Tyrosine Phosphorylation of p130Cas Is Involved in Actin Organization in Osteoclasts*

Integrin-mediated interaction with the extracellular matrix plays a critical role in the function of osteoclasts, the bone-resorbing cells. This study examines the role of p130Cas (Crk-associatedsubstrate (Cas)) in actin organization in osteoclasts. Multinucleated osteoclast-like cells (OCLs) were obtained in a co-culture of murine bone marrow cells and primary osteoblasts. After plating on culture dishes, OCLs formed a ringlike structure consisting of F-actin dots at cell periphery (actin ring). The percentage of OCLs with actin rings and its diameter increased with time and cell spreading. Tyrosine phosphorylation of a protein (p130) increased with actin ring formation. Treatment with cytochalasin D disrupted actin rings and reduced tyrosine phosphorylation of p130. Using specific antibodies, p130 was identified as Cas. By immunocytochemistry, Cas was localized to the peripheral regions of OCLs and its distribution overlapped that of F-actin. In OCLs derived from Src(−/−) mice, in which osteoclast activity is severely compromised, tyrosine phosphorylation of Cas was markedly reduced. Moreover, Cas was diffusely distributed in the cytoplasm and actin ring formation is not observed. These findings suggest that Src-dependent tyrosine phosphorylation of Cas is involved in the adhesion-induced actin organization associated with osteoclast activation.

Integrins are a major family of cell surface receptors that play crucial roles in cell-cell and cell-extracellular matrix (ECM) 1 interactions (1). Integrin/ECM protein interactions participate in a variety of biological processes including embryonic development, wound healing, tumor metastases, and immune responses (1,2). It is now established that, in addition to mediating cell adhesion, integrins activate multiple signaling pathways. These include elevation of intracellular Ca 2ϩ , lipid turnover, and tyrosine phosphorylation, leading to cytoskeletal rearrangement and de novo gene expression (3). The proteins that are tyrosine-phosphorylated by ECM-integrin interactions include focal adhesion kinase (FAK) (4), paxillin (5), tensin (6), and cortactin (7).
Osteoclasts, the bone-resorbing cells, play a critical role in bone remodeling (8 -10). Their adhesion to the bone surface induces the cytoskeletal reorganization associated with activation. The recognition of extracellular matrix components is, therefore, an important step in the initiation of osteoclast function. Several studies have demonstrated that ␣ v ␤ 3 integrins play a central role in osteoclast adhesion (11)(12)(13)(14)(15)(16). Using murine osteoclast-like multinucleated cells formed in vitro, we have recently reported that integrin-mediated cell adhesion to ECM molecules, such as vitronectin, fibronectin or type I collagen, induces the formation of a ringlike structure of F-actin dots at the cell periphery (17). This ringlike organization, the actin ring, is formed by the assembly of podosomes that precedes the formation of the sealing zone (18 -20) and has been considered to be a marker of osteoclast activation (17,21,22). Actually, various inhibitory agents of osteoclast function disrupt this actin ring (23).
In this study, we examined protein-tyrosine phosphorylation occurring during the adhesion-induced actin organization in osteoclasts, and identified p130 Cas (Crk-associated substrate (Cas)) as a molecule that participates in the signaling cascade of actin ring formation. Cas was originally described as a major tyrosine-phosphorylated protein in cells transformed by either v-src (24 -26) or v-crk (27)(28)(29). The recent molecular cloning of Cas has shown that Cas contains an N-terminal SH3 domain, a substrate domain, a proline-rich region, and several tyrosine residues near the C terminus (30,31). The SH3 domain of Cas is known to bind to FAK (32,33), FAK-related nonkinase (33), and PTP1B (protein-tyrosine phosphatase 1B) (34). The substrate domain, which has 15 potentially phosphorylated tyrosine residues, binds to v-Crk (35,36). The proline-rich region near the C terminus and Tyr-762 provide the binding sites for the SH3 and SH2 domains of Src kinase, respectively (36). These structural characteristics indicate that Cas is an adapter molecule, which can transmit cellular signals via interaction with the SH2 and SH3 domains of various signaling molecules. It has already been reported that Cas undergoes tyrosine phosphorylation upon integrin-mediated cell adhesion in fibroblasts (7,37,38). Evidence presented here shows that tyrosine phosphorylation of Cas is involved in adhesion-induced actin organization in osteoclasts and is absent in the compromised osteoclasts of Src(Ϫ/Ϫ) mice. eruption. All animals were cared and housed under conditions as stated in the Institutional Animal Care and Use Committee (IACUC) Guide for the Care and Use of Laboratory Animals, and the studies were reviewed and approved by the Merck Research Laboratories Institutional Animal Care and Use Committee.
