Oncogenic RET Kinase domain mutations perturb the autophosphorylation trajectory by enhancing substrate presentation in trans

To decipher the molecular basis for RET kinase activation and oncogenic deregulation, we defined the temporal sequence of RET autophosphorylation by label-free quantitative mass spectrometry. Early autophosphorylation sites map to regions flanking the kinase domain core, while sites within the activation loop only form at later time points. Comparison with oncogenic RET kinase revealed that late autophosphorylation sites become phosphorylated much earlier than wild-type RET, which is due to a combination of an enhanced enzymatic activity, increased ATP affinity, and surprisingly, by providing a better intermolecular substrate. Structural analysis of oncogenic M918T and wild-type RET kinase domains reveal a cis-inhibitory mechanism involving tethering contacts between the glycine-rich loop, activation loop, and αC-helix. Tether mutations only affected substrate presentation but perturbed the autophosphorylation trajectory similar to oncogenic mutations. This study reveals an unappreciated role for oncogenic RET kinase mutations in promoting intermolecular autophosphorylation by enhancing substrate presentation

Identification of the molecular basis of the lacrimo-auriculo-dento-digital (LADD) syndrome

Lacrimo-auriculo-dento-digital (LADD) syndrome, also known as Levy-Hollister syndrome, is a rare autosomal dominant developmental disorder, mainly characterized by abnormalities of the lacrimal system and salivary glands, ears and hearing, teeth and distal limb development. Besides these cardinal features, facial dysmorphism and malformations of the kidney and the respiratory system have been reported. In this study, the LADD1 locus was mapped to chromosome 10q26 by genome wide linkage analysis using the Affymetrix GeneChip 10K array in three large LADD families. In all three LADD families and in one sporadic case, heterozygous missense mutations were found in exon 16 of the gene encoding the fibroblast-growth-factor-receptor 2 (FGFR2). After exclusion of the FGFR2 locus by haplotype analysis in two additional LADD families, one missense mutation was identified in FGFR3 and one mutation was found in the fibroblast-growth-factor 10 (FGF10), a known ligand of FGFR2 [Rohmann et al., 2006]. The functional properties of FGF10 LADD and FGFR2 LADD mutants were analyzed and compared to the activities of their normal counterparts. Protein expression in BL21 cells and binding studies showed that each of the three analyzed FGF10 mutations demonstrated severely impaired activity by different mechanisms. Transient and stable expression studies exhibited that the FGFR2 mutations possess a reduced autophosphorylation and a weaker tyrosine kinase activity. Mutations also lead to diminished phosphorylation activity in FGFR2-mediated substrates (e. g. FRS2 and Shc) and to a decreased downstream signaling pathway, as shown by MAPK activity. While tested FGF10 LADD mutations caused haploinsufficiency, the FGFR2 LADD mutants could exert a dominant-negative effect on normal FGFR2 protein [Shams and Rohmann et al., 2007]. An in vitro kinase assay and crystallization of both, FGFR2 WT and the p.A628T missense mutation in the catalytic part of the tyrosine kinase domain, demonstrated that the A628T LADD mutation disrupts the catalytic activity due to conformational changes, leading to LADD syndrome. In addition, the newly described crystal structure of FGFR2 in comparison to FGFR1 revealed that the FGFR2 utilizes a less stringent mode of autoinhibition [Lew, Bae and Rohmann et al., 2007]

Structure-Functional Prediction and Analysis of Cancer Mutation Effects in Protein Kinases

A central goal of cancer research is to discover and characterize the functional effects of mutated genes that contribute to tumorigenesis. In this study, we provide a detailed structural classification and analysis of functional dynamics for members of protein kinase families that are known to harbor cancer mutations. We also present a systematic computational analysis that combines sequence and structure-based prediction models to characterize the effect of cancer mutations in protein kinases. We focus on the differential effects of activating point mutations that increase protein kinase activity and kinase-inactivating mutations that decrease activity. Mapping of cancer mutations onto the conformational mobility profiles of known crystal structures demonstrated that activating mutations could reduce a steric barrier for the movement from the basal low activity state to the active state. According to our analysis, the mechanism of activating mutations reflects a combined effect of partial destabilization of the kinase in its inactive state and a concomitant stabilization of its active-like form, which is likely to drive tumorigenesis at some level. Ultimately, the analysis of the evolutionary and structural features of the major cancer-causing mutational hotspot in kinases can also aid in the correlation of kinase mutation effects with clinical outcomes

