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Tyrosine Phosphorylation of p130Cas Is Involved in Actin Organization in Osteoclasts

1998; Elsevier BV; Volume: 273; Issue: 18 Linguagem: Inglês

10.1074/jbc.273.18.11144

1083-351X

Ichiro Nakamura, Eijiro Jimi, Le T. Duong, Takahísa Sasaki, Naoyuki Takahashi, Gideon A. Rodan, Tatsuo Suda,

Protease and Inhibitor Mechanisms

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. 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) 1The abbreviations used are: ECM, extracellular matrix; Cas, Crk-associated substrate; TRAP, tartrate-resistant acid phosphatase; FAK, focal adhesion kinase; PTP, protein-tyrosine phosphatase; SH, Src homology; OCL, osteoclast-like cell; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TBS-T, Tris-buffered saline with Tween 20. 1The abbreviations used are: ECM, extracellular matrix; Cas, Crk-associated substrate; TRAP, tartrate-resistant acid phosphatase; FAK, focal adhesion kinase; PTP, protein-tyrosine phosphatase; SH, Src homology; OCL, osteoclast-like cell; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TBS-T, Tris-buffered saline with Tween 20. interactions (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar). Integrin/ECM protein interactions participate in a variety of biological processes including embryonic development, wound healing, tumor metastases, and immune responses (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar, 2Juliano R.L. Haskill S. J. Cell Biol. 1993; 120: 577-585Crossref PubMed Scopus (1528) Google Scholar). It is now established that, in addition to mediating cell adhesion, integrins activate multiple signaling pathways. These include elevation of intracellular Ca2+, lipid turnover, and tyrosine phosphorylation, leading to cytoskeletal rearrangement and de novo gene expression (3Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2809) Google Scholar). The proteins that are tyrosine-phosphorylated by ECM-integrin interactions include focal adhesion kinase (FAK) (4Guan J.L. Shalloway D. Nature. 1992; 358: 690-692Crossref PubMed Scopus (723) Google Scholar), paxillin (5Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1175) Google Scholar), tensin (6Bockholt S.M. Burridge K. J. Biol. Chem. 1993; 268: 14565-14567Abstract Full Text PDF PubMed Google Scholar), and cortactin (7Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar).Osteoclasts, the bone-resorbing cells, play a critical role in bone remodeling (8Athanasou N.A. J. Bone Joint Surg. 1996; 78A: 1096-1112Crossref Scopus (131) Google Scholar, 9Roodman G.D. Endocr. Rev. 1996; 17: 308-332PubMed Google Scholar, 10Suda T. Udagawa N. Takahashi N. Principles of Bone Biology. Academic Press, San Diego1996: 87-102Google Scholar). 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 αvβ3 integrins play a central role in osteoclast adhesion (11Davies J. Warwick J. Totty N. Philp R. Helfrich M. Horton M. J. Cell Biol. 1989; 109: 1817-1826Crossref PubMed Scopus (319) Google Scholar, 12Reinholt F.P. Hultenby K. Oldberg A. Heinegard D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4473-4475Crossref PubMed Scopus (698) Google Scholar, 13Lakkakorpi P.T. Horton M.A. Helfrich M.H. Karhukorpi E.K. Väänänen H.K. J. Cell Biol. 1991; 115: 1179-1186Crossref PubMed Scopus (156) Google Scholar, 14Lakkakorpi P.T. Helfrich M.H. Horton M.A. Väänänen H.K. J. Cell Sci. 1993; 104: 663-670Crossref PubMed Google Scholar, 15Nesbitt S. Nesbit A. Helfrich M. Horton M. J. Biol. Chem. 1993; 268: 16737-16745Abstract Full Text PDF PubMed Google Scholar, 16Nakamura I. Gailit J. Sasaki T. Cell Tissue Res. 1996; 286: 507-515Crossref PubMed Scopus (50) Google Scholar). 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 (17Nakamura I. Takahashi N. Sasaki T. Jimi E. Kurokawa T. Suda T. J. Bone Miner. Res. 1996; 11: 1873-1879Crossref PubMed Scopus (92) Google Scholar). This ringlike organization, the actin ring, is formed by the assembly of podosomes that precedes the formation of the sealing zone (18Zambonin-Zallone A. Teti A. Carano A. Marchisio P.C. J. Bone Miner. Res. 1988; 5: 517-523Google Scholar, 19Kanehisa J. Yamanaka T. Doi S. Turksen K. Heersche J.N.M. Aubin J.E. Takeuchi H. Bone. 1990; 11: 287-293Crossref PubMed Scopus (121) Google Scholar, 20Lakkakorpi P.T. Väänänen H.K. J. Bone Miner. Res. 1991; 6: 817-826Crossref PubMed Scopus (203) Google Scholar) and has been considered to be a marker of osteoclast activation (17Nakamura I. Takahashi N. Sasaki T. Jimi E. Kurokawa T. Suda T. J. Bone Miner. Res. 1996; 11: 1873-1879Crossref PubMed Scopus (92) Google Scholar, 21Teti A. Marchisio P.C. Zambonin-Zallone A. Am. J. Physiol. 