Stable Association of PYK2 and p130Cas in Osteoclasts and Their Co-localization in the Sealing Zone*

Bone resorption is initiated by osteoclast attachment to the mineralized matrix, cytoskeletal reorganization, cellular polarization, and the formation of the sealing zone. The present study examines the interaction between PYK2 and p130Cas (Crk-associatedsubstrate), suggested to be part of the signaling pathway initiated by osteoclast adhesion. Using murine osteoclast-like cells (OCLs) and their mononuclear precursors (pOCs), generated in a co-culture of bone marrow and osteoblastic MB1.8 cells, we show that: 1) p130Cas is tyrosine-phosphorylated upon adhesion of pOCs to vitronectin or ligation of β3 integrins; 2) p130Cas colocalizes with PYK2 and the cytoskeletal proteins F-actin, vinculin, and paxillin in the podosomal-rich ring-like structures of OCLs plated on glass and in the sealing zone in actively resorbing OCLs on bone; 3) p130Cas and PYK2 form a stable complex in pOCs, independent of tyrosine phosphorylation of either molecule, and this complex is present in Src (−/−) OCLs, in which neither protein is phosphorylated or associated with the osteoclast adhesion structure; 4) the association of p130Cas and PYK2 is mediated by the SH3 domain of p130Cas and the C-terminal domain of PYK2. These findings suggest that p130Cas and its association with PYK2 may play an important role in the adhesion-dependent signaling that leads to cytoskeletal reorganization and formation of the sealing zone during osteoclast activation.

Adhesion of cells to the extracellular matrix (ECM) 1 initiates signaling pathways that lead to cellular spreading and migration as well as to modulation of growth and differentiation. Integrins belong to a major class of adhesion receptors that mediate these cellular functions. Integrin engagement induces a cascade of tyrosine phosphorylation and the recruitment of structural and signaling molecules to multimeric complexes associated with the actin cytoskeleton, called focal adhesion contacts (1,2).
Proline-rich tyrosine kinase 2 (PYK2, also known as RAFTK and CAK␤) (3)(4)(5) is related to FAK, known to play an important role in cell adhesion (6). Similar to FAK, PYK2 lacks a transmembrane region and SH2 and SH3 domains but has two proline-rich regions in its C terminus. PYK2 is highly expressed in brain and various hematopoietic cells (3). In PC12 cells, PYK2 tyrosine phosphorylation and activation are stimulated by neuronal stimuli and stress signals, leading to modulation of a potassium channel and activation of the JNK signaling pathway (4,7,8). In addition, stimulation of Gprotein-coupled receptors induces tyrosine phosphorylation of PYK2 and complex formation between PYK2 and Src via the SH2 domain of Src, leading to activation of the MAP kinase signaling pathway (9). Similar to FAK, PYK2 is tyrosine-phosphorylated and is activated by adhesion-mediated signaling in platelets and B cells (10,11). In addition, PYK2 interacts with and phosphorylates the focal adhesion-associated protein paxillin in vitro (12). PYK2 is thus suggested to participate in the transfer of signals from the cell surface to the cytoskeleton.
p130 Cas was first identified as a major tyrosine-phosphorylated protein in v-src and v-crk transformed cells (13,14). p130 Cas contains an N-terminal SH3 domain, a substrate domain, a proline-rich region, and a C-terminal domain with multiple tyrosine residues (14). This unique structure suggested that p130 Cas may serve as a docking protein for multiple SH2 and SH3 domain-containing molecules. In addition, p130 Cas is tyrosine-phosphorylated during integrin-mediated adhesion and localizes to focal adhesions in fibroblasts (15)(16)(17). p130 Cas was shown to bind to FAK both in vitro and in vivo (15,18). Integrin-dependent p130 Cas phosphorylation was absent in c-Src deficient fibroblasts (19). Recently, PYK2 was shown to be tyrosine phosphorylated and associated with p130 Cas upon B cell adhesion to fibronectin and stimulation of the antigen receptor (10).
