1 Department of Cell and Developmental Biology SUNY Upstate Medical University, 750 East Adams St, Syracuse, NY 13210, USA
2 Biosource International, Hopkinton, MA 01748, USA
* Author for correspondence (e-mail: blystons{at}mail.upstate.edu)
Accepted 13 November 2003
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Summary |
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Key words: Cytoskeleton, PSSA, Pyk2, Actin
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Introduction |
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ß3 integrins play important functional roles in hematopoietic cell types including platelets, monocytes and osteoclasts. IIbß3 integrins mediate platelet aggregation and clot retraction with receptor dysfunction resulting in Glanzmann's thrombasthenia (Bajt et al., 1992
).
Vß3 is the major adhesion receptor in osteoclasts and mediates adhesion to various bone matrix proteins. During osteoclast-mediated bone resorption,
Vß3 regulates cytoskeletal organization required for cell migration and formation of the sealing zone (McHugh et al., 2000
).
Vß3 also modulates
Lß2-dependent migration along ICAM-1, suggesting an important role for ß3 in transendothelial migration of macrophages (Weerasinghe et al., 1998
).
Adhesive structures resulting from changes in actin dynamics have been well characterized in numerous cell types (Nobes and Hall, 1995). Actin stress fiber formation in fibroblast cell lines occurs as a result of Rho activation, whereas Rac and Cdc42 induce the formation of lamellipodia and filopodia, respectively (Hall, 1998
). Cells of hematopoietic lineage form neither focal contacts nor adhesions, but rather exhibit smaller, biochemically similar adhesive structures, termed podosomes, that contain the appropriate signaling and adaptor proteins to achieve membrane extension (Evans et al., 2003
; Worthylake and Burridge, 2001
). In addition, leukocytes need an additional stimulus to achieve firm adhesion through actin-dependent behaviors. Ligand-induced phosphorylation on ß3 Y747 and activation of protein kinase C (PKC) are required for Rho activation and firm adhesion of leukocytes to vitronectin (Butler et al., 2003
). This phosphorylation event results in receptor clustering and subsequent direct and indirect recruitment of a signaling and adaptor protein complex to the cytoplasmic tail, including Pyk2, Src, Syk, phosphoinositide 3-kinase, vinculin, talin and
-actinin proteins (van der Flier and Sonnenberg, 2001
). The interactions among these signaling molecules, in conjunction with the Arp2/3 complex of proteins and the cellular actin machinery, result in coordinated changes in actin dynamics necessary to achieve inflammatory phenotypes.
Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor protein tyrosine kinase that is closely related to focal adhesion kinase and serves many of the same functions in hematopoietic cells (Sasaki et al., 1995). It has been shown to be a proximal link between integrin and chemokine signaling in natural killer cells and has been implicated in the regulation of the Rac pathway leading to lamellipodial formation and transendothelial migration (Gismondi et al., 2003
). In osteoclasts, Pyk2 plays an important role in the cell adhesion and spreading that is imperative for active bone resorption. Pyk2 phosphorylation at tyrosine 402 (Y402), believed to result in Pyk2 activation, has been shown to play a crucial regulatory role in adhesion-dependent cytoskeletal organization in osteoclasts, notably in the formation of the sealing zone, an
Vß3-dependent structure (Lakkakorpi et al., 2003
).
Integrin-mediated adhesion, phagocytosis and migration require a coordinated rearrangement of the actin cytoskeleton. Actin nucleation and polymerization occur as a result of active signaling complexes recruiting the appropriate cellular machinery for cytoskeletal rearrangement. The Arp2/3 complex of proteins mediates initiation and growth of actin filaments. Activation of the complex results in an open conformation, permitting the Arp2 and Arp3 subunits to form a template actin filament for nucleation (Robinson et al., 2001). The Arp3 subunit contains residues that interact with ATP on actin. It has been shown that the C-terminal VCA domains of WASp/Scar proteins, which are downstream of Rho GTPases, interact with the Arp2/3 complex to activate actin nucleation, regulating filament formation and extension (Machesky et al., 1999
). DeMali et al. have recently shown that the Arp2/3 complex localizes to adhesion contacts that contain active integrin complexes, presenting a functional link between integrin engagement and actin polymerization (DeMali et al., 2002
). In these studies, vinculin was shown to mediate the link between Arp2/3 and the integrin.