Cell Culture-Murine osteoclast-like multinucleated cells (OCLs) were obtained from co-cultures of primary osteoblasts and bone marrow cells from ddY mice in the presence of 10 nM 1␣,25-dihydroxyvitamin D 3 (Wako Pure Chemical Co., Osaka, Japan) (crude preparations of OCLs) and purified by 0.001% Pronase (Calbiochem Co., La Jolla, CA) (purified preparations of OCLs) (39 -41). Src(Ϫ/Ϫ) and Src(ϩ/?) OCLs were also obtained from co-cultures of primary osteoblasts derived from normal murine calvaria and spleen cells from either c-Src-deficient mice or their normal littermates and purified as described above.
Immunofluorescent Analyses-Cells were cultured for 2 h on glass coverslips and fixed for 15 min at room temperature with 4% paraformaldehyde. Cells were then washed with PBS and treated for 10 min with 0.2% Triton X-100 to permeate cell membranes. After incubating for 30 min with 5% skim milk to block nonspecific binding, the cells were incubated for 30 min at 37°C with mouse anti-Cas monoclonal antibodies (Transduction Laboratories, Lexington, KY) or rabbit anti-Cas polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100 with 5% skim milk. Negative control cells were incubated with non-immune mouse or rabbit serum (1:100 dilution with 5% skim milk). The cells were washed with PBS and incubated for 30 min at 37°C with a second antibody (fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulins or Cy3-conjugated goat anti-rabbit immunoglobulins). For identification of F-actin, rhodamine-conjugated phalloidin or fluorescein isothiocyanate-conjugated phalloidin (Molecular Probes, Inc., Eugene, OR) diluted to 1:100 with 5% skim milk was added to the second antibody solution.
Double staining for tartrate-resistant acid phosphatase (TRAP), a marker enzyme for osteoclasts and F-actin, was performed as described previously (17).
Western Blot Analyses-Cells on culture plates were washed twice with ice-cold PBS and lysed with 1ϫ sample buffer for SDS-PAGE. Samples were denatured by boiling for 5 min and electrophoresed on 7.5% SDS-polyacrylamide gels. Proteins were transferred onto Immobilon-P (Millipore Co., Bedford, MA), and nonspecific binding sites on the membrane were blocked by incubating at 4°C overnight in 2% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 (TBS-T). The membranes were then probed for 2 h with anti-phosphotyrosine (Tyr(P)) monoclonal antibody (Upstate Biotechnology Inc., Lake Placid, NY) in 2% bovine serum albumin at a dilution of 1:1000, washed with TBS-T three times, and incubated for 1 h with horseradish peroxidase-conjugated sheep anti-mouse immunoglobulins. After washing with TBS-T, the membranes were developed using enhanced chemiluminescence (ECL, Amersham International plc., Amersham Place, United Kingdom).
