Figure 5.

The GEF Function of Ca Vav3 Is Required for Ligand-Independent Activation of AR

A, PC3 cells were transfected with AR, PSA-Luc, and either Ca Vav3, Ca Vav3 mutants lacking GEF activity [Ca Vav3 ISO III (mutant 1), Ca Vav3 QIF (mutant 2)], Ca Vav3 containing a PH domain mutation (Ca Vav3 W493L), or equimolar amounts of the empty vector. Cells were treated with vehicle or R1881 (1 nm). Luciferase activity was determined 48 h after transfection. The means of triplicate determinations from four independent experiments are plotted as a percent of vector control (RLU ± sem). Lysates from A were immunoblotted to assess relative Ca Vav3 and mutant expression levels. Actin is shown as a loading control. B. PC3 cells were transfected with AR, PSA-luc, Vav3 (full length), or Vav3 harboring analogous mutations as in panel A in the DH (GEF) or PH domains (W493L). Luciferase activity was determined as described in panel A. Significance was determined using a one-way ANOVA and represents differences from untreated, empty vector transfected cells (A) or from androgen-treated empty vector transfected cells (B) (**, P < 0.01; ***, P < 0.001). RLU, Relative light units.