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Originally published In Press as doi:10.1074/jbc.M609983200 on January 15, 2007

J. Biol. Chem., Vol. 282, Issue 11, 8454-8463, March 16, 2007
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Changes in the Balance between Caldesmon Regulated by p21-activated Kinases and the Arp2/3 Complex Govern Podosome Formation*Formula

Tsuyoshi Morita, Taira Mayanagi, Toshiyuki Yoshio, and Kenji Sobue1

From the Department of Neuroscience (D13), Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita, Osaka 565-0871, Japan

Received for publication, October 24, 2006 , and in revised form, December 26, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The podosomes found in monocyte-derived cells, osteoclasts, and smooth muscle cells are protrusions from the ventral surface of the plasma membrane and are highly dynamic structures of cell adhesion (1-5). The similar adherent protrusions formed in Rous sarcoma virus (RSV)2-transformed fibroblasts were named invadopodia by Chen (6). Invadopodia degrade the extracellular matrix (ECM) with matrix metallo-proteinases, which implicates remodeling of ECM (6). As podosomes and invadopodia appear to be similar in morphology, functions, and molecular compositions, they are considered to be related structures with different cellular contexts. The podosomes are composed of actin, its associated proteins, and signaling molecules (2-4). Within the podosome, a central core of actin filaments is surrounded by a juxtamembranous ring that is enriched in vinculin (7), {alpha}-actinin (7, 8), talin (7), and non-erythroid spectrin (8, 9). Neural WASP (N-WASP) and the Arp2/3 complex are also localized to the podosomes (10-12). These proteins function with a variety of other actin-associated proteins, such as cortactin, Cdc42, profilin, and cofilin, to mediate actin polymerization and actin network formation toward the plasma membrane (4). In fact, blocking the functions of N-WASP and the Arp2/3 complex using dominant-negative mutants and RNA interference inhibits podosome formation (11-13), suggesting that N-WASP and the Arp2/3 complex are crucial for the formation and function of these adhesive structures.

Caldesmon (CaD), which is an actin- and calmodulin-binding protein, controls smooth muscle and non-muscle actin-myosin interactions (14, 15). Two different molecular weight (Mr) isoforms of CaD are identified: high molecular weight CaD (h-CaD; 120-150 kDa) and low molecular weight CaD (l-CaD; 70-80 kDa). They are generated from a single gene by alternative splicing. h-CaD is exclusively expressed in smooth muscle cells, but l-CaD is widely distributed in non-muscle cells (15). In addition to controlling actin-myosin interactions, l-CaD in collaboration with tropomyosin (TM) stabilizes parallel actin filaments (16). In vitro reconstitution experiments further revealed that CaD competes with filamin, Arp2/3 complex, and cofilin to bind to F-actin (17-19). In a variety of cultured cells, l-CaD and TM are co-distributed along stress fibers (20, 21). We reported previously that in RSV-transformed fibroblasts, l-CaD is mostly concentrated in the F-actin core of podosomes but is excluded from the focal adhesions of normal fibroblasts (22). The expression of l-CaD is down-regulated in certain transformed and cancer cells (20, 22, 23). The significance of the expression and localization, however, remained unclear. During the preparation of this manuscript, Eves et al. (24) have reported a role of CaD in the negative regulation of podosome formation in smooth muscle A7r5 cells. Here we provided additional information that extends the understanding of the molecular mechanism of CaD-regulated podosome formation. We clearly demonstrated that changes in the balance between CaD regulated by p21-activated kinases (PAKs) and the N-WASP-Arp2/3 pathway dictate the formation of podosomes in RSV-transformed cells.


Figure 1
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  		FIGURE 1.
Expression and localization of CaD, TM, N-WASP, and the Arp2/3 complex in 3Y1 and BY1 cells. A, expression levels of the indicated proteins in 3Y1 and BY1 cells compared by immunoblot. B, 3Y1 and BY1 cells were stained with indicated antibodies (green) and phalloidin (red). Insets show Z projections of podosomes in BY1 cells. Because the expression levels of CaD and TM were low in BY1 cells, we changed the exposure time to detect the immunofluorescence for CaD and TM in BY1 cells. Scale bar, 20 µm. C, panel a, trace amounts of pEGFP-Arp2/3 (green) and pDsRed-CaD (red) were expressed in BY1 cells cultured on Matrigel-coated coverslips. Panel b, Z projection of the region indicated by the white line in a. Scale bar, 20 µm.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—BY1 is a clonal line of RSV-transformed 3Y1 cells derived from Fisher rat embryos (25). These cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.