1991; 261: C1-C7Crossref PubMed Google Scholar, 22Väänänen H.K. Horton M. J. Cell Sci. 1995; 108: 2729-2732Crossref PubMed Google Scholar). Actually, various inhibitory agents of osteoclast function disrupt this actin ring (23Suda T. Nakamura I. Jimi E. Takahashi N. J. Bone Miner. Res. 1997; 12: 869-879Crossref PubMed Scopus (336) Google Scholar).In this study, we examined protein-tyrosine phosphorylation occurring during the adhesion-induced actin organization in osteoclasts, and identified p130Cas (Crk-associatedsubstrate (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 (24Reynolds A.B. Kanner S.B. Wang H.-C.R. Parsons J.T. Mol. Cell. Biol. 1989; 9: 3951-3958Crossref PubMed Scopus (135) Google Scholar, 25Kanner S.B. Reynolds A.B. Vines R.R. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3328-3332Crossref PubMed Scopus (396) Google Scholar, 26Kanner S.B. Reynolds A.B. Wang H.-C.R. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar) or v-crk (27Mayer B.J. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2638-2642Crossref PubMed Scopus (105) Google Scholar, 28Matsuda M. Mayer B.J. Fukui Y. Hanafusa H. Science. 1990; 248: 1537-1539Crossref PubMed Scopus (283) Google Scholar, 29Birge R.B. Fajardo J.E. Mayer B.J. Hanafusa H. J. Biol. Chem. 1992; 267: 10588-10595Abstract Full Text PDF PubMed Google Scholar). 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 (30Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Nishida J. Yazaki Y. Hirai H. J. Biol. Chem. 1994; 269: 32740-32746Abstract Full Text PDF PubMed Google Scholar, 31Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). The SH3 domain of Cas is known to bind to FAK (32Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 33Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), FAK-related nonkinase (33Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), and PTP1B (protein-tyrosine phosphatase 1B) (34Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The substrate domain, which has 15 potentially phosphorylated tyrosine residues, binds to v-Crk (35Burnham M.R. Harte M.T. Richardson A. Parsons J.T. Bouton A.H. Oncogene. 1996; 12: 2467-2472PubMed Google Scholar, 36Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). 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 (36Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). 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 (7Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,37Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 38Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). 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(−/−) mice.DISCUSSIONWe have reported previously that actin ring formation in osteoclasts is dependent on the interaction of integrins and matrix proteins (17Nakamura I. Takahashi N. Sasaki T. Jimi E. Kurokawa T. Suda T. J. Bone Miner. Res. 1996; 11: 1873-1879Crossref PubMed Scopus (92) Google Scholar). 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 (24Reynolds A.B. Kanner S.B. Wang H.-C.R. Parsons J.T. Mol. Cell. Biol. 1989; 9: 3951-3958Crossref PubMed Scopus (135) Google Scholar, 25Kanner S.B. Reynolds A.B. Vines R.R. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3328-3332Crossref PubMed Scopus (396) Google Scholar, 26Kanner S.B. Reynolds A.B. Wang H.-C.R. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar) or v-Crk (27Mayer B.J. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2638-2642Crossref PubMed Scopus (105) Google Scholar, 28Matsuda M. Mayer B.J. Fukui Y. Hanafusa H. Science. 1990; 248: 1537-1539Crossref PubMed Scopus (283) Google Scholar, 29Birge R.B. Fajardo J.E. Mayer B.J. Hanafusa H. J. Biol. Chem. 1992; 267: 10588-10595Abstract Full Text PDF PubMed Google Scholar), 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 (31Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). Moreover, several studies have reported that tyrosine phosphorylation of Cas is induced by cell adhesion (7Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 37Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 38Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar), and that Cas is localized to focal adhesions (33Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar,38Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar).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 (19Kanehisa J. Yamanaka T. Doi S. Turksen K. Heersche J.N.M. Aubin J.E. Takeuchi H. Bone. 1990; 11: 287-293Crossref PubMed Scopus (121) Google Scholar, 20Lakkakorpi P.T. Väänänen H.K. J. Bone Miner. Res. 1991; 6: 817-826Crossref PubMed Scopus (203) Google Scholar, 21Teti A. Marchisio P.C. Zambonin-Zallone A. Am. J. Physiol. 