title: AF-6 controls integrin-mediated cell adhesion by regulating Rap1 activation through the specific recruitment of Rap1GTP and SPA-1.
abstract: In the present study, we showed that SPA-1, a Rap1 GTPase-activating protein (GAP), was bound to a cytoskeleton-anchoring protein AF-6. SPA-1 and AF-6 were co-immunoprecipitated in the 293T cells transfected with both cDNAs as well as in normal thymocytes. In vitro binding studies using truncated fragments and their mutants suggested that SPA-1 was bound to the PDZ domain of AF-6 via probable internal PDZ ligand motif within the GAP-related domain. The motif was conserved among Rap1 GAPs, and it was shown that rapGAP I was bound to AF-6 comparably with SPA-1. RapV12 was also bound to AF-6 via the N-terminal domain, and SPA-1 and RapV12 were co-immunoprecipitated only in the presence of AF-6, indicating that they could be brought into close proximity via AF-6 in cells. Immunostaining analysis revealed that SPA-1 and RapV12 were co-localized with AF-6 at the cell attachment sites. In HeLa cells expressing SPA-1 in a tetracycline-regulatory manner, expression of AF-6 inhibited endogenous Rap1GTP and beta(1) integrin-mediated cell adhesion to fibronectin in SPA-1-induced conditions, whereas it affected neither of them in SPA-1-repressed conditions. These results suggested that AF-6 could control integrin-mediated cell adhesion by regulating Rap1 activation through the recruitment of both SPA-1 and Rap1GTP via distinct domains.
authors.affiliation: Department of Immunology and Cell Biology, Graduate School of Medicine and Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Li
authors.initials: L
authors.lastname: Su
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hattori
authors.forename: Masaki
authors.initials: M
authors.lastname: Moriyama
authors.forename: Norihito
authors.initials: N
authors.lastname: Murata
authors.forename: Masashi
authors.initials: M
authors.lastname: Harazaki
authors.forename: Kozo
authors.initials: K
authors.lastname: Kaibuchi
authors.forename: Nagahiro
authors.initials: N
authors.lastname: Minato
mesh_terms: D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000502:physiology; D016547:Kinesins / Q000378:metabolism / Q000502:physiology*; D008214:Lymphocytes / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D009218:Myosins / Q000378:metabolism / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013950:Thymus Gland / Q000166:cytology; D014162:Transfection; D014407:Tumor Cells, Cultured; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: C095516:AFDN protein, human; C490657:Afdn protein, mouse; D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins; D016023:Integrins; D009687:Nuclear Proteins; C490403:SIPA1 protein, human; C091108:Sipa1 protein, mouse; D009218:Myosins; D016547:Kinesins; D020827:rap1 GTP-Binding Proteins