Expression Vectors and Transfection—Coding regions of rat Arp3, PAK1, and PAK2 were amplified by PCR using 3Y1 cDNA as the template. The coding region of human l-CaD used was as described previously (26). The HA tag or FLAG tag sequence was fused to the 5' end of the coding sequences of these genes by PCR. Each fragment was inserted into the mammalian expression vector pcDNA3.1(+) (Invitrogen). The pEGFP-Arp3 and pDsRed-CaD vectors were constructed by inserting the coding sequence of rat Arp3 or human l-CaD into pEGFP-C2 Vector or pDsRed-Monomer-C1 Vector (Clontech). The pcDNA3.1(+)-GFP-actin vector was constructed by inserting the coding sequence of human beta-actin into pEGFP-C2. pcDNA3.1(+)-HA-CaD(S479/509A), pcDNA3.1(+)-HA-CaD(S479/509D), pcDNA3.1(+)-HA-PAK1(K298R), pcDNA3.1(+)-HA-PAK4(K352/353A), and pcDNA3.1(+)-HA-PAK1(T422E) were constructed by site-directed mutagenesis. pCAGGS-myc-Rac1-V12, pCAGGS-myc-Rac1-N17, pCAGGS-myc-Cdc42-V12, and pCAGGS-myc-Cdc42-N17 were prepared as described previously (27). BY1 cells were transfected with these expression vectors using Optifect transfection reagent (Invitrogen). To establish stable cell lines expressing HA-CaD, BY1 cells were transfected with pcDNA3.1(+)-HA-CaD. These cells were cultured with 100 µg/ml G418, and the drug-resistant clones were isolated.

Immunofluorescence Microscopic and Time-lapse Image Analyses—Cells grown on coverslips were fixed using 4% paraformaldehyde in phosphate-buffered saline for 30 min at room temperature and then permeabilized with 0.2% Triton X-100 in phosphate-buffered saline. The cells were incubated with primary antibody, followed by the appropriate secondary antibody. To visualize actin filaments, Alexa 568- or Alexa 350-conjugated phalloidin (Molecular Probes) was added with the secondary antibody solution. When endogenous CaD and PAK1 were doubly stained, anti-CaD antibody was labeled using the Zenon Alexa Fluor 488 rabbit IgG labeling kit (Molecular Probes). Because the expression levels of endogenous PAK1 and PAK2 were too low in BY1 cells to be detected by immunostaining, immunoreaction-enhanced Can Get Signal immunostain (Toyobo) was used as a dilution solution for the antibodies. Antibodies used in this study are listed in supplemental Table 1. For time-lapse image analysis, BY1 cells were transfected with CaD siRNA (CaD-depleted cells) or scrambled siRNA (control cells). After 2 days of transfection, the cells were transfected with pcDNA3.1(+)-GFP-actin, and GFP fluorescence was observed every 20 s under the Axiovert 200 (Carl Zeiss).


Figure 2
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  		FIGURE 2.
Effects of overexpression and depletion of CaD on podosome formation. A, HA-CaD was transiently expressed in BY cells. The cells were stained with anti-HA antibody (green) and phalloidin (red). Scale bar, 20 µm. B, expression levels of endogenous CaD and exogenous HA-CaD in BY1-CaD stable cell lines (clones C4 and C11) shown by immunoblot using anti-CaD antibody. C, 3Y1, BY1, and BY1-CaD cells (clones C4 and C11) stained with phalloidin. Scale bar, 20 µm. D, depletion of CaD in BY1 cells using CaD siRNA shown by immunoblot. E, BY1 cells transfected with scrambled siRNA (control cells) or CaD siRNA (CaD-depleted cells) were stained with phalloidin. Scale bar, 20 µm. F, time-lapse image analysis of podosome dynamics in control and CaD-depleted BY1 cells co-transfected with GFP-actin to monitor podosome dynamics. Scale bars, 10 µm. G, lifetimes of podosomes in control (black bars) and CaD-depleted BY1 (white bars) cells.

 
Actin Binding Assay—F-actin was polymerized in buffer (20 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM dithiothreitol, 4 µM phalloidin, 0.2 mM ATP) for 40 min at 30 °C. After polymerization, a 0.3 or 0.6 µM C-terminal fragment of CaD (CaD39 (28)) was added, and the mixtures were incubated for 30 min at 30 °C. Next, 10 nM Arp2/3 complex (Cytoskeleton Inc.) and 50 nM GST-VCA (Cytoskeleton Inc.) were added, and the mixtures were incubated for 20 min at 30 °C. After the incubation, the samples were spun (105,000 x g, 2 h) to collect the F-actin associated with CaD and the Arp2/3 complex. The proteins in the pellets were dissolved in SDS sample buffer, separated by SDS-gel electrophoresis, and detected by immunoblot using the respective antibody.

