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• W1995006965 abstract "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. 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. 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 (1Tarone G. Cirillo D. Giancotti F.G. Comoglio P.M. Marchisio P.C. Exp. Cell Res. 1985; 159: 141-157Crossref PubMed Scopus (341) Google Scholar, 2Sobue K. Neurosci. Res. 1990; 13: S80-S91Google Scholar, 3Linder S. Aepfelbacher M. Trends Cell Biol. 2003; 13: 376-385Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 4Buccione R. Orth J.D. McNiven M.A. Nat. Rev. Mol. Cell Biol. 2004; 5: 647-657Crossref PubMed Scopus (486) Google Scholar, 5Destaing O. Saltel F. Geminard J.C. Jurdic P. Bard F. Mol. Biol. Cell. 2003; 14: 407-416Crossref PubMed Scopus (366) Google Scholar). The similar adherent protrusions formed in Rous sarcoma virus (RSV) 2The 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. 2The 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.-transformed fibroblasts were named invadopodia by Chen (6Chen W.T. J. Exp. Zool. 1989; 251: 167-185Crossref PubMed Scopus (269) Google Scholar). Invadopodia degrade the extracellular matrix (ECM) with matrix metallo-proteinases, which implicates remodeling of ECM (6Chen W.T. J. Exp. Zool. 1989; 251: 167-185Crossref PubMed Scopus (269) Google Scholar). 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 (2Sobue K. Neurosci. Res. 1990; 13: S80-S91Google Scholar, 3Linder S. Aepfelbacher M. Trends Cell Biol. 2003; 13: 376-385Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 4Buccione R. Orth J.D. McNiven M.A. Nat. Rev. Mol. Cell Biol. 2004; 5: 647-657Crossref PubMed Scopus (486) Google Scholar). Within the podosome, a central core of actin filaments is surrounded by a juxtamembranous ring that is enriched in vinculin (7Marchisio P.C. Cirillo D. Teti A. Zambonin-Zallone A. Tarone G. Exp. Cell Res. 1987; 169: 202-214Crossref PubMed Scopus (166) Google Scholar), α-actinin (7Marchisio P.C. Cirillo D. Teti A. Zambonin-Zallone A. Tarone G. Exp. Cell Res. 1987; 169: 202-214Crossref PubMed Scopus (166) Google Scholar, 8Sobue K. Kanda K. Miyamoto I. Iida K. Yahara I. Hirai R. Hira-gun A. Exp. Cell Res. 1989; 181: 256-262Crossref PubMed Scopus (14) Google Scholar), talin (7Marchisio P.C. Cirillo D. Teti A. Zambonin-Zallone A. Tarone G. Exp. Cell Res. 1987; 169: 202-214Crossref PubMed Scopus (166) Google Scholar), and non-erythroid spectrin (8Sobue K. Kanda K. Miyamoto I. Iida K. Yahara I. Hirai R. Hira-gun A. Exp. Cell Res. 1989; 181: 256-262Crossref PubMed Scopus (14) Google Scholar, 9Sobue K. Fujio Y. Kanda K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 482-486Crossref PubMed Scopus (47) Google Scholar). Neural WASP (N-WASP) and the Arp2/3 complex are also localized to the podosomes (10Linder S. Nelson D. Weiss M. Aepfelbacher M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9648-9653Crossref PubMed Scopus (356) Google Scholar, 11Mizutani K. Miki H. He H. Maruta H. Takenawa T. Cancer Res. 2002; 62: 669-674PubMed Google Scholar, 12Yamaguchi H. Lorenz M. Kempiak S. Sarmiento C. Coniglio S. Symons M. Segall J. Eddy R. Miki H. Takenawa T. Condeelis J. J. Cell Biol. 2005; 168: 441-452Crossref PubMed Scopus (541) Google Scholar). 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 (4Buccione R. Orth J.D. McNiven M.A. Nat. Rev. Mol. Cell Biol. 2004; 5: 647-657Crossref PubMed Scopus (486) Google Scholar). In fact, blocking the functions of N-WASP and the Arp2/3 complex using dominant-negative mutants and RNA interference inhibits podosome formation (11Mizutani K. Miki H. He H. Maruta H. Takenawa T. Cancer Res. 2002; 62: 669-674PubMed Google Scholar, 12Yamaguchi H. Lorenz M. Kempiak S. Sarmiento C. Coniglio S. Symons M. Segall J. Eddy R. Miki H. Takenawa T. Condeelis J. J. Cell Biol. 2005; 168: 441-452Crossref PubMed Scopus (541) Google Scholar, 13Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), 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 (14Sobue K. Muramoto Y. Fujita M. Kakiuchi S. