Abnormal signaling by FGFR3-K650M mutant associated with SADDAN disease targets paxillin causing cytoskeleton disorganization.

Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene cause chondrodysplasias. Change of lysine 650 to methionine (K650M) leads to Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN), a disease characterized by constitutive FGFR3 tyrosine kinase activity. We previously showed that autophosphorylation occurs early during protein biosynthesis, hampering complete maturation of SADDAN receptors. A similar behaviour was observed when lysine 650 was sustituted by glutamic acid (K650E), a mutation associated with Thanatophoric Dysplasia type II (TDII). Both mutants accumulate in the ER/Golgi as highly phosphorylated immature receptors from where they trigger an abnormal signalling. Based on the observation that SADDAN-FGFR3 causes alterations in cell morphology, we analyzed whether this mutant could affect cytoskeletal organization through paxillin (PXN) function. PXN is a focal adhesion-associated protein playing a key role in cytoskeletal organization. A critical event for PXN activation is phosphorylation at tyrosine 118 by FAK and Src proteins. Our data show that SADDAN-FGFR3 enhanced PXN phosphorylation at tyrosine 118 causing cell morphology changes. The SADDAN-KD (kinase dead) mutant, lacking kinase activity, did not change PXN phosphorylation, implying the requirement of receptor enzymatic activity. Interestingly, the kinase active TDII-FGFR3, had no effect on PXN phosphorylation, suggesting that PXN is a specific target of SADDAN-FGFR3. Finally, the SADDAN-Y760F mutant abolishing the interaction with PLCγ1 rescued the effect on PXN, suggesting a role for PLCγ1 in PXN activation.The results of this study will contribute to clarify the molecular events leading to actin cytoskeletal disorganization by SADDAN-FGFR3.