ADP-ribosylation factors (Arfs) are members of the Ras superfamily that coordinated membrane and actin remodeling, which integral to a number of cellular functions, including cell movement, endocytosis and exocytosis, and mitosis and are central to pathological processes such as tumor cell invasion and metastasis. In our studies of the regulation of Arfs, we discovered the Arf GTPase-activating proteins (GAPs), which facilitate the hydrolysis of GTP bound to Arf, converting Arf-GTP to Arf-GDP. The first GAP we discovered, ASAP1, is composed of a BAR, PH, Arf GAP, Ankyrin repeat, proline rich, E/DLPPKP repeat and SH3 domains. It regulates remodeling of the actin cytoskeleton and associated focal adhesions. Consistent with these biochemical activities, it has been implicated in regulating differentiation and has also been implicated as a regulator of cancer cell behaviors, including invasion and metastasis. Furthermore, ASAP1 is overexpressed in a number of cancers, including childhood rhabdomyosarcomas and overexpression correlates with poor prognosis in a number of cancers, which has motivated our recent focus on ASAP1. We study three aspects of ASAP1 biochemistry and biology, with progress in all three areas in the past year. First, we are working towards determining the mechanism of regulated catalysis by the Arf GAP domain. In the past year, we have discovered that the PH domain is an integral part of the catalytic pocket, necessary for function of the Arf GAP domain and, in collaboration with Dr. R. Andrew Byrd, have discovered that the PH domain binds directly to an N-terminal extension of the substrate Arf-GTP. We are currently extending the work to define mechanism at atomic resolution. In the second area of study, we are examining the link between oncoproteins to which ASAP1 binds and the actin remodeling that it mediates. In the past year, we have discovered direct binding of the BAR and PH domain of ASAP1 to F-actin, which drives bundling of the F-actin. In ongoing studies, we are determining the contribution to ASAP1-driven actin bundling to remodeling of actin in stress fibers, invadopodia and circular dorsal ruffles. In a third area of work in collaboration with Dr. Marielle Yohe of Pediatric Oncology Branch, we are examining the contribution of ASAP1 to the behavior of fusion-negative rhabdomyosarcoma (FN-RMS), an ideal model for the function of ASAP1 in cancer cells. First, as for other cancer, ASAP1 is overexpressed in fusion-negative rhabdomyosarcoma. Second, while ASAP1 has been found to affect both differentiation of nontransformed cells and proliferation of cancer cells, fusion-negative rhabdomyosarcoma has a defect in differentiation of myoblasts. In the past year, we have discovered that ASAP1 regulates differentiation pathways in both myoblasts and rhabdomyosarcoma cell lines and that there are differences in effects on myoblasts and fusion-negative rhabdomyosarcoma. In ongoing studies, we are examining how these differences affect differentiation, proliferation, invasion and metastasis.