan (Yale Flow Cytometry Center). Resulting cell populations were maintained in culture medium supplemented with 500 µg ml-1 Zeocin.
Immunoprecipitation
GD25 transfected and untransfected cells were washed in cold phosphate buffered saline (PBS) and the cell surface proteins labeled with 0.25 mg ml-1 Sulfo-NHS-biotin (Pierce) for 40 minutes at room temperature. Cells were then washed once with serum free DMEM and three times with PBS. They were then lysed in buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 1 mM CaCl2, 0.5% sodium deoxycholate, 1% NP-40 and 2 mM PMSF in the presence of a protease inhibitor cocktail (10 µg ml-1 leupeptin, 10 µg ml-1 pepstatin, 10 µg ml-1 aprotinin, 10 µg ml-1 benzamidine) for 1 hour on ice. Lysates were precleared by centrifugation at 13,000g for 15 minutes. Samples were then incubated with 10 µg of anti-chicken ß1 antibody (W1B10) or 10 µg of anti-mouse 6 antibody (GOH3) overnight at 4°C. The samples were then incubated with 50 µl protein G beads for 1 hour at 4°C with constant agitation. The bound material was then washed three times with precipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM CaCl2, 1% NP-40) and once with 1 M Tris-buffered saline pH 7.4 (TBS). The unbound fraction (resulting supernatant) was reserved for use as a loading control. Samples were solubilized in 2x Laemmli buffer, boiled for 5 minutes and the proteins resolved by SDS -PAGE. Proteins resolved by SDS-PAGE were electrophoretically transferred to Immobilon-P (Millipore) and the membranes blocked for 1 hour at room temperature with TBS containing 0.03% Tween-20 (TBST) and 3% dry milk. The blots were incubated for 1 hour at room temperature with HRP-conjugated strepavidin diluted in TBST (1:1000). For the loading control, blots were incubated with mouse anti-
-fodrin (spectrin), using manufacturers specifications, for one hour at room temperature followed by five washes with TBST and then a 1 hour incubation with anti-mouse IgG-HRP (1:10,000) at room temperature. Blots were then processed by enhanced chemiluminescence, and visualized on Kodak XAR film.
Immunofluorescence
GD25 transfected cells were fixed for 10 minutes in 3.7% formaldehyde and then washed twice with PBS and permeabilized with 0.5% Triton-X100. Cells were then washed twice in PBS and blocked for 30 minutes at 37°C in PBS supplemented with 2% BSA. Cells were then washed with PBS/BSA and incubated for 1 hour at 37°C with 20 µg ml-1 W1B10 and 20 µg ml-1 rabbit anti-vinculin. Cells were then washed five times in PBS for 5 minutes. Cells were then incubated with FITC-conjugated goat anti-mouse IgG (1:100) or rhodamine-conjugated goat anti rabbit IgG (1:100) for 1 hour at room temperature followed by five 5-minute washes with PBS. Cells were washed, mounted and examined using a Nikon fluorescence microscope and images captured using a SPOT digital camera and Adobe Photoshop software.
Cell attachment assay
96-well tissue culture plates were coated with 30 µg ml-1 laminin overnight at room temperature. Control wells were coated with 2% BSA. The plates were then washed with PBS and blocked for 2 hours with 2% BSA at 37°C. Plates were then washed and then cells were added and allowed to attach for 1 hour as previously described (Stephens et al., 1993). After washing the wells the extent of attachment was determined by colorimetric detection at 595 nm using Bradford Assay Reagent (Pierce). For those experiments involving F9 cells, endogenous ß1 function was inhibited by incubating the cells with the function-blocking antibody CD29 HA2/5 (Research Diagnostics) for 1 hour prior to seeding. The level of attachment of GD25 transfected cells was determined by subtracting the residual attachment to BSA from attachment to laminin. All samples were analyzed in quadruplicate.
Cell spreading assay
24-well tissue culture plates were coated with 30 µg ml-1 laminin-1 overnight at room temperature. Equal quantities of cells were seeded and the cells allowed to attach and spread for 4-6 hours. Cells were then washed with PBS and photographed. For the F9 spreading assays, cells were allowed to attach for 1 hour before 20 µg ml-1 CD29 HA2/5 was added to the media. All samples were analyzed in triplicate.