Osteoclasts are highly differentiated bone resorbing cells. Osteoclast activation is initiated by adhesion to bone matrix and formation of the sealing zone, a specialized adhesion structure responsible for the tight attachment of osteoclasts to mineralized bone matrix (20,21). We found that PYK2 is highly expressed in osteoclasts and is tyrosine-phosphorylated upon integrin-mediated adhesion and is activated by osteoblastic MB1.8 cells, which are essential for osteoclast function in vitro (22,23). Furthermore, PYK2 localizes to podosomes, the primary adhesion structures in osteoclasts which become the sealing zone in actively resorbing osteoclasts (22). We also showed that tyrosine phosphorylation of p130 Cas is involved in the organization of the podosome-rich ring structures in osteoclasts (24). Tyrosine phosphorylation of p130 Cas was markedly reduced in osteoclasts derived from Src(Ϫ/Ϫ) mice, in which osteoclast activity is severely compromised (24).
In this study, we demonstrate that p130 Cas participates in adhesion-mediated signaling in osteoclasts. p130 Cas is tyrosine-phosphorylated upon ␤ 3 -integrin engagement by ligand binding or antibody-induced clustering. In osteoclasts, p130 Cas is stably associated with PYK2, via the SH3 domain of p130 Cas and the C-terminal domain of PYK2, independent of tyrosine phosphorylation. Furthermore, p130 Cas co-localizes with PYK2 in the sealing zone of resorbing osteoclasts on bone. These findings suggest that the engagement of ␤ 3 -integrin initiates the activation of the p130 Cas ⅐PYK2 complex which plays a role in osteoclast activation.
Animals-Heterozygote Src(ϩ/Ϫ) mice were obtained from Jackson Laboratory (Bar Harbor, ME), and Src(Ϫ/Ϫ) mice were phenotypically distinguished from their Src(ϩ/?) siblings by lack of tooth eruption. All animals were cared for according to Institutional Animal Care and Use Committee (IACUC) Guide.
Cell Adhesion-After isolation, pOCs (10 5 cells/plate) were washed twice with serum-free ␣-minimal essential medium containing 0.1% bovine serum albumin (Sigma) and kept in suspension or allowed to A, pOCs were kept in suspension or allowed to attach to vitronectin-coated plates in the absence of serum at 37°C for indicated times, 5-60 min. Cell lysates were subjected to p130 Cas immunoprecipitation, followed by blotting for phosphotyrosine (PY) and p130 Cas , as described under "Experimental Procedures." B and C, in the parallel cultures, pOCs were fixed and stained for TRAP at the indicated times, and the number and area of attached TRAP(ϩ) cells were quantitated as described under "Experimental Procedures." Data are expressed as the means Ϯ S.E. of four fields in panel B and as the means as Ϯ S.E. of more than 300 pOCs in panel C.
attach to polystyrene dishes, coated with ECM proteins (fetal bovine serum, fibronectin (25 g/ml), vitronectin (10 g/ml), osteopontin (50 g/ml), laminin (25 g/ml), type I or type IV collagen (25 g/ml)). After 5-60 min at 37°C, an equal volume of 2ϫ TNE lysis buffer (20 mM Tris, pH 7.8, 300 mM NaCl, 2 mM EDTA, 2% Nonidet P-40, 2 mM NaVO 3 , 20 mM NaF, 20 g/ml leupeptin, 1 trypsin inhibitory units/ml aprotinin and 2 mM phenylmethylsulfonyl fluoride) was added to the plates. Clarified lysates were subjected to immunoprecipitation and -blotting. Alternatively, pOCs were allowed to attach to vitronectin-coated plates in serum-free medium for the indicated times and then fixed and stained for TRAP as described (24). Numbers and area of attached pOCs were measured using the Empire Imaging Analyzing Systems (Milford, NJ). Results are expressed as the means (Ϯ S.E.) of four fields for the number of attached cells and of more than 300 cells for the area of pOCs.
Immunoblotting and Immunoprecipitation-Lysates were separated on a 4 -20% gradient or 8% SDS-PAGE (Novex, San Diego, CA), and electrotransferred to Immobilon-P membrane (Millipore, Bedford, MA) overnight. After blocking with 100 mM NaCl, 10 mM Tris, 0.1% Tween 20, 1% bovine serum albumin, the membrane was incubated with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies, and detected with the ECL chemiluminescence system (Amersham Pharmacia Biotech). For immunoprecipitation, the lysates were precleared with Sepharose-4B and precipitated with anti-PYK2 polyclonal antibodies or anti-p130 Cas monoclonal antibody for 1 h at 4°C followed by protein G-Sepharose for 1 h at 4°C, respectively. Immunoprecipitated proteins were washed with lysis buffer (5ϫ), followed by SDS-PAGE, blotted, and stained as described above.
