Figure 5
RSK1 and RSK2 kinase activity is impaired by the substitution of Arg108 in RSK1 and Arg114 in RSK2. (A,B) HeLa cells were transfected with pDEST27 plasmid coding for wild type RSK1 and RSK2, the phosphomimetic mutant (S221D for RSK1 and S227D for RSK2), the mutants in the amino acids shown to interact with the AL phosphate (K210A and R108A for RSK1, K216A and R114A for RSK2), the kinase dead mutant (K100A in RSK2) and the constitutively active mutant (Y707A) fused with GST. As a control, we used a pDEST27 expressing GST only. Such cells were kept overnight with normal culture medium. Then, HeLa cells were starved for 3 h with normal culture medium without FCS. After that, these 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. (C-D) Quantification of the YB-1 phosphorylation on the Ser 102 normalized on β-actin level. Data were plotted as the mean ± s.d of three independent experiments.
