(A) Left panel shows activation of endogenous RSK2 as assessed by phosphorylation at Ser386 in cells expressing TEL-FGFR3. Ba/F3 cells in the presence or absence of IL-3 were included as controls. Right panel shows that RSK2 is further activated in Ba/F3 cells stably expressing FGFR3 TDII in the presence of aFGF ligand.
(B) Tyrosine kinase activity is required for TEL-FGFR3-dependent activation of RSK2. Left: Ba/F3 cells stably expressing TEL-FGFR3 were cultured in the presence of increasing concentrations of PKC412. Right: Cells expressing the kinase-defective mutant TEL-FGFR3 (T/F) K508R were included as a negative control.
(C) Expression of myc-RSK2 enhanced FGFR3 TDII-dependent activation of RSK2. Myc-tagged RSK2 was stably transduced into Ba/F3 cells expressing FGFR3 TDII. Phosphorylation level of myc-RSK2 at Ser386 was assessed by western blotting.
(D) Expression of myc-RSK2 enhanced FGFR3 TDII conferred IL-3-independent proliferation of Ba/F3 cells. Cells were cultured in absence of IL-3 and counted daily; control Ba/F3 cells were included. The data are presented as means ± SD (n = 3).
(E) FGFR3 activates RSK2 through the MEK/ERK pathway. Control RSK2 ΔC20 mutant harbors a deletion of 20 amino acids at the C terminus that are required for ERK binding.
(F) Treatment of MEK inhibitor U0126 abolishes FGFR3-dependent RSK2 activation. Ba/F3 cells stably expressing FGFR3 TDII were treated with serum and IL-3 withdrawal and aFGF stimulation for 4 hr followed by 90 min treatment of increasing concentrations of U0126 (left) or 10 μM U0126 with PI3K inhibitors wortmannin and LY294002 as negative controls (right). Western blotting was performed to detect phosphorylation and expression levels of RSK2 and ERK.

(A) Y529F mutation attenuates inactive ERK binding to RSK2. Ba/F3 cells stably expressing FGFR3 TDII and distinct myc-tagged RSK2 variants were cultured in the absence of serum for 4 hr in the presence of U0126 (10 μM) prior to coimmunoprecipitation.
(B) Global structure of RSK2 proteins is not altered by point mutations, which was determined by similar partial protease digestion patterns of (His)₆-RSK2 WT and variants. Recombinant proteins (2.5 μg) were incubated with 0.5 unit of chymotrypsin for 30 min at 37°C.
(C) RSK2 Y529 is specifically phosphorylated in cells expressing FGFR3, detected by an antibody specifically recognizes phospho-Y529 of RSK2.
(D) FGFR3 directly phosphorylates RSK2 at Y529. Purified recombinant RSK2 (rRSK2) C-terminal kinase domain (CTD) and CTD-Y529F proteins were incubated with recombinant FGFR3 kinase domain (rFGFR3) that is constitutively activated. Phosphorylation at Y529 in rRSK2 CTD was detected by specific antibody pRSK2 (Y529).
(E) RSK2 Y529 is specifically phosphorylated by FGFR3, which consequently facilitates inactive ERK binding. GST-tagged RSK2 wild-type or Y529F mutant were pulled down by beads from transfected 293T cell lysates and treated with protein tyrosine phosphatase (PTP), followed by treatment of activated rFGFR3. The beads were then incubated with U0126-treated 293T cell lysates. Reconstituted Y529 phosphorylation and inactive ERK in the complex of bead-bound GST-RSK2 were detected by immunoblotting.
(F) Y529F mutation does not intrinsically affect the kinase activation of RSK2. Purified rRSK2 CTD variants were incubated with or without activated recombinant ERK in an in vitro kinase assay. The phosphorylation of the specific CTD-tide peptide substrate in each reaction was normalized to readings from reaction of rRSK2 CTD WT with activated ERK (1.0) (mean ± SD).

(A) RSK2 is specifically tyrosine-phosphorylated at Y529 and activated in FGFR3-expressing, t(4;14)-positive human myeloma cell lines.
(B) Inhibition of FGFR3 by PKC412 abolishes Y529 phosphorylation as well as activation of RSK2 in t(4;14)-positive KMS11 and OPM1 cells.
(C) Targeted downregulation of RSK2 but not RSK1 induces apoptotic cell death in KMS11 cells. Left: Transfection of siRNA targeting RSK1 or RSK2 specifically decreases RSK1 or RSK2 protein expression in KMS11 cells, respectively. β-actin was detected as a loading control. Middle: Induction of apoptosis by siRNA targeting RSK2 but not RSK1 in KMS11 cells. Cells were transfected with distinct siRNA for 48 hr and 72 hr prior to annexin V staining and flow cytometry analysis. The apoptotic population was characterized as annexin V positive cells. Right: KMS11 cells were transfected with distinct siRNAs for indicated periods. The relative cell viability was normalized to the viability of cells transfected by a nonspecific siRNA. The data were presented as mean ± SD (n = 3).
(D) All of the 4 individual siRNA in the RSK2 siRNA pool in Figure 6C significantly downregulated RSK2 protein expression and induced apoptosis in KMS11 cells (mean ± SD).