1991; 261: C1-C7Crossref PubMed Google Scholar), 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 (34Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) and PTP-PEST (51Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar), 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 (52Sasaki T. Debari K. Udagawa N. Calcif. Tissue Int. 1993; 53: 217-221Crossref PubMed Scopus (9) Google Scholar). 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 (31Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). 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 (31Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). 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 non-transformed 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 (7Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,37Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 38Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). Moreover, recent reports indicate that FAK can bind to the SH3 domain of Cas in vivo (32Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 33Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). On the other hand, the C-terminal portion of Cas can also bind directly the SH2 and SH3 domains of Src kinase (36Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). 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 (47Hamasaki K. Mimura T. Morino N. Furuya H. Nakamoto T. Aizawa S. Morimoto C. Yazaki Y. Hirai H. Nojima Y. Biochem. Biophys. Res. Commun. 1996; 222: 338-343Crossref PubMed Scopus (116) Google Scholar,48Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar). 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(−/−) OCLs, whereas the expression of Cas was not suppressed (Fig. 8). Moreover, actin rings did not form in Src(−/−) 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(−/−) mice, osteoclast activity is severely compromised, resulting in osteopetrosis (42Soriano P. Montgomery C. Geske R. Bradley A. Cell. 1991; 64: 693-702Abstract Full Text PDF PubMed Scopus (1793) Google Scholar, 43Boyce B.F. Yoneda T. Lowe C. Soriano P. Mundy G.R. J. Clin. Invest. 1992; 90: 1622-1627Crossref PubMed Scopus (516) Google Scholar) and, as shown here, Src(−/−) 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(−/−) mice. To prove this hypothesis, rescue experiments of Src(−/−) mice using Cas or Cas related molecules are required, and are now in progress.Recently, Nakamoto et al. (53Nakamoto T. Sakai R. Honda H. Ogawa S. Ueno H. Suzuki T. Aizawa S. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 3884-3897Crossref PubMed Scopus (135) Google Scholar) 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. Integrins are a major family of cell surface receptors that play crucial roles in cell-cell and cell-extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; Cas, Crk-associated substrate; TRAP, tartrate-resistant acid phosphatase; FAK, focal adhesion kinase; PTP, protein-tyrosine phosphatase; SH, Src homology; OCL, osteoclast-like cell; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TBS-T, Tris-buffered saline with Tween 20. 1The abbreviations used are: ECM, extracellular matrix; Cas, Crk-associated substrate; TRAP, tartrate-resistant acid phosphatase; FAK, focal adhesion kinase; PTP, protein-tyrosine phosphatase; SH, Src homology; OCL, osteoclast-like cell; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TBS-T, Tris-buffered saline with Tween 20. interactions (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar). Integrin/ECM protein interactions participate in a variety of biological processes including embryonic development, wound healing, tumor metastases, and immune responses (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar, 2Juliano R.L. Haskill S. J. Cell Biol. 1993; 120: 577-585Crossref PubMed Scopus (1528) Google Scholar). It is now established that, in addition to mediating cell adhesion, integrins activate multiple signaling pathways. These include elevation of intracellular Ca2+, lipid turnover, and tyrosine phosphorylation, leading to cytoskeletal rearrangement and de novo gene expression (3Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2809) Google Scholar). The proteins that are tyrosine-phosphorylated by ECM-integrin interactions include focal adhesion kinase (FAK) (4Guan J.L. Shalloway D. Nature. 1992; 358: 690-692Crossref PubMed Scopus (723) Google Scholar), paxillin (5Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1175) Google Scholar), tensin (6Bockholt S.M. Burridge K. J. Biol. Chem. 1993; 268: 14565-14567Abstract Full Text PDF PubMed Google Scholar), and cortactin (7Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Osteoclasts, the bone-resorbing cells, play a critical role in bone remodeling (8Athanasou N.A. J. Bone Joint Surg. 1996; 78A: 1096-1112Crossref Scopus (131) Google Scholar, 9Roodman G.D. Endocr. Rev. 1996; 17: 308-332PubMed Google Scholar, 10Suda T. Udagawa N. Takahashi N. Principles of Bone Biology. Academic Press, San Diego1996: 87-102Google Scholar). 