Actin Polymerization Assay—Actin polymerization assay was performed using the actin polymerization biochem kit (Cytoskeleton Inc.). Briefly, 0.4 mg/ml pyrene-labeled actin was mixed with 15 nM Arp2/3 complex, 50 nM GST-VCA, and 0-1 µM recombinant CaD39 in general actin buffer, in which 10 mM dithiothreitol was added to prevent the dimerization of CaD protein. The pyrene fluorescence signal was measured by Spectra Max Gemini microplate spectrofluorometer (Molecular Devices) every 7 s from just after the addition of actin polymerization buffer.

Co-immunoprecipitation—The cells were lysed with buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 50 mM NaF, 1 mM Na3VO4, 1 mM beta-glycerophosphate, and protease inhibitor mixture tablets (Roche Applied Science) (pH 7.5)) and incubated on ice for 15 min. The lysates were spun at 10,000 x g for 30 min. The resulting supernatants were incubated with the indicated antibodies for 4 h at 4 °C. After incubation, protein A-Sepharose or protein G-Sepharose (Amersham Biosciences) was added to the mixtures, which were incubated overnight at 4 °C. To elute the immunocomplexes, the Sepharose beads were washed four times with lysis buffer, and SDS-sample buffer was then added.


Figure 3
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  		FIGURE 3.
Competitive effects of CaD and the Arp2/3 complex on podosome formation. A, localization of the p34 Arc subunit of the Arp2/3 complex in CaD-depleted BY1 cells. The cells were stained with anti-p34 Arc antibody (green) and phalloidin (red). Scale bars, 20 µm. B, depletion of p34 Arc or CaD/p34 Arc in BY1 cells by their respective siRNA is shown by immunoblot. C, CaD-, p34 Arc-, and CaD/p34 Arc-depleted BY1 cells stained with phalloidin. Scale bar, 20 µm. D, p34 Arc-depleted BY1 cells were double-stained with anti-CaD antibody (green) and phalloidin (red). Scale bar, 20 µm. E, co-sedimentation assay of F-actin with CaD and the Arp2/3 complex. The amounts of Arp2/3 complex co-precipitated with F-actin were determined by immunoblot with anti-p34 Arc antibody. The precipitated actin was visualized by staining with Ponceau S. The ratios of p34 Arc and actin in the precipitates are expressed as the amounts of p34 Arc per unit of actin. Statistical analysis was carried out for three independent experiments. *, p < 0.05. F, the inhibitory effect of CaD on Arp2/3-induced actin polymerization was examined by monitoring pyrene fluorescence. The conditions of actin polymerization were as follows: 0.4 mg/ml pyrene-labeled actin, 15 nM Arp2/3, 50 nM GST-VCA, and 0-1.0 µM CaD.

 
RNA Interference—Sequences of siRNAs used in this study are listed in supplemental Table 2. Cells were transfected with these siRNAs using HiPerFect transfection reagent (Qiagen) and cultured for 3 days before analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Localization of Caldesmon, Tropomyosin, N-WASP, and the Arp2/3 Complex in Fibroblasts and Their RSV Trans-formants—CaD and TM bind to parallel actin filaments, stabilizing their alignment (16), whereas the N-WASP-Arp2/3 pathway induces the branching of actin filaments (29). To investigate the role of these actin-associated proteins in podosome formation, we compared their expression and localization in a rat fibroblast cell line (3Y1) and its RSV-transformant cell line (BY1). Consistent with our previous findings (22), l-CaD and high molecular weight TMs (TM1 and TM2) proteins were markedly reduced in BY1 cells compared with parental 3Y1 cells. Low molecular weight TM was expressed at the same levels in both cell lines. The N-WASP protein expression was greater in BY1 cells than in 3Y1 cells, whereas Cdc42, an activator of N-WASP, and the Arp2/3 complex (p34 Arc and p21 Arc subunits), a downstream target of N-WASP, were expressed at the same levels in both lines (Fig. 1A). Because the 3Y1 and BY1 cells examined expressed only l-CaD, we refer to l-CaD as CaD throughout this study, unless otherwise noted.


Figure 4
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  		FIGURE 4.
Expression and subcellular localization of PAKs. A, expression levels of PAK1, PAK2, and PAK3 in 3Y1 and BY1 cells shown by immunoblot. Anti-PAK3 antibody ({gamma}PAK (N-19)) cross-reacted weakly with PAK2 but did not detect PAK3 in either cell line. B, BY1 cells were doubly stained with indicated antibodies (PAK1/2, red; CaD, green) and phalloidin (blue). Anti-CaD antibodies were labeled using Zenon Alexa Fluor 488 rabbit IgG when double staining with anti-PAK1 antibody, because both antibodies were raised in rabbit. Scale bar, 30 µm.

