The pretreatment of ARVMs with 3 μM FMK attenuates the increase in Ser386 phosphorylation, but it has no inhibitory effect on the increase in Thr577 phosphorylation . FMK inhibits relatively few protein kinases in the panel, although it does inhibit protein tyrosine kinases, such as Src, Lck, Yes and Eph-A2, as well as S6K1. FMK will not inhibit RSK if the N-terminal kinase domain is activated by a mechanism that is independent of the C-terminal domain. Fmk potently inactivates the CTD auto-kinase activity of RSK1 and RSK2 with high specificity in mammalian cells. Targeting RSK2 by FMK attenuates FGFR3-induced cytokine-independent growth in Ba/F3 cells. FMK inhibits cytokine-independent proliferation of Ba/F3 cells conferred by FGFR3 .
To determine the ability of FGFR3 to phosphorylate RSK2, 500 ng of purified recombinant RSK2 variants are incubated with 500 ng of recombinant active FGFR3 in 10 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM DTT, 0.01% Triton-X-100, 10 mM MnCl2, and 200 μM ATP for 30 min at 30°C. Phosphorylation of Y529 RSK2 is detected by specific phospho-antibody. To determine kinase activity of RSK2 CTD variants, purified recombinant RSK2 CTD proteins (500 nM) are incubated with 500 nM of active ERK in 20 mM HEPES [pH 8.0], 10 mM MgCl2, 2 mM tris-(2-carboxyethyl)-phosphine (TCEP), and 200 μM ATP for 1 hr at 30°C. Kinase reactions are initiated by the addition of 5 μCi of [γ-32P] ATP and 100 μM peptide substrate (CTD-tide), followed by incubation for 20 min at room temperature. Kinase activity is determined using the standard disk phospho-cellulose assay .
RSK2 expressing Ba/F3 cell lines are generated by retroviral transduction as described by using Ba/F3 cells stably expressing FGFR3 TDII with pMSCV-puro plasmids encoding myc-tagged RSK2 variants, followed by antibiotic selection. For cell viability assays, 1×10^5 Ba/F3 cells stably expressing FGFR3 are cultured in 24-well plates with media containing increasing concentrations of FMK, acidic FGF (10 nM), and heparin (30 μg/mL) in the absence of IL-3. The relative cell viability at each experimental time point is determined by using the Celltiter96AQueous One solution proliferation kit .