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 αvβ3 integrins play a central role in osteoclast adhesion (11Davies J. Warwick J. Totty N. Philp R. Helfrich M. Horton M. J. Cell Biol. 1989; 109: 1817-1826Crossref PubMed Scopus (319) Google Scholar, 12Reinholt F.P. Hultenby K. Oldberg A. Heinegard D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4473-4475Crossref PubMed Scopus (698) Google Scholar, 13Lakkakorpi P.T. Horton M.A. Helfrich M.H. Karhukorpi E.K. Väänänen H.K. J. Cell Biol. 1991; 115: 1179-1186Crossref PubMed Scopus (156) Google Scholar, 14Lakkakorpi P.T. Helfrich M.H. Horton M.A. Väänänen H.K. J. Cell Sci. 1993; 104: 663-670Crossref PubMed Google Scholar, 15Nesbitt S. Nesbit A. Helfrich M. Horton M. J. Biol. Chem. 1993; 268: 16737-16745Abstract Full Text PDF PubMed Google Scholar, 16Nakamura I. Gailit J. Sasaki T. Cell Tissue Res. 1996; 286: 507-515Crossref PubMed Scopus (50) Google Scholar). 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 (17Nakamura I. Takahashi N. Sasaki T. Jimi E. Kurokawa T. Suda T. J. Bone Miner. Res. 1996; 11: 1873-1879Crossref PubMed Scopus (92) Google Scholar). This ringlike organization, the actin ring, is formed by the assembly of podosomes that precedes the formation of the sealing zone (18Zambonin-Zallone A. Teti A. Carano A. Marchisio P.C. J. Bone Miner. Res. 1988; 5: 517-523Google Scholar, 19Kanehisa J. Yamanaka T. Doi S. Turksen K. Heersche J.N.M. Aubin J.E. Takeuchi H. Bone. 1990; 11: 287-293Crossref PubMed Scopus (121) Google Scholar, 20Lakkakorpi P.T. Väänänen H.K. J. Bone Miner. Res. 1991; 6: 817-826Crossref PubMed Scopus (203) Google Scholar) and has been considered to be a marker of osteoclast activation (17Nakamura I. Takahashi N. Sasaki T. Jimi E. Kurokawa T. Suda T. J. Bone Miner. Res. 1996; 11: 1873-1879Crossref PubMed Scopus (92) Google Scholar, 21Teti A. Marchisio P.C. Zambonin-Zallone A. Am. J. Physiol. 1991; 261: C1-C7Crossref PubMed Google Scholar, 22Väänänen H.K. Horton M. J. Cell Sci. 1995; 108: 2729-2732Crossref PubMed Google Scholar). Actually, various inhibitory agents of osteoclast function disrupt this actin ring (23Suda T. Nakamura I. Jimi E. Takahashi N. J. Bone Miner. Res. 1997; 12: 869-879Crossref PubMed Scopus (336) Google Scholar). In this study, we examined protein-tyrosine phosphorylation occurring during the adhesion-induced actin organization in osteoclasts, and identified p130Cas (Crk-associatedsubstrate (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 (24Reynolds A.B. Kanner S.B. Wang H.-C.R. Parsons J.T. Mol. Cell. Biol. 1989; 9: 3951-3958Crossref PubMed Scopus (135) Google Scholar, 25Kanner S.B. Reynolds A.B. Vines R.R. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3328-3332Crossref PubMed Scopus (396) Google Scholar, 26Kanner S.B. Reynolds A.B. Wang H.-C.R. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar) or v-crk (27Mayer B.J. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2638-2642Crossref PubMed Scopus (105) Google Scholar, 28Matsuda M. Mayer B.J. Fukui Y. Hanafusa H. Science. 1990; 248: 1537-1539Crossref PubMed Scopus (283) Google Scholar, 29Birge R.B. Fajardo J.E. Mayer B.J. Hanafusa H. J. Biol. Chem. 1992; 267: 10588-10595Abstract Full Text PDF PubMed Google Scholar). 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 (30Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Nishida J. Yazaki Y. Hirai H. J. Biol. Chem. 1994; 269: 32740-32746Abstract Full Text PDF PubMed Google Scholar, 31Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). The SH3 domain of Cas is known to bind to FAK (32Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 33Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), FAK-related nonkinase (33Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), and PTP1B (protein-tyrosine phosphatase 1B) (34Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The substrate domain, which has 15 potentially phosphorylated tyrosine residues, binds to v-Crk (35Burnham M.R. Harte M.T. Richardson A. Parsons J.T. Bouton A.H. Oncogene. 1996; 12: 2467-2472PubMed Google Scholar, 36Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). 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 (36Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). 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 (7Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,37Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 38Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). 