Trp(56) of rac1 specifies interaction with a subset of guanine nucleotide exchange factors.
Signaling specificity of Rho GTPase pathways is achieved in part by selective interaction between members of the Dbl family guanine nucleotide exchange factors (GEFs) and their Rho GTPase substrates. For example, Trio, GEF-H1, and Tiam1 are a subset of GEFs that specifically activate Rac1 but not the closely related Cdc42. The Rac1 specificity of these GEFs appears to be governed by Rac1-GEF binding interaction. To understand the detailed mechanism underlying the GEF specificity issue, we have analyzed a panel of chimeras made between Rac1 and Cdc42 and examined a series of point mutants of Rac1 made at the switch I, switch II, and beta(2)/beta(3) regions for their ability to interact with and to be activated by the GEFs. The results reveal that Rac1 residues of both the switch I and switch II regions are involved in GEF docking and GEF-mediated nucleotide disruption, because mutation of Asp(38), Asn(39), Gln(61), Tyr(64), or Arg(66)/Leu(67) into Ala results in the loss of GEF binding, whereas mutation at Tyr(32), Asp(65), or Leu(70)/Ser(71) leads to the loss of GEF catalysis while retaining the binding capability. The region between amino acids 53-72 of Rac1 is required for specific recognition and activation by the GEFs, and Trp(56) in beta(3) appears to be the critical determinant. Introduction of Trp(56) to Cdc42 renders it fully responsive to the Rac-specific GEF in vitro and in cells. Further, a polypeptide derived from the beta(3) region of Rac1 including the Trp(56) residue serves as a specific inhibitor for Rac1 interaction with the GEFs. Taken together, these results indicate that Trp(56) is the necessary and sufficient determinant of Rac1 for discrimination by the subset of Rac1-specific GEFs and suggest that a compound mimicking Trp(56) action could be explored as an interfering reagent specifically targeting Rac1 activation.
Gao
Y
Y
Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Xing
J
J
Streuli
M
M
Leto
T L
TL
Zheng
Y
Y
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D013997:Time Factors; D014162:Transfection; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D020663:ras Guanine Nucleotide Exchange Factors / Q000378:metabolism
D000596:Amino Acids; D018076:DNA, Complementary; D020662:Guanine Nucleotide Exchange Factors; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C513260:Tiam1 protein, mouse; D020663:ras Guanine Nucleotide Exchange Factors; D006639:Histidine; D014364:Tryptophan; D005982:Glutathione Transferase; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein