SADDAN-FGFR3 causes cytoskeleton disorganization and paxillin hyperphosphorylation by Src

chondrodysplasias. FGFR3 is a tyrosine kinase (TK) receptor playing a key role in skeletal development. In this study, we analyzed mutations of FGFR3 associated with Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN) and with Thanatophoric Dysplasia type II (TDII), carrying the K650M and K650E substitutions, respectively. Both substitutions affect the TK-domain functions, resulting in a strong ligand-independent constitutive FGFR3 activation. The highly phosphorylated SADDAN and TDII receptors fail to reach full maturation and accumulate in their immature isoforms in the endoplasmic reticulum, from where they induce abnormal signalling. Objective: This study aimed to investigate whether the SADDAN-FGFR3 signalling could affect cytoskeletal organization through paxillin phosphorylation. Paxillin is a focal adhesion-associated protein playing an important role in cytoskeletal organization, cell morphology regulation, migration and proliferation. Paxillin is phosphorylated at Tyr118 by FAK and Src proteins. Methods: Paxillin phosphorylation was analyzed in HEK293 cells expressing FGFR3 mutants receptors by immunoprecipitation with anti-paxillin antibodies and immunoblotting with anti-phospho-paxillin (Tyr118) antibodies. Cytoskeletal changes and paxillin localization were analyzed in HeLa cells by immunofluorescence. Results: SADDAN-FGFR3 enhances paxillin phosphorylation at Tyr118 causing cell morphology alterations, and partially colocalizes with phosphorylated paxillin. The SADDAN-KD mutant, lacking kinase activity, does not affect paxillin phosphorylation, indicating the requirement of receptor enzymatic activity. Conversely, the TDII-FGFR3 mutant, although highly auto-phosphorylated, does not affect paxillin phosphorylation. Interestingly, PLC-γ1 plays a key role in paxillin hyperphosphorylation since the SADDAN-Y754F double mutant, abolishing the binding to PLC-γ1, does not enhance paxillin phosphorylation. Finally, the Src kinases inhibitor PP2 downregulates paxillin hyperphosphorylation, suggesting a role for Src in paxillin phospho-alterations.Conclusions: Paxillin is recruited by SADDAN-FGFR3 and hyperphosphorylated through Src kinases. The results of this study will contribute to clarify the molecular events leading to actin cytoskeletal disorganization by SADDAN-FGFR3.