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(−/−) mice. DISCUSSIONWe have reported previously that actin ring formation in osteoclasts is dependent on the interaction of integrins and matrix proteins (17Nakamura I. Takahashi N. Sasaki T. Jimi E. Kurokawa T. Suda T. J. Bone Miner. Res. 1996; 11: 1873-1879Crossref PubMed Scopus (92) Google Scholar). 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 (24Reynolds A.B. Kanner S.B. Wang H.-C.R. Parsons J.T. Mol. Cell. Biol. 1989; 9: 3951-3958Crossref PubMed Scopus (135) Google Scholar, 25Kanner S.B. Reynolds A.B. Vines R.R. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3328-3332Crossref PubMed Scopus (396) Google Scholar, 26Kanner S.B. Reynolds A.B. Wang H.-C.R. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar) or v-Crk (27Mayer B.J. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2638-2642Crossref PubMed Scopus (105) Google Scholar, 28Matsuda M. Mayer B.J. Fukui Y. Hanafusa H. Science. 1990; 248: 1537-1539Crossref PubMed Scopus (283) Google Scholar, 29Birge R.B. Fajardo J.E. Mayer B.J. Hanafusa H. J. Biol. Chem. 1992; 267: 10588-10595Abstract Full Text PDF PubMed Google Scholar), 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 (31Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). Moreover, several studies have reported that tyrosine phosphorylation of Cas is induced by cell adhesion (7Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 37Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 38Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar), and that Cas is localized to focal adhesions (33Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar,38Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar).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 (19Kanehisa J. Yamanaka T. Doi S. Turksen K. Heersche J.N.M. Aubin J.E. Takeuchi H. Bone. 1990; 11: 287-293Crossref PubMed Scopus (121) Google Scholar, 20Lakkakorpi P.T. Väänänen H.K. J. Bone Miner. Res. 1991; 6: 817-826Crossref PubMed Scopus (203) Google Scholar, 21Teti A. Marchisio P.C. Zambonin-Zallone A. Am. J. Physiol. 1991; 261: C1-C7Crossref PubMed Google Scholar), 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 (34Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) and PTP-PEST (51Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar), 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 (52Sasaki T. Debari K. Udagawa N. Calcif. Tissue Int. 1993; 53: 217-221Crossref PubMed Scopus (9) Google Scholar). 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 (31Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). 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 (31Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). 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 non-transformed 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 (7Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,37Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 38Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). Moreover, recent reports indicate that FAK can bind to the SH3 domain of Cas in vivo (32Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 33Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). On the other hand, the C-terminal portion of Cas can also bind directly the SH2 and SH3 domains of Src kinase (36Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). 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 (47Hamasaki K. Mimura T. Morino N. Furuya H. Nakamoto T. Aizawa S. Morimoto C. Yazaki Y. Hirai H. Nojima Y. Biochem. Biophys. Res. Commun. 1996; 222: 338-343Crossref PubMed Scopus (116) Google Scholar,48Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar). 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(−/−) OCLs, whereas the expression of Cas was not suppressed (Fig. 8). Moreover, actin rings did not form in Src(−/−) 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(−/−) mice, osteoclast activity is severely compromised, resulting in osteopetrosis (42Soriano P. Montgomery C. Geske R. Bradley A. Cell. 1991; 64: 693-702Abstract Full Text PDF PubMed Scopus (1793) Google Scholar, 43Boyce B.F. Yoneda T. Lowe C. Soriano P. Mundy G.R. J. Clin. Invest. 1992; 90: 1622-1627Crossref PubMed Scopus (516) Google Scholar) and, as shown here, Src(−/−) 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(−/−) mice. To prove this hypothesis, rescue experiments of Src(−/−) mice using Cas or Cas related molecules are required, and are now in progress.Recently, Nakamoto et al. (53Nakamoto T. Sakai R. Honda H. Ogawa S. Ueno H. Suzuki T. Aizawa S. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 3884-3897Crossref PubMed Scopus (135) Google Scholar) 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. We have reported previously that actin ring formation in osteoclasts is dependent on the interaction of integrins and matrix proteins (17Nakamura I. Takahashi N. Sasaki T. Jimi E. Kurokawa T. Suda T. J. Bone Miner. Res. 1996; 11: 1873-1879Crossref PubMed Scopus (92) Google Scholar). 