In Vitro Protein Association Assays-These experiments were performed with GST-fusion proteins containing the SH3 domains of Fyn, Lyn, Src, PI3-kinase, and p130 Cas or the kinase and N-and C-terminal domains of PYK2. OCLs lysates (1 mg/ml) were incubated with GSTfusion protein coupled with glutathione-Sepharose beads for 2 h at 4°C. The beads were washed (3ϫ) with lysis buffer and with phosphate-buffered saline (1ϫ), and precipitated proteins were separated by SDS-PAGE and subjected to immunoblot analysis using anti-PYK2 or anti-p130 Cas antibody.
Immunofluorescence-pOCs were seeded on glass coverslips or on bone slices together with 1␣,25(OH) 2 D 3 -treated MB1.8 cells. At the indicated times, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline. Cells were stained using polyclonal anti-PYK2, poly-or monoclonal anti-p130 Cas , anti-vinculin and anti-paxillin antibodies, followed with the appropriate secondary antibodies, or with FITC-or rhodamine-conjugated phalloidin. Immunofluorescent stainings were viewed with a fluorescence microscope or with a confocal laser scanning microscope (Leica, Heidelberg, Germany), equipped with a multiline Omnichrome argon-crypton laser (Chino, California) (26).

Adhesion-dependent Tyrosine Phosphorylation of p130 Cas -
Expression of p130 Cas and PYK2 in osteoclasts was examined using Western blot analysis. Both pOCs and multinucleated OCLs express significant levels of p130 Cas and PYK2 similar to that of IC-21 macrophages (Fig. 1A). We have previously found that the osteoblastic MB1.8 cells do not express detectable levels of PYK2 (22).
The effect of adhesion on p130 Cas tyrosine phosphorylation was examined in isolated pOCs, which can be reseeded on various ECM proteins in the absence of serum. Attachment to vitronectin, osteopontin, fibronectin, and serum stimulated p130 Cas tyrosine phosphorylation, whereas lower levels of tyrosine phosphorylation were detected in pOCs adhering to type I or type IV collagen, or to laminin (Fig. 1B). Relative to pOCs maintained in suspension, p130 Cas was tyrosine-phosphorylated in a time-dependent manner upon attachment to vitronectin. An increase in p130 Cas tyrosine phosphorylation was detected within 5 min of seeding and reached the maximum around 30 min (Fig. 2A). Interestingly, pOC attachment to vitronectin-coated plates appears to precede the peak phosphorylation of p130 Cas (Fig. 2B), suggesting that tyrosine phosphorylation of p130 Cas may play a role in the cytoskeletal organization of osteoclast precursors upon adhesion. Furthermore, tyrosine phosphorylation parallels to the time course of osteoclast spreading (Fig. 2C).
␤ 3 -Integrin Clustering Induces Tyrosine Phosphorylation of p130 Cas -Matrix preferences for inducing p130 Cas phosphorylation suggested a selective integrin-mediated phenomenon. Osteoclasts express ␣ v ␤ 3 -, ␣ v ␤ 1 -, and ␣ 2 ␤ 1 -integrins (26 -29). Immature osteoclast precursors were shown to express ␤ 2 -and ␤ 5 -integrins (30,31). Integrin involvement and specificity for p130 Cas tyrosine phosphorylation was examined by antibody-mediated clustering of integrins in pOCs in suspension. Clustering with anti-␤ 3 -antibody strongly induced p130 Cas tyrosine phosphorylation, whereas clustering with anti-␤ 1 -or anti-␤ 2antibody induced low levels of p130 Cas phosphorylation in pOCs (Fig. 3), indicating that the vitronectin receptor ␣ v ␤ 3 is the major integrin responsible for this effect. Clustering of ␤ 5 -integrin was not examined in this study because antibodies that recognize the extracellular domain of the murine ␤ 5 -integrin are not available.
