Abstract
ASAP1 is an Arf GAP with a PH domain immediately N-terminal to the catalytic Arf GAP domain. PH domains are thought to regulate enzymes by binding to specific phosphoinositide lipids in membranes, thereby recruiting the enzyme to a site of action. Here, we have examined the functional relationship between the PH and Arf GAP domains. We found that GAP activity requires the cognate PH domain of ASAP1, leading us to hypothesize that the Arf GAP and PH domains directly interact to form the substrate binding site. This hypothesis was supported by the combined results of protection and hydrodynamic studies. We then examined the role of the PH domain in the regulation of Arf GAP activity. The results of saturation kinetics, limited proteolysis, FRET and fluorescence spectrometry support a model in which regulation of the GAP activity of ASAP1 involves a conformational change coincident with recruitment to a membrane surface, and a second conformational change following the specific binding of phosphatidylinositol 4,5-bisphosphate.
MeSH terms
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ADP-Ribosylation Factor 1 / metabolism
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Adaptor Proteins, Signal Transducing / chemistry
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Adaptor Proteins, Signal Transducing / genetics
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Adaptor Proteins, Signal Transducing / metabolism*
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Amino Acid Sequence
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Binding Sites / genetics
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Binding, Competitive / drug effects
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Biotin / analogs & derivatives
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Biotin / chemistry
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Blood Proteins / metabolism
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Catalytic Domain
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Fluorescence Resonance Energy Transfer
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GTPase-Activating Proteins / metabolism
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Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
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Guanosine 5'-O-(3-Thiotriphosphate) / pharmacology
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Guanosine Triphosphate / metabolism
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Guanosine Triphosphate / pharmacology
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Humans
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Isoenzymes / genetics
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Isoenzymes / metabolism
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Kinetics
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Liposomes / metabolism
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Liposomes / pharmacology
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Lysine / chemistry
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Models, Molecular
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Molecular Sequence Data
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Mutation / genetics
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Peptide Fragments / chemistry
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Peptide Fragments / genetics
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Peptide Fragments / metabolism
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Phosphatidic Acids / metabolism
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Phosphatidylinositol 4,5-Diphosphate / analogs & derivatives
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Phosphatidylinositol 4,5-Diphosphate / metabolism
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Phospholipase C delta
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Phospholipids / metabolism*
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Phospholipids / pharmacology
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Phosphoproteins / metabolism
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Plasmids / genetics
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Protein Binding / drug effects
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Protein Conformation
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Sequence Homology, Amino Acid
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Succinimides / chemistry
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Type C Phospholipases / genetics
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Type C Phospholipases / metabolism
Substances
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ASAP1 protein, human
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Adaptor Proteins, Signal Transducing
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Blood Proteins
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GTPase-Activating Proteins
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Isoenzymes
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Liposomes
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Peptide Fragments
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Phosphatidic Acids
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Phosphatidylinositol 4,5-Diphosphate
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Phospholipids
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Phosphoproteins
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Recombinant Fusion Proteins
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Succinimides
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biotinyl N-hydroxysuccinimide ester
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platelet protein P47
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Guanosine 5'-O-(3-Thiotriphosphate)
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Biotin
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Guanosine Triphosphate
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Type C Phospholipases
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Phospholipase C delta
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ADP-Ribosylation Factor 1
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Lysine