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Updated: Aug 15, 2026

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
Published on: March 9, 2012
AF-6 controls integrin-mediated cell adhesion by regulating Rap1 activation through the specific recruitment of
Li Su1, Masakazu Hattori, Masaki Moriyama
1Department of Immunology and Cell Biology, Graduate School of Medicine and Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
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.
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