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The highly conserved RasGEF1 family of proteins contain a C-terminal CDC25-Ras exchange motif domain and an N-terminal RasGEF-N domain, and are of unknown function and specificity. Using purified Ras-GEF1A and RasGEF1B proteins, as well as Ras family proteins, we estab-lished that RasGEF1A and RasGEF1B function as very specific exchange factors for Rap2, a member of the Rap subfamily of Ras-like G-proteins. | ỊFEBS Journal RasGEFIA and RasGEFIB are guanine nucleotide exchange factors that discriminate between Rap GTP-binding proteins and mediate Rap2-specific nucleotide exchange Elif Yaman1 z Raphael Gasper2 z Carolin Koerner2 Alfred Wittinghofer2 and Uygar H. Tazebay1 1 Department of Molecular Biology and Genetics Bilkent University Ankara Turkey 2 Abteilung Strukturelle Biologie Max-Planck-Institut fur Molekulare Physiologie Dortmund Germany Keywords G-proteins guanine nucleotide exchange Rap2 Ras family RasGEF1 Correspondence U. H. Tazebay Department of Molecular Biology and Genetics Bilkent University 06800 Bilkent Ankara Turkey Fax 90 312 2665097 Tel 90 312 2902419 E-mail tazebay@fen.bilkent.edu.tr These authors contributed equally to this work Received 28 April 2009 revised 18 June 2009 accepted 22 June 2009 doi 10.1111 j.1742-4658.2009.07166.x The highly conserved RasGEFl family of proteins contain a C-terminal CDC25-Ras exchange motif domain and an N-terminal RasGEF-N domain and are of unknown function and specificity. Using purified Ras-GEFlA and RasGEFlB proteins as well as Ras family proteins we established that RasGEFlA and RasGEFlB function as very specific exchange factors for Rap2 a member of the Rap subfamily of Ras-like G-proteins. They do not act on Rapl or other members of the Ras subfamily. Although Rap2 was implicated in the regulation of cell adhesion the establishment of cell morphology and the modulation of synapses in neurons no specific guanine nucleotide exchange factor for Rap2 was previously identified. Using reciprocal site-directed mutagenesis we analyzed residues that allow RasGEFl proteins to discriminate between Rapl and Rap2 and we were able to identify Phe39 in the switch I region of Rap2 as a specificity residue. Mutation of the corresponding Ser39 in Rapl changed the specificity and allowed the nucleotide exchange of Rapl S39F to be stimulated by RasGEFlB. Introduction Rap proteins are members of the Ras family of proteins which .
