Human congenital skeletal dysplasias are caused by mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. FGFR3 is a glycosylated transmembrane tyrosine kinase receptor exposed on the plasma membrane and plays a key role in skeletal development. Mutations associated with severe skeletal dysplasias cause an abnormal intracellular signaling by the receptor. In this study, we analyzed the mutations associated to Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN) and with Thanatophoric Dysplasia type II (TDII). In these two disorders, FGFR3 carries the K650 M/E substitutions located in the activation loop of the tyrosine kinase domain. Both substitutions result in a strong ligand-independent constitutive receptor auto-phosphorylation. As previously described, the highly phosphorylated SADDAN and TDII receptors fail to reach full maturation and accumulate in their immature high-mannose-rich isoforms in the endoplasmic reticulum (ER) [1].In this study, we have analyzed whether the FGFR3-SADDAN mutant could affect the cytoskeletal organization by altering actin fibers through paxillin activation. Paxillin is a multidomain focal adhesion-associated protein that plays a key role in integrin signaling and in the control of cytoskeletal organization, thus exerting a central role in the regulation of cell morphology. A critical site for paxillin activation is phosphorylation of Tyrosine (Tyr) 118 through FAK (Focal Adhesion Kinase) and Src proteins.By immunoprecipitation (IP) analysis with specific anti-paxillin antibodies, we assayed whether the phosphorylation of paxillin at Tyr 118 was induced by the SADDAN receptor. For this purpose, we have performed transient transfection in HEK293 cells with plasmids carrying the murine FGFR3-wt-HA, SADD-HA, TDII-HA and other FGFR3 mutant cDNAs. We observed that the level of paxillin phosphorylation at Tyr 118 was highly induced by the FGFR3-SADDAN. Interestingly, the TDII mutant, although highly auto-phosphorylated, it did not increase the paxillin phosphorylation level, suggesting that paxillin is a specific target of SADDAN-FGFR3.To investigate on a possible interaction between paxillin and SADDAN-FGFR3, we have performed co-immunoprecipitation experiments. Our data indicate that paxillin does not co-immunoprecipitate the FGFR3 mutant. The FGFR3-SADDAN-KD (kinase dead) mutant, lacking kinase activity, did not affect paxillin phosphorylation at Tyr-118, suggesting that the enzymatic activity is required for such activation. Interestingly, the double mutant SADDAN-754, abolishing the binding with PLC-γ1, an effector of FGFR3 signaling from the ER [2], did not affect paxillin phosphorylation, suggesting a role for this protein in paxillin activation. Furthermore, by confocal analysis, we showed in HeLa cells that the actin stress fibers are disorganized in cells expressing the FGFR3-SADDAN, but not in cells expressing wild-type FGFR3 or TDII receptors. In addition, our analysis reveals that paxillin is organized in peripheral area in FGFR3-wt cells while in SADDAN cells paxillin is distributed through the cytoplasm. A further observation is that phospho-paxillin co-localizes with FGFR3-SADDAN mutant but not with FGFR3-wt.In conclusion we show evidences that paxillin is associated with the ER and strongly phosphorylated by FGFR3-SADDAN. Our future studies will be focused on determining the paxillin interaction with other structural and signaling proteins of the focal adhesion structures (such as FAK and Src) to reveal the molecular events leading to actin cytoskeletal disorganization by FGFR3-SADDAN.[1] Lievens, P. M-J., Mutinelli, C., Baynes, D. & Liboi, E. The kinase activity of fibroblast growth factor receptor 3 with activation loop mutations affects receptor trafficking and signaling. J. Biol. Chem. 279, 43254–43260 (2004).[2] Lievens, P. M.-J., Roncador, A., and Liboi, E. K644E/M FGFR3 Mutants Activate Erk1/2 from the Endoplasmic Reticulum through FRS2a and PLCg-independent Pathways. J. Mol. Biol. 357, 783–792 (2006).