Rho is a small GTP-binding protein in the signaling pathway that leads to leukocyte adhesion and stress fiber formation. Rho cycles between a GDP-bound inactive state and GTP-bound active state, and when in its active conformation, it activates downstream signaling pathways. In leukocytes, activation of Rho is required for Vß3-mediated adhesion to vitronectin. This Rho activation is dependent on the phosphorylation of ß3 Y747 (Butler et al., 2003
). Rho kinase (ROCK) is a downstream Rho effector implicated in numerous cellular processes. In NIH 3T3 cells, activation of the Rho-ROCK pathway results in myosin light chain (MLC) phosphorylation from suppression of MLC phosphatase, initiating changes in actin dynamics (Kimura et al., 1996
). In leukocytes, inhibition of ROCK prevents retraction of trailing edge adhesive contacts (Alblas et al., 2001
).
To better understand early adhesion events preceding actin stress fiber formation, we visualized the localization and behavior of ß3 Y747 phosphorylation using a phosphorylated state-specific antibody (PSSA) specific to the phosphorylated form of ß3 Y747. KVß3 WT cells adherent to vitronectin presented progressively decreasing ß3 Y747 PSSA staining along with increasing Arp3 organization as adhesion sites matured and actin stress fiber formation occurred. Importantly, Arp3 failed to organize in K
Vß3 Y747,759F cells, suggesting that phosphorylation on ß3 Y747 is important for the downstream Arp2/3 organization that leads to actin assembly. To disrupt the normal progression of adhesion, we inhibited ROCK. This resulted in sustained ß3 Y747 PSSA staining throughout the adhesion time course in K
Vß3 WT cells, suggesting the presence of a feedback regulation of ß3 Y747 phosphorylation. Arp3 failed to organize in K
Vß3 WT cells when ROCK was inhibited, introducing a rate-limiting step for actin stress fiber formation. In addition, competitive inhibition of phosphorylated ß3 with integrin tail phosphomimetic peptides disrupted Arp3 organization in K
Vß3 WT cells. Our results confirm the requirement of ß3 Y747 phosphorylation for
Vß3-mediated adhesion to vitronectin. Furthermore, our data suggest that ß3 Y747 phosphorylation is required for the appropriate localization of the Arp2/3 complex and its subsequent assembly of actin.
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Materials and Methods |
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Cell adhesion assays
96 well-plates (Immulon II, Dynatech, Chantilly, VA) were coated with vitronectin (1.5 µg/ml) in PBS overnight at 4°C. Plates were washed twice with PBS, followed by the addition of 100 µl casein blocker per well and incubation at room temperature for 30 minutes. Overlapping peptides corresponding to the tyrosine-phosphorylated ß3 cytoplasmic tail were constructed as cell permeant fusions with the HIV-TAT leader (TAT-Yp: YGRKKRRQRRR G DTANNPL Yp KEATSTFT-COOH and TAT-Y/F: YGRKKRRQRRR G DTANNPL F KEATSTFT-COOH, where the leader sequence is underlined and the position of Y747 of ß3 is in bold).
Phorbol myristate acetate (PMA; 10 ng/ml), TAT-fusion proteins (10 mM) and/or anti-ß3 antibodies (5 µg/ml) were added to empty wells, followed by cells (1x105 in 100 µl of Hanks' Balanced Salt Solution (HBSS) with 1.0 mM Ca2+ and 1 mM Mg2+ added (HBSS++)). Plates were incubated for 1 hour at 37°C. Following two washes with HBSS-, cells were fixed with 5% formaldehyde in PBS, washed, stained with 0.5% crystal violet, and washed and lysed with 200 µl methanol. Adhesion was quantitated by absorbance at 570 nm using a Molecular Devices microplate reader (Sunnyvale, CA).