The blots were stripped for 40 min at 55°C in 62.5 mM Tris-HCl (pH . After culture for another 1 h, total cell lysates were collected from the purified OCLs. Total cell lysates were separated by 7.5% SDS-PAGE, transferred onto Immobilon-P, and probed with antiphosphotyrosine antibody. The molecular masses of marker proteins are indicated in kilodaltons on the left. Arrowheads show the positions of highly tyrosine-phosphorylated proteins. b, total proteins were stained with Coomassie Brilliant Blue to confirm equal loading. 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, re-equilibrated in TBS-T, blocked, and reprobed separately with anti-Cas monoclonal antibody at a dilution of 1:500, anti-c-Cbl polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:300, or anti-Src (mAb327) monoclonal antibody (Oncogene Science Inc., Manhasset, NY) at a dilution of 1:1000 in the manner described above.
Immunoprecipitation-All procedures were performed at 4°C. Cells were washed twice with ice-cold PBS, then lysed in cold lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 10 mg/ml aprotinin, 2 mM Na 3 VO 4 , and 1 mM phenylmethylsulfonyl fluoride. Lysates were clarified by centrifugation at 12,000 ϫ g for 20 min and precleared by incubation with protein G-or protein A-Sepharose beads (Zymed Laboratories Inc.) for 1 h. Proteins were immunoprecipitated by incubation with anti-Cas, anti-Tyr(P), anti-Src, or anti-paxillin monoclonal antibody (Transduction Laboratories) for 2 h, followed by addition of protein G-or protein A-Sepharose beads, and incubated for another 1 h. Immunoprecipitates were washed five times with lysis buffer, extracted in 2ϫ SDS sample buffer, then separated using 7.5% SDS-polyacrylamide gels and analyzed by Western blotting with anti-Tyr(P) antibody, followed by reblotting with anti-Cas, anti-Src, or anti-paxillin antibody.

Time Course of Actin Ring Formation in OCLs Induced by
Cell Adhesion-When crude preparations of OCLs were plated on culture plates in the presence of 10% fetal bovine serum, OCLs began to form actin rings at the cell periphery within 10 min (Fig. 1, a and b). The percentage of OCLs with actin rings and the diameter of the rings increased with time (Fig. 1, c-g), peaking 2 h after cells were plated. At this time, more than 80% of OCLs had formed actin rings, the average diameter of which attained 175 m (Fig. 1, h and i). This state of actin ring formation was maintained at least for another 10 h.
We next examined whether actin rings of OCLs were affected by the removal of osteoblasts. One hour after osteoblasts were removed, almost all purified OCLs had actin rings (Fig. 2). A similar number of OCLs had actin rings after 3 h (data not shown).
Involvement of Protein-tyrosine Phosphorylation in Actin Ring Formation-Tyrosine kinases, such as c-Src, were shown to be involved in osteoclastic bone resorption (42)(43)(44). We examined, therefore, by Western blot analysis the general pattern of tyrosine phosphorylation during actin ring formation. In crude preparations of OCLs, tyrosine phosphorylation of several proteins was enhanced in a time-dependent manner after plating (Fig. 3a, lanes 1-5) for equal gel loading (Fig. 3b, lanes  1-5). This time-dependent increase in tyrosine phosphorylation correlated with adhesion-induced actin ring formation in OCLs (Fig. 1). To determine which tyrosine-phosphorylated proteins were derived from OCLs, total cell lysates from purified OCL preparations were examined. In purified OCLs cultured for 3 and 7 h, we detected four highly tyrosine-phosphorylated proteins with molecular mass values of around 130, 89, 85, and 74 kDa (p130, p89, p85, p74) (Fig. 3a, lanes 6 and 7, arrowheads). The tyrosine phosphorylation of these four proteins was much less pronounced in the crude OCL preparation kept in suspen- Candidates for the p130 protein that is tyrosine-phosphorylated during actin ring formation in OCLs. Purified OCL preparations were obtained from crude OCL preparations by removing osteoblasts after culture for 2 h. After culture for another 1 h, total cell lysates were collected, separated by 7.5% SDS-PAGE, transferred onto Immobilon-P, and probed with anti-phosphotyrosine antibody (lanes 1,  2, 5, and 6). The same membrane was stripped and reprobed with anti-c-Cbl antibody (lanes 3 and 4) or anti-Cas antibody (lanes 7 and 8). sion (Fig. 3a, lane 1). Moreover, cytochalasin D, an inhibitor of actin polymerization, also disrupted the actin rings of OCLs (Fig. 4, a and b) and markedly reduced tyrosine phosphorylation of p130 in purified OCLs (Fig. 4, c and d). Tyrosine phosphorylation of other proteins was less affected by cytochalasin D. These results suggest that tyrosine phosphorylation of p130 is closely associated with actin organization in osteoclasts.
