Recognition and activation of Rho GTPases by Vav1 and Vav2 guanine nucleotide exchange factors.
Vav proteins are Rho GTPase-specific guanine nucleotide exchange factors (GEFs) that are distinguished by the tandem arrangement of Dbl homology (DH), Pleckstrin homology (PH), and cysteine rich domains (CRD). Whereas the tandem DH-PH arrangement is conserved among Rho GEFs, the presence of the CRD is unique to Vav family members and is required for efficient nucleotide exchange. We provide evidence that Vav2-mediated nucleotide exchange of Rho GTPases follows the Theorell-Chance mechanism in which the Vav2.Rho GTPase complex is the major species during the exchange process and the Vav2.GDP-Mg(2+).Rho GTPase ternary complex is present only transiently. The GTPase specificity for the DH-PH-CRD Vav2 in vitro follows this order: Rac1 > Cdc42 > RhoA. Results obtained from fluorescence anisotropy and NMR chemical shift mapping experiments indicate that the isolated Vav1 CRD is capable of directly associating with Rac1, and residues K116 and S83 that are in the proximity of the P-loop and the guanine base either are part of this binding interface or undergo a conformational change in response to CRD binding. The NMR studies are supported by kinetic measurements on Rac1 mutants S83A, K116A, and K116Q and Vav2 CRD mutant K533A in that these mutants affect both the initial binding event of Vav2 with Rac1 (k(on)) and the rate-limiting dissociation of Vav2 from the Vav2.Rac1 binary complex (thereby influencing the enzyme turnover number, k(cat)). The results suggest that the CRD domain in Vav proteins plays an active role, affecting both the k(on) and the k(cat) for Vav-mediated nucleotide exchange on Rho GTPases.
Heo
Jongyun
J
Department of Biochemistry and Biophysics, University of North Carolina, 530 Mary Ellen Jones Building, Chapel Hill, North Carolina 27599-7260, USA.
Thapar
Roopa
R
Campbell
Sharon L
SL
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000378:metabolism; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000502:physiology*; D003545:Cysteine / Q000378:metabolism; D004789:Enzyme Activation; D005454:Fluorescence Polarization; D020662:Guanine Nucleotide Exchange Factors / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000302:isolation & purification / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification / Q000502:physiology*; D051567:Proto-Oncogene Proteins c-vav; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D001798:Blood Proteins; D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; C057811:MCF2 protein, human; D015513:Oncogene Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D015682:Retroviridae Proteins, Oncogenic; C496618:VAV1 protein, human; C093651:VAV2 protein, human; C039077:platelet protein P47; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D003545:Cysteine