PAK1 induces podosome formation in A7r5 vascular smooth muscle cells in a PAK-interacting exchange factor-dependent manner

Bradley A. Webb, Robert Eves, Scott W. Crawley, Shutang Zhou, Graham P. Côté, and Alan S. Mak

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Remodeling of the vascular smooth muscle cytoskeleton is essential for cell motility involved in the development of diseases such as arteriosclerosis and restenosis. The p21-activated kinase (PAK), which is an effector of the Rho GTPases Rac and Cdc42, has been shown to be involved in cytoskeletal remodeling and cell motility. We show herein that expression of cytoskeletally active constructs of PAK1 is able to induce the formation of dynamic, podosome-like F-actin columns in the A7r5 vascular smooth muscle cell line. Most of these actin columns appear at the junctions between stress fibers and focal adhesions and contain several known podosomal protein markers, such as cortactin, Arp2/3, {alpha}-actinin, and vinculin. The kinase activity of PAK plays a role in the regulation of the turnover rates of these actin columns but is not essential for their formation. The ability of PAK to interact with the PAK-interacting exchange factor (PIX) but not with Rac or Cdc42, however, is required for the formation of the actin columns as well as for the translocation of PIX and G protein-coupled receptor kinase-interacting protein (GIT) to focal adhesions adjacent to the actin columns. These findings suggest that interaction between PAK and PIX, as well as the recruitment of PIX and GIT to focal adhesions, plays an important role in the formation of actin columns that resemble podosomes induced by phorbol ester in vascular smooth muscle cells.

actin cytoskeleton; p21-activated kinase


PODOSOMES ARE HIGHLY DYNAMIC, actin filament-based structures that function as sites of cell adhesion and active extracellular matrix remodeling (28). Podosomes have classically been found in highly motile, invasive cells such as macrophages and osteoclasts, Rous sarcoma virus-transformed fibroblasts, and carcinoma cell lines. More recently, treatment with phorbol esters has been shown to induce the formation of podosome in A7r5 cells, a cell line derived from embryonic rat vascular smooth muscle (18).

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 {alpha}-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 (PAK1–PAK3) 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 {beta}-PIX in podosome formation is highlighted by the fact that overexpression of {beta}-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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
DNA constructs. A plasmid encoding for enhanced green fluorescent protein (EGFP)-{beta}-actin was purchased from Clontech BD Biosciences (Palo Alto, CA). The cDNA for Myc-tagged human PAK1, H83,86L/T422E (LL/TE), and H83,86L/K299R (LL/KR) in the pCMV6 plasmid were gifts from Dr. W. T. Gerthoffer (University of Nevada, Reno, NV) (12, 43). To generate wild-type, P192G/R193A, and H83,86L PAK1, the above plasmids were used as templates for site-directed mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The full-length cDNA-encoding mouse FLAG-tagged {beta}-PIX variant 1 in the pCMV6 plasmid was a gift from Dr. R. T. Premont (Duke University, Durham, NC) (3). For the construction of Myc-tagged {beta}-PIX, the cDNA of {beta}-PIX was subcloned into a modified pcDNA3.1 vector (Invitrogen, Burlington, ON, Canada) in which a linker was ligated into the start of the multiple coding site and encoded for an initiating Met codon followed by a Myc epitope tag (EQKLISEEDL). The identity of all constructs was confirmed by conducting DNA sequence analysis. The size and integrity of all constructs was confirmed using Western blot analysis of lysates of transiently transfected COS-1 and A7r5 cells by probing with antibodies specific to each protein and epitope tag (data not shown).

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 {Delta}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 {beta}-PIX- and EGFP-{beta}-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 {beta}-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 (48–72 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-{beta}-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 {beta}-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 [{gamma}-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 20–60 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-{beta}-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 {Delta}T dishes were transfected with pEGFP-{beta}-actin alone or cotransfected with pEGFP-{beta}-actin and either pCMV6 LL/TE or LL/KR PAK1. {Delta}T dishes were moved to a Bioptechs {Delta}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 = Poe–kt, 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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Fig. 1. Kinase activity and p21-activated kinase (PAK)-interacting exchange factor (PIX)-binding activities of p21-activated kinase 1 (PAK1) mutants. A: comparison of the kinase activity of PAK1 mutants. A7r5 cells were transiently transfected with vector alone (Mock), H83,86L (LL), H83,86L/T422E (LL/TE), or H83,86L/K298R (LL/KR) PAK1. PAK1 was immunoprecipitated and assayed for in vitro kinase activity using myelin basic protein (MBP) as a substrate. An autoradiogram (AR) identified 32P-labeled MBP. Relative specific activity was determined by normalizing the intensity of radioactive bands to Coomassie blue (CB)-stained protein bands. The mutations LL and LL/TE enhanced the catalytic activity of PAK1, while LL/KR effectively inhibited catalytic activity. B: comparison of PIX binding of LL/KR and LL/KR/PIX mutants. COS-1 cells were transfected with plasmids encoding for FLAG-{beta}-PIX, Myc-LL/KR, Myc-LL/KR/PIX, or empty vector as indicated. FLAG-{beta}-PIX were immunoprecipitated (IP) from cell lysates and probed with anti-Myc antibody to detect PAK1 mutants. The flow-through from the precipitation was also examined using Western blot analysis to ensure that both LL/KR PAK1 and LL/KR/PIX PAK1 were expressed in approximately equal amounts. Mutation of the PIX binding site of PAK1 effectively prevented association of PAK with PIX.

 


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Fig. 4. Actin columns induced by PAK1 contain podosomal marker proteins. Shown are confocal images of A7r5 cells cotransfected with plasmids expressing LL/KR PAK1 and stained for the presence of known podosomal marker proteins (Marker) cortactin (A), Arp3 (B), caldesmon (C), and {alpha}-actinin (D). Left to right: fluorescent staining of LL/KR PAK1, phalloidin-labeled actin, protein markers, and overlay of three channels (PAK, blue; actin, red; podosomal marker protein, green). AD, insets, top right column: x4 magnification of area within smaller white rectangle in each image. Bars, 20 µm.

 

    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
PAK1 mutants defective in kinase activity and PIX binding. To assess the effect of the kinase activity of PAK1 on cytoskeletal rearrangements in A7r5 smooth muscle cells, we used a kinase-dead mutant, LL/KR, as well as two constitutively active mutants, LL and LL/TE, known to have moderate and high kinase activity, respectively (see Supplementary Fig. 1) (17, 32). The three mutants were expressed in A7r5 cells, and their in vitro kinase activity was measured. As shown in Fig. 1, both immunoprecipitated Myc-tagged LL and LL/TE mutants from A7r5 cells were active toward MBP; the additional T422E mutation at the active loop of the kinase domain increased the specific activity by ~50% (23, 44). A K299R mutation at the kinase subdomain II effectively abolished kinase activity (Fig. 1A).

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 {beta}-PIX from COS-1 cells that coexpressed both proteins, indicating that the P192,193A mutation effectively abolished interaction between PAK1 and {beta}-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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Fig. 2. PAK1 functional mutants induce formation of actin columns. AE: confocal images showing A7r5 cells expressing vector control (A), wild-type PAK1 (B), LL-PAK1 mutant (C), constitutively kinase-active LL/TE PAK1 mutant (D), and kinase-defective LL/KR-PAK1 mutant (E). Left: Myc-tagged PAK1 mutants; right: phalloidin-labeled actin filaments. CE, right-corner insets: x2 magnification of the area within the smaller white squares. F: x-z profiles of actin columns induced by LL/KR PAK1 (left) and phorbol ester (right). G: podosomes induced by stimulation of A7r5 cells with 1 µM phorbol ester phorbol 12,13-dibutyrate (PDBu) for 30 min. Bars, 20 µm.

 
As shown in Fig. 2C, A7r5 cells overexpressing both kinase-active LL and LL/TE mutants, which have impaired binding to Cdc42/Rac and lack autoinhibitory ability, generally assumed a more rounded cell shape, with a marked increase in peripheral ruffles and lamellipodia, and demonstrated a significant but not complete loss of actin stress fibers as observed previously in fibroblasts (17). The highly kinase-active LL/TE PAK1 produced a greater number of large, polarized lamellipodia than the moderately active LL PAK1. However, in contrast to fibroblasts, overexpression of LL and LL/TE PAK1 in smooth muscle cells also caused the formation of many punctate F-actin-containing structures (Fig. 2, C and D, insets) in ~50% of the transfected A7r5 cells (Supplementary Fig. 2), while the other 50% of transfected cells that contained few or no actin columns generally retained most of the stress fibers and tended to produce more lamellipodia and ruffles (see Fig. 2C, right). Vertical cross-sections of the punctate actin structures reconstructed using confocal microscopic imaging revealed cone-shaped columns of filamentous actin with diameters between 0.5 and 2 µm (Fig. 2F). The actin columns extended from the layer of actin stress fibers, located within 0.5 µm of the ventral surface, to a height of 1.4–2 µm. Morphologically, the actin columns resembled the podosome-like structures induced in A7r5 cells upon phorbol ester treatment as shown in Fig. 2, F and G (18, 19, 21).