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 (24Reynolds A.B. Kanner S.B. Wang H.-C.R. Parsons J.T. Mol. Cell. Biol. 1989; 9: 3951-3958Crossref PubMed Scopus (135) Google Scholar, 25Kanner S.B. Reynolds A.B. Vines R.R. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3328-3332Crossref PubMed Scopus (396) Google Scholar, 26Kanner S.B. Reynolds A.B. Wang H.-C.R. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar) or v-Crk (27Mayer B.J. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2638-2642Crossref PubMed Scopus (105) Google Scholar, 28Matsuda M. Mayer B.J. Fukui Y. Hanafusa H. Science. 1990; 248: 1537-1539Crossref PubMed Scopus (283) Google Scholar, 29Birge R.B. Fajardo J.E. Mayer B.J. Hanafusa H. J. Biol. Chem. 1992; 267: 10588-10595Abstract Full Text PDF PubMed Google Scholar), 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 (31Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). Moreover, several studies have reported that tyrosine phosphorylation of Cas is induced by cell adhesion (7Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 37Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 38Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar), and that Cas is localized to focal adhesions (33Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar,38Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). 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 (19Kanehisa J. Yamanaka T. Doi S. Turksen K. Heersche J.N.M. Aubin J.E. Takeuchi H. Bone. 1990; 11: 287-293Crossref PubMed Scopus (121) Google Scholar, 20Lakkakorpi P.T. Väänänen H.K. J. Bone Miner. Res. 1991; 6: 817-826Crossref PubMed Scopus (203) Google Scholar, 21Teti A. Marchisio P.C. Zambonin-Zallone A. Am. J. Physiol. 1991; 261: C1-C7Crossref PubMed Google Scholar), 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 (34Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) and PTP-PEST (51Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar), 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 (52Sasaki T. Debari K. Udagawa N. Calcif. Tissue Int. 1993; 53: 217-221Crossref PubMed Scopus (9) Google Scholar). 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 (31Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). 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 (31Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). 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 non-transformed 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 (7Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,37Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 38Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). Moreover, recent reports indicate that FAK can bind to the SH3 domain of Cas in vivo (32Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 33Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). On the other hand, the C-terminal portion of Cas can also bind directly the SH2 and SH3 domains of Src kinase (36Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). 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 (47Hamasaki K. Mimura T. Morino N. Furuya H. Nakamoto T. Aizawa S. Morimoto C. Yazaki Y. Hirai H. Nojima Y. Biochem. Biophys. Res. Commun. 1996; 222: 338-343Crossref PubMed Scopus (116) Google Scholar,48Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar). 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(−/−) OCLs, whereas the expression of Cas was not suppressed (Fig. 8). Moreover, actin rings did not form in Src(−/−) 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(−/−) mice, osteoclast activity is severely compromised, resulting in osteopetrosis (42Soriano P. Montgomery C. Geske R. Bradley A. Cell. 1991; 64: 693-702Abstract Full Text PDF PubMed Scopus (1793) Google Scholar, 43Boyce B.F. Yoneda T. Lowe C. Soriano P. Mundy G.R. J. Clin. Invest. 1992; 90: 1622-1627Crossref PubMed Scopus (516) Google Scholar) and, as shown here, Src(−/−) 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(−/−) mice. To prove this hypothesis, rescue experiments of Src(−/−) mice using Cas or Cas related molecules are required, and are now in progress. Recently, Nakamoto et al. (53Nakamoto T. Sakai R. Honda H. Ogawa S. Ueno H. Suzuki T. Aizawa S. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 3884-3897Crossref PubMed Scopus (135) Google Scholar) 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. We are grateful to Drs. Yasuhisa Fukui and Sayoko Ihara (University of Tokyo) for their help in Western blot analyses and to Lorraine Lipfert (Merck Research Laboratories) for critical reading and fruitful discussion.