Figure 3.

myr 5 expressed in Sf9 insect cells specifically stimulates the in vitro GTP hydrolysis of RhoA but not of Cdc42Hs or Rac1. Sf9 insect cells were infected with a baculovirus encoding myr 5. After 48 h, soluble lysates of the cells were prepared and incubated with purified GTPases preloaded with [γ-³²P]GTP. After 15 min at 30°C (RhoA and Cdc42Hs) or 25°C (Rac1), remaining radioactivity was determined in a filter binding assay. Lysate of uninfected cells was used as a control. Remaining radioactive GTP bound to the different GTPases in assays incubated only with lysis buffer was taken as 100% values. Although lysates of uninfected cells (□) and cells infected with virus encoding myr 5 (▪) show no difference in their GAP activity for Cdc42Hs and Rac1, an increased GAP activity for RhoA is detected in lysates of cells infected with myr 5 virus.