Department of Biochemistry and Protein Function Discovery Program, Queen's University, Kingston, Ontario, Canada
Submitted 4 March 2005 ; accepted in final form 2 June 2005
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ABSTRACT |
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actin cytoskeleton; p21-activated kinase
The molecular structure of podosomes is not clearly defined and, indeed, seems to differ somewhat from cell type to cell type. However, a podosome typically consists of a central membrane invagination surrounded by an actin filament core that in turn is encircled by a larger, ring-shaped structure containing focal adhesion proteins such as -actinin and vinculin and cytoskeletal proteins such as myosin II and tropomyosin (18, 21, 36, 39, 47). Analysis of phorbol ester-stimulated A7r5 cells has shown that podosomes arise at discrete microdomains located at the ventral surface of the plasma membrane near the sites where stress fibers insert into adhesion plaques (24). In A7r5 cells, the recruitment of p190RhoGAP, cortactin, neuronal Wiskott-Aldrich syndrome protein (N-WASp), and the actin-related protein 2/3 (Arp2/3) complex have been identified as early steps in podosome formation (8, 24). The accumulation of p190RhoGAP has led to the proposal that podosome formation is favored by local inhibition of RhoA activity accompanied by the loss of contractile myosin II bipolar filaments. The Arp2/3 complex activated by N-WASp and/or cortactin promotes actin polymerization at branch points of actin filaments at lamellipodia (51). Recent studies have indicated that activation of the Arp2/3 complex represents a key initial step in podosome assembly in which actin filament assembly and disassembly occur continuously in the core of the podosome (13, 24, 29, 30, 34). The recruitment of focal adhesion proteins, myosin II, and tropomyosin to podosomes marks a later stage in podosome development and is accompanied by the dissociation of focal adhesions (8, 24).
The Rho family of GTPases has been implicated in the regulation of podosome biogenesis involving RhoA, Rac, and Cdc42 in a cell type-dependent manner. For example, constitutively active Rac1 disrupts podosome formation in chicken osteoclast-like cells, although it does not affect podosome formation in human dendritic cells (9, 38). Microinjection of active or inactive mutants of Cdc42 impairs podosome formation in human macrophages and dendritic cells (9, 28, 30). As such, podosome formation may thus depend on the integrated activities of several Rho family GTPases (28).
In this study, we have examined the roles that the p21-activated kinase (PAK), a key downstream effector of activated Rac and Cdc42, and its binding partner PAK-interacting exchange factor (PIX), a guanine nucleotide exchange factor (GEF) for Rac, play in podosome formation in A7r5 cells. The three closely related mammalian group I PAKs (PAK1PAK3) consist of a COOH-terminal Ser/Thr protein kinase domain fused to a proline-rich NH2-terminal region that harbors a conserved p21-binding domain (PBD) that binds GTP-Rac and GTP-Cdc42 (6). Inactive PAK is a homodimer in which the PBD domain of one monomer crosses over to autoinhibit the catalytic domain of the second monomer (27). Upon binding GTP-Cdc42/Rac, PAK is converted into an extended monomer that autophosphorylates to attain maximal kinase activity. Catalytically active forms of PAK can mediate the disassembly of actin stress fibers and focal adhesions through the phosphorylation of a variety of substrates (6). However, kinase-defective versions of PAK remain able to promote cell spreading, membrane ruffling, and lamellipodia formation, effects that have been ascribed to the ability of PAK to activate the exchange factor activity of PIX (35).
PAK and PIX are localized to focal adhesions through association with a family of serine- and tyrosine-phosphorylated proteins termed paxillin kinase linker (PKL) (7, 22, 48), Cool-associated tyrosine (CAT1 or CAT2)-phosphorylated (3), or G protein-coupled receptor kinase interactor (GIT1 or GIT2) (40, 41) Arf6 GTPase-activating protein domain-containing proteins. PKL mediates the localization of PAK/PIX to focal adhesions via its interaction with PIX and paxillin (7). It appears likely that the activities of PAK mediated via the PIX-GIT complex are important components of the dynamics that contribute to localized cytoskeletal regulation during changes in cell shape and motility (6).
