Figure 5.

Nm23H1 transphosphorylates to Rac1 in vitro. (a) GDP-loaded GST-Rac1 on glutathione Sepharose (1 μg) was UV-irradiated as described in Materials and Methods, followed by incubation with 10 μCi [γ-³²P]ATP in the presence of 0.1 μg GST-nm23H1, GST-nm23H1 (H118C), or control GST, as indicated above the lanes. The reactants were washed and resolved by 12% SDS/PAGE. Precipitated GST-tagged Rac1 was detected by Coomassie stain (Bottom). (b) 293T cells were transfected with pEBG-Rac1 in combination with either mock vector, wild type (WT), H118C mutant of nm23H1 or wild-type Tiam1, as indicated above. Cells were labeled with ³²P_i for 2 h. The labeled guanine nucleotides bound to GST-tagged GTPases were separated by TLC. Expression of GST-tagged Rac1 in individual lysates was confirmed by immunoblotting (Bottom). Similar results were obtained in three independent experiments.