Elucidation of a four-site allosteric network in fibroblast growth factor receptor tyrosine kinases.

Receptor tyrosine kinase (RTK) signaling is tightly regulated by protein allostery within the intracellular tyrosine kinase domains. Yet the molecular determinants of allosteric connectivity in tyrosine kinase domain are incompletely understood. By means of structural (X-ray and NMR) and functional characterization of pathogenic gain-of-function mutations affecting the FGF receptor (FGFR) tyrosine kinase domain, we elucidated a long-distance allosteric network composed of four interconnected sites termed the 'molecular brake', 'DFG latch', 'A-loop plug', and 'αC tether'. The first three sites repress the kinase from adopting an active conformation, whereas the αC tether promotes the active conformation. The skewed design of this four-site allosteric network imposes tight autoinhibition and accounts for the incomplete mimicry of the activated conformation by pathogenic mutations targeting a single site. Based on the structural similarity shared among RTKs, we propose that this allosteric model for FGFR kinases is applicable to other RTKs.

The N550K/H mutations in FGFR2 confer differential resistance to PD173074, dovitinib, and ponatinib ATP-competitive inhibitors.

We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2(mutant) endometrial cancers (ECs). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2(mutant) cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2(N550K) in JHUEM-2 cells (FGFR2(C383R)) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2(N550K) can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2(N550K). We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

The matrix peptide exporter HAF-1 signals a mitochondrial UPR by activating the transcription factor ZC376.7 in C. elegans.

Genetic analyses previously implicated the matrix-localized protease ClpP in signaling the stress of protein misfolding in the mitochondrial matrix to activate nuclear-encoded mitochondrial chaperone genes in C. elegans (UPR(mt)). Here, we report that haf-1, a gene encoding a mitochondria-localized ATP-binding cassette protein, is required for signaling within the UPR(mt) and for coping with misfolded protein stress. Peptide efflux from isolated mitochondria was ATP dependent and required HAF-1 and the protease ClpP. Defective UPR(mt) signaling in the haf-1-deleted worms was associated with failure of the bZIP protein, ZC376.7, to localize to nuclei in worms with perturbed mitochondrial protein folding, whereas zc376.7(RNAi) strongly inhibited the UPR(mt). These observations suggest a simple model whereby perturbation of the protein-folding environment in the mitochondrial matrix promotes ClpP-mediated generation of peptides whose haf-1-dependent export from the matrix contributes to UPR(mt) signaling across the mitochondrial inner membrane.