GD25 cell migration
Cell migration assays were performed as previously described (Sakai et al., 1998). Briefly, assays were performed in modified Boyden chambers containing Nucleopore polycarbonate membranes (5 µm pore size, Costar). Filters were coated overnight in 30 µg ml-1 laminin. The filters were rinsed with PBS supplemented with 2% BSA and placed into the chambers. 500 µl PBS containing 2% BSA and 100 µM epidermal growth factor (EGF) (Upstate Biotechnology) was added to the chamber. Cells were suspended in PBS with 2% BSA and added to the upper chamber and then incubated overnight. The cells on the upper side of the filter were removed with a cotton swab to ensure that only those cells that had passed through the membrane were counted, and the filters were then washed with PBS and stained with Crystal Violet. Cells on the underside of the filter were then counted. All samples were analyzed in quadruplicate.
F9 cell migration and parietal endoderm outgrowth assays
F9 parietal endoderm outgrowth assays were performed as previously described (Jiang and Grabel, 1995). Briefly, F9 cells were kept in suspension culture in the presence of 0.1 µM retinoic acid until mature visceral endoderm was present on the outer edge (9-11 days). Mature embryoid bodies were then plated on laminin-coated 33 mm tissue culture dishes and allowed to attach for 16 hours. After attachment, 20 µg ml-1 CD29 HA2/5 was added to the cultures and the outgrowth photographed after 5 hours and 20 hours. The migration distance of the outgrowth for each embryoid body was measured in ten locations and the average migration distance used as one data point. A minimum of ten embryoid bodies from each cell line were used to quantify migration of parietal endoderm.
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RESULTS |
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Role of S785 in cell attachment of F9 and GD25 cell lines
To address the functional role of the different chicken ß1 proteins in F9 stem cells, the contribution of the endogenous mouse ß1 was inhibited by the addition of CD29-HA2/5, a function-blocking antibody specific for mouse ß1. In each experiment, the ability to attach to laminin was determined for cells expressing the different forms of the chicken ß1 protein. (The role of the ß1 subunit was assayed using laminin substrates, because attachment to laminin relies heavily if not totally upon the ß1-containing integrin 6ß1 (Jiang and Grabel, 1995; Wennerberg et al., 1996).) All F9 cell lines attached well to laminin in the absence of antibody (Fig. 2). Increasing concentrations of the CD29 HA2/5 antibody progressively inhibited attachment of untransfected F9 cells to laminin because the antibody selectively blocks the endogenous mouse ß1. By contrast, F9-S785S cells demonstrate residual attachment in the presence of the antibody owing to expression of the chicken protein. F9-S785D cells can also attach to laminin in the presence of the antibody but F9-S785M cells cannot. Attachment of F9-S785S cells to laminin can be completely inhibited if the cells are incubated with both the mouse-specific CD29 HA2/5 and the chicken-ß1-specific W1B10 (data not shown). This supports our conclusion that residual attachment observed in the presence of the antibody is due to the chicken ß1. These experiments were repeated with F9-derived parietal endoderm from all cell lines and the data are consistent with those presented for stem cells in Fig. 2A (data not shown). These data suggest that mutations mimicking the dephosphorylated form of chicken ß1 interfere with attachment to laminin.
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Role of serine 785 in cell spreading
For cells to spread on an ECM substrate, the integrin ß subunit must bind the ECM as well as engage the actin-based cytoskeleton. To facilitate an examination of spreading and not attachment behavior, F9 cells were allowed to attach for 1 hour prior to addition of the antibody. In the absence of the antibody, all the F9 cells spread on laminin. We inhibited cell spreading of untransfected F9 cells with the addition of the CD29 HA2/5 antibody (Fig. 3A). Both the F9-S785S and F9-S785M cell lines had residual spreading activity in the presence of the antibody. Although expression of an aspartate at 785 allowed cells to attach (Fig. 2), these cells failed to spread and retained a rounded morphology in the presence of CD29HA2/5, as observed for the untransfected F9 cells (Fig. 3A). Spreading of F9-S785 cells could be completely inhibited by the addition of both CD29 HA2/5 and W1B10 antibodies (Fig. 3B). Similar results were obtained when these experiments were repeated using F9 parietal endoderm (data not shown).
