FIG. 10.

The cortactin SH3 domain is required for cortactin to promote S-phase entry and RhoA activation. (A) FaDu control and cortactin knockdown cells, along with cortactin knockdown cells expressing shRNA-resistant myc-tagged wild-type murine cortactin, 3YF or ΔSH3 mutant versions, were synchronized into G₁ phase by serum starvation and hydroxyurea (HU) treatment and then released into complete medium for the time periods indicated. Expression of the indicated proteins was then analyzed by Western blotting. Data shown are representative of three independent experiments. (B) Effect of cortactin mutants on cell cycle progression. Entry into S phase after a 6-h release was determined by uptake of BrdU during a 2-h pulse. The incorporation of BrdU was measured by flow cytometry. Data shown represent the mean ± standard error of three independent experiments. **, P < 0.01. (C) Cortactin lacking the SH3 domain does not rescue RhoA activity. Active RhoA was isolated from synchronized cells using Rhotekin RBD-GST beads. Active and total levels of RhoA were then determined by Western blotting. Activity was normalized relative to the level of total protein in each cell type and is expressed relative to the level of activity in control cells, which was arbitrarily set at 100%. Data shown represent the mean ± standard error of at least three independent experiments. *, P < 0.05.