Tyrosine Phosphorylation of p130Cas Is Involved in Actin Organization in Osteoclasts*
- Ichiro Nakamura‡§,
- Eijiro Jimi‡,
- Le T. Duong§,
- Takahisa Sasaki‡,
- Naoyuki Takahashi‡,
- Gideon A. Rodan§ and
- Tatsuo Suda‡¶
- From the ‡Departments of Biochemistry and Oral Anatomy, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan and the §Department of Bone Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
Abstract
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.
Footnotes
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↵* This work was supported in part by Grants-in-aid 07557118 and 08557101 from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom reprint requests should be addressed: Dept. of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan.
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↵1 The 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.
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- Received December 23, 1997.
- Revision received February 25, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
