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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Substrate specificities and activities of AZAP family Arf GAPs in vivo

Department of Cell Biology, University of Virginia Health System, Charlottesville, Virginia

Submitted 9 July 2007 ; accepted in final form 7 November 2007

The ADP-ribosylation factor (Arf) GTPases are important regulators of vesicular transport in eukaryotic cells. Like other GTPases, the Arfs require guanine nucleotide exchange factors to facilitate GTP loading and GTPase-activating proteins (GAPs) to promote GTP hydrolysis. Whereas there are only six mammalian Arfs, the human genome encodes over 20 proteins containing Arf GAP domains. A subset of these, referred to as AZAPs (Randazzo PA, Hirsch DS. Cell Signal 16: 401–413, 2004), are characterized by the presence of at least one NH₂-terminal pleckstrin homology domain and two or more ankyrin repeats following the GAP domain. The substrate specificities of these proteins have been previously characterized by using in vitro assay systems. However, a limitation of such assays is that they may not accurately represent intracellular conditions, including posttranslational modifications, or subcellular compartmentalization. Here we present a systematic analysis of the GAP activity of seven AZAPs in vivo, using an assay for measurement of cellular Arf-GTP (Santy LC, Casanova JE. J Cell Biol 154: 599–610, 2001). In agreement with previous in vitro results, we found that ACAP1 and ACAP2 have robust, constitutive Arf6 GAP activity in vivo, with little activity toward Arf1. In contrast, although ARAP1 was initially reported to be an Arf1 GAP, we found that it acts primarily on Arf6 in vivo. Moreover, this activity appears to be regulated through a mechanism involving the NH₂-terminal sterile- motif. AGAP1 is unique among the AZAPs in its specificity for Arf1, and this activity is dependent on its NH₂-terminal GTPase-like domain. Finally, we found that expression of AGAP1 induces a surprising reciprocal activation of Arf6, which suggests that regulatory cross talk exists among Arf isoforms.

adenosine 5'-diphosphate ribosylation factors; guanosine 5'-triphosphatase-activating protein; ACAP; ASAP; AGAP; ARAP