Figure 8.

RhoA activity is necessary and sufficient for phosphorylation of moesin. R12 or NIH3T3 cells were transfected with vector only, HA-moesin only, HA-moesin and RhoAV14, or HA-moesin and C3 transferase, as indicated. Cells were then serum starved and metabolically labeled with [³²P]orthophosphate; cell lysates were immunoprecipitated as in Figure 7. To ensure that equivalent levels of HA-moesin were expressed and immunoprecipitated, immunoprecipitated samples were transferred to PVDF after SDS-PAGE and immunoblotted with 12CA5 antibody; signal was detected by enhanced chemiluminescence. Moesin is indicated by arrowhead at left. The presence of RhoA increased the basal level of moesin phosphorylation by 7-fold in R12 cells and 2.5-fold in NIH3T3 cells, while C3 treatment reduced the basal level of phosphorylation by at least 50% in both cell types (top panel). Protein levels were approximately equivalent in all conditions tested (bottom panel). The data shown are representative of three independent experiments.