SADDAN-FGFR3 signalling involves paxillin phosphorylation and causes cytoskeleton disorganization.

Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene cause chondrodysplasias. FGFR3 is a tyrosine kinase (TK) receptor playing a key role in skeletal development as a negative regulator of bone growth. In this study, we analyzed gain-of-function mutations associated with Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN) and with Thanatophoric Dysplasia type II (TDII). In these two severe dwarfisms, FGFR3 carries the K650M and K650E substitutions, respectively, located in the activation loop of the TK-domain. Both substitutions result 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 high mannose-rich forms in the endoplasmic reticulum, from where they induce abnormal signalling. We analyzed whether the SADDAN-FGFR3 mutant could affect cytoskeletal organization through paxillin (PXN) activation. PXN is a focal adhesion-associated protein playing a key role in cytoskeletal organization and cell morphology regulation. A critical site for PXN activation is phosphorylation at tyrosine (Tyr) 118 through FAK and Src proteins. Our data show that SADDAN-FGFR3 enhances PXN phosphorylation at Tyr-118 and causes cell morphology changes. The SADDAN-KD (kinase dead) mutant, lacking kinase activity, did not affect PXN phosphorylation, indicating the requirement of receptor enzymatic activity. Similar results were obtained with the SADDAN-Y754F double mutant abolishing the interaction with PLCγ1, suggesting a role for this effector in PXN activation. Interestingly, the TDII-FGFR3 mutant, although highly auto-phosphorylated, did not increase PXN phosphorylation, suggesting that PXN is a specific target of SADDAN-FGFR3. The results of this study will contribute to clarify the molecular events leading to actin cytoskeletal disorganization by SADDAN-FGFR3.