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 30–40% 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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Table 1. Properties of podosomes induced by catalytically active and inactive PAK1

 
To investigate whether PAK-induced actin columns share dynamic properties similar to those of podosomes, we produced time-lapse images of A7r5 cells either transfected with a plasmid encoding for EGFP-{beta}-actin alone or cotransfected with EGFP-{beta}-actin and LL/KR or LL/TE PAK1 (see movies in Supplementary Figs. 3 and 4). Control cells expressing EGFP-{beta}-actin alone appeared to be polarized, with membrane ruffling occurring at one edge of the cell, and contained prominent stress fibers that remained fairly static within 30 min (Fig. 3A; Supplementary Fig. 3). In contrast, cells expressing LL/KR PAK1 were marked by membrane ruffling around the entire periphery of the cell and by the presence of transient punctate actin columns (Fig. 3B; Supplementary Fig. 4). The actin columns arose from areas on or near stress fibers and were dynamic in nature, having a mean lifetime of 1.71 ± 0.18 min (Table 1), which is similar to that of podosomes in osteoclasts (13) and in A7r5 cells treated with phorbol ester (data not shown; Ref. 19).



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Fig. 3. Actin columns induced by PAK1 are highly dynamic. A and B: selected frames from a time-lapse sequence (please refer to the Supplementary Material for this article to view movies) of live A7r5 cell cotransfected with plasmids expressing either enhanced green fluorescent protein (EGFP)-{beta}-actin alone (A) or the kinase-defective LL/KR PAK1 and EGFP-{beta}-actin (B). The elapsed time is indicated below the individual frames. Inset in B is magnified x2 and shown at right. Cells expressing EGFP-{beta}-actin have strong stress fibers and polarized membrane ruffling. In contrast, cells expressing both EGFP-{beta}-actin and LL/KR PAK1 show ruffling around the entire cell and the formation of actin columns throughout the cell. These podosome-like actin columns rapidly assemble and disassemble, with an average lifetime of ~2 min. Bars, 50 µm.

 
As shown in Table 1, LL/KR-transfected cells produced significantly more actin columns per cell than its kinase-active counterpart, LL/TE. This observation suggests that PAK kinase activity may play a part in the observed difference in the number of actin columns at steady state by affecting the dynamics of podosome formation. Comparison of time-lapse images of cells expressing LL/TE and LL/KR indeed showed that actin columns induced by the kinase-active PAK have significantly higher turnover rate (0.017 s–1), shorter lifetime (1.35 min), and shorter (41.4 s) than those of the kinase-dead PAK1 (0.012 s–1, 1.71 min, and 58.7 s, respectively).

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 {alpha}-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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Fig. 5. Actin columns induced by PAK1 localize in close proximity to the stress fiber-focal adhesion interfaces. A: confocal images of A7r5 cells cotransfected with plasmids expressing LL/KR PAK1 and vinculin (scale bar, 20 µm); left to right: Myc-tagged LL/KR PAK1, phalloidin-labeled F-actin, vinculin, and composite overlay image showing LL/KR (blue), actin (red), and vinculin (green). B: x4 magnification of the white circle area shown in A, right, overlay image. C: x-z profiles of the rectangle in B showing actin columns next to vinculin-containing focal adhesions. D: x4 magnification of the white square area shown in A, right, overlay image. E: x-z profiles of the rectangle in D showing that some actin columns appear to be surrounded by a ring of vinculin. The x-z profiles in C and E also show that LL/KR PAK1 was enriched in and around the areas occupied by the actin columns. Because PAK enrichment at actin columns generally covers a broader area than that surrounding the columns, it becomes obvious only in x-z profiles.

 
The bulk of LL/KR PAK1 was distributed fairly evenly in the cytoplasm and was concentrated at ruffles and lamellipodia (Fig. 5, A and B). Enrichment of the PAK mutant could also be observed at and around the actin columns, covering a broader and more diffuse area than that occupied by the actin columns. This is shown more clearly in vertical x-z sections of actin columns (Fig. 5, C and E).

