title: Distinct phosphoinositide binding specificity of the GAP1 family proteins: characterization of the pleckstrin homology domains of MRASAL and KIAA0538.
abstract: GAP1, one of the Ras GTPase-activating protein families, includes four distinct genes (GAP1(m), GAP1(IP4BP), MRASAL (murine Ras GTPase-activating-like), and KIAA0538). It contains an amino-terminal tandem C2 domain, a GAP-related domain, and a carboxyl-terminal pleckstrin homology (PH) domain. Although the PH domains of GAP1(m) and GAP1(IP4BP) have been shown to be essential for membrane targeting via binding of specific phospholipids, little is known about the functions of the PH domains of MRASAL and KIAA0538. Herein, we show that the PH domain of MRASAL has binding activity toward PI(4,5)P(2) and PI(3,4,5)P(3), while the PH domain of KIAA0538 does not bind these phospholipids due to an amino acid substitution at position 592 (Leu-592). Mutation of the corresponding position of MRASAL (Arg-to-Leu substitution at position 591) resulted in loss of the phospholipid binding activity. MRASAL proteins were localized at the plasma membrane in NIH3T3 cells, and this plasma membrane association was unchanged even after cytochalasin B or wortmannin treatment. By contrast, KIAA0538 and MRASAL (R591L) proteins were present in the cytosol. Our data indicate that the distinct phosphoinositide binding specificity of the PH domain is attributable to the distinct subcellular localization of the GAP1 family.
authors.affiliation: International Joint Project "Calcium Oscillation,", Japan Science and Technology Corporation, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Minagawa
authors.forename: M
authors.initials: M
authors.lastname: Fukuda
authors.forename: K
authors.initials: K
authors.lastname: Mikoshiba
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D020703:ras GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001798:Blood Proteins; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D020033:Protein Isoforms; C491144:RASA4 protein, human; C039077:platelet protein P47; D020703:ras GTPase-Activating Proteins