In this study, we have used the A7r5 vascular smooth muscle cell line as a study model to investigate the role of PAK1 in the organization of the actin cytoskeleton in smooth muscle cells. We show that the expression of cytoskeletally active PAK1 mutants in A7r5 cells results in the loss of stress fibers and the concomitant formation of F-actin columns. We conclude that these actin columns are podosomes because they share similar physical properties, dynamics, and protein composition. Podosome formation does not require PAK1 kinase activity; however, catalytic activity regulates the dynamics of podosomes. The ability to bind PIX, localizing both PIX and GIT to focal adhesions, is essential for podosome formation. Abolition of the PIX-binding ability of PAK prevents both the translocation of PIX and GIT to focal adhesions and the formation of podosomes. The essential role for -PIX in podosome formation is highlighted by the fact that overexpression of
-PIX by itself can induce the formation of podosomes. These data indicate that PAK and PIX are able to induce podosome formation in the A7r5 vascular smooth muscle cell line.
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MATERIALS AND METHODS |
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Cell culture and transfection.
A7r5 rat smooth muscle cells (American Type Culture Collection, Manassas, VA) were grown in low-glucose (1 g/l) Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum and penicillin/streptomycin (Invitrogen). Cells were plated at a density of 2 x 104 cells per 12-mm-diameter glass coverslips precoated with 10 µg/ml fibronectin (Roche Applied Science, Laval, QC, Canada). For live cell imaging, cells were plated at the same density on fibronectin-coated T dishes (Bioptechs, Butler, PA). Cells were transfected 16 h after plating with the use of LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's recommendations, with 0.2 µg of DNA for PAK1-encoding plasmids and 0.4 µg of DNA for
-PIX- and EGFP-
-actin-encoding plasmids.
Coimmunoprecipitation.
COS-1 cells were plated at a density of 1 x 106 cells per 100-mm dish 16 h before cotransfection with 3 µg of either pcDNA FLAG -PIX or pcDNA FLAG vector control and 1 µg of either pCMV6 LL/KR PAK1 or pCMV6 LL/KR/PIX PAK1 using LipofectAMINE Plus reagent (Invitrogen) in accordance with the manufacturer's recommendations. After transfection (4872 h), cells were washed three times in ice-cold, phosphate-buffered saline (PBS; in mM: 138 NaCl, 26 KCl, 84 Na2HPO4, and 14 KH2PO4, pH 7.4 maintained using HCl) and scraped into 1 ml of buffer A [50 mM HEPES, pH 7.4, 1% (vol/vol) Triton X-100, 150 mM NaCl, 1 mM EDTA, and 1 mM sodium orthovanadate] supplemented with protease inhibitor cocktail for mammalian cells (Sigma-Aldrich). Cells were incubated on ice for 30 min, after which cellular debris were pelleted by performing centrifugation at 10,000 g for 15 min. The lysates were transferred to a 1.5-ml Eppendorf tube, and FLAG-
-PIX was precipitated by addition of 30 µl of a 50% anti-FLAG M2-agarose affinity gel (Sigma-Aldrich). After 2-h incubation at 4°C with shaking, the beads were sedimented by performing centrifugation, and the flow-through was removed to a separate tube. The beads were washed three times in buffer A, and proteins were recovered by boiling the beads at 100°C for 5 min after the addition of 50 µl of 2x SDS sample buffer. Samples were separated on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. Immunoblot analysis was performed using rabbit anti-c-Myc at a 1:1,000 dilution (Sigma-Aldrich) to detect PAK1, a horseradish peroxidase-conjugated secondary antibody, and enhanced chemiluminescence (PerkinElmer, Boston, MA). Membranes were stripped and reprobed with anti-FLAG M2 antibody at a 1:5,000 dilution (Sigma-Aldrich) to detect
-PIX. Lysates were also probed to ensure equal expression of the PAK1 constructs.
In vitro kinase activity assays.
The kinase activity of all PAK constructs was determined using an in vitro kinase assay with myelin basic protein (MBP) as a substrate after immunoprecipitation of PAK1 from transiently transfected A7r5 cells, essentially as described by Sells et al. (44) and Frost et al. (17). Briefly, A7r5 cells plated at a density of 1 x 106 cells per 100-mm dish were transfected using 4 µg of plasmid DNA encoding for the various Myc-tagged PAK1 constructs. Lysates were precleared with protein A-agarose beads, and PAK was immunoprecipitated by the addition of 2 µg of rabbit anti-c-Myc IgG followed by protein A-agarose slurry. Beads were extensively washed, and immunoprecipitated proteins were subjected to an in vitro kinase assay using MBP as a substrate in the presence of [-32P]ATP for 30 min. The proteins were separated using SDS-PAGE, and the dried gel was scanned by performing autoradiography using an Imaging Screen (Bio-Rad, Hercules, CA) for 16 h. Western blot analysis was performed to ensure that an equal amount of kinase was used in each sample. Data were collected for three independent experiments.