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Role of serine phosphorylation in F9 cell migration
To determine the role of serine phosphorylation in F9 cell migration, we used parietal endoderm outgrowth assays. Previous work in our laboratory has shown that migration on laminin is mediated by the 6ß1 integrin (Jiang and Grabel, 1995). When mature embryoid bodies are plated on laminin in the presence of an antibody directed against
6ß1 (GOH3), the extent of outgrowth is inhibited (Jiang and Grabel, 1995). To determine the effects of expressing ß1 with different residues at position 785, we performed these outgrowth assays on laminin substrates in the presence of the ß1 function-blocking antibody, CD29 HA2/5, using the transfected cell lines. Note that all the F9 cell lines produce outgrowth in the absence of the function blocking antibody (Fig. 4A). Embryoid bodies derived from untransfected controls as well as F9-S785D failed to produce substantial parietal endoderm outgrowth in the presence of the antibody. However, embryoid bodies from both F9-S785S and F9-S785M cells produced extensive outgrowth. We calculated the average distance of outgrowth migration at ten points for ten embryoid bodies from each cell line (see Methods) (Fig. 4B). In the presence of the antibody, embryoid bodies derived from both the F9-S785S and F9-S785M display somewhat decreased migration distances than their untreated controls, although the difference is not statistically significant. By contrast, the migration of parietal endoderm from F9-S785D derived embryoid bodies is inhibited by 66% in the presence of the antibody. In further support for a role for ß1 phosphorylation in parietal endoderm migration, we have shown that parietal endoderm from untransfected F9 embryoid bodies show a marked decrease in migration distance when cultured in the presence of okadaic acid, a treatment that promotes a twofold increase in ß1 phosphorylation levels (Barreuther and Grabel, 1996) (data not shown).
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Localization of chicken ß1 with site substituted mutations
We have previously shown that ß1 from F9-S785D cells failed to localize to focal adhesion sites, whereas ß1 containing a serine or methionine residue at this site was able to localize to the focal adhesion (Barreuther and Grabel, 1996). We have extended those studies to determine whether these amino acid substitutions have the same effect on ß1 localization in GD25 cells. We examined the localization of the chicken ß1 protein in the transfected GD25 cell lines using indirect immunofluorescence (Fig. 5). To facilitate focal adhesion formation by all the cell lines, cells were plated on fibronectin because untransfected GD25 cells can assemble these structures on fibronectin using other integrins, such as Vß3 (Fassler et al., 1995; Wennerberg et al., 1996). The presence of ß1 containing focal adhesions was assayed by double labeling for vinculin and ß1. As previously reported for the transfected F9 cell lines, we show that the chicken ß1 with an aspartate substitution fails to localize to the focal adhesion, whereas wildtype or methionine-substituted ß1 colocalizes with vinculin to focal adhesions in these cells. Taken together with our previous F9 work, these data suggest dephosphorylation at S785 is important for the placement of ß1 in focal adhesion sites.
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DISCUSSION |
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The results from our attachment and spreading assays were unexpected, in that distinct phenotypes resulted from the same mutation. Expression of an aspartate at 785, mimicking a phosphorylated residue, results in attachment levels similar to or better than wild-type S785 in both the F9 and GD25 cell lines, whereas the same mutation was unable to support spreading or migration. By contrast, expressing a methionine at this site results in reduced attachment levels but can support spreading and migration. This result is less surprising when taken in the context of previous work, which suggests that integrin-mediated attachment and spreading are two separable behaviors that can be distinguished by whether or not the integrin links to the cytoskeleton (Tawil et al., 1993). Integrins may mediate attachment without subsequent cytoskeletal linkage. This is exemplified by the fact that and ß isoforms can have identical extracellular domains, which facilitate attachment, but alternate cytoplasmic domains, which may or may not effectively associate with the actin-based cytoskeleton. For example, after attachment the ß1A isoform is found predominantly in focal adhesions, whereas the ß1C isoform mediates attachment but does not localize to an adhesion site, suggesting an inability to connect to the cytoskeleton (Fornaro and Languino, 1997). The
6A and
6B isoforms both promote attachment to laminin but only the
6A isoform makes stable interactions with the cytoskeleton and can promote cell migration (Jiang and Grabel, 1995; Shaw et al., 1995). Our data suggest that phosphorylation, in addition to isoform switching, can regulate the ability of the integrin subunit to interact with the cytoskeleton.
The reduced attachment levels observed for the cells expressing chicken ß1 with S785M may be attributable to a conformational change in the integrin subunit, resulting in reduced ligand binding. Integrins with similar mutations introduced at the two threonine residues (T788, 789A) display reduced attachment but can promote focal adhesion formation and FAK activation, two processes requiring cytoskeletal interactions. It was proposed that this mutation leads to a shift in conformation of the extracellular domain to a non-ligand-binding form of the protein (Wennerberg et al., 1998).