Immunoprecipitation
K562 cells expressing wild-type, Y747F, Y759F or Y747,759F mutant Vß3 were incubated either with or without (untreated) MnCl (2 mM) and sodium pervanadate (100 µM) for 10 minutes at 37°C. Cells were lysed in PBS buffer containing 1% Nonidet P-40, phenylmethylsulfonyl fluoride (1 mM), sodium orthovanadate (100 µM) and iodoacetimide (1.85 mg/ml). ß3 was immunoprecipitated from cleared lysates using mAb, 7G2, as previously described (Butler et al., 2003
). Whole cell lysates and IP samples were separated on 7.5% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blotted with ß3 Y747 PSSA antibodies and HRP-conjugated anti-rabbit secondary antibody (Pierce Biotechnology, Rockford, IL). Reactivity was visualized by enhanced chemiluminescence (ECL, Amersham Biosciences, Piscataway, NJ).
Fluorescent microscopy
12-mm glass coverslips were coated overnight at 4°C with vitronectin (1 µg/ml) in PBS. 8x104 K562 cells expressing wild-type or mutant (Y747,759F) Vß3 were adhered to coverslips in the presence of either PMA (10 ng/ml), Y27632 (ROCK inhibitor; 10 µM) or both for indicated times at 37°C in Iscove's Modified Dulbecco's Media (IMDM). For TAT-peptide experiments, cells were pre-incubated with TAT-Yp or TAT-Y/F peptide (100 µM) for 15 minutes at 37°C, centrifuged at low speed and resuspended in IMDM for adhesion to vitronectin in the presence of PMA (10 ng/ml). Cells were washed twice with PBS. Following fixation with 3.7% formaldehyde for 1 hour, cells were permeabilized with 0.005% NP-40 for 15 seconds at 4°C followed by two washes in PBS. For ß3 Y747 PSSA staining, cells were incubated with phosphorylated state-specific antibody (0.387 µg/ml) in PBS. For total ß3 staining, 400 µl of 1A2 tissue culture supernatant was used per well. Other antibodies were diluted 1:500 in PBS. Primary antibody incubation was for 30 minutes at 37°C. Cells were washed three times in PBS. Anti-rabbit IgG, FITC-conjugated secondary antibody (1:1000), anti-mouse IgG, TRITC-conjugated secondary antibody (1:1000) and/or rhodamine-phalloidin (1:1200) were diluted in PBS containing 0.3% goat serum. Secondary antibody incubation was for 30 minutes at 37°C, followed by five washes in PBS. Coverslips were reversed onto o-phenylenediamine in glycerol. Fluorescence was visualized on a Nikon Eclipse E800 fluorescent microscope (Nikon, Melville, NY). Images were digitally captured with a Hamamatsu ORCA-ER digital camera (Bridgewater, NJ) and processed with Simple PCI and Adobe PhotoShop 5.5 software.
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Results |
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ß3 Y747 phosphorylation is an early adhesion event
The ß3 cytoplasmic tail contains a tyrosine at position 747 that is required for Vß3-mediated adhesion. To determine when ß3 Y747 phosphorylation is required, K
Vß3 WT (Fig. 2A-C,E-G) and K
Vß3 Y747,759F (Fig. 2D,H) cells were adhered to vitronectin in the presence of PMA for 10 (Fig. 2A,D,E,H), 30 (Fig. 2B,F) or 70 (Fig. 2C,G) minutes and stained for rhodamine phalloidin (Fig. 2A-D) and ß3 Y747 PSSA (Fig. 2E-H) as described in Materials and Methods. At the 10 minute adhesion time point, K
Vß3 WT cells exhibited ß3 Y747 PSSA staining in punctate form, indicative of podosomes (Fig. 2E). This staining was absent in K
Vß3 Y747,759F cells (Fig. 2H). Organization of actin into stress fibers was detected with rhodamine phalloidin; as stress fiber formation progressed, ß3 Y747 PSSA levels in K
Vß3 WT cells decreased (Fig. 2G) to amounts comparable to those in ß3 Y747,759F mutant cells (Fig. 2H). More interestingly, at the 70 minute time point, K
Vß3 WT cells containing mature stress fibers were completely devoid of ß3 Y747 PSSA staining, compared with those cells in an early adhesive state (Fig. 2I,J).