Localization of p130 Cas in Podosomes and in the Sealing Zone of Osteoclasts and Co-localization with PYK2-Because ␤ 3 -mediated adhesion is thought to be involved in osteoclast activation, the subcellular localization of p130 Cas was examined in OCLs plated on glass coverslips and on bone. On glass coverslips, p130 Cas was localized in podosomes (Fig. 4) and, along with F-actin, organized in a typical podosomal-rich adhesion structure at the periphery of OCLs (24). Double staining of p130 Cas and PYK2 in OCLs on glass showed that p130 Cas also co-localized with PYK2 (Fig. 4). On mineralized bone matrix, osteoclasts polarize and form the sealing zone that circumscribes the resorptive ruffled border membrane, where protons and proteases are secreted. Sealing zone formation can be followed using vinculin immunostaining to visualize the transition from small rings in podosomes to a double circle structure in the mature sealing zone (21). Similarly, on bone, p130 Cas localized in OCLs to podosomes and to the nascent sealing zone, as well as to the double circle structure in the mature sealing zone (Fig. 5A). p130 Cas also co-localized with the cytoskeletal proteins F-actin, vinculin, and paxillin (Fig. 6). The distribution of paxillin (Fig. 6h), shown here for the first time in osteoclasts on bone, follows the staining pattern of vinculin. However, in resorbing OCLs, p130 Cas also co-localized with PYK2 in the nascent sealing zone, as well as in the mature sealing zone (Fig. 5B). This morphological evidence for the co-localization of p130 Cas and PYK2 in osteoclasts in vivo supports a role for p130 Cas in the morphological changes associated with osteoclast activity.
Association of p130 Cas with PYK2 in Osteoclasts-Because p130 Cas colocalizes with PYK2 in the sealing zone of active osteoclasts, we investigated the interaction of the two proteins in these cells. Immunoprecipitation and immunoblotting experiments show association of p130 Cas with PYK2 in pOCs in situ (Fig. 7). Furthermore, this association was present both in suspended and attached cells (Fig. 7), suggesting that it is independent of tyrosine phosphorylation.
c-Src Is Not Required for the Association of p130 Cas and PYK2-This was confirmed in Src(Ϫ/Ϫ) osteoclasts. Src has been implicated in p130 Cas and PYK2 tyrosine phosphorylation, and osteoclast function is severely compromised in Src(Ϫ/Ϫ) mice (9,32). As observed previously, the level of tyrosine phosphorylation of PYK2 immunoprecipitated from Src(Ϫ/Ϫ) OCLs is markedly reduced, although the same level of PYK2 is expressed in Src(Ϫ/Ϫ) and Src(ϩ/?) OCLs (Fig. 8) (22). Interestingly, a comparable level of p130 Cas was immunopre-cipitated with PYK2 from both Src(Ϫ/Ϫ) and Src(ϩ/?) OCLs (Fig. 8, middle panel). We previously reported the expression of both p130 Cas isoforms (Cas A and B) in Src(Ϫ/Ϫ) OCLs, as compared with the predominant expression of Cas B in Src(ϩ/?) OCLs. Both isoforms of p130 Cas appeared to associate with PYK2 in Src(Ϫ/Ϫ) OCL lysates (Fig. 8, middle panel). Furthermore, treatment of Src(ϩ/?) OCLs with cytochalasin D for 20 min caused dephosphorylation of PYK2 and dissociation of the Src and PYK2 complex (Fig. 8, bottom panel) but had no significant effect on the association of p130 Cas with PYK2 (Fig. 8,  middle panel). These results indicate that the association of p130 Cas and PYK2 is not dependent on an intact cytoskeleton or on c-Src function in osteoclasts.
