Cyclin A2 potentiates RhoA GEF exchange activity. (A) Interaction of Cyclin A2 with either nucleotide-free GST RhoA or GST RhoA loaded, respectively, with GDP or GTP (left) and quantification of retained Cyclin A2 (right). Molecular markers are given in kilodaltons. (B–G) Kinetics of Mant-GTP loading by Dbl (B) and Tgat (D). Results are expressed as fluorescence units versus time. The reactions performed in the absence of GEFs and with irrelevant proteins (MBP or GST) reflect the spontaneous exchange activity of RhoA. In F and G, the N- and C-terminal halves as well as Cyclin E1 are included in the assay. C, E, and G represent a normalized quantitative analysis of the initial velocity measured in six independent experiments (B and C) and three independent experiments (D–G). Data are represented as means ± SEM (*, P = 0.03; **, P = 0.0081). WB, Western blot; N-ter, N-terminal; C-ter, C-terminal.