Figure 8.

SGEF preferentially binds and exchanges RhoG in vitro. (A) Myc-SGEF DH/PH/SH3 (aa 431-871) was expressed in fibroblasts and then pulled down with the indicated GST-Rho fusion protein containing a point mutation that disrupts binding of guanine nucleotides. Myc-SGEF DH/PH/SH3 bound nucleotide-free RhoG. (B). GST-GTPase fusion proteins (Rac1, Rac3, RhoG, or Cdc42) were incubated with Mant-GTP and the rate of nucleotide incorporation assessed over time (Δ460 nm). SGEF DH/PH (red), Vav2 DH/PH/CRD (blue), or GEF elution buffer (black) were added to the reactions at the indicated time (GEF with arrow) and the resulting change in the rate of Mant-GTP incorporation monitored. SGEF exchanged nucleotide on RhoG and, to a lesser extent, Cdc42 in vitro.