Role of SADDAN-FGFR3 mutant in cytoskeletal disorganization from the endoplasmic reticulum

Montone, Rosa;BARUZZI, Anna;ROMANELLI, Maria;LIBOI, Elio Maria;LIEVENS, Patricia
2014-01-01

Abstract

Human congenital skeletal dysplasias are caused by mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. FGFR3 is a glycosylated transmembrane tyrosine kinase receptor exposed on the plasma membrane and plays a key role in skeletal development. Mutations associated with severe skeletal dysplasias cause an abnormal intracellular signaling by the receptor. In this study, we analyzed the mutations associated to Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN) and with Thanatophoric Dysplasia type II (TDII). In these two disorders, FGFR3 carries the K650 M/E substitutions located in the activation loop of the tyrosine kinase domain. Both substitutions result in a strong ligand-independent constitutive receptor auto-phosphorylation. As previously described, the highly phosphorylated SADDAN and TDII receptors fail to reach full maturation and accumulate in their immature high-mannose-rich isoforms in the endoplasmic reticulum (ER) [1].In this study, we have analyzed whether the FGFR3-SADDAN mutant could affect the cytoskeletal organization by altering actin fibers through paxillin activation. Paxillin is a multidomain focal adhesion-associated protein that plays a key role in integrin signaling and in the control of cytoskeletal organization, thus exerting a central role in the regulation of cell morphology. A critical site for paxillin activation is phosphorylation of Tyrosine (Tyr) 118 through FAK (Focal Adhesion Kinase) and Src proteins.By immunoprecipitation (IP) analysis with specific anti-paxillin antibodies, we assayed whether the phosphorylation of paxillin at Tyr 118 was induced by the SADDAN receptor. For this purpose, we have performed transient transfection in HEK293 cells with plasmids carrying the murine FGFR3-wt-HA, SADD-HA, TDII-HA and other FGFR3 mutant cDNAs. We observed that the level of paxillin phosphorylation at Tyr 118 was highly induced by the FGFR3-SADDAN. Interestingly, the TDII mutant, although highly auto-phosphorylated, it did not increase the paxillin phosphorylation level, suggesting that paxillin is a specific target of SADDAN-FGFR3.To investigate on a possible interaction between paxillin and SADDAN-FGFR3, we have performed co-immunoprecipitation experiments. Our data indicate that paxillin does not co-immunoprecipitate the FGFR3 mutant. The FGFR3-SADDAN-KD (kinase dead) mutant, lacking kinase activity, did not affect paxillin phosphorylation at Tyr-118, suggesting that the enzymatic activity is required for such activation. Interestingly, the double mutant SADDAN-754, abolishing the binding with PLC-γ1, an effector of FGFR3 signaling from the ER [2], did not affect paxillin phosphorylation, suggesting a role for this protein in paxillin activation. Furthermore, by confocal analysis, we showed in HeLa cells that the actin stress fibers are disorganized in cells expressing the FGFR3-SADDAN, but not in cells expressing wild-type FGFR3 or TDII receptors. In addition, our analysis reveals that paxillin is organized in peripheral area in FGFR3-wt cells while in SADDAN cells paxillin is distributed through the cytoplasm. A further observation is that phospho-paxillin co-localizes with FGFR3-SADDAN mutant but not with FGFR3-wt.In conclusion we show evidences that paxillin is associated with the ER and strongly phosphorylated by FGFR3-SADDAN. Our future studies will be focused on determining the paxillin interaction with other structural and signaling proteins of the focal adhesion structures (such as FAK and Src) to reveal the molecular events leading to actin cytoskeletal disorganization by FGFR3-SADDAN.[1] Lievens, P. M-J., Mutinelli, C., Baynes, D. & Liboi, E. The kinase activity of fibroblast growth factor receptor 3 with activation loop mutations affects receptor trafficking and signaling. J. Biol. Chem. 279, 43254–43260 (2004).[2] Lievens, P. M.-J., Roncador, A., and Liboi, E. K644E/M FGFR3 Mutants Activate Erk1/2 from the Endoplasmic Reticulum through FRS2a and PLCg-independent Pathways. J. Mol. Biol. 357, 783–792 (2006).
2014
condrodysplasia, FGFR, signal transduction
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/936048
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