Fluid shear stress is generated as a result of interstitial fluid that moves within the bone upon exposure to mechanical stimulation. Osteoblasts respond to this fluid shear stress by controlling RAD001 in vivo expression of proteins involved in bone formation and bone resorption such as cyclooxygenase-2 and prostaglandin E2 in a process defined as mechanotransduction. Our lab has proposed that changes in gene expression result from unique signaling complexes called mechanosomes that originate at sites of adhesion with the extracellular matrix and with other bone cells. Focal adhesions, which are composed of integrins, vinculin, a-actinin, actin filaments and several other focal adhesion associated proteins, are proposed as likely mechanosensors in bone cells and are ideal launching sites for mechanosomes. Focal adhesion kinase is a non-receptor tyrosine kinase that associates with integrins at focal adhesions, and association of FAK with integrins at the focal adhesion results in an autophosphorylation event at tyrosine 397 which provides a binding site for Src and other signaling molecules. In addition, the C-terminal domain of FAK can associate with talin and paxillin which connects the focal adhesion with the actin cytoskeleton, and the ability of FAK to associated with several downstream effectors makes it a key component of the focal adhesion. Our previous studies reported FAK to function as a part of a mechanosome complex that is required for FSSinduced mechanotransduction in osteoblasts. Proline- rich tyrosine kinase 2 is another member of the FAK family of non-receptor tyrosine kinases that can also localize to focal adhesions. FAK and Pyk2 exhibit,48% amino acid sequence identity and share a similar domain structure. Both contain a unique N terminus, a protein tyrosine kinase domain and two proline-rich regions at the C terminus. Unlike FAK which is ubiquitously expressed, Pyk2 expression is restricted with the highest levels of expression in the brain and hematopoietic cells. Pyk2 is also highly expressed in osteoclasts in which it is primarily found in podosomes, actin-rich structures that mediate cell attachment and migration. Like FAK, Pyk2 has also been implicated in the regulation of bone health and mechanotransduction. Decreased expression of Pyk2 in murine osteoclast-like multinucleated cells exhibited impaired spreading and inhibited osteoclast bone resorption. Pyk2 can be also be found at focal contacts in ROS 17/2.8 osteoblast-like cells, and Pyk2 moves away from focal contacts in response to cyclic strain, which makes Pyk2 a likely candidate for functioning as part of a mechanosome. Additionally, mechanical strain of ROS 17/ 2.8 osteoblast-like cells resulted in the autophosphorylation of Pyk2 and FAK. The goal of this study was to better understand the role of the FAK and Pyk2 tyrosine kinases during osteoblast mechanotransduction.