Abstract
The Ras family small GTPase Rap is regulated by an array of specific guanine nucleotide exchange factors (GEFs) in response to upstream stimuli. RA-GEF-1 was identified as a novel Rap GEF, which possesses a Ras/Rap1-associating (RA) domain. Here we report a protein closely related to RA-GEF-1, named RA-GEF-2. Like RA-GEF-1, a putative cyclic nucleotide monophosphate-binding domain, a Ras exchanger motif, a PSD-95/DlgA/ZO-1 domain, and an RA domain in addition to the GEF catalytic domain are found in RA-GEF-2. However, RA-GEF-2 displays a different tissue distribution profile from that of RA-GEF-1. RA-GEF-2 stimulates guanine nucleotide exchange of both Rap1 and Rap2, but not Ha-Ras. The RA domain of RA-GEF-2 binds to M-Ras in a GTP-dependent manner, but not to other Ras family GTPases tested, including Ha-Ras, N-Ras, Rap1A, Rap2A, R-Ras, RalA, Rin, Rit, and Rheb, in contrast to the RA domain of RA-GEF-1, which specifically binds to Rap1. In accordance with this, RA-GEF-2 colocalizes with activated M-Ras in the plasma membrane in COS-7 cells, suggesting a role of RA-GEF-2 in the regulation of Rap1 and Rap2, particularly in the plasma membrane. In fact, an increase in the level of the GTP-bound form of plasma membrane-located Rap1 was observed when coexpressed with RA-GEF-2 and activated M-Ras. Thus, RA-GEF-2 acts as a GEF for Rap1 and Rap2 downstream of M-Ras in the plasma membrane, whereas RA-GEF-1 exerts Rap GEF function in perinuclear compartments including the Golgi apparatus.
Highlights
The Ras family small GTPase Rap1 participates in the regulation of a wide variety of cellular responses, including proliferation, differentiation, lymphocyte aggregation, T-cell anergy, and platelet activation [1, 2]
Unlike RA-guanine nucleotide exchange factors (GEFs)-1, the RA domain of RA-GEF-2 binds to M-Ras1⁄7GTP, which causes the translocation of RA-GEF-2 to the plasma membrane, where M-Ras exists
Cloning of RA-GEF-2—Through a BLAST search of the GenBank data base, we identified a genomic clone (AC004227) encoding an open reading frame closely related to RA-GEF-1, designated RA-GEF-2
Summary
The Ras family small GTPase Rap1 participates in the regulation of a wide variety of cellular responses, including proliferation, differentiation, lymphocyte aggregation, T-cell anergy, and platelet activation [1, 2]. RA-GEF-2 colocalizes with activated M-Ras in the plasma membrane in COS-7 cells, suggesting a role of RA-GEF-2 in the regulation of Rap1 and Rap2, in the plasma membrane. Rap1A1⁄7GTP and HaRas1⁄7GTP were affinity-precipitated by GST-RalGDS-RID and GST-Raf-1-RBD, respectively, from COS-7 cells that expressed increasing amounts of RA-GEF-2 and either Rap1A or Ha-Ras, and were detected by immunoblotting.
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