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Increased ß3 Y747 phosphorylation with ROCK inhibition
We have previously shown that in KVß3 WT cells, Rho is needed for actin assembly and that its activity is dependent on ß3 Y747 phosphorylation (Butler et al., 2003
). In the Rho-ROCK signaling pathway leading to actin assembly and stress fiber formation, ROCK has been shown to lie upstream of actin reorganization (Maekawa et al., 1999
). We utilized the ROCK inhibitor, Y27632, to disrupt actin related events downstream of ß3 Y747 phosphorylation. Interestingly, ROCK inhibition resulted in sustained levels of ß3 Y747 phosphorylation at late adhesion time points (Fig. 3A-C). This suggests a feedback mechanism of ß3 Y747 phosphorylation involving either ROCK or molecules downstream of ROCK.
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As the human and murine ß3 cytoplasmic tail sequences are identical, wild-type murine BMMs were adhered to vitronectin under equivalent conditions to compare ß3 Y747 PSSA staining with our K562 cell model. ß3 Y747 PSSA staining was present in murine BMMs adhering to vitronectin in the presence of PMA (Fig. 4A) in similarly appearing adhesion contacts as those observed in KVß3 WT cells. As seen in K
Vß3 WT cells, the presence of phosphorylated ß3 Y747 decreased over the duration of murine BMM adhesion to vitronectin in the presence of PMA (Fig. 4B) and was sustained during ROCK inhibition (Fig. 4C,D).
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Arp3 organizes in adherent KVß3 WT cells
Actin cytoskeletal rearrangement has been observed following leukocyte adhesion to a vitronectin matrix in the presence of PMA. On ß3 Y747 phosphorylation in the presence of PMA, KVß3 WT cells show evidence of actin organization over time of adhesion to vitronectin (Fig. 2A-C). The Arp2/3 complex is a docking site involved in actin polymerization upon which free actin monomers bind to form filaments and fibers. In K
Vß3 WT cells, we observed an increase in Arp3 detection over adhesion time (Fig. 5E-G). Arp3 organized into adhesion contacts analogous to those containing ß3 Y747 PSSA, although we did not observe direct colocalization of Arp3 with ß3. Arp3 detection was minimal in K
Vß3 Y747,759F cells (Fig. 5A-C). These data suggest a requirement of ß3 Y747 phosphorylation for Arp3 recruitment to early adhesion contacts.
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To further explore this event, we studied the effect of ROCK inhibition on Arp3 organization. In adherent KVß3 WT cells, inhibition of ROCK with Y27632 resulted in a sustained presence of phosphorylated ß3 (Fig. 3). Arp3 failed to organize in K
Vß3 WT cells at the 70 minute adhesion time point in the presence of Y27632 (Fig. 5H), illustrating a requirement for ROCK in Arp3 organization. This indicates that phosphorylation of ß3 is necessary, but not sufficient, for Arp2/3 sequestration to podosomes.
We have previously shown that Rho activation is required for adhesion to both fibronectin and vitronectin (Butler et al., 2003). The striking difference between
Vß3-mediated adhesion to these two ligands is that adhesion to vitronectin requires phosphorylation on Y747, and activation of both PKC and phosphoinositide 3-kinase, whereas these events are not required for adhesion to fibronectin. Taking this into account, we adhered K
Vß3 Y747,759F cells to fibronectin and stained for Arp3. Arp2/3 complex formation and organization is thought to lie between Rho activation and downstream actin stress fiber formation. We observed equivalent Arp3 organization in K
Vß3 Y747,759F cells as compared to K
Vß3 WT cells when cells were adhered to fibronectin in the presence of PMA (Fig. 5D), reinforcing our previous observation that the requirement for ß3 Y747 phosphorylation is ligand selective.
