Profound Ligand-Independent Kinase Activation of Fibroblast Growth Factor Receptor 3 by the Activation Loop Mutation Responsible for a Lethal Skeletal Dysplasia, Thanatophoric Dysplasia Type II

Abstract

Thanatophoric dysplasia type II (TDII) is a neonatal lethal skeletal dysplasia caused by a recurrentLys-6503Glu mutation within the highly conserved activation loop of the kinase domain of fibroblast growth factor receptor 3 (FGFR3). We demonstrate here that this mutation results in profound constitutive activation of the FGFR3 tyrosine kinase, approximately 100-fold above that of wild-type FGFR3. The mechanism of FGFR3 activation in TDII was probed by constructing various point mutations in the activation loop. Substitutions at position 650 indicated that not only Glu but also Asp and, to a lesser extent, Gln and Leu result in pronounced constitutive activation of FGFR3. Additional mutagenesis within the ␤10-␤11 loop region (amino acids Tyr-647 to Leu-656) demonstrated that amino acid 650 is the only residue which can activate the receptor when changed to a Glu, indicating a specificity of position as well as charge for mutations which can give rise to kinase activation. Furthermore, when predicted sites of autophosphorylation at Tyr-647 and Tyr-648 were mutated to Phe, either singly or in combination, constitutive kinase activity was still observed in response to the Lys-6503Glu mutation, although the effect of these mutations on downstream signalling was not investigated. Our data suggest that the molecular effect of the TDII activation loop mutation is to mimic the conformational changes that activate the tyrosine kinase domain, which are normally initiated by ligand binding and autophosphorylation. These results have broad implications for understanding the molecular basis of other human developmental syndromes that involve mutations in members of the FGFR family. Moreover, these findings are relevant to the study of kinase regulation and the design of activating mutations in related tyrosine kinases.Fibroblast growth factor receptors (FGFRs) play an important role in regulating biological processes, including proliferation, differentiation, angiogenesis, and embryonic development. The four members of the FGFR family are highly related at the amino acid level, with each protein having an extracellular ligand-binding domain composed of three immunoglobulin-like domains, a transmembrane spanning region, and a cytoplasmic tyrosine kinase domain that is split by the kinase insert region. In response to ligand binding by members of the fibroblast growth factor family, FGFRs dimerize, resulting in autophosphorylation of the kinase domain and interaction with and phosphorylation of effector signalling proteins (for reviews, see references 11 and 12).Recently, several human congenital skeletal disorders have been shown to result from point mutations in members of the FGFR family. For instance, a variety of mutations mapping to the region between the second immunoglobulin loop and the transmembrane domain of FGFR2 cause the highly related craniosynostosis disorders known as Crouzon, Jackson-Weiss, Apert, and Pfeiffer syndromes. Pfeiffer syndrome also results from a mutation at a similar position in FGFR1 (for a revi...

Acidic substitution of the activation loop tyrosines in TrkA supports nerve growth factor-independent cell survival and neuronal differentiation

“…While substitution of activation loop residues does not always generate active enzymes (Robbins et al, 1993;Weinmaster and Pawson, 1986;Webster et al, 1996;Ra oni et al, 1998;Alessi et al, 1994), success will depend on understanding how each kinase functions. In this regard, it is clear that there are di erences in the auto-inhibitory mechanisms regulating di erent RTK's.…”

“…For example, the catalytic core tyrosines of the FGF receptor (FGFR) are not required for auto-inhibition in the absence of ligand; rather, auto-inhibition involves amino acids C-terminal to the activation loop (Mohammadi et al, 1996). Thus, constitutive activation of the FGFR3 is achieved by introducing negative or positive charges carboxy-terminal to the activation loop tyrosines rather than substitution of the activation loop tyrosines themselves (Webster et al, 1996).…”

Germline mutation of RET codon 883 in two cases of de novo MEN 2B

“…Further receptor tyrosine kinase mutations in this region (D820G in c-kit and V1238I, D1246N, D1246H, Y1248C, Y1248H, M1268T in c-met; Figure 3) are thought to be activating. The FGFR3 K650E mutation has been suggested to alter conformation of the`activation loop' and stabilise it in a non-inhibitory conformation such that the dimerisation-induced phosphorylation of the two tyrosine residues in this region is no longer required for activation (Webster et al, 1996;Mohammadi et al, 1996a,b). If this model can be extended to RET, phosphorylation of Y905 following dimerisation (by ligand or MEN 2A/FMTC cysteine codon mutation) would be required for kinase activity, however phosphorylation of Y905 would not be required for the activity of MEN 2B mutants.…”

“…The M918T mutation lies within the substrate binding pocket of the tyrosine kinase domain (Carlson et al, 1995;Hanks et al, 1988), and whereas a M residue is seen at the equivalent position in most (Piao et al, 1996;Pignon et al, 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al, 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al, 1997); RET: M918T and A883F in MEN 2B). The position of the catalytic and activation loops is shown, and Y residues believed to be involved in activation are marked * (Webster et al, 1996;Mohammadi et al, 1996a,b).…”

“…Activating mutations in the kinase domain of the receptor tyrosine kinases c-kit (D814Y; equivalent to RET codon 898) and FGFR3 (K650E; equivalent to RET codon 908) which also alter the catalytic substrate speci®city have recently been reported (Webster et al, 1996;Piao et al, 1996;Su et al, 1997), and in the case of the c-kit mutation the substrate speci®city change is related to that seen in M918T-RET. Consequently we decided to analyse this region of RET (exon 15) in three cases of clinically clear MEN 2B (two de novo and one familial) which do not harbour the M918T mutation.…”