THE PATHOGENIC K650M MUTATION IN THE TYROSINE KINASE DOMAIN OF FGFR3 AFFECTS CYTOSKELETON ORGANIZATION

Fibroblast growth factor receptor 3 (FGFR3) is a transmembrane tyrosine kinase receptor, exposed on cell surface as monomer that dimerizes upon specific binding with fibroblast growth factors (FGFs). The FGF-FGFR3 interaction results in tyrosine (Tyr) autophosphorylation within activation loop of FGFR3 tyrosine kinase domain, crucial event for the recruitment and activation of several signalling proteins. FGFR3 plays a key role in skeletal development as negative regulator of bone elongation by inhibiting the proliferation and differentiation of chondrocytes during the endochondral ossification. Accordingly, specific germline activating mutations in the FGFR3 gene cause several skeletal dysplasias. In the present study, we examined the Lys650Met (K650M) and Lys650Glu (K650E) amino acid substitutions associated with two severe skeletal disorders, respectively, severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN) and thanatophoric dysplasia type II (TDII). In these disorders, both substitutions are located in the FGFR3 kinase activation loop domain and lead to ligand-independent/constitutive activation of the receptor. Previous studies have shown that the highly phosphorylated mutant receptor does not complete its biosynthesis resulting in the accumulation of the 120-kDa immature isoform in the endoplasmic reticulum, from where an aberrant signalling is triggered. Since several experimental evidences indicated that cells transfected with SADDAN-FGFR3 construct show an abnormal cellular morphology, we hypothesized that the anomalous FGFR3 signalling could affect actin cytoskeleton organization. Accordingly, the aim of this study was to define, in vitro, the molecular mechanism triggered by SADDAN-FGFR3 signalling. To identify SADDAN-FGFR3 molecular partners involved in cytoskeleton alterations, we focused on paxillin, a focal adhesion-associated protein playing a crucial role in the control of cell morphology changes and cytoskeleton reorganization both required for cell migration and proliferation. We observed that the expression of the SADDAN-FGFR3 mutant causes drastic changes in actin cytoskeleton organization, event associated with an increase of paxillin phosphorylation at Tyr118, a well-known target of FAK and c-Src kinases. In addition, by immunofluorescence analysis we revealed that the SADDAN receptor partially colocalizes with phosphorylated paxillin. Moreover, we showed that paxillin hyper-phosphorylation requires the kinase activity of the SADDAN mutant receptor. Interestingly, we observed that paxillin is a specific target for SADDAN-FGFR3 since TDII-FGFR3 does not increase paxillin phosphorylation. Furthermore, we showed that PLC-γ1, a downstream effector of FGFR3 signalling, plays a role in paxillin hyper-phosphorylation. Interestingly, our results indicate that SADDAN-FGFR3 receptor enhances paxillin phosphorylation through c-Src and FAK activation. Overall our findings contribute to elucidate the molecular events leading to actin cytoskeleton disorganization by SADDAN-FGFR3 signalling pathway.