To further explore the requirement of ß3 Y747 phosphorylation for Arp3 organization into podosomes, we utilized TAT-fusion peptides to competitively inhibit ß3 phosphorylation-dependent events. TAT-Yp and TAT-Y/F peptides used in Fig. 1 were pre-incubated with KVß3 WT cells, followed by adhesion to vitronectin. TAT-Yp reduced Arp3 localization to podosomes and limited actin stress fiber formation (Fig. 5I,K). Minimal reduction in Arp3 organization was seen in cells pre-incubated with TAT-Y/F peptide, perhaps attributable to inhibitory effects of other ß3 cytoplasmic motifs in the TAT-peptide.
Phosphorylated Pyk2 colocalizes with total ß3 over course of adhesion to vitronectin
ß3 Y747 PSSA staining to vitronectin decreased over adhesion time in the presence of PMA (Fig. 2). We proposed that this decrease was due to the masking of the Y747 phosphorylation site by adaptor or signaling proteins that form a receptor complex on ß3 Y747 phosphorylation. We double-labeled KVß3 WT cells with anti-ß3 antibody, 1A2, paired with numerous antibodies to proteins downstream of integrin activation, such as talin,
-actinin, vinculin, Vav and phosphoinositide 3-kinase. We detected organization of each protein into adhesion contacts but did not observe strong colocalization of any of these proteins with ß3. By contrast, active Pyk2 did colocalize with ß3. Pyk2 is a nonreceptor protein tyrosine kinase closely related to focal adhesion kinase that has been shown to couple integrins to their downstream effectors, including
Vß3 in osteoclasts (Duong et al., 2000
). Pyk2 Y402 phosphorylation is required for integrin-mediated Rac activation (Gismondi et al., 2003
). We examined the relationship between ß3 and active Pyk2 in K
Vß3 WT and K
Vß3 Y747,759F cells adherent to vitronectin in the presence of PMA. We double-labeled cells with anti-ß3 monoclonal antibody, 1A2 (Fig. 6A-C,J-L), and Pyk2 Y402 PSSA (Fig. 6D-F,M-O) and observed aggregation of Y402 phosphorylated Pyk2 to adhesion contacts comparable to those containing Y747-phosphorylated ß3 after 10 minutes of adhesion (Fig. 2E). Over the adhesion time course, an increase in Pyk2 Y402 PSSA staining (Fig. 6F) was seen along with colocalization of total ß3 (Fig. 6I). These data suggest that active Pyk2 is recruited to adhesion contacts containing phosphorylated ß3 and may be in closest proximity to ß3. Over an identical adhesion time course with K
Vß3 Y747,759F cells, Y402-phosphorylated Pyk2 neither organized into podosomes (Fig. 6O) nor colocalized with ß3 (Fig. 6R). These data provide evidence for ß3 Y747 phosphorylation-dependent organization of Y402 phosphorylated Pyk2 into podosomes and colocalization with ß3.
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Pyk2, Arp3 and vinculin present in adhesion contacts
Unlike fibroblast cell lines that form focal adhesions, leukocytes form podosomes - smaller adhesive structures that we have shown to contain Y747 phosphorylated ß3. After 70 minutes of adhesion to vitronectin in the presence of PMA, we observed actin stress fiber formation (Fig. 2C), Pyk2 Y402 PSSA organization and colocalization with total ß3 (Fig. 6F,I) and Arp3 organization (Fig. 5G). Here, we double-labeled KVß3 WT cells to determine the proximity of proteins within the signaling complex on ß3. Vinculin was present in adhesion contacts throughout the course of adhesion, but was especially evident in mature contacts in the cell periphery (Fig. 7A,G).