FGFR3 phosphorylates RSK2 at Y529 to facilitate recruitment of the inactive form of ERK to RSK2 (Step 1), which subsequently promotes the phosphorylation and activation of RSK2 by ERK when activated by FGFR3 (Step 2).

(A) Schematic diagram of p90RSK2 shows domain structure and residues phosphorylated during RSK2 activation by ERK and PDK1 (upper panel). N/CTD, N/C-terminal kinase domain. S386 is autophosphorylated by activated CTD. The two phosphorylated tyrosine residues (Y488 and Y529) identified in the proteomics studies are indicated. Lower panel shows MS spectra of phospho-Tyr peptide fragments of RSK2 with xCorr = 4.1592 for peptide containing Y488 and xCorr = 4.8611 for peptide containing Y529.
(B) TEL-FGFR3 requires its tyrosine kinase activity to induce tyrosine phosphorylation of RSK2 in Ba/F3 cells. Ba/F3 cells stably expressing TEL-FGFR3 were cultured in media with serum and IL-3 withdrawal in the presence or absence of PKC412 for 4 hr prior to harvest. Ba/F3 cells treated with IL-3 withdrawal were included as controls.
(C) RSK2 is tyrosine phosphorylated in Ba/F3 cells expressing TEL-FGFR3 or FGFR3 TDII but not in cells expressing the kinase-defective mutants TEL-FGFR3 K508R or FGFR3 TDII FF4F.

(A) Substitution of Y529 on RSK2 attenuates ERK-dependent phosphorylation and activation of RSK2.
(B) Y529F decreases RSK2 kinase activity in the presence of FGFR3 TDII. Ba/F3 cells stably expressing FGFR3 TDII and distinct myc-tagged RSK2 variants were cultured in the presence of aFGF with withdrawal of IL-3 and serum for 4 hr. Immunocomplexes of RSK2 variants were isolated and incubated with equal amount of S6-peptide (top) or MBP (middle) as exogenous substrates, as well as [γ-³²P] ATP. The phosphorylation of S6 peptide was normalized to readings from reactions using RSK2 immunocomplexes from cells stably expressing FGFR3 TDII alone (0.0) and cells coexpressing TDII and myc-RSK2 wild-type (1.0). The data were presented as mean ± SD; the p values were determined by Student’s t test; ns = not significant; 2F = Y488/529F. Bottom: Parallel immunoprecipitation and western blotting results confirmed equal amounts of myc-RSK2 proteins in each kinase reaction.

(A) Schematic representation of the specific RSK inhibitor, fmk (Cohen et al., 2005).
(B) Dose response analysis of Ba/F3 cells stably expressing FGFR3 TDII or TEL-FGFR3 to fmk. The relative cell viability was normalized to the viability of cells in the absence of fmk (mean ± SD). Right panel shows the cellular IC₅₀ (μM) of distinct cell lines.
(C) Inhibitory effects of fmk on phosphorylation of RSK2 in Ba/F3 cells stably expressing TEL-FGFR3 or FGFR3 TDII.

(A) fmk treatment inhibits RSK2 activation in t(4;14)-positive KMS11 and OPM1 cells in a dose-dependent manner.
(B) fmk effectively induces apoptosis in t(4;14)-positive KMS11 cells in a dose-dependent manner. Cells were treated with increasing concentrations of FGFR3 inhibitor PKC412 or fmk for 6 hr prior to FACS analysis. The apoptotic population was characterized as the fraction of annexin V positive cells of total treated cells (left; mean ± SD). Cleaved PARP was detected by western blotting (right).
(C) fmk induces significant apoptosis in human t(4;14)-positive OPM1, LP1, and KMS18 myeloma cells (mean ± SD).
(D) fmk induces significant apoptotic cell death in primary t(4;14)-positive multiple myeloma cells. Freshly isolated bone marrow mononuclear cells (BMNCs) from t(4;14)-positive and negative patients were incubated in the absence or presence of fmk for 16 and 24 hr. Cells were stained with annexin V-FITC and analyzed by flow cytometry. Myeloma cells were identified by CD138 labeling. The apoptotic population was characterized as the fraction of annexin V positive cells of total CD138 positive or negative cells (mean ± SD).