The isolated catalytic hairpin of the Ras-specific guanine nucleotide exchange factor Cdc25Mm retains nucleotide dissociation activity but has impaired nucleotide exchange activity.
Cdc25Mm is a mammalian Ras-specific guanine nucleotide exchange factor (GEF). By homology modeling we show that it shares with Sos-GEF the structure of the putative catalytic HI hairpin where the dominant negative T1184E mutation is located. Similarly to Cdc25MmT1184E, the isolated wild-type and mutant hairpins retain the ability to displace Ras-bound nucleotide, originate a stable Ras/GEF complex and downregulate the Ras pathway in vivo. These results indicate that nucleotide re-entry and Ras/GEF dissociation--final steps in the GEF catalytic cycle--require GEF regions different from the HI hairpin. GEF down-sizing could lead to development of novel Ras inhibitors.
Sacco
Elena
E
Dipartimento di Biotechnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
Fantinato
Sonia
S
Manzoni
Romilde
R
Metalli
David
D
De Gioia
Luca
L
Fantucci
Piercarlo
P
Alberghina
Lilia
L
Vanoni
Marco
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002021:Buffers; D002384:Catalysis; D020134:Catalytic Domain; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D018360:Crystallography, X-Ray; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D005799:Genes, Dominant; D017930:Genes, Reporter; D011905:Genes, ras; D018698:Glutamic Acid / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006720:Homozygote; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D062367:ortho-Aminobenzoates / Q000378:metabolism; D020666:ras-GRF1 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D002021:Buffers; D062367:ortho-Aminobenzoates; D020666:ras-GRF1; C065197:3'-(methylanthraniloyl)-2'-deoxy-guanosine diphosphate; D006153:Guanosine Diphosphate; D018698:Glutamic Acid; D006160:Guanosine Triphosphate; D008156:Luciferases