 
In 3Y1 cells, CaD and TM were localized along stress fibers, whereas N-WASP and a subunit (p34 Arc) of the Arp2/3 complex were diffusely distributed in the cytoplasm, with a limited accumulation at the ruffling membrane (Fig. 1B). In BY1 cells, dot-shaped F-actin clusters were predominant in the podosomes, replacing stress fibers. CaD, N-WASP, and p34 Arc were concentrated in the core of podosomes, and TM was diffusely distributed within podosomes (Fig. 1B). Three-dimensional reconstruction along the z axis revealed that CaD and TM were preferentially localized to the tip domain of the podosomes, whereas N-WASP and p34 Arc were concentrated at their base (Fig. 1B, insets). To further compare the localizations of the Arp2/3 complex and CaD, we co-expressed trace amounts of GFP-Arp3 and DsRed-CaD in BY1 cells, and we determined their localizations by three-dimensional reconstruction. They partially overlapped each other within the podosomes located at the ventral side of BY1 cells. GFP-Arp3 was mainly concentrated at the podosomal base, whereas DsRed-CaD was preferentially localized to the tip domain (Fig. 1C). These distinct localizations support a recent finding (13) that N-WASP- and Arp2/3-mediated nucleation and branching network formation of actin initially occurs at the base, followed by the elongation of parallel actin filaments within the invadopodia.

Caldesmon as a Repressor of Podosome Formation—Because CaD expression was markedly down-regulated in BY1 cells, we examined the effect of HA-tagged CaD overexpression on podosome formation. HA-CaD reduced the number of podosomes and induced a flattened cell morphology and the formation of cortical actin bundles with thin stress fibers (Fig. 2A). We then cloned stable cell lines expressing HA-CaD from the transfected BY1 cells (BY1-CaD cells). The total amounts of exogenous and endogenous CaD in two BY1-CaD (C4 and C11) clones were nearly equivalent to that of endogenous CaD in 3Y1 cells (Fig. 2B). Consistent with this, these two clones showed a reduced number of podosomes (the average numbers of podosomes per cell: 31.2 ± 2.2 in BY1 cells, 12.4 ± 4.7 in BY1-CaD (C4) cells, and 10.9 ± 4.3 in BY1-CaD(C11) cells), flattened cell morphology, and formation of thin actin bundles (Fig. 2C).

We examined the effect of CaD depletion on podosome formation using siRNA (Fig. 2, D and E). The CaD-depleted BY1 cells had numerous small-sized podosomes at the ventral surface (the average numbers of podosomes per cell: 34.7 ± 2.6 in control BY1 cells and 219.4 ± 29.1 in CaD-depleted BY1 cells). They frequently formed belt-like structures along the cell periphery. Unlike osteoclasts, these structures never formed sealing zones. Compared with podosomes of control cells, small podosomes in CaD-depleted cells were highly dynamic; the positions of small podosomes changed dynamically from the cell periphery to the center of the ventral cell surface and vice versa (Fig. 2F and supplemental Videos 1 (control cells) and 2 (CaD-depleted cells)). The lifetime of small podosomes in CaD-depleted cells, as determined by following individual podosomes visualized by GFP-actin, was shorter than that in control cells (average lifetimes of podosomes: 52.0 ± 6.4 s in CaD-depleted cells and 184.8 ± 10.6 s in control cells) (Fig. 2G).

Competition between CaD and the Arp2/3 Complex Regulates Podosome Formation—CaD-depleted cells had numerous small podosomes in which the p34 Arc subunit of the Arp2/3 complex was highly concentrated (Fig. 3A). In agreement with a previous report (12), the depletion of p34 Arc from BY1 cells resulted in the disassembly of podosomes and the formation of cortical actin bundles that co-localized with CaD (Fig. 3, B-D). This phenotype is similar to that of BY1 cells expressing exogenous CaD, as shown in Fig. 2A. Depletion of both CaD and p34 Arc partially reversed this effect (Fig. 3, B and C), suggesting that the remaining Arp2/3 complex can form podosomes under CaD-depleted conditions. These combined results suggest that the balance between the relative expression levels of CaD and the Arp2/3 complex is critical for podosome formation. In connection with this, Yamakita et al. (18) reported that CaD reduces the affinity of the Arp2/3 complex for F-actin, thereby preventing the Arp2/3-dependent nucleation of actin in vitro. To confirm the competition between CaD and the Arp2/3 complex for actin binding and nucleation, we performed an in vitro co-sedimentation assay and actin polymerization assay using recombinant CaD and the Arp2/3 complex. We found that CaD inhibited both Arp2/3 complex binding to F-actin and Arp2/3 complex-mediated actin polymerization in a dose-dependent manner (Fig. 3, E and F).