Fixed cell imaging.
Cells were fixed for 10 min in 3.2% paraformaldehyde (Sigma-Aldrich) and then permeabilized for 5 min in 0.2% Triton X-100. Cells were blocked with 3% bovine serum albumin (BSA; Bioshop) in PBS for 2060 min before being incubated with primary antibodies, and they were washed extensively with PBS between staining steps. Cells were stained for either 1 h at room temperature or overnight at 4°C using the following antibodies diluted in PBS containing 3% BSA: rabbit anti-Arp3 at a 1:50 dilution (Sigma-Aldrich), rabbit anti--PIX at a dilution of 1:50 (Chemicon International, Temecula, CA), mouse anti-l-caldesmon at a 1:200 dilution (BD Biosciences, Lexington, KY), mouse anti-paxillin at a 1:100 dilution (BD Biosciences), mouse anti-cortactin at a dilution of 1:200 (4F11; Upstate USA, Charlottesville, VA), mouse anti-vinculin at a dilution of 1:200 (hVIN-1; Sigma-Aldrich), mouse anti-c-Myc at a 1:50 dilution (Upstate), and rabbit anti-c-Myc at a 1:500 dilution (Sigma-Aldrich). A monoclonal antibody raised against p95PKL (BD Biosciences) that reacts with both GIT1 and GIT2 was used at a 1:50 dilution to identify GIT. Actin filaments were stained using tetramethylrhodamine isothiocyanate-phalloidin at a dilution of 1:500 (Sigma-Aldrich) in PBS containing 3% BSA. Secondary antibodies raised against either mouse or rabbit IgG and conjugated to Alexa 488 or Alexa 568 were used at a 1:500 dilution, and Alexa 350-conjugated antibodies were used at a 1:200 dilution (Molecular Probes, Eugene, OR). Coverslips were mounted on glass slides with fluorescent mounting medium (Dako, Carpinteria, CA). Immunofluorescent images were acquired using a Zeiss Axiovert S100 microscope equipped with a Plan-Neofluar x40 magnification/0.75 numerical aperture (NA) lens objective, a Plan-Apochromat x63 magnification/1.40 NA oil-immersion lens objective, and a high-performance charge-coupled device camera (SensiCam; Cooke, Auburn Hills, MI). Cells were also imaged using a Leica TCS-SP2 RS confocal laser-scanning microscope equipped with a PlanApo x100 magnification/1.40 NA oil-immersion lens objective. Three-dimensional reconstructions of confocal optical sections were created using Leica confocal software, ImageJ software, and Corel PhotoPaint software. Quantification of the percentage and SD of transfected cells with punctate actin structures was assessed by counting >100 cells in each of three independent experiments. To quantify differences in the average number of podosomes induced per cell by catalytically active and inactive versions of PAK1, the total number of podosomes (defined as punctate F-actin structures of 0.5- to 2-µm diameter) was counted for a total of 20 randomly chosen cells. Differences were considered significant at P < 0.05 as determined using Student's t-test.
Live cell imaging.
Live cell imaging was performed 2 days after cells plated on T dishes were transfected with pEGFP-
-actin alone or cotransfected with pEGFP-
-actin and either pCMV6 LL/TE or LL/KR PAK1.
T dishes were moved to a Bioptechs
TC3 culture dish microobservation temperature control system (Bioptechs) mounted on the Zeiss Axiovert S100 microscope to maintain the temperature at 35°C. Images were obtained using the Plan-Neofluar x40 magnification/0.75 NA lens objective every 30 s for a total of 30 min. Images were stored and analyzed using Slidebook image analysis software (Intelligent Imaging Innovations) and ImageJ version 1.30 software (National Institutes of Health).