Association of p130 Cas and PYK2 Is Mediated by SH3 Domains of p130 Cas and the C-terminal Domain of PYK2-To characterize the domains that mediate the association of p130 Cas with PYK2, GST-fusion proteins encoding for SH3 domains from various signaling molecules, as well as the kinase and N-and C-terminal domains of PYK2 were incubated with lysates prepared from OCLs. GST-fusion protein encoding the SH3 domain of p130 Cas binds to PYK2 from OCL lysates, whereas the GST-fusion proteins encoding the SH3 domains of Src, Lyn, Fyn, or PI3-kinase or GST alone do not bind to PYK2 in OCL lysates (Fig. 9A). On the other hand, the GST-fusion protein encoding the C-terminal domain of PYK2, but not the kinase or N-terminal domains or GST alone, interacts with p130 Cas (Fig. 9B). Lysates from attached OCLs were used for this experiment, and the p130 Cas that associated with the C-terminal domain of PYK2 was tyrosine-phosphorylated, suggesting that tyrosine-phosphorylated p130 Cas can associate with PYK2.

DISCUSSION
Bone resorbing osteoclasts are highly differentiated cells specialized in the digestion of mineralized matrix. Osteoclast activation is initiated by recognition of and adhesion to the bone surface, followed by cellular polarization and formation of the sealing zone, a specialized membrane structure mediating tight adhesion between the osteoclast cell membrane and the bone surface. It is well documented from pharmacological studies that ␣ v ␤ 3 , the major integrin in osteoclasts, is important for osteoclast function (27,33,34). ␣ v ␤ 3 mediates osteoclast adhesion to several RGD matrix proteins including vitronectin, osteopontin, bone sialoprotein, and fibronectin (35). Although ␣ v ␤ 3 localizes to podosomes, the initial adhesion structure in osteoclasts (26,36), the presence of this integrin in the mature sealing zone in resorbing osteoclasts is controversial (26,34,37,38). The signaling mechanisms regulating the adhesion-dependent activation of osteoclasts are not well understood. We have recently found that PYK2 is the major adhesion kinase in osteoclasts, which undergoes tyrosine phosphorylation upon ␤ 3 -integrin engagement and that localizes to the sealing zone (22).
Similarly, p130 Cas tyrosine phosphorylation correlates with the formation of the podosomal-rich adhesion structures in osteoclasts plated on glass or on plastic culture dishes (24). These findings suggest that both PYK2 and p130 Cas may be involved in the adhesion-dependent signaling that mediates cytoskeletal organization in osteoclasts. This is further supported by the present study which shows that both p130 Cas and PYK2 are tyrosine-phosphorylated upon pOC adhesion to the same ECM proteins and with a similar time course. In addition, p130 Cas , similar to PYK2, is highly tyrosine-phosphorylated in pOCs upon ␤ 3 -integrin clustering by either ligand engagement or antibody cross-linking. However, whereas PYK2 appears to be selectively tyrosine-phosphorylated upon clustering of the ␤ 3 -integrin (22), p130 Cas tyrosine phosphorylation is also induced, albeit to a lower level by other osteoclast integrins including ␤ 1 and ␤ 2 -integrins.
The adhesion-dependent tyrosine phosphorylation of p130 Cas in osteoclasts is consistent with findings in other cell types although the kinetics are slightly different. In fibroblasts (17) and primary chicken embryo cells (15), maximal phosphorylation was seen at 15-20 min, whereas in osteoclasts, optimal phosphorylation is not reached until after 30 min, paralleling the time course of osteoclast spreading. The preferences for ECM proteins is also different, osteopontin, vitronectin, and fibronectin in osteoclasts rather than primarily fibronectin in fibroblasts. ␤ 3 seems to be the major integrin that mediates these effects in osteoclasts.
We further examined the localization of p130 Cas in osteoclasts and found it to be present in podosomes, where it co-localizes with PYK2 and cytoskeletal proteins. More importantly, it colocalizes with PYK2 in the sealing zone. During bone resorption, osteoclast adhesion to bone matrix leads to reorganization of cytoskeletal structures and formation of the sealing zone, a tight attachment between the osteoclast plasma membrane and bone matrix, with specific organization of Factin and associated proteins, such as vinculin and talin (21). Initial formation and accumulation of podosomes precedes these changes (21). In this study, we localized p130 Cas by confocal microscopy in resorbing osteoclasts on bone and found it is present in the newly forming sealing zone and mainly at the edges (the double circle structure) of mature sealing zones. In these structures, p130 Cas colocalizes with vinculin and paxillin and as mentioned with PYK2. Together with our recent results on PYK2 (22), these findings strongly suggest that p130 Cas and the p130 Cas ⅐PYK2 complex play a role in the cytoskeletal reorganization and formation of the sealing zone during osteoclast activation.