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In Fig. 5, Arp3 exhibited progressive organization into podosomes over the course of adhesion. After 70 minutes of adhesion, we detected colocalization of Arp3 with both vinculin (Fig. 7C) and Pyk2 (Fig. 7F) in KVß3 WT cells. Total Pyk2 staining was largely diffuse (Fig. 7D) compared with vinculin and Arp3. In addition, we observed strong colocalization between Pyk2 and Arp3 (Fig. 7F). However, we believe that the subset of Pyk2 that colocalized with Arp3 (Fig. 7F) was the Y402-phosphorylated form. Owing to antibody incompatibility, we could not double-label cells for Pyk2 Y402 PSSA and Arp3. Instead, we double-labeled cells with Pyk2 Y402 PSSA and vinculin, revealing colocalization (Fig. 7I). Combining these data with total ß3/Pyk2 Y402 PSSA colocalization (Fig. 6I), we propose that vinculin, Arp3 and Y402-phosphorylated Pyk2 are present in ß3-containing adhesion contacts. Importantly, ß3 Y747 phosphorylation appears to play a crucial role in the assembly of this adhesion complex into podosomes during adhesion to vitronectin.
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Discussion |
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Hematopoietic cell types exhibit a regulated adhesion, requiring multiple input signals to prevent inappropriate thromboses or unwarranted inflammation. Unique signaling pathways and hematopoietic-specific isomers of various signaling molecules contribute to the regulation of integrin-mediated adhesion in these cell types. We have characterized one event in these regulated adhesion pathways. Phosphorylation of Y747 of the ß3 cytoplasmic tail occurs as a result of integrin-ligand interaction. In leukocytes, this phosphorylation is required for PKC-dependent adhesion to vitronectin. Mutation of this tyrosine prevents hematopoietic cell adhesion both in vitro and in vivo, but has no effect on the adhesion of nonhematopoietic cell types and does not affect normal development. The defective adhesion is due to a failure of the ligand-occupied mutant integrin to activate Rho. Accordingly, these cells do not form actin stress fibers. Interestingly, the Y747F mutation of ß3 does not affect the ability of the receptor to support cell adhesion to fibronectin, with concordant activation of Rho and stress fiber formation. This suggests a very selective regulatory event in the process of leukocyte adhesion. Furthermore, the Y747F mutant ß3 provides an excellent tool to evaluate integrin regulation of the actin cytoskeleton as the affinity regulation of the receptor does not differ from wild type and the defective assembly of an actin cytoskeleton is restricted to the ligand vitronectin. In this study, we have used phosphorylation-specific ß3 antibodies and phosphorylation-deficient ß3 integrins to show a selective pathway for the recruitment of actin assembly machinery to the Vß3 adhesion site.
We have previously shown that KVß3 Y747,759F cells exhibit neither actin stress fibers nor achieve firm adhesion on vitronectin (Blystone et al., 1997
). However, these cells do possess the appropriate signaling molecules and actin assembly machinery for adhesion as can be observed on their adhesion to fibronectin (Butler et al., 2003
). To better understand the role of ß3 Y747 phosphorylation for adhesion to vitronectin, we began by localizing this phosphorylation event in adhering cells. Although K
Vß3 WT cells displayed a firmly adherent phenotype after 1 hour of adhesion to vitronectin in the presence of PMA, ß3 Y747 phosphorylation was an early event, occurring within minutes of exposure to ligand. Phosphorylated ß3 appeared in punctate structures typical of leukocyte integrin adhesion contacts and were distributed widely throughout the cell. At early time points there was a concentration of phosphorylated ß3 at the cell periphery, forming an incomplete ring at the base of filopodial extensions. These punctate structures, termed podosomes, are hematopoietic integrin adhesion structures and analogous to focal contacts. Podosomes contain many of the same molecules as focal contacts, including
-actinin and vinculin, but also contain hematopoietic-specific signaling proteins, such as Vav1 and Pyk2. As adhesion progressed, ß3 Y747 phosphorylation diminished in direct opposition to the formation of actin stress fibers. In primary cells, phosphorylated ß3 appeared in analogous contacts. These cells, however, have a shorter time course to achieve firm adhesion and exhibit ß3 phosphorylation at earlier time points accordingly. As in the K
Vß3 WT cell line, ß3 phosphorylation diminished over time of adhesion in murine BMMs. No significant reactivity of the ß3 Y747 PSSA was detected in K
Vß3 Y747,759F cells under any conditions.
