FIGURE 5.

Ubiquitination of RSK2 is required for RSK2 activity. A, ubiquitin is required for RSK2 activity. RSK2-WT or RSK2-K345R,K364R with Ub-Lys-63 and TRAF2 were transfected into 293 cells. At 36 h after transfection, RSK2-Xpress was immunoprecipitated by anti-Xpress for an in vitro kinase assay using GST-IκBα as substrate as described under “Experimental Procedures.” Phosphorylated IκBα was detected using the phosphorylation IκBα (Ser-32) antibody to demonstrate RSK2 activity. Immunoblotting (IB) was performed using the indicated antibodies. B, mutant RSK2-K345R,K364R attenuates RSK2 activity. Xpress-RSK2-WT and Xpress-RSK2- K345R,K364R were transfected into HeLa cells. At 20 h after transfection, cells were starved for 16 h and then treated with EGF (50 ng/ml) for various times as indicated. Immunoblotting was performed using the indicated antibodies. C, Xpress-RSK2-WT and Xpress-RSK2- K345R,K364R were transfected into HeLa cells. At 20 h after transfection, cells were starved for 16 h and then treated with EGF (50 ng/ml) for various times as indicated. RSK2-Xpress was immunoprecipitated with anti-Xpress for a ³²P-labeled γ-ATP in vitro kinase assay using GST-IκBα as substrate as described under “Experimental Procedures.” Phosphorylated IκBα was detected by auto-exposure (auto-ex) to show RSK2 activity. CBS, Coomassie Blue Staining. D, overexpression of RSK2-K345R,K364R attenuates cell proliferation. Stable HaCaT cells overexpressing RSK2-WT or RSK2-K345R,K364R (left panel) were constructed as described under “Experimental Procedures.” HaCaT cells expressing Mock, RSK2-WT, or RSK2-RR were seeded (1 × 10³ per well/in 100 μl) into 96-well plates, and proliferation was assessed using the CellTiter96 Aqueous One Solution detection kit. Cell viability was estimated by reading the absorbance (A₄₉₀). The graph shows data from multiple experiments expressed as the means ± S.D. The asterisks (*) indicate a significant difference (p < 0.05 Student's t test) (right panel). E, overexpression of mutant RSK2-K345R,K364R attenuates EGF-induced anchorage-independent cell transformation. HaCaT cells expressing Mock, RSK2-WT, or RSK2-RR was exposed to EGF (20 ng/ml) in 0.3% BME agar containing 10% FBS. The cultures were maintained in a 37 °C, 5% CO₂ incubator for 15 days, and then colonies were counted using a microscope and the Image-Pro PLUS (v.6) computer software program. The graph shows data from multiple experiments expressed as the means ± S.D. The asterisks (*) indicate a significant difference (p < 0.05 Student's t test). F, the effect of mutant RSK2-K345R,K364R on the EGF signaling pathway is shown. HaCaT cells expressing Mock, RSK2-WT, or RSK2-RR were starved for 36 h and treated with EGF (20 ng/ml) for various times as indicated. Immunoblotting was performed using the indicated antibodies.