Identification of guanine nucleotide exchange factors (GEFs) for the Rap1 GTPase. Regulation of MR-GEF by M-Ras-GTP interaction.
Although the Ras subfamily of GTPases consists of approximately 20 members, only a limited number of guanine nucleotide exchange factors (GEFs) that couple extracellular stimuli to Ras protein activation have been identified. Furthermore, no novel downstream effectors have been identified for the M-Ras/R-Ras3 GTPase. Here we report the identification and characterization of three Ras family GEFs that are most abundantly expressed in brain. Two of these GEFs, MR-GEF (M-Ras-regulated GEF, KIAA0277) and PDZ-GEF (KIAA0313) bound specifically to nucleotide-free Rap1 and Rap1/Rap2, respectively. Both proteins functioned as Rap1 GEFs in vivo. A third GEF, GRP3 (KIAA0846), activated both Ras and Rap1 and shared significant sequence homology with the calcium- and diacylglycerol-activated GEFs, GRP1 and GRP2. Similarly to previously identified Rap GEFs, C3G and Smg GDS, each of the newly identified exchange factors promoted the activation of Elk-1 in the LNCaP prostate tumor cell line where B-Raf can couple Rap1 to the extracellular receptor-activated kinase cascade. MR-GEF and PDZ-GEF both contain a region immediately N-terminal to their catalytic domains that share sequence homology with Ras-associating or RalGDS/AF6 homology (RA) domains. By searching for in vitro interaction with Ras-GTP proteins, PDZ-GEF specifically bound to Rap1A- and Rap2B-GTP, whereas MR-GEF bound to M-Ras-GTP. C-terminally truncated MR-GEF, lacking the GEF catalytic domain, retained its ability to bind M-Ras-GTP, suggesting that the RA domain is important for this interaction. Co-immunoprecipitation studies confirmed the interaction of M-Ras-GTP with MR-GEF in vivo. In addition, a constitutively active M-Ras(71L) mutant inhibited the ability of MR-GEF to promote Rap1A activation in a dose-dependent manner. These data suggest that M-Ras may inhibit Rap1 in order to elicit its biological effects.
Department of Biochemistry and Molecular Biology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
J F
JF
Rebhun
A F
AF
Castro
L A
LA
Quilliam
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D004075:Diglycerides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009419:Nerve Tissue Proteins; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D050807:ets-Domain Protein Elk-1; D020668:rap GTP-Binding Proteins / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*; D020663:ras Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004075:Diglycerides; C494112:ELK1 protein, human; D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; D011518:Proto-Oncogene Proteins; C115955:RAP1GDS1 protein, human; C403525:RAPGEF2 protein, human; C417242:RAPGEF5 protein, human; C413212:RASGRP3 protein, human; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; C040985:phosphatidylinositol receptors; D020663:ras Guanine Nucleotide Exchange Factors; D019908:Proto-Oncogene Proteins c-raf; C116756:RAP2A protein, human; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins; D002118:Calcium