To assess the influence of kinase activity on podosome dynamics, the lifetimes of podosomes induced by constitutively active PAK1 (LL/TE) and kinase-dead PAK1 (LL/KR) were compared. The lifetimes of 300 individual podosomes from cells transfected with each PAK1 plasmid were determined using movies obtained from at least three independent cell cultures. Please refer to the Supplementary Material for this article to view movies (see Supplementary Figs. 14).1 An F-test showed that the variation in the lifetimes for each construct in all of the movies was not significantly different. To compare the turnover rate of podosomes induced by LL/TE and LL/KR, a simple exponential decay model, P = Poekt, was used, with
= ln(2)/k, where P is the number of podosomes remaining at time t, P0 is the initial number of podosomes, k is the first-order rate constant for podosome turnover, and
is the half-life. The mean ± SD of k was calculated on the basis of data derived from the movies (see Supplementary Figs. 14), and statistical significance was determined using a two-sided t-test.
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RESULTS |
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To study the effect of PIX binding of PAK1 on the cytoskeletal organization of A7r5 cells, we also generated two PAK1 mutants that do not bind PIX. They were created in the LL and LL/KR background of PAK1 by mutating P192,193A at the noncanonical proline-rich PIX-binding site. As shown in Fig. 1B, the Myc-tagged LL/KR/PIX protein was not coprecipitated with FLAG-tagged -PIX from COS-1 cells that coexpressed both proteins, indicating that the P192,193A mutation effectively abolished interaction between PAK1 and
-PIX as previously shown by other researchers (33).
PAK1 induces the formation of podosome-like actin columns in A7r5 cells. Cultured A7r5 vascular smooth muscle cells grown on fibronectin displayed a highly organized and prominent array of actin stress fibers reminiscent of a contractile phenotype characteristic of differentiated vascular smooth muscle cells (Fig. 2A) (25). Overexpression of wild-type PAK1 had no significant effect on the overall morphology of the actin cytoskeleton of A7r5 cells (Fig. 2B) as has been observed in other cell types (17, 26, 32, 44, 56). The lack of effect of wild-type PAK1 on the actin cytoskeleton suggests that PAK activity is tightly controlled by regulatory factors such as GTPases, and the normal cell is able to adjust and maintain optimal PAK activity in spite of overexpression of the regulatable wild-type enzyme. Thus, to gain insight into the roles of various functional domains of PAK, it is necessary to perturb the functional homeostasis of this enzyme in the cell by overexpressing unregulatable mutants, which often produce dramatic remodeling of the cytoskeleton (4, 17, 43, 44).
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Expression of the kinase-dead LL/KR PAK1 induced dramatic actin cytoskeletal reorganization similar to that observed in LL PAK1- and LL/TE PAK1-transfected cells (Fig. 2E), i.e., cell rounding with significant loss of actin stress fibers and the formation of multiple lamellipodia. In addition, the kinase-dead PAK1 mutant induced the formation of similar actin columns in 50% of the transfected cells, indicating that kinase activity is not essential for actin column formation. However, there was one major difference in the morphology of cells overexpressing the catalytically inactive LL/KR PAK1 compared with its catalytically active counterparts: The inhibition of kinase activity caused cells to spread out and extend numerous lamellipodia. This result is similar to findings in other cell types (17, 44).
PAK1 catalytic activity regulates the dynamics of podosome-like actin columns. As shown in Table 1, while the catalytic activity did not affect the diameter or height of the podosome-like actin columns, the kinase-dead mutant induced 3040% more actin columns per cell than the kinase active mutants, which may be indicative of a role played by PAK kinase activity on the dynamics of actin columns.
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PAK-induced actin columns contain podosome marker proteins. Because the actin columns induced by the overexpression of LL PAK1 mutants have size, shape, and dynamics similar to those of podosomes, we next determined whether known marker proteins of podosomes can be found in the actin columns.
As shown in Fig. 4, LL/KR PAK1-induced actin columns contained cortactin and the Arp2/3 complex, which are members of the actin polymerization machinery. Cortactin appears in two major regions: membrane ruffles at the periphery of the cell, as commonly observed in fibroblasts and other cell types (37, 52, 53), and punctate structures that practically colocalize with all of the actin columns (Fig. 4A). The punctate appearance of cortactin shown in Fig. 4 is consistent with observations reported previously in that cortactin is present at sites at which podosomes develop (13) and appears to cluster at "microdomains" located at the stress fiber-focal adhesion interface before podosome formation in A7r5 cells (8, 24).
