Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

Abstract

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3. INTRODUCTIONThe fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases (RTKs) mediates growth, differentiation, and cellular migration in a diverse range of cell types. These receptors function as high-affinity binding proteins for the FGF family of ligands, of which there are nearly 20 members (Szebenyi and Fallon, 1999). Ligand binding to the extracellular domain of FGFRs results in receptor dimerization and transphosphorylation of tyrosine residues in the intracellular domain.Many human disorders have been linked to mutations in FGFR3 (reviewed in Webster and Donoghue, 1997b;Burke et al., 1998). For example, mutations in the extracellular domain of FGFR3 such as R248C and G370C result in thanatophoric dysplasia type I (TDI), a lethal skeletal disorder. Mutations in the FGFR3 transmembrane domain can also lead to constitutive receptor activation, such as the G380R mutation responsible for most cases of achondroplasia, or skeletal dwarfism. Mutations in the FGFR3 kinase domain are responsible for developmental disorders that range in severity from the relatively mild hypochondroplasia (N540K) to the neonatal lethal disorder TDII (K650E). A different mutation at this same position, K650M, leads to a second syndrome, SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans), which involves skeletal malformations, CNS disturbances, and skin dysplasia (Tavormina et al., 1999).An exciting link between FGFR3 and cancer has recently been uncovered. The G375C and K650E mutations involved in skeletal malformation syndromes have also been identified in some patients with multiple myeloma, a proliferative B cell disorder (Chesi et al., 1997;Richelda et al., 1997). The K650E kinase domain mutation, as well as the R248C, S249C, and G370C extracellular domain mutati...

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

“…An inhibitory phosphorylation site has been shown to occur in FGFR3 WT at Y770 which, upon phosphorylation, inhibits cell transformation (40). Interestingly, the residue Y770 has been removed from the FGFR3-TACC3 fusion, but the significance of this change was not explored here.…”

“…This mutation has been exploited in many studies to examine signaling pathways utilized by FGFR3 (14,(39)(40)(41)(42)(43). However, the K650E mutation has not yet been identified together with the FGFR3-TACC3 oncogenic gene fusion in human cancer, and thus represents an artificial construct not yet found in nature.…”

“…2). Residue Y724 has been shown to be critical for activation of downstream signaling pathways, such as MAPK, STAT, and PI3K, as well as cell transformation (40). In the fusion protein FGFR3-TACC3, Y647 was the major site of phosphorylation within the activation loop, and phosphorylation of additional sites such as Y577, Y599, Y607, and Y798 was also observed.…”

“…In addition, Y760 may contribute to maximal STAT activation (40). The removal of this site from FGFR3-TACC3 may be contributing to the absence of STAT activation.…”

Immortalisation of Normal Human Urothelial Cells Compromises Differentiation Capacity

“…The most common mutation is a S249C substitution, which allows ligand-independent dimerisation and constitutive auto-phosphorylation. As a result, FGFR3 enhances proliferation by activating Ras-Raf-Extracellular signal-Related Kinase (ERK), Phosphatidylinositol 3-Kinase (PI3-K) and Signal Transducer and Activator of Transcription (STAT) proteins [4]. The p53/p21 and p16/Retinoblastoma (Rb) tumour suppressor pathways are frequently altered in muscle-invasive disease, with p53 loss associated with muscle-invasive disease and p16/Rb loss linked to tumours of higher metastatic potential (reviewed in [5]).…”