P-Rex1 is a dominant GEF for PIP3-driven Rac activation. (A) Schematics for experiments described in C–E. Stimulation using chemoattractant and opto-PI3K is represented by a red circle and red lightning bolt, respectively. Red lines indicate links in the cascade where Rac GEFs participate in signal transduction. By delivering an input specifically at the level of PIP3 (right), only PIP3-stimulated Rac GEFs (dark red line) should participate in Rac activation. (B) P-Rex1 antibody immunoblots of WT and PREX1-null cells. GAPDH was used as a loading control. (C) MS analysis of PIP3 in WT or PREX1-null cells. Cells were collected before treatment (“Unstimulated”) after exposure to red light for 2 min (“opto-PI3K”) or incubation with 10 nM fMLP for 1 min (“Chemoattractant”) and were processed as described in Fig. 4 A. Each bar represents a mean of three independent trials. Loss of P-Rex1 did not significantly affect PIP3 production. Error bars indicate SD. n.s., P > 0.05 by unpaired t test. For both WT and PREX1-null cells, the difference in PIP3 levels between unstimulated cells and cells stimulated with either fMLP or opto-PI3K is significant (P < 0.05 by unpaired t test). (D and E) WT (black curves) or PREX1-null (red dashed curves) cells were treated with either red light (D) or 10 nM fMLP (E) for 10 min. Rac* was measured as in Fig. 4 C. Each point represents a mean of three independent experiments. Rac activation was significantly decreased in PREX1-null cells. Error bars indicate SEM. *, P < 0.05 by unpaired t test. Immunoblots depict one representative experiment. Molecular masses are given in kilodaltons.
