RA-GEF-1, a guanine nucleotide exchange factor for Rap1, is activated by translocation induced by association with Rap1-GTP and enhances rap1-dependent B-Raf activation

We previously identified RA-GEF-1, a novel guanine nucleotide exchange fact or (GEF) for Rap1 with the ability to associate with Rap1.GTP at its Ras/Ra p1-associating (RA) domain. Because it possesses a PSD-95/DlgA/ZO-1 (PDZ) d omain, it was also named PDZ-GEF. In this report, we have examined the role of the RA domain of this protein in Rap1-mediated cellular responses. A mu tant of RA-GEF-1 (RA-GEF-1 Delta RA) carrying a 21-residue deletion at its RA domain fully retains the in vitro GEF activity toward Rap1 but completel y loses the Rap1 binding activity. In contrast, RA-GEF-1 Delta RA, expresse d in COS-7 cells, exhibits a 3-fold reduction in its in vivo GEF activity t oward Rap I compared with wild-type RA-GEF-1 as examined by the Rap1 pull-d own assay. Correspondingly, when coexpressed with wild-type Rap1, RA-GEF-1 Delta RA is unable to further activate B-Raf, whereas RA-GEF-1 stimulates B -Raf as efficiently as activated Rap1. Consistent with these observations, coexpression of activated Rap1 induces translocation of RA-GEF-1, which is otherwise located in the cytoplasm, to the perinuclear compartment, where R ap1 is also predominantly localized. This localization almost coincides wit h that of the Golgi apparatus, which was detected by anti-trans-Golgi-netwo rk 38 antibody. RA-GEF-1 Delta RA fails to show the translocation. These re sults indicate that RA-GEF-1 defines a novel category of GEF that is transl ocated to a particular subcellular compartment by association with the GTP- bound form of a small GTPase and catalyzes activation of the GDP-bound form present in the compartment, thereby causing an amplification of cellular r esponses induced by the small GTPase.