The ability of a cell to spread or migrate is dependent on the association of the integrin cytoplasmic domain with the actin-based cytoskeleton. It has been suggested that migrating cells must make and subsequently break contacts between the ECM and the plasma membrane (Sheetz et al., 1998). At the leading edge of the cell, extensions or lamellipodia elongate on the substrate and integrins make contacts with the underlying ECM (Regen and Horwitz, 1992; Schmidt et al., 1993). A contractile force is then generated from both ends of the cell towards the nucleus as these integrins engage the ECM, as a result of their linkage to the cytoskeleton (Felsenfeld et al., 1996). Receptor release at the rear of the cell causes forward movement (Palecek et al., 1998; Palecek et al., 1996). The appearance of shed whole integrin molecules in the tracks of migrating cells suggests that this release occurs at the integrin-cytoskeleton link (Schmidt et al., 1993). We recently proposed a model for migration in which phosphorylation modulates the integrin-cytoskeleton interactions. In a dephosphorylated form, the integrin interacts with the actin-based cytoskeleton and focal adhesion proteins to create a focal adhesion site. We further proposed that phosphorylation at S785 results in the breaking of this integrin-cytoskeleton link. Migration is promoted, in part, by the dynamic regulation of the phosphorylation state of S785, which regulates the integrin actin connection.
In support of our model, we now show that expression of S785D in both F9 and GD25 cell lines inhibits migration, whereas the expression of S785M results in intermediate level of migration in the GD25 cell line. The intermediate levels of migration observed for a substitution of phenylalanine, mimicking a dephosphorylated residue, for either of the two tyrosine (Y783, Y795) residues (Sakai et al., 1998) is consistent with our results. If the ability of a cell to migrate is modulated by subsequent changes in the phosphorylation state of the integrin, one might expect inhibitory effects from a methionine substitution, a mutation that could enhance or strengthen integrin-cytoskeleton interactions. In our studies with the transfected cell line GD25-S785M2, we show an intermediate level of migration, between those observed for GD25-S785S4 and GD25-S785D2. This form of the integrin can make the necessary contacts with the cytoskeleton and spread, but the breaking of the integrin-cytoskeleton link might be inhibited. If this is the case, how do GD25-S785M2 cells migrate? The fact that they can suggests that alternate mechanisms exist for disengaging the integrin from the cytoskeleton. It has been proposed that proteolytic cleavage of integrin extracellular domains may be involved in migration (Alfandari et al., 1997). Alternatively, the integrin-cytoskeleton interaction might be broken by the removal of an actin-binding protein.
Additional support for an alternate mechanism for disengaging the integrin from the cytoskeleton comes from our observation that expression of S785M in F9 cells results in levels of migration similar to wild-type cells. The difference between the ability of GD25-S785M2 and F9-S785M cells to migrate may be attributable to the differences between the two assays. Although both assays measure directed migration, the Boyden chamber assays are more complex, in that the cells must migrate through a filter towards a chemotactic agent. By contrast, parietal endoderm outgrowth assays only measure the ability of the cells to migrate away from the embryoid body. The distinction between these assays is supported by work investigating the chemotactic versus chemokinetic responses by Y to F mutations in the cytoplasmic domain of ß1. In these experiments, the Y to F mutation was more inhibitory in chemotactic assays than in chemokinetic assays (Sakai et al., 1998).
Our data supporting a role for S785 phosphorylation suggest that the regulation of phosphorylation at this site is important for ß1-dependent functions. The F9 system provides an excellent model for examining the significance of phosphorylation of the ß1 integrin subunit, because phosphorylation of S785 is developmentally regulated (Dahl and Grabel, 1989). In an effort to determine the kinase and phosphatase that regulate this phosphorylation event, we have identified a candidate kinase (ILK) and phosphatase (PP2A), both of which colocalize and coimmunoprecipitate with ß1 in F9 cells (Mulrooney et al., 2000).
How might phosphorylation modulate ß1 function? One possibility is that phosphorylation causes a conformational change of the cytoplasmic tail, which results in altered protein-protein interactions. Previous work designed to elucidate the structure of the ß3 cytoplasmic domain suggests that the two NPXY domains result in ß reverse turns, forming a hairpin loop (Haas and Plow, 1997). The sequence homology between the cytoplasmic domains of ß1 and ß3 suggests that they are structurally similar. The serine, two tyrosine and two threonine residues are located within this loop formed by the two NPXY domains. The introduction of a phosphate group on one of these residues could change the secondary structure of the cytoplasmic tail. This change could, in turn, expose or hide interaction domains necessary for cytoskeletal linkage or integrin activation. Interestingly, these NPXY motifs correspond with the binding sites for many focal adhesion and signaling proteins (Dedhar and Hannigan, 1996; LaFlamme et al., 1997). Support for this notion comes from investigations of a naturally occurring serine-to-proline mutation in the ß3 tail. This proline substitution, known to introduce dramatic changes in conformation, results in inhibited platelet activation (Chen et al., 1992). The results presented here are consistent with this model in that the distinct phenotypes that are attributable to altered phosphorylation states appear to be related to whether or not the integrin can engage the cytoskeleton.
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