title: Structure-function relationships of the mouse Gap1m. Determination of the inositol 1,3,4,5-tetrakisphosphate-binding domain.
abstract: Gap1(IP4BP), one of a member of Ras GTPase-activating proteins, has been identified as a specific inositol 1,3,4,5-tetrakisphosphate (IP4)-binding protein (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 386, 527-530). In this paper we describe Gap1(m), which is closely related to Gap1(IP4BP), to also be an IP4-binding protein and show that the pleckstrin homology domain (PH) is the central IP4-binding domain by expressing fragments of the mouse Gap1(m) in Escherichia coli as fusion proteins and examining their activities. However, in addition to the PH domain, an adjacent GAP-related domain and carboxyl terminus are required for high affinity specific IP4 binding. The PH domain is highly conserved in the Gap1 family and also has striking homology to the amino-terminal region of Bruton's tyrosine kinase. Substitution of Cys for Arg at position 628 in the PH domain corresponding to the mutation of Bruton's tyrosine kinase observed in X-linked immunodeficiency mice results in a dramatic reduction of IP4 binding activity as well as phospholipid binding capacity of Gap1(m). This mutant also showed the GAP activity against Ha-Ras to be similar to that of the wild type Gap1(m). Our results suggest that the PH domain of Gap1(m) functions as a modulatory domain of GAP activity by binding IP4 and phospholipids.
authors.affiliation: Molecular Neurobiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Fukuda
authors.forename: K
authors.initials: K
authors.lastname: Mikoshiba
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001798:Blood Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D020690:GTPase-Activating Proteins; D007295:Inositol Phosphates / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins; D017354:Point Mutation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D020703:ras GTPase-Activating Proteins
chemical_list: D001798:Blood Proteins; D017931:DNA Primers; D020690:GTPase-Activating Proteins; D007295:Inositol Phosphates; D010750:Phosphoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; D020703:ras GTPase-Activating Proteins; C081655:inositol-1,3,4,5-tetrakisphosphate