Modification of CaD Function in Podosome Formation by PAK Phosphorylation—It has been well documented that CaD is highly phosphorylated by many kinds of kinases, such as Cdc2 kinase (30), ERK (31), casein kinase II (32), protein kinase C (33), cAMP-dependent protein kinase (34), and p38 MAPK (35). Recently, PAK has been also reported as an important kinase for modification of CaD function (36, 37). In BY1 cells, the expression levels of PAK1 and PAK2 were low compared with those in 3Y1 cells (Fig. 4A). PAK3 was not detected in either cell line. Additionally, PAK1 and PAK2 were mainly colocalized in the core region of podosomes with CaD and also in the more peripheral area of podosomes (Fig. 4B). From these results, we investigated the relationship between CaD and PAKs in podosome formation. Foster et al. (36) determined two serine sites of CaD as phosphorylation sites by PAK. To analyze the functional significance of CaD phosphorylation by PAK, we constructed expression vectors of mutant CaD proteins in which the two serine sites were replaced with alanine (CaD(AA)) or aspartate (CaD(DD)). The expression of CaD(DD) in BY1 cells inhibited podosome formation to a similar extent as wild-type CaD (CaD (WT)), whereas CaD(AA) showed a less significant effect (Fig. 5, A and B). Additionally, CaD(WT) and CaD(DD) rescued the aberrant podosome formation in CaD-depleted BY1 cells, whereas CaD(AA) scarcely reduced the number of podosomes in CaD-depleted BY1 cells (Fig. 5C). These data indicate that the phosphorylation of CaD by PAK1 and/or PAK2 enhances the CaD-induced inhibition of podosome formation.


Figure 5
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  		FIGURE 5.
The mutations of PAK phosphorylation sites of CaD. A, BY1 cells transfected with GFP, HA-CaD, HA-CaD(AA), or HA-CaD(DD) were stained with anti-HA antibody (green) and phalloidin (red). Scale bar, 20 µm. B, BY1 cells expressing GFP, HA-CaD, HA-CaD(AA), or HA-CaD(DD) were counted and classified according to their phenotypes as follows: no effect (black bars), compared with nontransfected BY1 cells; weak effect (white bars), slightly decreased number of podosomes; strong effect (gray bars), markedly disassembled podosomes and new formation of cortical actin bundles with thin stress fibers. Statistical analysis was carried out for three independent experiments. One hundred cells per sample were counted in each experiment. C, BY1 cells were transfected with CaD siRNA. After 2 days of transfection, cells were respectively transfected with HA-CaD(WT), HA-CaD(AA), or HA-CaD(DD) and cultured for 24 h. The cells were then stained with anti-HA antibody (green) and phalloidin (red). Scale bar, 20 µm. D, whole cell extracts of BY1 cells transfected with HA-CaD, HA-CaD(AA), or HA-CaD(DD) were prepared, and immunoprecipitation (IP) was performed using anti-HA antibody. The precipitates were analyzed by immunoblot (IB) using the indicated antibodies. E, amounts of p34 Arc and actin in the precipitates were determined from immunoblot (D) and expressed as the amounts of p34 Arc per unit of actin. Statistical analysis was carried out for three independent experiments. *, p < 0.05.

 
As shown in Fig. 3, CaD competes with the Arp2/3 complex for actin binding in vitro and podosome formation. To analyze whether this competition is further modulated by the PAK-dependent phosphorylation of CaD, we transfected HA-tagged CaD(WT), CaD(AA), or CaD(DD) into BY1 cells and collected the CaD-actin-Arp2/3 complex from the extracts of transfected cells by co-immunoprecipitation using anti-HA antibody. When HA-CaD(WT) or HA-CaD(DD) was expressed in BY1 cells, only a little Arp2/3 complex was co-immunoprecipitated with actin. In contrast, the amount of Arp2/3 complex bound to actin in BY1 cells expressing HA-CaD(AA) was 5-fold higher than that in the HA-CaD(WT)- or HA-CaD(DD)-expressing cells (Fig. 5, D and E). These data suggest that the phosphorylation of CaD by PAKs enhances its inhibitory effect on podosome formation by increasing its ability to compete with the Arp2/3 complex.