Actin columns induced by the kinase-active PAK1 mutants, LL PAK1 or LL/TE PAK1, have staining patterns for cortactin, Arp3, caldesmon, {alpha}-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), {beta}-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 {beta}-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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Fig. 6. PAK1 induces the translocation of {beta}-PIX, G protein-coupled receptor kinase-interacting protein (GIT), and paxillin to focal adhesions in a PIX-binding dependent manner. AF: confocal images of A7r5 cells expressing LL/KR PAK1 (AC) or the PIX-binding-deficient LL/KR/PIX PAK1 (DF). AF, left to right: fluorescent staining for either LL/KR (AC) or LL/KR/PIX (DF), fluorescent staining of phalloidin-labeled actin, fluorescent staining using primary antibodies (Antibody) to identify {beta}-PIX (A and D), GIT (B and E), or paxillin (C and F), and overlays (far right) of 3 channels (PAK mutants, blue; actin, red; protein stain, green). G: x2 magnification of the area within the white box in B, far right, showing the relative location of GIT, actin, and LL/KR PAK1. H: x-z profiles of the area shown within the rectangle in G. Bars, 20 µm.

 
Next, we determined whether interaction between PAK and PIX is required for the targeting of PIX and GIT to focal adhesion complexes and the formation of actin columns. As shown in Fig. 6, DF, transfection of PIX-binding mutants of PAK1, LL/PIX, and LL/KR/PIX failed to induce the translocation of PIX and GIT to focal adhesion complexes. In addition, these constructs are unable to induce the formation of actin columns in A7r5 cells. To further study the role of PIX in actin column formation, we tested whether overexpression of {beta}-PIX alone could induce the formation of actin columns in A7r5 cells (Fig. 7). About 40% of the A7r5 cells expressing {beta}-PIX displayed the formation of actin columns at the cell periphery, and, unlike cells transfected with PAK mutants, most of the PIX-transfected cells retained stress fibers similar to those of untransfected or vector-transfected cells (Fig. 7). {beta}-PIX was diffuse throughout the cytoplasm but also was concentrated in actin columns at the periphery of the cell. These findings strongly suggest that interaction between PIX and PAK1 is essential for the formation of actin columns in A7r5 cells as well as for the translocation of PIX and PKL to the focal adhesions abutting the actin columns.



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Fig. 7. PIX alone promotes podosome formation as shown in images of A7r5 cells expressing Myc-tagged {beta}-PIX. Cells were stained with anti-Myc (left) and tetramethylrhodamine isothiocyanate-phalloidin (right). {beta}-PIX colocalized with actin columns and enhanced the formation of these structures. Bar, 20 µm.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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PAK-induced actin columns in A7r5 smooth muscle cells resemble podosomes. We have demonstrated that overexpression of either kinase-active or kinase-inactive PAK1 mutants drives the formation of dynamic F-actin columns in A7r5 vascular smooth muscle cells. These actin columns closely resemble podosomes in general and, in particular, podosomes in A7r5 smooth muscle cells induced by phorbol ester with respect to their physical, chemical, and dynamic properties (18, 19, 21, 28). Both PDBu- and PAK1-induced structures contained a vertical core of actin filaments 0.5–2 µm in diameter that extended vertically from the base of the cell to close to the dorsal surface (Figs. 2F and 5, C and E) (18). Both structures assembled at sites adjacent to focal adhesions and the ends of stress fibers (Fig. 5) (8, 24). The actin columns induced by PAK have an ~2-min lifetime, similar to that of podosomes found in A7r5 cells (19) and osteoclasts (13). PAK1-induced actin columns contain actin-binding proteins known to be present in podosomes, including cortactin, the Arp2/3 complex, {alpha}-actinin, and caldesmon (8, 9, 18, 24, 29, 34, 39, 47). Some PAK1-induced actin columns also recruit the focal adhesion protein vinculin (Fig. 5) to a ringlike structure surrounding the dense F-actin core similar to that observed in podosomes in phorbol ester-treated A7r5 cells (21), osteoclasts (2), and Src-transformed fibroblasts (45). On the basis of these similarities, it seems likely that PAK1-induced actin columns are indeed the podosome-like structures previously found in A7r5 cells upon PDBu stimulation (18, 19, 21).

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.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
This work was supported by grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Ontario (HSFO; to A. S. Mak) and from HSFO (to G. P. Côté). B. A. Webb was supported by an Ontario Graduate Doctoral Scholarship. S. W. Crawley was supported by a HSFO Doctoral Research Scholarship.


    ACKNOWLEDGMENTS
 
We thank Dr. R. T. Premont and Dr. W. T. Gerthoffer for the plasmids used in this study and the Protein Function Discovery Facility for the use of the imaging facilities. Dr. Lilly Jia is gratefully acknowledged for technical support.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. S. Mak, Dept. of Biochemistry, Queen's Univ., Botterell Hall, Room 616, Kingston, ON, Canada K7L 3N6 (e-mail: maka{at}post.queensu.ca)

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. Back


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