Enhanced Signaling and Morphological Transformation by a Membrane-Localized Derivative of the Fibroblast Growth Factor Receptor 3 Kinase Domain†

Abstract

Fibroblast growth factor (FGF) receptors (FGFRs) are membrane-spanning tyrosine kinase receptors that mediate regulatory signals for cell proliferation and differentiation in response to FGFs. We have previously determined that the Lys6503Glu mutation in the activation loop of the kinase domain of FGFR3, which is responsible for the lethal skeletal dysplasia thanatophoric dyplasia type II (TDII), greatly enhances the ligand-independent kinase activity of the receptor. Here, we demonstrate that expression of this construct induces a c-fos promoter construct approximately 10-fold but does not lead to proliferation or morphological transformation of NIH 3T3 cells. In contrast, the isolated kinase domain of activated FGFR3, targeted to the plasma membrane by a myristylation signal, is able to stimulate c-fos expression by 40-fold, induce proliferation of quiescent cells, and morphologically transform fibroblasts. This result suggests that the extracellular and transmembrane domains of FGFRs exert a negative regulatory influence on the activity of the kinase domain. Targeting of the activated kinase domain to either the cytoplasm or the nucleus does not significantly affect biological signaling, suggesting that signals from FGFR3 resulting in mitogenesis originate exclusively from the plasma membrane. Furthermore, our novel observation that expression of a highly activated FGFR3 kinase domain is able to morphologically transform fibroblasts suggests that dysregulation of FGFR3 has the potential to play a role in human neoplasia.Fibroblast growth factor (FGF) receptors (FGFRs) are high-affinity membrane-spanning receptors for FGFs. FGFRs are normally catalytically inactive in the absence of FGF ligands. The binding of FGF to the extracellular domain of FGFRs, in the presence of heparan sulfate proteoglycans, induces the dimerization of two receptor molecules, allowing transphosphorylation of tyrosines within the activation loop of the intracellular tyrosine kinase domains. Activation loop phosphorylation greatly enhances the ability of FGFRs to autophosphorylate as well as to phosphorylate substrates which transmit biological signals into the cell leading to cell proliferation, differentiation, angiogenesis, or embryogenesis (4,11,21,23). Although growth factor receptor-mediated signaling has traditionally been assumed to initiate from the plasma membrane, FGF-induced cell proliferation requires prolonged exposure to ligand (63), during which activated FGFRs relocalize to the perinuclear and/or nuclear compartments of the cell (35,43,44).Point mutations in different domains of three of the four highly related FGFRs have been identified as causing human developmental abnormalities, including skeletal and cranial malformation syndromes (38,40,58). Recent work suggests that the biochemical mechanism underlying these syndromes is ligand-independent activation of the FGFR tyrosine kinase activity (58), and this constitutive signal transduction is postulated to cause premature and abnormal maturation of the affected long bo...

The Cytoplasmic Tyrosine Kinase Pyk2 as a Novel Effector of Fibroblast Growth Factor Receptor 3 Activation

“…FGFR Constructs-The full-length FGFR3 wild type, kinase-active (TD;K650E), and myristoylated forms of FGFR3 have been previously described (31,32) The FGFR3-⌬C-term was constructed by inserting a linker from the XhoI site in FGFR3 (Leu 457 -Glu…”

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

“…FGFR3, FGFR3(K650E), and FGFR3(K508R) were developed as described previously (9). To construct FGFR3-TACC3 fusion gene, a unique ClaI site was introduced by PCR-based site-directed mutagenesis after residue 758 in FGFR3 and before residue 648 in TACC3.…”

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

“…To facilitate mutagenesis of FGFR3, additional silent restriction sites were engineered into the myristylated wild-type (WT) FGFR3 construct PM-Kin(WT) described previously (Webster and Donoghue, 1997a). The basic organization of the WT construct, with location of restriction sites relative to the relevant tyrosine codons, is as follows:…”

“…NIH3T3, COS-1, CHO-K1, and 293T cells were transfected using a modified calcium phosphate transfection protocol (Chen and Okayama, 1987). Transformation assays were performed using NIH3T3 cells as described previously (Webster and Donoghue, 1997a). Transfection efficiencies were determined by G418-resistant colonies on parallel plates.…”