Inhibitory Function of PAK in Podosome Formation—To further analyze the involvement of PAK1 and PAK2 in podosome formation, we depleted or overexpressed these kinases in BY1 cells. Like CaD-depleted cells, PAK1- and/or PAK2-depleted cells had numerous small podosomes. Most cells displayed belt-like organization or large clusters of small podosomes (Fig. 6, A and B). The expression of HA-PAK1 or HA-PAK2 markedly induced podosome disassembly and the formation of cortical actin bundles (Fig. 6, C and D). Although exogenous PAK3 expression also impaired podosome formation, its efficiency was much lower than that of PAK1 and PAK2. The formation of cortical actin bundles was less significant in PAK3-transfected BY1 cells (Fig. 6, C and D). We also confirmed that the expression level of endogenous CaD was not altered by PAK1/2 overexpression and PAK1/2 depletion (Fig. 6E). These data indicate that both PAK1 and PAK2 are, like CaD, repressors of podosome formation.


Figure 6
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  		FIGURE 6.
Involvement of PAKs in podosome formation. A, effect of siRNAs on the expression of PAK1 and PAK2 in BY1 cells shown by immunoblot. B, BY1 cells transfected with control siRNA, or PAK1, and/or PAK2 siRNAs were stained with phalloidin. Scale bar, 20 µm. C, BY1 cells transfected with GFP, HA-PAK1, HA-PAK2, or HA-PAK3 were stained with anti-HA antibody (green) and phalloidin (red). Arrowheads indicate newly formed cortical actin bundles. Scale bar, 20 µm. D, BY1 cells expressing GFP, HA-PAK1, HA-PAK2, or HA-PAK3 were counted and classified as described in Fig. 5B: no effect (black bars), weak effect (white bars), and strong effect (gray bars). Statistical analysis was carried out for three independent experiments. One hundred cells per sample were counted in each experiment. E, BY1 cells were transfected with HA-PAK1, HA-PAK2, or siRNAs against PAK1 and PAK2, respectively. Total proteins were extracted from these cells, and Western analyses were performed using indicated antibodies.

 
To assess the necessity of PAK kinase activity in podosome formation, we used a kinase-dead K298R mutant of PAK1 (PAK1-KD). Exogenous PAK1-KD was localized in podosomes and interacted with substrata CaD similar to WT PAK1 in BY1 cells (Fig. 7, A and B). BY1 cells transfected with PAK1-KD showed a phenotype similar to that seen in PAK1- and 2-depleted cells, namely formation of numerous small-sized podosomes. These results indicate that PAK1-KD competes with endogenous PAKs to interact with a downstream kinase target CaD, and forms nonproductive complexes with CaD, thereby approaching the dominant-negative effect as reported previously (38). Additionally, disassembly of podosomes by exogenous CaD expression was markedly reverted by the co-expression of PAK1-KD (Fig. 7, C and D), supporting the idea that PAK1 and/or PAK2 phosphorylate CaD and modulate the CaD-induced inhibition of podosome formation.

Involvement of Rac1/Cdc42 in Podosome Formation—PAKs are downstream targets of Rho family GTPases Rac1 and Cdc42, and the N-WASP-Arp2/3 signaling is also regulated by Cdc42 (39, 40). Rac1 and Cdc42, respectively, regulate lamellipodia and filopodia formation through activation of Arp2/3-mediated actin nucleation and polymerization (41). To examine whether Rac1 and Cdc42 regulate podosome formation in BY1 cells, constitutively active Rac1 or Cdc42 (Rac1-V12 or Cdc42-V12) was expressed in BY1 cells. The ectopic expression of Rac1-V12 or Cdc42-V12 in BY1 cells resulted in the disappearance of podosomes (Fig. 8, A and B). We also transfected dominant-negative Rac1 or Cdc42 (Rac1-N17 and Cdc42-N17) into BY1 cells, and we observed that they also inhibited podosome formation (Fig. 8, A and B).


