Fibroblast growth factor (FGF) receptor 3 (FGFR3) belongs to a family of receptor tyrosine kinases (RTKs) responding to FGF with four members (FGFR1 to -4) that share a conserved structure and a high level of amino acid homology (56 to 71% overall identity) (15
). Each FGFR is composed of an extracellular ligand-binding domain, a transmembrane domain, and a split cytoplasmic tyrosine kinase domain (17
). FGFR3 is activated by oligomerization induced by ligand binding, followed by autophosphorylation at multiple tyrosine residues that are believed to provide docking sites for signaling factors through their respective Src homology 2 (SH2) phosphotyrosine binding domains. This, in turn, is required for stimulation of the intrinsic catalytic activity and activation of downstream signaling modules, including the phosphatidylinositol 3-kinase (PI3K)/AKT and phospholipase C-γ (PLC-γ) pathways (13
The t(4;14) translocation has been identified in approximately 15% of multiple myeloma (MM) patients (3
) and results in overexpression of wild-type (WT) FGFR3. MM is among the most common hematologic malignancies in patients over 65 years of age and is currently incurable. The t(4;14) MM is associated with a particularly poor clinical prognosis using conventional treatment strategies. In some t(4;14) MM cases, the translocated FGFR3 gene contains an activating mutation, K650E, that, when present in the germ line, causes thanatophoric dysplasia type II (TDII) (30
). Moreover, expression of a constitutively activated fusion tyrosine kinase, TEL-FGFR3, is associated with t(4;12)(p16;p13) acute myeloid leukemia (33
). Thus, the pathogenic role of FGFR3 makes it an attractive therapeutic target. We and others have demonstrated the therapeutic efficacy of small molecule tyrosine kinase inhibitors, including PKC412, PD173074, SU5402, and TKI258, which effectively inhibit FGFR3, in murine hematopoietic Ba/F3 cells; FGFR3-expressing t(4;14)-positive human MM cell lines (HMCLs), including KMS11, KMS18, and OPM-2; and as in bone marrow (BM) transplant (BMT) and xenograft murine models (2
FGFR3 has been demonstrated to activate multiple signaling components. Identification and characterization of critical downstream signaling effectors of FGFR3 will inform not only molecular mechanisms underlying FGFR3-induced transformation but also development of novel therapeutic strategies to treat FGFR3-associated human malignancies. We have performed mass spectrometry-based phospho-proteomics studies (18
) to comprehensively identify potential downstream substrates/effectors that are tyrosine phosphorylated in hematopoietic cells transformed by oncogenic FGFR3 mutants. We identified p90 ribosomal S6 kinase 2 (RSK2) as a substrate and signaling effector of FGFR3. RSK family members are Ser/Thr kinases and substrates of the Ras/extracellular signal-regulated kinase (ERK) pathway. RSK plays an essential role in a number of cellular functions, including regulation of gene expression, cell cycle, and survival by phosphorylating downstream substrates/signaling effectors.
While the C-terminal kinase (CTK) domain (CTD) is believed to be responsible for autophosphorylation and the N-terminal kinase (NTK) domain phosphorylates exogenous RSK substrates (8
), the precise mechanism of RSK activation remains elusive. The current model suggests that ERK-dependent activation of RSK contains a series of sequential events. First, inactive ERK binds to the C terminus of RSK in quiescent cells, and this interaction is an absolute requirement for activation of RSK (10
). Upon mitogen stimulation, ERK becomes activated and phosphorylates RSK at Thr577 (murine RSK2 numbering) in the activation loop of the CTD and Ser369 and Thr365 in the linker region between the two kinase domains, leading to activation of the RSK CTD. Second, activation of the CTD results in autophosphorylation of S386 in the linker region, which provides a docking site for 3-phosphoinositide-dependent protein kinase 1 (PDK1) (9
). Third, PDK1 in turn phosphorylates Ser227 in the activation loop of the NTK domain, allowing RSK to phosphorylate its downstream targets (16
). Finally, the activated NTK domain autophosphorylates Ser749 at the RSK CTD, which results in dissociation of active ERK from RSK (25
We recently proposed a novel two-step model in which leukemogenic FGFR3 activates RSK2 by both tyrosine phosphorylation of RSK2 and activation of the MEK/ERK pathway (18
). The first step involves tyrosine phosphorylation at Y529 of RSK2 by FGFR3, which facilitates binding of the inactive form of ERK to RSK2 in the initial step of ERK-dependent RSK2 activation. This binding, which is required for phosphorylation and activation of RSK2 by ERK, in turn promotes the second step where ERK is activated via the Ras/Raf/MEK/mitogen-activated protein kinase (MAPK) pathway downstream of FGFR3, leading to phosphorylation and activation of RSK2 by ERK. We also demonstrated that phosphorylation at Y529 of RSK2 is not a specific requirement of FGFR3 signaling in hematopoietic cells and that it may represent a more general mechanism for RSK2 activation (19
). We found that upon treatment of EGF, RSK2 is tyrosine phosphorylated at Y529 and activated in 293T and COS7 cells that do not express FGFR3. However, this phosphorylation was not mediated directly by activated receptor tyrosine kinase epidermal growth factor (EGF) receptor (EGFR), but by Src tyrosine kinase family members. Phosphorylation at Y529 by Src facilitates ERK binding to RSK2, which represents a general requirement for RSK2 activation by EGF through the MEK/ERK pathway.
In this paper, we identified an additional tyrosine site in RSK2, Y707, that when phosphorylated by FGFR3 contributes to RSK2 activation. Phosphorylation at Y707 may disrupt the autoinhibitory αL-helix in the C terminus of RSK2 to activate RSK2 CTD (21
), unlike Y529 phosphorylation, which facilitates ERK binding (18
). Moreover, we found that FGFR3 interacts with RSK2 and that this association is critical for FGFR3-dependent tyrosine phosphorylation at Y529 and Y707 of RSK2 as well as its subsequent activation. Furthermore, we demonstrated that RSK2 is important for FGFR3-induced hematopoietic transformation in vivo in our murine model of leukemia.