Stable association of PYK2 and p(130)Cas in their co-localization in the sealing zone

Bone resorption is initiated by osteoclast attachment to the mineralized ma trix, cytoskeletal reorganization, cellular polarization, and the formation of the sealing zone. The present study examines the interaction between PY K2 and p130(Cas) ((C) under bar rk-(a) under bar ssociated (s) under bar ub strate), suggested to be part of the signaling pathway initiated by osteocl ast adhesion. Using murine osteoclast-like cells (OCLs) and their mononucle ar precursors (pOCs), generated in a co-culture of bone marrow and osteobla stic MB1.8 cells, we show that: 1) p130(Cas) is tyrosine-phosphorylated upo n adhesion of pOCs to vitronectin or ligation of beta(3) 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 phosph orylation of either molecule, and this complex is present in Src (-/-) OCLs , in which neither protein is phosphorylated or associated with the osteocl ast adhesion structure; 4) the association of p130(Cas) and PYK2 is mediate d by the SH3 domain of p130(Cas) and the C-terminal domain of PYK2. These f indings suggest that p130(Cas) and its association with PYK2 may play an im portant role in the adhesion-dependent signaling that leads to cytoskeletal reorganization and formation of the sealing zone during osteoclast activat ion.