Figure 7
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  		FIGURE 7.
The effect of kinase-dead PAK1 on podosome formation. A, trace amount of HA-PAK1-KD was transfected in BY1 cells. The cells were stained with anti-HA antibody (green) and phalloidin (red). Scale bar, 20 µm. B, BY1 cells were co-transfected with the indicated combinations of FLAG-CaD, HA-PAK1, or HA-PAK1-KD. Whole cell extracts of the cells were prepared, and co-immunoprecipitation (IP) was performed using the indicated antibodies. The proteins in the precipitates were detected by immunoblot (IB) using the indicated antibodies. C, BY1 cells transfected with HA-PAK1-KD and/or FLAG-CaD were stained with anti-HA or anti-FLAG antibody and phalloidin. Scale bar, 20 µm. D, BY1 cells expressing HA-PAK1-KD and/or HA-CaD were counted and classified according to the criteria described in Fig, 5B: no effect (black bars), weak effect (white bars), and strong effect (gray bars). Statistical analysis was carried out for three independent experiments. One hundred cells per sample were counted in each experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Mounting evidence suggests that the molecules responsible for podosome formation are actin and its associated proteins as listed in the Introduction. CaD controls actin-myosin interactions and stabilizes actin filaments (15). The expression levels of CaD are decreased in several transformed and cancer cells (20, 22, 23), and CaD is localized to the central core of podosomes (Fig. 1) (22). Despite these findings, the role of CaD in transformed and cancer cells has remained unclear. Most recently, Eves et al. (24) have reported that CaD suppressed podosome formation in smooth muscle A7r5 cells. Consistent with this study, we demonstrated here that CaD is a repressor of podosome formation in RSV-transformed cells (BY1) (Fig. 2). We further uncovered the regulatory mode of CaD in podosome formation, in which CaD competes with the Arp2/3 complex for actin binding and thereby podosome formation (Figs. 3 and 5), and the PAK-mediated phosphorylation of CaD enhances this inhibitory mode of podosome formation (Figs. 5, 6 and 7). In BY1 cells, the expressions of CaD, PAK1, and PAK2 are down-regulated, whereas N-WASP is highly expressed (Fig. 1), leading to enhancement of the podosome formation. In this connection, Mizutani et al. (11) reported that ectopic expression of N-WASP in 3Y1-src cells resulted in large podosome-like accumulations of F-actin.

WASP family proteins initiate actin nucleation and the formation of a branching actin network through activation of the Arp2/3 complex (42, 43). Among these proteins, N-WASP, along with the Arp2/3 complex, has been identified in the podosomes (10, 11). RNA interference and dominant-negative mutant expression analyses revealed that N-WASP and the Arp2/3 complex are necessary for podosome formation (10-12). Lorenz et al. (13) clearly demonstrated by using a biosensor technique that N-WASP plays a role in the initiation of invadopodium formation, in which the activation of N-WASP and the Arp2/3 complex are restricted to the base of invadopodia. Consistent with these findings, we demonstrated that N-WASP and an Arp2/3 subunit, p34 Arc, are localized at the base of podosomes, and depletion of p34 Arc inhibits podosome formation in BY1 cells. Conversely, depletion of both p34 Arc and CaD partially restored podosome formation (Fig. 5). Taking these results together, we hypothesize that CaD inhibits the initial stage of podosome formation by competition with the Arp2/3 complex.

The depletion of CaD by RNA interference enhanced the formation of numerous small sized podosomes; however, these podosomes had a significantly shorter lifetime than in control cells (Fig. 2). Thus, our data raise the possibility that CaD displays dual actions, namely inhibition of initiation of podosome formation and stabilization of the mature ones. Supporting this idea, Lorenz et al. (13) reported that the newly nucleated parallel actin filaments are elongated and must push the plasma membrane forward into the ECM in the maturation process of invadopodia. CaD is localized in the tip domain of mature podosomes but not in the base (Fig. 1). CaD and TM are known to protect parallel actin filaments from gelsolin-induced severing (16, 44). Burgstaller and Gimona (45) have reported that in phorbol 12,13-dibutyrate-treated A7r5 cells, TM is not localized to dynamic podosomes at the initiation stage of podosome formation, but it is enriched in mature stable ones. Considering all of these reports, CaD and TM might stabilize the parallel organization of actin filaments at the tip of mature podosomes.

Our present data using RNA interference and exogenous expression of PAK1, PAK2, and kinase-dead PAK1 clearly demonstrated that PAK1 and PAK2 act as repressors of podosome formation in BY1 cells (Figs. 6 and 7). However, Webb et al. (46) reported that ectopic expression of PAK1 in A7r5 cells induces podosome formation in its kinase activity-independent manner. We have not yet resolved this discrepancy, but we think that this discrepancy is caused by a difference in cellular context. They also described that kinase-dead PAK1 induced a larger number of podosomes than highly kinase-active PAK1. These data partly support our idea that the inhibitory function of PAK1/2 on podosome formation depends on its kinase activity through phosphorylation of CaD (Figs. 6 and 7).


Figure 8
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  		FIGURE 8.
Effects of Rac1-V12, Rac1-N17, Cdc42-V12, and Cdc42-N17 on podosome formation. A, GFP, myc-Rac1-V12, myc-Rac1-N17, myc-Cdc42-V12, or myc-Cdc42-N17 were transfected into BY1 cells, respectively. The cells were stained with anti-Myc antibody (green) and phalloidin (red). Scale bar, 20 µm. B, the numbers of podosomes per cell were counted in BY1 cells expressing GFP, myc-Rac1-V12, myc-Rac1-N17, myc-Cdc42-V12, or myc-Cdc42-N17. At least 50 cells were evaluated, and statistical analyses were performed.