For adhesion to vitronectin, ß3 Y747 phosphorylation is required for Rho activation (Butler et al., 2003). Rho GTPase activity initiates the signaling events that result in actin stress fiber formation. To determine if the ß3 Y747 phosphorylation-dependent activation of Rho was responsible for the failure of K
Vß3 Y747,759F cells to form stress fibers on vitronectin, we monitored the organization of actin assembly machinery with respect to ß3 Y747 phosphorylation. Surprisingly, we found that Arp3 organized into podosomes over time of adhesion to vitronectin in cells expressing wild-type
Vß3 but failed to organize in K
Vß3 Y747,759F cells. This defective Arp3 organization was solely due to the ß3 Y747F mutation and the failure of this receptor to activate Rho on vitronectin, as K
Vß3 Y747,759F adhesion to fibronectin, which results in active Rho, resulted in Arp3 organization. In addition, Arp3 organization, as well as actin stress fiber formation, was prevented by the presence of peptide mimetics of the phosphorylated ß3 cytoplasmic tail. These data suggest that the presence of phosphorylated ß3 in podosomes is a prerequisite for Arp3 localization.
Rho kinase (ROCK) is a Rho GTPase effector that is required for the initiation of actin assembly and presumably lies between Rho and Arp2/3 in this signaling pathway. Inhibition of ROCK resulted in the complete failure of Arp3 to organize into adhesion contacts. Interestingly, ROCK inhibition also induced a sustained phosphorylation of ß3 for the duration of the time course. This suggests that ROCK activity contributes to feedback mechanisms to dephosphorylate ß3. Alternatively, ß3 Y747 phosphorylation may recruit molecules to the ß3 cytoplasmic tail, progressively blocking epitope availability. If this is the case, recruitment of molecules downstream of ROCK are likely to be responsible for this steric hindrance. In mature podosomal structures, we observed colocalization of Arp3 with Pyk2 in KVß3 WT cells. Pyk2 plays regulatory and adaptor roles between integrins and their downstream signaling partners in osteoclast-like cells (Pfaff and Jurdic, 2001
). Interestingly, over time of K
Vß3 WT cell adhesion to vitronectin, we observed increased Pyk2 Y402 PSSA organization into podosomes and colocalization with ß3 that did not occur in K
Vß3 Y747,759F cells. Vinculin, an integrin-associated and actin-binding protein (Jockusch and Isenberg, 1981
), colocalized with both Arp3 and Pyk2 Y402 in mature adhesive contacts. In our biochemical studies, vinculin coprecipitates with ß3 from adherent K
Vß3 WT cells at equivalent levels throughout an adhesion time course to vitronectin (data not shown). Similarly, by immunofluorescence we were unable to visualize any significant changes in vinculin organization over the adhesion time course. Although this does not preclude a vinculin dependence for Arp2/3 organization as reported by DeMali et al. (DeMali et al., 2002
), it suggests that Arp2/3 translocation to the podosome requires additional events.
With this combined evidence of colocalization, we propose that vinculin, Arp3 and Y402-phosphorylated Pyk2 are present in adhesive contacts containing Y747 phosphorylated ß3 integrin. As Arp3 presents a ß3 Y747 phosphorylation dependency for organization into podosomes and is part of a large protein complex, it is a strong candidate for the steric masking of Y747-phosphorylated ß3 epitopes on completion of adhesion site signaling. The requirement of ß3 Y747 phosphorylation for Vß3-mediated adhesion to vitronectin appears to lie in the activation and recruitment of actin nucleators for cytoskeletal assembly. Examination of the ß3 Y747-phosphorylation-independent recruitment of Arp2/3 on
Vß3 attachment to fibronectin will be of future interest.
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References |
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