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5652-5655Crossref PubMed Scopus (364) Google Scholar, 15Sobue K. Sellers J.R. J. Biol. Chem. 1991; 266: 12115-12118Abstract Full Text PDF PubMed Google Scholar). 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 (15Sobue K. Sellers J.R. J. Biol. Chem. 1991; 266: 12115-12118Abstract Full Text PDF PubMed Google Scholar). In addition to controlling actin-myosin interactions, l-CaD in collaboration with tropomyosin (TM) stabilizes parallel actin filaments (16Ishikawa R. Yamashiro S. Matsumura F. J. Biol. Chem. 1989; 264: 7490-7497Abstract Full Text PDF PubMed Google Scholar). In vitro reconstitution experiments further revealed that CaD competes with filamin, Arp2/3 complex, and cofilin to bind to F-actin (17Nomura M. Yoshikawa K. Tanaka T. Sobue K. Maruyama K. Eur. J. Biochem. 1987; 163: 467-471Crossref PubMed Scopus (29) Google Scholar, 18Yamakita Y. Oosawa F. Yamashiro S. Matsumura F. J. Biol. Chem. 2003; 278: 17937-17944Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 19Yonezawa N. Nishida E. Maekawa S. Sakai H. Biochem. J. 1988; 251: 121-127Crossref PubMed Scopus (38) Google Scholar). In a variety of cultured cells, l-CaD and TM are co-distributed along stress fibers (20Koji-Owada M. Hakura A. Iida K. Yahara I. Sobue K. Kakiuchi S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3133-3137Crossref PubMed Scopus (93) Google Scholar, 21Bretscher A. Lynch W. J. Cell Biol. 1985; 100: 1656-1663Crossref PubMed Scopus (115) Google Scholar). 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 (22Tanaka J. Watanabe T. Nakamura N. Sobue K. J. Cell Sci. 1993; 104: 595-606Crossref PubMed Google Scholar). The expression of l-CaD is down-regulated in certain transformed and cancer cells (20Koji-Owada M. Hakura A. Iida K. Yahara I. Sobue K. Kakiuchi S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3133-3137Crossref PubMed Scopus (93) Google Scholar, 22Tanaka J. Watanabe T. Nakamura N. Sobue K. J. Cell Sci. 1993; 104: 595-606Crossref PubMed Google Scholar, 23Ross D.T. Scherf U. Eisen M.B. Perou C.M. Rees C. Spellman P. Iyer V. Jeffrey S.S. Van de Rijn M. Waltham M. Pergamenschikov A. Lee J.C. Lashkari D. Shalon D. Myers T.G. Weinstein J.N. Botstein D. Brown P.O. Nat. Genet. 2000; 24: 227-235Crossref PubMed Scopus (1821) Google Scholar). The significance of the expression and localization, however, remained unclear. During the preparation of this manuscript, Eves et al. (24Eves R. Webb B.A. Zhou S. Mak A.S. J. Cell Sci. 2006; 119: 1691-1702Crossref PubMed Scopus (51) Google Scholar) 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. Cell Culture—BY1 is a clonal line of RSV-transformed 3Y1 cells derived from Fisher rat embryos (25Kimura G. Itagaki A. Summers J. Int. J. Cancer. 1975; 15: 694-706Crossref PubMed Scopus (278) Google Scholar). 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 (26Hayashi K. Yano H. Hashida T. Takeuchi R. Takeda O. Asada K. Takahashi E. Kato I. Sobue K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12122-12126Crossref PubMed Scopus (73) Google Scholar). 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 β-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 (27Konno D. Yoshimura S. Hori K. Maruoka H. Sobue K. J. Biol. Chem. 2005; 280: 5082-5088Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). 