Identification of Tyrosine-phosphorylated p130 in Actin Ring Formation-To identify the tyrosine-phosphorylated p130, total cell lysates from purified OCLs were blotted with anti-Tyr(P) antibody (Fig. 5, lanes 1, 2, 5, and 6), then reprobed with several antibodies containing anti-c-Cbl (Fig. 5, lanes 3 and 4) and anti-Cas (Fig. 5, lanes 7 and 8) antibodies. A band was recognized by anti-c-Cbl antibody (Fig. 5, arrowhead), but its molecular weight differed from that of tyrosine-phosphorylated p130. c-Cbl, the product of the c-cbl proto-oncogene, has been reported to be a tyrosine-phosphorylated c-Src substrate in OCLs (45). In this study, c-Cbl was also tyrosine-phosphorylated in c-Cbl immunoprecipitates from purified OCLs (data not shown), although we could not detect tyrosine phosphorylation of c-Cbl in total cell lysates. Positive bands were also recognized during reblotting with anti-FAK and with anti-Src substrate p120 antibodies, but neither were identical to the tyrosine-phosphorylated p130 (data not shown). On the other hand, two bands (Cas A and Cas B) were recognized by anti-Cas antibody, and one (Cas B) was the same as that of the tyrosine-phosphorylated p130 (Fig. 5, lanes 5-8, arrows). It has been reported that, in normal fibroblasts, Cas is detected as two bands at 125 and 130 kDa (Cas A and Cas B, respectively) (31). These results suggest that Cas is a candidate for the 130-kDa tyrosine-phosphorylated protein. Moreover, in immunoprecipitates with anti-Cas antibody from total cell lysates of purified OCLs, Cas B was tyrosine-phosphorylated (Fig. 6,  lanes 1-4). Cas B was also detected in immunoprecipitates with anti-Tyr(P) antibody (Fig. 6, lanes 5 and 6). In addition, Cas B immunoprecipitated from purified OCLs pretreated with cytochalasin D was not tyrosine-phosphorylated (Fig. 6, lanes  7-10). Taken together, these findings indicate that the p130, which is tyrosine-phosphorylated during adhesion-induced actin rearrangement in OCLs, is indeed Cas.
Intracellular Localization of Cas in OCLs-By immunohistochemistry, Cas was localized at perinuclear and peripheral regions in OCLs (Fig. 7a), the later distribution of which overlaps exactly with that of F-actin at the cell periphery (Fig. 7, b  and c). When OCLs were treated with cytochalasin D at 5 M, Cas was re-distributed throughout the cytoplasm (Fig. 7d). No immunolabeling was detected, with a nonspecific immunoglobulins used as the first antibody (Fig. 7e). These findings suggest that Cas may play a role in actin ring formation or maintenance in OCLs.

FIG. 7. Intracellular localization of Cas in OCLs.