Furthermore, co-immunoprecipitation of p130 Cas and PYK2 in osteoclasts shows stable association of the two proteins. This resembles the association of p130 Cas with FAK (15,18) and is consistent with recent findings in B cells (10). Using GSTfusion proteins, this association was found to be mediated by the SH3 domain of p130 Cas and the C-terminal domain of PYK2, similar to the association of p130 Cas with FAK (15). This is in agreement with recent findings by Ohba et al. (39), showing that the first proline-rich sequence in the C-terminal domain of PYK2 can bind SH3-domain of p130 Cas . Interestingly, SH3 domains of Src, Fyn, Lyn, or PI3-kinase did not bind to  PYK2, suggesting preferential interaction of the SH3 domain of p130 Cas with PYK2. These results suggest that tyrosine phosphorylation of p130 Cas and PYK2 are not necessary for the association of these two molecules.
This was further confirmed using osteoclasts derived from Src(Ϫ/Ϫ) mice, in which tyrosine phosphorylation of PYK2 (22) and p130 Cas (24) are markedly reduced, but p130 Cas still associates with PYK2. Interestingly, p130 Cas association with FAK was not readily detected in lysates from Src(Ϫ/Ϫ) fibroblasts. This association was found to be mediated by the N-terminal fragment of c-Src which includes its SH3 and SH2 domains (40), suggesting that c-Src serves as an adapter for the p130 Cas -FAK interaction (40). Our results indicate that in osteoclasts p130 Cas and PYK2 associate in the absence of c-Src but do not localize to the podosomes and ring-like structures (22,24). These observations suggest that c-Src may be necessary in osteoclasts for the integrin-mediated activation of the p130 Cas ⅐PYK2 complex required for its recruitment to the adhesion structures. Tyrosine phosphorylation of the p130 Cas ⅐PYK2 complex could regulate and possibly enhance downstream signaling events initiated by ␤ 3 -integrins ligation in osteoclasts.
In osteoclasts, c-Src is highly expressed and is essential for osteoclast function (32). Src-deficient mice have an osteopetrotic phenotype caused by nonfunctional osteoclasts (32). The majority of c-Src is localized in the osteoclast ruffled border and in vacuoles rather than in the sealing zone (41,42), suggesting that the interaction of c-Src and the p130 Cas ⅐PYK2 complex precedes formation of the actin ring and sealing zone. However, the direct involvement of c-Src in this process is clearly indicated by our recent findings showing adhesion-induced association of PYK2 with c-Src in osteoclasts (22) and PYK2 phosphorylation by c-Src in vitro. In addition, both PYK2 and c-Src were shown to translocate to the cytoskeleton upon osteoclast adhesion (22,43), pointing to Src interaction with PYK2 in vivo. Furthermore, integrin-mediated p130 Cas tyrosine phosphorylation is substantially diminished in both Src-deficient fibroblasts and osteoclasts (19,24). In addition, c-Src was shown to bind directly to p130 Cas (44), suggesting that p130 Cas could be a substrate of c-Src in vivo. In human B cells, PYK2 phosphorylates p130 Cas following ␤ 1 -mediated stimulation (45). Taken together, these observations suggest that cooperation between PYK2 and Src kinases may be required for the complete phosphorylation of p130 Cas , which is probably involved in downstream signaling.
In summary, the findings presented here show adhesion-and ␤ 3 -integrin-mediated, Src-dependent tyrosine phosphorylation of p130 Cas in osteoclasts with similar kinetics as recently shown for PYK2. Furthermore, p130 Cas and PYK2 are stably associated via the SH3 domain of p130 Cas and the proline-rich C-terminal domain of PYK2, independent of tyrosine phosphorylation or c-Src. Finally, p130 Cas localizes to podosomes and the sealing zone, the functionally important adhesion structures in osteoclasts, where it co-localizes with PYK2. Taken together these findings suggest a role for p130 Cas and the p130 Cas ⅐PYK2 complex in the c-Src and adhesion-dependent signaling that leads to osteoclast activation, cytoskeletal reorganization, and formation of the sealing zone.