Regulation of ADP-ribosylation factor

Division Of Basic Sciences - Nci

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Abstract

ADP-ribosylation factors (Arfs) are members of the Ras superfamily. They coordinate membrane and actin remodeling, which are integral to normal and pathologic cellular behaviors, including cancer cell invasion and metastasis. 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 migration and has also been implicated in cancer invasion and metastasis. Furthermore, the ASAP1 gene is amplified in a number of cancers and expression correlates with poor prognosis in a number of cancers. The potential link to cancer progression has motivated our 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, through a site opposite the phosphoinositide binding site, 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 two years, we have discovered direct binding of the BAR and PH domain of ASAP1 to F-actin, which drives bundling of the F-actin and is necessary for remodeling of actin in stress fibers, invadopodia and circular dorsal ruffles. We have begun to define the binding site between ASAP1 and F-actin. In a third area of work, we are examining the contribution of ASAP1 to the behavior of fusion-negative rhabdomyosarcoma (FN-RMS), a model for the function of ASAP1 in cancer cells. First, as for other cancer, ASAP1 is amplified in fusion-negative rhabdomyosarcoma. Second, ASAP1 has been found to affect both differentiation of nontransformed cells and proliferation of cancer cells and 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 in vitro and in vivo studies, including intravital studies, we are examining how these differences affect differentiation, proliferation, invasion and metastasis.