After crude OCL preparations were cultured for 2 h, cells were treated with (d) or without (a-c and e) 5 M cytochalasin D for 20 min and fixed. a, cells were stained with anti-Cas monoclonal antibody. In OCLs (arrow), Cas is present in the peripheral and perinuclear regions. b, the same field as in a, double-stained with rhodamine-conjugated phalloidin to visualize F-actin. c, the overlaid image of a and b. Note that Cas (green) and F-actin (red) overlap in the peripheral region, appearing as yellow structures. d, cells treated with cytochalasin D were stained with anti-Cas monoclonal antibody. Note that Cas is distributed throughout the cytoplasm in an OCL treated with cytochalasin D (arrow). e, when non-immune mouse serum was used as the first antibody, no specific immunolabeling was detected. In b, d, and e, the position of the nuclei is indicated by DAPI staining. Bars ϭ 20 m. in Src(Ϫ/Ϫ) OCLs-Recently, several lines of evidence have shown that c-Src is involved in Cas tyrosine phosphorylation in the integrin-mediated signaling pathway (46 -49). We examined, therefore, the relationship of c-Src and Cas to actin ring formation in osteoclasts. For this purpose, we prepared Src(Ϫ/Ϫ) OCLs using the co-culture of Src(Ϫ/Ϫ) spleen cells and normal primary osteoblasts. In Src(Ϫ/Ϫ) OCLs, tyrosine phosphorylation of Cas was markedly reduced (Fig. 8b, top  panel). The expression of Cas protein in Src(Ϫ/Ϫ) OCLs was actually elevated, as shown by Western blotting (Fig. 8, a, and  b, top panel), but the mechanism for this change is not known. A similar phenomenon was reported in Src(Ϫ/Ϫ) fibroblasts (50). Importantly, the reduced phosphorylation of Cas in Src(Ϫ/Ϫ) OCLs appears to be protein specific, because the tyrosine phosphorylation of paxillin was not significantly different between Src(ϩ/?) and Src(Ϫ/Ϫ) OCLs (Fig. 8b, middle  panel). Moreover, in Src(Ϫ/Ϫ) OCLs, Cas was diffusely distributed in the cytoplasm and actin rings were not formed (Fig. 9, b and d-f). Whereas 83.1% of Src(ϩ/?) OCLs (167 out of 201) formed actin rings, none of Src(Ϫ/Ϫ) OCLs (0 out of 203) did. Instead of actin rings, small focal adhesion contacts were formed around the cell periphery and underneath the nuclei (Fig. 9d). The number of TRAP-positive OCLs formed was not significantly different between Src(Ϫ/Ϫ) and Src(ϩ/?) culture (data not shown). These findings suggest that Src-dependent tyrosine phosphorylation of Cas is involved in actin ring formation and in the localization of Cas in actin rings. DISCUSSION We have reported previously that actin ring formation in osteoclasts is dependent on the interaction of integrins and matrix proteins (17). In this study, we examined the involvement of protein-tyrosine phosphorylation in the formation of actin rings in osteoclasts. The findings clearly show that tyrosine phosphorylation of Cas, a novel adaptor molecule, is involved in adhesion-induced actin organization. Cas was originally identified as a highly tyrosine-phosphorylated protein during cellular transformation by v-Src (24 -26) or v-Crk (27)(28)(29), and was shown to form stable complexes with these oncoproteins. Recent molecular cloning of Cas identified it as a novel SH3-containing signaling molecule with a cluster of multiple putative SH2-binding motifs (31). Moreover, several studies have reported that tyrosine phosphorylation of Cas is induced by cell adhesion (7,37,38), and that Cas is localized to focal adhesions (33,38).
As shown in this study, Cas is also tyrosine-phosphorylated in OCLs after cell adhesion, and co-localizes with F-actin at the cell periphery as a ringlike structure, "the actin ring," Because the actin ring is considered to be an assembly of podosomes (19 -21), our results suggest that tyrosine phosphorylation of Cas plays a role in podosome formation. Moreover, treatment of OCLs with cytochalasin D disturbed the intracellular localization of Cas and reduced Cas tyrosine phosphorylation (Figs. 6 and 7). Possibly, cytosolic protein-tyrosine phosphatases (PTP), such as PTP-1B (34) and PTP-PEST (51), may play a role in the dephosphorylation of Cas that is translocated to the cytoplasm by treatment with cytochalasin D. We also reported that the disruption of cytoskeletal organization by cytochalasin D treatment induced the inhibition of osteoclast function (52). These findings suggest the close relationship between Cas phosphorylation and actin organization, which is related to osteoclast activity.
