We have identified the presence of leupaxin (LPXN), which belongs to the paxillin extended family of focal adhesion-associated adaptor proteins, in prostate cancer cells. Previous studies have demonstrated that LPXN is a component of the podosomal signaling complex found in osteoclasts, where LPXN was found to associate with the protein tyrosine kinases Pyk2 and c-Src and the cytosolic protein tyrosine phosphatase-proline-, glutamate-, serine-, and threonine-rich sequence (PTP-PEST). In the current study, LPXN was detectable as a 50-kDa protein in PC-3 cells, a bone-derived metastatic prostate cancer cell line. In PC-3 cells, LPXN was also found to associate with Pyk2, c-Src, and PTP-PEST. A siRNA-mediated inhibition of LPXN resulted in decreased in vitro PC-3 cell migration. A recombinant adenoviral-mediated overexpression of LPXN resulted in an increased association of Pyk2 with LPXN, whereas a similar adenoviral-mediated overexpression of PTP-PEST resulted in decreased association of Pyk2 and c-Src with LPXN. The overexpression of LPXN in PC-3 cells resulted in increased migration, as assessed by in vitro Transwell migration assays. On the contrary, the overexpression of PTP-PEST in PC-3 cells resulted in decreased migration. The overexpression of LPXN resulted in increased activity of Rho GTPase, which was decreased in PTP-PEST-overexpressing cells. The increase in Rho GTPase activity following overexpression of LPXN was inhibited in the presence of Y27632, a selective inhibitor of Rho GTPase. In conclusion, our data demonstrate that LPXN forms a signaling complex with Pyk2, c-Src, and PTP-PEST to regulate migration of prostate cancer cells.PC-3; protein tyrosine phosphatase-proline-, glutamate-, serine-, and threonine-rich sequence; c-Src; migration LEUPAXIN (LPXN), which was originally characterized as a member of the paxillin extended family of multifunctional docking/adaptor phosphoproteins in cells of hematopoietic origin, was further identified as an additional component of the sealing zone in osteoclasts (7,25,40,51). In both hematopoietic cells and the osteoclast, LPXN was found to associate with the nonreceptor protein tyrosine kinase (PTK) Pyk2 and the cytoplasmic protein tyrosine phosphatase-proline-, glutamate-, serine-, and threonine-rich sequence (PTP-PEST) (25,40,72).More recently, we have demonstrated that, in osteoclasts,