Differences between AGAP1, ASAP1 and Arf GAP1 in substrate recognition: interaction with the N-terminus of Arf1.
The Arf GAPs are a structurally diverse group of proteins that catalyze the hydrolysis of GTP bound to Arf1. Here, we directly compare the role of amino acids 2-17 of Arf1, a GTP- and phospholipid-sensitive switch, for interaction with three Arf GAPs: Arf GAP1, AGAP1 and ASAP1. Sequestration of amino acids 2-17 with an antibody inhibited interaction with the three tested Arf GAPs. Examination of Arf1 mutants also indicated that [2-17]Arf1 is a critical structural determinant of interaction with all three Arf GAPs; however, the effect of specific mutations differed among the GAPs. Compared to wild-type Arf1, Arf1 with the amino terminal 13 ([Delta13]Arf1) and 17 amino acids ([Delta17]Arf1) deleted had 200- and 4000-fold reduced interaction with ASAP1 and 150-fold reduced interaction with AGAP1. In contrast, deletion of the amino terminus of Arf reduced interaction with Arf GAP1 by 5-fold. By analysis of point mutants, we found that lysines 15 and 16 had a greater contribution to productive interaction between Arf1, ASAP1 and AGAP1 than between Arf1 and Arf GAP1. Leucine 8 contributed to the interaction with Arf GAP1 but not with ASAP1 and AGAP1. Amino acids 2-17 of Arf1, isolated from the protein, inhibited GAP activity of Arf GAP1, ASAP1 and AGAP1 and bound directly to ASAP1. Taken together, our results indicate that (i) Arf GAPs interact with amino acids 2-17 of Arf1 and (ii) each subgroup of Arf GAPs has a unique interface with Arf1.
Yoon
Hye-Young
HY
Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Building 37, Room 4118, National Institutes of Health, Bethesda, MD 20892, USA.
Jacques
Kerry
K
Nealon
Beth
B
Stauffer
Stacey
S
Premont
Richard T
RT
Randazzo
Paul A
PA
D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D004789:Enzyme Activation / Q000187:drug effects; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D007106:Immune Sera / Q000494:pharmacology; D051379:Mice; D016296:Mutagenesis; D041681:NIH 3T3 Cells; D011485:Protein Binding; D021381:Protein Transport; D013379:Substrate Specificity
D048868:Adaptor Proteins, Signal Transducing; C503238:Arfgap1 protein, mouse; C503241:Arfgap3 protein, mouse; D020690:GTPase-Activating Proteins; D007106:Immune Sera; D020558:GTP Phosphohydrolases; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors