K650M-FGFR3 mutant causes cytoskeleton alteration and paxillin hyperphosphorylation through c-Src and FAK activation.

Fibroblast growth factor receptor 3 (FGFR3) is a transmembrane tyrosine kinase (TK) receptor playing key roles in skeletal development. Activating mutations in FGFR3 gene cause skeletal dysplasias. In our study, we examined the K650M and K650E substitutions associated with Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN) and with Thanatophoric Dysplasia type II (TDII), respectively. Both mutations, affecting the TK-domain, lead to a strong ligand-independent and constitutive FGFR3 activation. The high kinase activity acquired by both SADDAN and TDII mutants hampers receptors full maturation, causing their accumulation in the endoplasmic reticulum (ER) from where an aberrant signalling is triggered. Since previous evidences indicate that cells transfected with SADDAN-FGFR3 mutant show cell morphology alterations, we investigated whether the anomalous signalling from the ER could affect cytoskeletal organization. We observed that SADDAN-FGFR3 causes actin disorganization. According with this result, we focused our studies on paxillin, a focal adhesion protein playing a crucial role in cytoskeletal organization and migration. We showed that SADDAN-FGFR3 enhances paxillin phosphorylation at tyrosine 118, a critical target of FAK and c-Src kinases. Conversely, TDII-FGFR3 does not affect paxillin phosphorylation, suggesting that paxillin is a specific target for SADDAN-FGFR3. Confocal microscopy analysis displayed that phosphorylated paxillin colocalizes with SADDAN receptor. Moreover, our results revealed that SADDAN-FGFR3 activates c-Src and FAK implying that both kinases may be involved in paxillin phospho-alterations. Finally, we hypothesize a role for PLC-γ1 in paxillin hyperphosphorylation, since the SADDAN-Y754F double mutant does not enhance phosphorylation at Tyr118. Overall our findings will contribute to elucidate the molecular mechanism that lead to actin cytoskeleton alteration by SADDAN-FGFR3.