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). 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 (28Novy R.E. Sellers J.R. Liu L.F. Lin J.J.C. Cell Motil. Cytoskeleton. 1993; 26: 248-261Crossref PubMed Scopus (70) Google Scholar)) 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 × 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 β-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 × 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. 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. 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 (16Ishikawa R. Yamashiro S. Matsumura F. J. Biol. Chem. 1989; 264: 7490-7497Abstract Full Text PDF PubMed Google Scholar), whereas the N-WASP-Arp2/3 pathway induces the branching of actin filaments (29Mullins R.D. Heuser J.A. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6181-6186Crossref PubMed Scopus (1030) Google Scholar). 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 (22Tanaka J. Watanabe T. Nakamura N. Sobue K. J. Cell Sci. 1993; 104: 595-606Crossref PubMed Google Scholar), 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. 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 (13Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) 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 (12Yamaguchi H. Lorenz M. Kempiak S. Sarmiento C. Coniglio S. Symons M. Segall J. Eddy R. Miki H. Takenawa T. Condeelis J. J. Cell Biol. 2005; 168: 441-452Crossref PubMed Scopus (541) Google Scholar), 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. (18Yamakita Y. Oosawa F. Yamashiro S. Matsumura F. J. Biol. Chem. 2003; 278: 17937-17944Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) 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 (30Yamashiro S. Yamakita Y. Hosoya H. Matsumura F. Nature. 1991; 349: 169-172Crossref PubMed Scopus (136) Google Scholar), ERK (31Childs T.J. Watson M.H. Sanghera J.S. Campbell D.L. Pelech S.L. Mak A.S. J. Biol. Chem. 1992; 267: 22853-22859Abstract Full Text PDF PubMed Google Scholar), casein kinase II (32Vorotnikov A.V. Shirinsky V.P. Gusev N.B. FEBS Lett. 1988; 236: 321-324Crossref PubMed Scopus (36) Google Scholar), protein kinase C (33Tanaka T. Ohta H. Kanda K. Tanaka T. Hidaka H. Sobue K. Eur. J. Biochem. 1990; 188: 495-500Crossref PubMed Scopus (62) Google Scholar), cAMP-dependent protein kinase (34Hettasch J.M. Sellers J.R. J. Biol. Chem. 1991; 266: 11876-11881Abstract Full Text PDF PubMed Google Scholar), and p38 MAPK (35Hedges J.C. Yamboliev I.A. Ngo M. Horowitz B. Adam L.P. Gerthoffer W.T. Am. J. Physiol. 1998; 275: C527-C534Crossref PubMed Google Scholar). Recently, PAK has been also reported as an important kinase for modification of CaD function (36Foster D.B. Shen L.H. Kelly J. Thibault P. Van Eyk J.E. Mak A.S. J. Biol. Chem. 2000; 275: 1959-1965Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 37Eppinga R.D. Li Y. Lin J.L. Mak A.S. Lin J.J. Cell Motil. Cytoskeleton. 2006; 63: 543-562Crossref PubMed Scopus (26) Google Scholar). 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. (36Foster D.B. Shen L.H. Kelly J. Thibault P. Van Eyk J.E. Mak A.S. J. Biol. Chem. 2000; 275: 1959-1965Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) 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 5The 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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" @default.
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• W1995006965 title "Changes in the Balance between Caldesmon Regulated by p21-activated Kinases and the Arp2/3 Complex Govern Podosome Formation" @default.
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