Fig. 1.

RSK2 activity is required for RhoA activation in response to diverse stimuli. (A) U87MG cells expressing HA-RhoA-WT were transfected with Flag-RSK2-Y707A vector or dominant negative (DN) Flag-RSK2-K100A. Flag-EV transfection was used as control. Cells were serum starved for 24 h before stimulation with FBS (10%, 10 min), TNFα (50 ng/mL, 5 min), PMA (100 ng/mL, 10 min), or EGF (100 ng/mL, 5 min). Five hundred micrograms of total cell lysates was subjected to GST-rhotekin-RBD precipitation. The levels of GTP-RhoA were determined by immunoblotting with anti-HA antibody. Equal loading of HA-RhoA proteins and expression levels of transfected Flag-RSK2 proteins are shown. Results are representative of seven independent experiments. (B) U87MG cells expressing HA-RhoA-WT were serum starved and then stimulated as indicated after pretreatment with BI-D1870 (10 μM). GTP-RhoA was detected by rhotekin assay as described above. Results are representative of four independent experiments.