Figure 2.

Functional properties of the recombinant mRSK2^NTKD.

(A) Inhibition by SL0101 of the catalytic activity of the phosphorylated protein. The kinase assays were performed in the presence of indicated amounts of SL0101 using myelin basis protein (MBP) as a substrate. Incorporated phosphate was analyzed by autoradiography of gel samples and proteins were visualized by Coomassie Blue staining (shown beneath corresponding autoradiography images). Lower panel: kinase activity data (averaged from duplicate measurements) were expressed as picomoles of incorporated phosphate and plotted against SL0101 concentration. The decreasing amount of phosphorylated mRSK2^NTKD following incubation with SL0101 suggests that autophosphorylation of the kinase is also subject to inhibition by SL0101.

(B) ITC profiles of the interaction of AMP-PNP and SL0101 with wild type mRSK2^NTKD. The individual dissipated heats were plotted against the molar ratio of interacting partners (squares) to estimate thermodynamic parameters. The data were fitted with a “one set of sites model” shown as a solid red line on lower panels. The best fits resulted in K_d = 50 µM, n = 0.85, ΔH = −9.1 kcal/mol and ΔS = −10.9 cal/mol/deg (TΔS = −3.2 kcal/mol) for AMP-PNP (ΔG = −5.9 kcal/mol) ; and K_d = 2.9 µM, n = 1.16, ΔH = −5.9 kcal/mol and ΔS = 4.9. cal/mol/deg (TΔS = 1.4 kcal/mol) for SL0101(ΔG = − 7.3 kcal/mol). Only representative experiments are shown out of several, which all yielded reproducible results.