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

doi:10.1074/jbc.274.8.4900

Stable Association of PYK2 and p130^Cas in Osteoclasts and Their Co-localization in the Sealing Zone*

From the ^‡Department of Bone Biology and Osteoporosis Research, Merck Research Laboratories, West Point, Pennsylvania 19486, the ^§Institute of Biomedicine, Department of Anatomy and Medicity Research Laboratories, University of Turku, FIN-20014 Turku, Finland, and the ^¶Department of Microbiology and Cancer Center, University of Virginia, Health Science Center, Charlottesville, Virginia 22908

Abstract

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 p130^Cas (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) p130^Cas is tyrosine-phosphorylated upon adhesion of pOCs to vitronectin or ligation of β₃ integrins; 2) p130^Cas 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) p130^Cas 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 p130^Cas and PYK2 is mediated by the SH3 domain of p130^Cas and the C-terminal domain of PYK2. These findings suggest that p130^Cas 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.

Footnotes

↵* 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.
↵‖ To whom correspondence should be addressed: Dept. of Bone Biology & Osteoporosis, Merck Research Laboratories, WP26A-1000, West Point, PA 19486. Tel.: 215-652-7574; Fax: 215-652-4328; E-mail:le_duong{at}merck.com.
Abbreviations:

ECM

extracellular matrix

Cas

Crk-associated substrate

FAK

focal adhesion kinase

PYK2

proline-rich kinase 2

SH

Src homology

OCL

osteoclast-like cell

pOC

prefusion osteoclast

mAb

monoclonal antibody

PI3

phosphatidylinositol 3

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

1α

25(OH)₂D₃, 1α,25-dihydroxy-vitamin D₃

RGD

arginine-glycine aspartic acid
- Received August 6, 1998.
- Revision received November 5, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.