The Arp2/3 complex has previously been shown to be essential for podosome formation in A7r5 cells in response to phorbol ester stimulation (24). As shown in Fig. 4, the Arp3 component of the Arp2/3 complex appears in two different locations. While Arp3 is clearly localized to actin columns induced by LL/KR PAK1, Arp3 is also diffusely cytoplasmic as observed previously in other cell types (54).
The actin-binding protein caldesmon has been shown to localize to podosomes in Rous sarcoma-transformed fibroblasts (47). As shown in Fig. 4C, caldesmon labeled stress fibers in the LL/KR PAK1-transfected A7r5 cells as well as the majority of actin columns, especially those situated at the end of stress fibers in the periphery of the cell.
The actin cross-linking protein -actinin, which has been shown to incorporate into podosomes in A7r5 cells in response to phorbol 12,13-dibutyrate (PDBu) treatment at an early stage of podosome development (18, 19, 24), was recruited to the LL/KR PAK1-induced actin columns (Fig. 4D).
The focal adhesion protein vinculin has been shown to be recruited to podosomes after focal adhesion dissolution in phorbol ester-treated cells (24). As shown in Fig. 5, A and B, most of the vinculin-labeled focal adhesions abut the ends of actin stress fibers. The majority of the actin columns arise next to vinculin-containing focal adhesions at the end of actin stress as clearly shown in x-y profiles (Fig. 5, B and C). It is of note that some actin columns also appeared to be enclosed by a ring of vinculin (Fig. 5, D and E).
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Actin columns induced by the kinase-active PAK1 mutants, LL PAK1 or LL/TE PAK1, have staining patterns for cortactin, Arp3, caldesmon, -actinin, and vinculin (data not shown) similar to those found in LL/KR cells. These data, taken together, show that overexpression of either kinase-active or kinase-dead PAK1 mutants induces actin columns that share similar molecular structures with one another and with phorbol ester-induced podosomes in A7r5 cells.
PAK1 induces the translocation of PIX and GIT to focal adhesion complexes. It has been shown that PAK can be targeted to focal adhesion complexes via its interaction with PIX (31, 46, 57). PIX interacts with GIT, which in turn associates with paxillin in the focal adhesion complex (7, 48). These findings and our observation that the majority of PAK-induced actin columns formed at the stress fiber-focal adhesion interface suggest that PAK plays a role in coordinating a change in the focal adhesion complexes leading to the formation of actin columns. We thus investigated whether the overexpression of actin column-promoting PAK1 mutants may play a role in the recruitment of PIX and GIT to focal adhesion complexes.
In nontransfected cells (Fig. 6) or in cells transfected with the empty vector (data not shown), -PIX and GIT diffuse throughout the cytoplasm, with a majority of cells showing little focal adhesion localization of these proteins. The majority of paxillin is localized to focal adhesions, although some is observed in the cytoplasm. In contrast, LL/KR PAK1 induces the translocation of
-PIX and GIT to regions corresponding to focal adhesions adjacent to actin columns (Fig. 6, AC) and to focal adhesion-like structures behind which actin columns arise (Fig. 6, G and H).
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DISCUSSION |
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The finding that the overexpression of mutant forms of PAK can produce podosomes has not been reported previously. However, active PAK2 has been noted to produce punctate actin-rich structures dispersed throughout the cytoplasm of endothelial cells (55). Whether the PAK2-induced actin structures in endothelial cells represent podosome-like structures has not been investigated to date.
Sites of podosome formation in A7r5 smooth muscle cells. The majority of PAK-induced podosomes were located at the ends of stress fibers adjacent to focal adhesions (Fig. 5), similarly to "microdomains" previously identified as sites of formation of phorbol ester-induced podosomes in A7r5 cells (24). The strategic juxtaposition of actin stress fibers, focal adhesions, and podosomes strongly implicates a direct association of the three structures and a coordinated structural remodeling of stress fibers and focal adhesions before the formation of podosomes. Thus, as shown in Fig. 5, enrichment of PAK to areas at and surrounding actin columns and focal adhesions may be involved in both translocation of PIX and GIT to focal adhesions and actin polymerization factors such as cortactin and the Arp2/3 complex to sites of podosome biogenesis. Localization of PAK to focal adhesions has been demonstrated previously in other cell types (31, 46, 57), although downstream events have not been elucidated.
