Fig. 6.

RSK2 activation of RhoA signaling in cell migration and invasion requires LARG. (A) RSK2-T577E activates LARG. U87MG cells were transfected with varying amounts of Flag-RSK2-T577E. Flag-EV transfection was used as a negative control. Cells were serum starved before cell lysate preparation. Activated LARG (ARHGEF12) was captured by GST-RhoA-G17A pulldown and detected by immunoblotting with anti-LARG antibody. The levels of LARG and Flag-RSK2 are shown. Results are representative of four independent experiments. (B) U87MG cells transfected with Flag-RSK2-T577A and serum starved were stimulated by FBS (10%, 10 min) or EGF (100 ng/mL, 5 min). Activated LARG was retrieved by GST-RhoA-G17A pulldown and detected by immunoblotting with anti-LARG antibody. Expression of LARG and Flag-RSK2-T577A levels is shown. (C) RSK2-T577E–induced U87MG cell migration and invasion requires LARG activity. U87MG cells were cotransfected with Flag-RSK2-T577E and Myc-LARG-S1288A or -DN-LARG. Flag-EV and Myc-EV transfection was used as controls. Transfected cells were serum starved before migration and invasion assays. Cell migration (Upper) and invasion (Middle) were assessed by Transwell assays. Expression levels of exogenous RSK2 and LARG proteins are shown (Lower). Refer to Dataset S1C for all P values. *P < 0.05. (D and E) Inhibition of LARG activity disrupts RSK2-T577E–triggered RhoA activation. U87MG cells expressing Myc-RhoA-WT were transfected with Myc-RSK2-T577E (1 μg) in the presence or absence of Myc-tagged DN-LARG (D) or LARG-S1288A (E). Cells were starved before lysate preparation and GST-rhotekin-RBD pulldown analysis. GTP-RhoA levels were determined by immunoblotting pulldown precipitate with anti-Myc antibody. Expression levels of Myc-RhoA-WT, Myc-RSK2, and Myc-LARG are determined by immunoblot and shown. Results are representative of three independent experiments.