In vitro assay

In vitro activity of human RSK CTKD was evaluated in the HTRF KinEASE assay (Cisbio Bioassays). For activity evaluation, RSK CTKD proteins (2 μM) were either untreated or pre-activated by ERK2 (0.1 μM) (SignalChem) in reaction buffer containing 200 μM ATP and 10 mM MgCl₂ in kinase buffer (50 mM HEPES, pH 7.0, 2 mM TCEP, 0.02% [wt/vol] NaN₃, 0.01% [wt/vol] BSA, and 0.1 mM orthovanadate) for 1 h at 30°C. Reactions were performed in 384-well plates for fluorescence (Greiner) with 10 ng RSK CTKD per well. The reactions were started by the addition of 100 μM ATP and 1 μM STK1 substrate, giving a 10 μl reaction solution. After 20 min at room temperature, the reaction was stopped with 5 μl anti-phospho STK antibody labeled with Eu³⁺-cryptate (FRET donor) and 5 μl streptavidin-XL665 (FRET acceptor), both prepared in EDTA. In inhibition studies, 10 ng constitutively active RSK CTKDs were mixed with DMSO or a concentration series of ruxolitinib, SB-203580, TG-100-115, or fmk in kinase buffer for a 30-min incubation prior kinase reaction (as described above). Evaluation of the time-dependent effect of fmk on RSK2-T577E-D694* was performed by incubation with a concentration series of fmk in kinase buffer (50 mM succinic acid, pH 6.0, 2.5 mM TCEP, 0.02% [wt/vol] NaN₃, 0.01% [wt/vol] BSA and 0.1 mM orthovanadate) for 1, 2, 4, 6 min or 2, 5, 15, 30 min (depending on concentration series) followed by kinase reaction. IC₅₀ values were calculated by non-linear regression using sigmoid concentration response. The observed rate constant of fmk inhibition, k_obs, at each concentration was determined from the slope of a semi-logarithmic plot of inhibition versus time. k_obs was re-plotted against inhibitor concentration and fitted to a hyperbolic equation, k_obs = k_inact[inhibitor]/(K_i + [inhibitor]) (22). Data analysis was performed using GraphPad Prism 9. All experiments were repeated at least three times (n = 3) and presented as means ± SD. The P-values were determined by t test.