Figure 2
RSK2 kinase activity is impaired by phosphomimetic substitution of Ser227 in the NTKD. (A) HeLa cells were transfected with a lentiviral vector containing the RSK2 coding sequence. These cells were starved overnight with normal culture medium without FCS and with or without the addition of the specific PDK1 inhibition GSK2334470 at 3 μM or the MEK inhibitor PD98059 at 50 μM or the RSK inhibitor BI-D1870 at 3 μM. Then, HeLa cells were stimulated with EGF (10 ng/ml) for 15 min and lysed. Western blot analysis was performed using the indicated primary antibodies. β-actin was used as a loading control. (B) Quantification of the YB-1 phosphorylation on the serine 102 compared to β-actin level. Data were plotted as the mean ± s.d. of three independent experiments. (C) 293T cells were transfected with pDEST27-RSK2WT plasmid. Then, cells were treated or not with the PDK1 inhibitor GSK2334470 (3 μM). After that, cells were lysed on ice. GST-RSK2WT was isolated from 293T cells extracts and assayed for the phosphorylation of a specific synthetic peptide as substrate. (D) HeLa cells were transfected with a lentiviral vector containing the wild type or mutated (S227D, Y707A, S227D/Y707A and K100A) RSK2 coding sequence or an empty vector (E.V.). Then, cells were serum-starved overnight and stimulated or not with PMA (60 ng/ml) or with EGF (10 ng/ml) for 5 min and lysed. Western blot staining was performed using the indicated primary antibodies. β-actin was used as a loading control. (E) Quantification of the YB-1 phosphorylation on the serine 102 compared to β-actin level. Data were plotted as the mean ± s.d. of three independent experiments. (F) 293T cells were transfected with pDEST27-RSK2WT or pDEST27-RSK2S227D plasmids and cultured without FCS (serum free, sf). Then, cells were lysed on ice. GST-RSK2WT and GST-RSK2S227D were isolated from 293T extracts and assayed for the phosphorylation of a peptide substrate.
