Critical but distinct roles for the pleckstrin homology and cysteine-rich domains as positive modulators of Vav2 signaling and transformation.
Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function. We evaluated the contribution of the PH domain and the CRD to Vav2 guanine nucleotide exchange, signaling, and transforming activity. Unexpectedly, we found that mutations of the PH domain impaired Vav2 signaling, transforming activity, and membrane association. However, these mutations do not influence exchange activity on Rac and only slightly affect exchange on RhoA and Cdc42. We also found that the CRD was critical for the exchange activity in vitro and contributed to Vav2 membrane localization. Finally, we found that phosphoinositol 3-kinase activation synergistically enhanced Vav2 transforming and signaling activity by stimulating exchange activity but not membrane association. In conclusion, the PH domain and CRD are mechanistically distinct, positive modulators of Vav2 DH domain function in vivo.
Booden
Michelle A
MA
Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. mbooden@med.unc.edu
Campbell
Sharon L
SL
Der
Channing J
CJ
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D051567:Proto-Oncogene Proteins c-vav; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014170:Transformation, Genetic
D001798:Blood Proteins; D020662:Guanine Nucleotide Exchange Factors; D015513:Oncogene Proteins; D010750:Phosphoproteins; D051567:Proto-Oncogene Proteins c-vav; C093651:VAV2 protein, human; C490565:Vav2 protein, mouse; C039077:platelet protein P47; D019869:Phosphatidylinositol 3-Kinases; D020558:GTP Phosphohydrolases