Functional and structural requirements of PAK for podosome formation. Our studies have shown that PAK1 mutants lacking catalytic activity as a result of the K299R mutation at the active site were as effective as the kinase-active mutants in generating podosomes. This finding is consistent with results reported for other cell types in which PAK exhibited kinase-dependent and/or kinase-independent functions, with the former being manifested in the phosphorylation of appropriate substrates and the latter being involved in the translocation of proteins to specific subcellular locations (11, 17, 44). Binding of active Rac/Cdc42 to PAK is thought to induce an "open" conformation, thus exposing the proline-rich domains to interact with adaptor proteins, such as PIX and Nck, and/or activate kinase activity by inhibiting interaction between the autoinhibitory domain and the catalytic site (6, 7, 31, 46). H82,83L mutation, as used in this work, impairs the interaction between the autoinhibitory domain with the kinase domain and presumably mimics the open conformation required for the binding of PIX and other interacting partners (44). Our finding that the interaction between PAK and PIX was required for podosome formation suggests that translocation of the PIX-GIT complex to the sites of podosome formation is critical for the initiation of podosome biogenesis. PIX is a multifunctional protein known to act as a GEF of Rac and thus potentially might be a part of a positive feedback loop for the activation of Rac and PAK (5, 15). Another possibility is that PIX acts as an adaptor protein that brings GIT and PAK to focal complexes adjacent to sites of podosome formation. Targeting of the PAK-PIX-GIT complex to focal complexes may be required for remodeling of the focal structures as well as activation of PAK by GIT as recently shown by Loo et al. (31).
While the catalytic activity of PAK1 was not required for actin column formation, it was found to regulate the dynamics of podosomes. The highly kinase-active construct of PAK1 induced fewer podosomes per cell than the kinase-inactive construct, in part because of an increased rate of podosome turnover (Table 1). PAK may initiate disassembly of actin columns directly by phosphorylating proteins in the columns, such as caldesmon (16) and cortactin (Webb BA, unpublished observations; Ref. 50), or indirectly by phosphorylating a number of other cytoskeletal targets, including myosin light-chain kinase (20, 42), LIM kinase (10, 14), and filamin A (49). In addition, PAK1 kinase activity is essential for the promotion of focal adhesion disassembly (1, 17, 32, 35), which may also play an active role in podosome dynamics (24).
Role of podosomes in vascular smooth muscle cells. Podosomes have been shown to be sites of ECM degradation, promoting the invasiveness of monocyte-derived cells and transformed cells (28). It is thus possible that PAK also induces a migratory, invasive phenotype in vascular smooth muscle cells that are major components of arterial remodeling in the development of vascular diseases such as atherosclerosis and restenosis. One of the early stages in the formation of atherosclerotic plaques involves the dedifferentiation of smooth muscle cells to a motile phenotype that migrates from the medial layer to the site of lesion in the blood vessel, a process that bears similarity to the migration of cancer cells. In this respect, it is interesting that Stofega et al. (46) recently showed PIX-dependent localization of constitutively active PAK in focal adhesions in breast cancer cells. Thus localization of the PAK-PIX-GIT complex to focal adhesions may represent a common mechanism leading to the induction of a migratory and invasive phenotype in normally docile cells. To further this study, we are investigating the role of podosomes in ECM degradation in vascular smooth muscle cells. In addition, because both overexpression of PAK1 mutants and stimulation of A7r5 cells with phorbol ester induce the formation of podosome-like structures, we are examining potential cross-talk between the PAK and PKC signaling pathways.
In conclusion, we have shown that the expression of PAK1 functional mutants in A7r5 smooth muscle cells induces the formation of podosomes similar to those induced by phorbol ester. Podosome biogenesis requires two highly coordinated events involving the remodeling of both focal complexes and actin stress fibers. This study suggests that PAK-PIX interaction plays a crucial role in this regulatory mechanism. We propose that remodeling of the focal complexes is initiated by the translocation of the PAK-PIX-GIT complex to existing focal complexes, thus eliciting the recruitment of actin polymerization factors such as Arp2/3, cortactin, and caldesmon for the formation of the actin core structures. Activation of PAK either through the positive feedback activation of Rac by PIX (5, 15) or by GIT (31) leads to subsequent phosphorylation of substrates in the actin columns and the nearby focal complexes as well as disassembly of the podosomes.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supplemental data for this article may be found at http://ajpcell.physiology.org/cgi/content/full/00095.2005/DC1.
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