In normal fibroblastic cells from rats (3Y1) or mice (NIH3T3), Cas has been detected as two bands, Cas A (125 kDa) and Cas B (130 kDa), respectively (31). On the other hand, when cells are transformed by v-Crk or v-Src, Cas A is decreased and another broad band of Cas C (130 -135 kDa) appears. Because tyrosine phosphorylation is found mostly in Cas C, Cas C may be a modified form of Cas A or Cas B with a retarded gel mobility, secondary to phosphorylation at multiple sites (31). In OCLs, however, Cas C was not detected, and the tyrosine-phosphorylated Cas was identified as Cas B (Fig. 6). This might be a result of the fact that osteoclasts are nontransformed cells.
The next question is: which tyrosine kinase(s) phosphorylates Cas? Focal adhesion kinase (FAK) is one of the candidates, as it is phosphorylated upon adhesion with similar kinetics to those of Cas (7,37,38). Moreover, recent reports indicate that FAK can bind to the SH3 domain of Cas in vivo (32,33). On the other hand, the C-terminal portion of Cas can also bind directly the SH2 and SH3 domains of Src kinase (36). In addition, two lines of evidence have demonstrated that the deficiency of c-Src, but not of FAK, completely abrogated integrin-mediated Cas phosphorylation in fibroblasts (47,48). These results suggest that tyrosine phosphorylation of Cas by integrin engagement is mediated by Src family kinases, especially c-Src, and FAK itself might not be necessary for Cas phosphorylation. This is supported by the findings of this study, which show that tyrosine phosphorylation of Cas was markedly reduced in Src(Ϫ/Ϫ) OCLs, whereas the expression of Cas was not suppressed (Fig. 8). Moreover, actin rings did not form in Src(Ϫ/Ϫ)  rings (a, arrows). c, the same field as in b, double-stained for TRAP, a marker enzyme for osteoclasts to identify osteoclasts (arrow). d-f, Src(Ϫ/Ϫ) OCLs were double-stained with fluorescein isothiocyanate-conjugated phalloidin to visualize F-actin (d) and with anti-Cas polyclonal antibody (e). f, overlaid image of d and e. Bars ϭ 10 m.
OCLs and Cas was localized throughout the cytoplasm (Fig. 9), suggesting that, in osteoclasts, c-Src plays an important role in Cas phosphorylation and in its localization. Considering that Cas phosphorylation is tightly associated with actin ring formation, Src may participate in the control of actin organization in osteoclasts via tyrosine phosphorylation of Cas. In Src(Ϫ/Ϫ) mice, osteoclast activity is severely compromised, resulting in osteopetrosis (42,43) and, as shown here, Src(Ϫ/Ϫ) OCLs do not form actin rings. Thus, the lack of Src-dependent Cas phosphorylation may be one of the causes for osteoclast inactivation in Src(Ϫ/Ϫ) mice. To prove this hypothesis, rescue experiments of Src(Ϫ/Ϫ) mice using Cas or Cas related molecules are required, and are now in progress.
Recently, Nakamoto et al. (53) reported that the association of Cas not only with Src kinase but also with FAK family kinases plays a pivotal role in the localization of Cas to focal adhesions in fibroblasts. FAK, which can bind to both Cas and Src family kinases, might recruit Src family kinases to phosphorylated Cas. Considering that Cas has multiple SH2-binding sites, a SH3 region, and a proline-rich region, Cas is likely to associate with various molecules such as Src family kinases, FAK family kinases, paxillin, tensin, and c-Crk, and thus might play a central role in podosome formation in osteoclasts. Further studies are required to determine the hierarchy among these molecules in the osteoclast polarization process. In conclusion, Src-dependent tyrosine phosphorylation of Cas appears essential for the signal transduction initiated by cell adhesion, leading to the formation of actin organization in osteoclasts.