FGFR3 Activates RSK2 To Mediate Hematopoietic Transformation through Both Tyrosine Phosphorylation of RSK2 and Activation of the MEK/ERK Pathway.

Abstract

Reccurent chromosomal translocation t(4;14)(p16.3;q32.3) that frequently occurs in patients with multiple myeloma is associated with ectopic overexpression of fibroblast growth factor receptor 3 (FGFR3), which sometimes contains the activation mutations such as K650E (TDII). To better understand the signaling properties of oncogenic FGFR3, we performed a mass spectrometry-based phospho-proteomics study to identify potential downstream signaling effectors that are tyrosine-phosphorylated due to expression of FGFR3. RSK2 (p90 ribosomal S6 kinase 2) was found to be specifically tyrosine-phosphorylated at Y488 and Y529 in hematopoietic Ba/F3 cells transformed by TEL-FGFR3 fusion, which is associated with t(4;12)(p16;p13) peripheral T-cell lymphoma. In addition, RSK2 was highly tyrosine-phosphorylated in t(4;14)-positive human myeloma cell lines expressing constitutively activated FGFR3 mutants. We found that expression of leukemogenic FGFR3 variants resulted in activation of RSK2 through the MEK/ERK pathway. The current model suggests that inactive ERK binds to the C-terminus of RSK2 in quiescent cells, prior to ERK-dependent phosphorylation and activation of RSK2 when ERK is activated. Interestingly, we found that FGFR3-dependent tyrosine phosphorylation at Y529 regulates the ERK-dependent activation of the serine/threonine kinase RSK2. Co-immunoprecipitation experiments revealed that substitution of Y529 significantly decreased inactive ERK binding to RSK2. This disrupted ERK-RSK2 association consequently attenuated phosphorylation and activation of RSK2 by ERK activated by FGFR3, in both in vitro kinase assays and an RSK2-specific CREB mediated transcription assay. Moreover, inhibition of RSK2 by specific siRNA or an RSK inhibitor fmk effectively induced apoptosis in FGFR3 transformed murine Ba/F3 and t(4;14) human myeloma cells, suggesting RSK2 is a critical signaling effector in FGFR3-induced hematopoietic transformation. Together, these data support a novel two-step model that leukemogenic FGFR3 activates RSK2 to mediate transformation signals by promoting both the ERK-RSK2 interaction and subsequent phosphorylation of RSK2 by ERK. Thus, RSK2 may represent an alternative therapeutic target in the treatment of diverse human malignancies associated with dysregulated FGFR3.