Purification of ARAP3 and characterization of GAP activities.
ARAP3 is a dual Arf and Rho GTPase activating protein (GAP) that was identified from pig leukocyte cytosol using a phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns[3,4,5]P3) affinity matrix in a targeted proteomics study. ARAP3's domain structure includes five PH domains, an Arf GAP domain, three ankyrin repeats, a Rho GAP domain, and a Ras association domain. ARAP3 is a PtdIns(3,4,5)P3-dependent GAP for Arf6 both in vitro and in vivo. It acts as a Rap-GTP-activated RhoA GAP in vitro, and this activation depends on a direct interaction between ARAP3 and Rap-GTP; in vivo PtdIns(3,4,5)P3 seems to be required to allow ARAP3's activation as a RhoA GAP by Rap-GTP. Overexpression of ARAP3 in pig aortic endothelial (PAE) cells causes the PI3K-dependent loss of adhesion to the substratum and interferes with lamellipodium formation. This overexpression phenotype depends on ARAP3's intact abilities to bind PtdIns(3,4,5)P3, to interact with Rap-GTP, and to be a catalytically active RhoA and Arf6 GAP.
Inositide Laboratory, The Babraham Institute, Cambridge, UK.
Sonja
S
Krugmann
Len
L
Stephens
Phillip T
PT
Hawkins
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000032:analysis / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D003600:Cytosol / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D020690:GTPase-Activating Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D007962:Leukocytes / Q000737:chemistry; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D018411:Spodoptera; D013552:Swine; D018631:ras Proteins / Q000302:isolation & purification; D020741:rho GTP-Binding Proteins / Q000302:isolation & purification
D000089982:ADP-Ribosylation Factor 6; C446512:ARAP3 protein, human; D048868:Adaptor Proteins, Signal Transducing; D020690:GTPase-Activating Proteins; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C085379:rho GTPase-activating protein; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D018631:ras Proteins; D020741:rho GTP-Binding Proteins