Common cancers, such as those of the breast, prostate and lung, frequently metastasize to bone and lead to bone disorder and untreatable consequences. Bone metastases contribute heavily to morbidity and mortality. About 75% of patients with advanced breast carcinoma developed significant tumor burden in the skeleton and resulted in severe bone osteolytic lesions. Current understanding of the molecular mechanism of bone metastasis is limited, however, studies indicate that bone microenvironment plays a role in osteolytic metastasis and involves a coupling between osteolysis and cancer cell growth through interactions between the tumor cells and the bone-resorbing osteoclasts. Proline-rich tyrosine kinase (PYK2), a cellular adhesion kinase, is expressed in high levels in osteoclasts and plays an important role in the adhesion-dependent, integrin-mediated signaling that leads to cytoskeletal reorganization and formation of the sealing zone during osteoclast activation. Autophosphorylation of PYK2 and interactions with Src kinase and CAS, a docking protein for SH2 and SH3-containg molecules, is required for the signaling. However, little is known concerning the molecular mechanism by which PYK2 regulates osteoclast function. Our long-term research goal is to understand the structural biology and function of PYK2 in osteoclast activation, explore the relationship among its structure, function, and dynamics, and exploit this relation for the design of specific inhibitors that alter PYK2's function required for osteolysis. Towards this goal, we will determine the structure of PYK2 in complex with substrate analogs and interacting protein domains. We hypothesize that interruption of this signaling pathway by inhibition of PYK2 activity prevents osteoclast activation and eventually disrupts the vicious cycle of osteolysis and cancer cell growth. Through a combination of structure and activity-based approaches, we will identify candidate leads of PYK2 specific inhibitors which not only serve as a probe to test this hypothesis and further study the mechanism by which PYK2 acts in osteoclast activation, but also lead to a potential therapeutic agent for cancer-induced bone resorption. We propose a comprehensive and collaborative effort by focusing initially on the following specific aims: 1) to express and purify active PYK2 in full length and separate domains, to characterize the kinase activity and binding activity of PYK2 with the SH2 domain of Src and the SH3 domain of CAS; 2) to crystallize PYK2 and determine the three-dimensional structure of PYK2 and its domains; 3) to use a combination of structure- and cellular activity-based approaches to identify candidate leads of specific PYK2 inhibitors that target the PYK2 kinase domain, possibly the protein-protein interactions that are required for its function in osteoclast activation.