Regulation of ADP-ribosylation factor

Division Of Basic Sciences - Nci

Investigators

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Abstract

We have been focusing on ASAP1, an ADP-ribosylation factor (Arf) GTPase-activating protein (GAP) that induces hydrolysis of GTP bound to Arf, controls the actin cytoskeleton and has been implicated in invasion and metastasis for a number of cancers. Our goals for the year have been to test hypotheses about catalytic mechanism of GTP hydrolysis and to test hypothesis about the molecular basis for ASAP1-regulated actin remodeling. We have been using an integrated biophysical/biochemical approach to understand catalytic mechanism. Because the proteins functions at a membrane surface, we used model membranes tof two types, nanodiscs and large unilamellar vesicles, to study the reactions. We employ NMR, EPR, structural MS, and molecular dynamic simulations together with mutagenesis, enzymology and biochemistry to examine structure/function relationships. We discovered a paradigm shifting mechanism in which a domain (PH), long thought to be a simple targeting domain, is the substrate binding site, directly contributing to a change towards the transition state. This finding is important because PH domains are found in 285 proteins in humans, many of which are oncoproteins. Hence, discovering a previously unrecognized function of PH domains might be of therapeutic relevance. In addition, we discovered that the Arf GAP domain, previously thought to the catalytic domain, is incomplete as a catalyst. This finding influences interpretation of the broader literature on Arf GTPases and their function in membrane and actin remodeling. We are continuing the work to further define the catalytic mechanism and determining how GTP hydrolysis is linked to large scale motion of ASAP1 outside of the Arf GAP and PH domains. This work is relevant to our second goal of understanding the mechanisms by which ASAP1 controls actin remodeling, crucial to cell migration and to pinocytosis of receptor tyrosine kinases and their recycling, cell functions that can contribute to oncogenesis. We have discovered that ASAP1 is a key regulator of the actin cytoskeleton, controlling actin stress fibers, circular dorsal ruffles (sites of receptor tyrosine kinase pinocytosis) and invadosomes. Our studies revealed that ASAP1 binds to nonmuscle myosin 2 and F-actin through its BAR domain under control of the GAP domain. It also controls membrane remodeling similarly through the BAR domain controlled by the GAP domain. Thus, the proteins that had been thought to function as stopping signals through Arf actually mediate the effects of Arf. Furthermore, the since the effects of Arf depend on the GAP mediated hydrolysis of GTP on Arf, inhibition of the GAP would block the function of Arf, which is completely opposed to the conventional wisdom and has important ramifications for therapeutic targeting of this pathway.