 
It is well documented that CaD is highly phosphorylated in vivo and in vitro by various kinases, for example Cdc2 kinase (30), ERK(31), caseinkinaseII(32), proteinkinaseC(33), cAMP-dependent protein kinase (34), and p38 MAPK (35). PAK3 is also reported to phosphorylate h-CaD in vitro (36). Most recently, Eppinga et al. (37) also reported the phosphorylation of l-CaD by PAK in vivo and in vitro. Thus, CaD is a favorable substrate for many kinases, including PAKs. We mutated the phosphorylation sites of l-CaD by PAKs by referring to the previous report (36). Foster et al. (36) previously reported that phosphorylation of CaD by PAK reduces its affinity to actin-tropomyosin in vitro. Eppinga et al. (37) confirmed the reduced actin binding of CaD by PAK phosphorylation using mutant proteins mimicking phosphorylated or unphosphorylated forms of CaD. The mutation sites of these CaD proteins were the same as that of CaD(AA) and CaD(DD) used in this study. Consistent with these in vitro experiments, unphosphorylated CaD(AA) bound to F-actin more tightly than CaD(WT) or CaD(DD) in BY1 cells (Fig. 5D), suggesting that the majority of CaD(WT) is phosphorylated by PAKs in BY1 cells. Despite the high affinity of CaD(AA) to F-actin, the exogenous CaD(AA) had little effect on podosome formation. On the other hand, CaD(WT) and CaD(DD) potently inhibited podosome formation through competition with the Arp2/3 complex for actin binding (Fig. 5, D and E). PAK phosphorylation sites on CaD are located near calmodulin (CaM)-binding sites, and therefore the phosphorylation of CaD by PAKs reduces its affinity to CaM (36, 37). In this connection, Ca2+/CaM reversed the inhibitory effect of CaD on Arp2/3-mediated actin polymerization (18). Eves et al. (24) reported that interaction between CaD and Ca2+/CaM is required for targeting CaD to podosomes. These data suggest that Ca2+/CaM further modulates the action of CaD in podosome formation.

The activity of PAKs is regulated by Rho family GTPases Rac1 and Cdc42 (40). Cdc42 also activates N-WASP, leading to the acceleration of Arp2/3-mediated actin nucleation and polymerization (39, 43). To assess the involvement of Rac1 and Cdc42 in podosome formation in BY1 cells, constitutively active or dominant-negative mutants of Rac1/Cdc42 were expressed in BY1 cells, and all constructs tightly suppressed podosome formation (Fig. 8). In other cell types, such as dendritic cells, macrophages, and osteoclast-like cells, both constitutively active and dominant-negative forms of Rac1/Cdc42 are known to impair or abolish podosome formation (10, 48, 49). On the other hand, Cdc42-V12 is reported to induce the ectopic formation of podosomes in endothelial cells (47). Taken together, our results and these reported data, we consider that Rac1 and Cdc42 display dual functions as positive and negative regulators for podosome formation depending upon the cellular contexts. In BY1 cells, the expression levels of PAKs and caldesmon were markedly reduced, whereas the expression of N-WASP was increased. Under these cellular balances, endogenous Cdc42 might predominantly activate N-WASP-Arp2/3 signaling, resulting in podosome formation in BY1 cells.

In summary, we conclude that changes in the balance between CaD regulated by PAK1 and -2 and the N-WASP-Arp2/3 pathway govern podosome formation in RSV-transformed cells. Future analyses are required to elucidate the molecular mechanism, which results in the down-regulation of CaD and PAKs in these cells. Our present study advances the molecular mechanism of podosome formation and dynamics.


   FOOTNOTES
 
* This work was supported by Grant-in-aid for Scientific Research 15GS0312 from the Ministry of Education, Science, Sports and Culture of Japan (to K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Videos 1 and 2. Back

1 To whom correspondence should be addressed: Dept. of Neuroscience (D13), Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3680; Fax: 81-6-6879-3689; E-mail: sobue{at}nbiochem.med.osaka-u.ac.jp.

2 The abbreviations used are: RSV, Rous sarcoma virus; CaD, caldesmon; TM, tropomyosin; PAK, p21-activated kinase; N-WASP, neural Wiskott-Aldrich syndrome protein; Arp2/3, actin-related proteins 2/3; ECM, extracellular matrix; Rac1, Ras-related C3 botulinum toxin substrate 1; Cdc42, cell division cycle 42; siRNA, short interfering RNA; HA, hemagglutinin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; CaM, calmodulin. Back


   ACKNOWLEDGMENTS
 
We thank Dr. T. Takenawa (Tokyo University) for the kind gift of the anti-N-WASP antibody.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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