Reciprocal regulation of the Fibroblast Growth Factor Receptor 2 (FGFR2) and the Transient Receptor Potential Ankyrin 1 (TRPA1) through their direct interaction

Tyrosine Kinase Receptors (RTKs) regulate fundamental cellular processes including cell proliferation, survival and invasion and thus, dysregulation of their activation is implicated in human malignancies. The canonical mechanism of activation is initiated by receptor activation through ligand-induced dimerization, and autophosphorylation of key tyrosine residues along the C-terminal cytoplasmic domain. It has been demonstrated in the past that FGFR2, an RTK, can dimerise in the absence of ligand resulting in signal activation or inhibition depending on the equilibrium of C-terminal-binding proteins i.e. GRB2 and PLCγ1. This study provided the first indication of a novel direct interaction between FGFR2 and a non-selective cation channel, TRPA1, through the C-terminal domain of FGFR2 and the Ankyrin repeat domain of TRPA1. The interaction was studied by protein interaction techniques in an overexpression system. The FGFR2-TRPA1 complex was also detected in a human lung adenocarcinoma cell line suggesting a possible implication in cancer. The regulatory effect of TRPA1 on FGFR2 was also investigated. In basal conditions, the interaction with TRPA1 exhibits an inhibitory effect on FGFR2 autophosphorylation which results in downstream PLCγ1 pathway inhibition. Co-immunoprecipitation experiments revealed a decrease in PLCγ1 binding to FGFR2 in the presence of TRPA1 explaining the reduction in the PLCγ1 pathway activation. Upon stimulating conditions, TRPA1-mediated receptor inhibition is raised, as shown by increase in p-FGFR2 however, binding of PLCγ1 to the C-terminal of FGFR2 is still impeded. Finally, a model of TRPA1-mediated regulation of the FGFR2 signalling was proposed in which TRPA1 prevents aberrant basal stimulation of the receptor while maintaining PLCγ1 pathway inhibition even in the presence of ligand. These findings provided the first evidence of direct interaction between an RTK and a TRP channel as well as proposed a novel regulatory mechanism of FGFR2 signalling that can facilitate in the development of therapeutic strategies for FGFR2-related diseases

Simulation & Experiment Learning From Kinases In Cancer

The decreasing cost of genome sequencing technology has lead to an explosion of informa- tion about which mutations are frequently observed in cancer, demonstrating an important role in cancer progression for kinase domain mutations. Many therapies have been devel- oped that target mutations in kinase proteins that lead to constitutive activation. However, a growing body of evidence points to the serious dangers of many kinase ATP competitive inhibitors leading to paradoxical activation in non-constitutively active proteins. The large number of observed mutations and the critical need to only treat patients harboring activat- ing mutations with targeted therapies raises the question of how to classify the thousands of mutations that have been observed. We start with an in depth look at the state of knowl- edge of the distribution and effects of kinase mutations. We then report on computational methods to understand and predict the effects of kinase domain mutations. Using molecular dynamics simulations of mutant kinases, we show that there is a switch-like network of la- bile hydrogen bonds that are often perturbed in activating mutations. This is paired with a description of a software platform that has been developed to streamline the execution and analysis of molecular dynamics simulations. We conclude by examining a machine learning method to demonstrate what kinds information derived from protein sequence alone have the most value in distinguishing activating and non-activating mutations

BCR-ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein

Patients with chronic myeloid leukemia in whom tyrosine kinase inhibitors (TKIs) fail often present mutations in the BCR-ABL catalytic domain. We noticed a lack of substitutions involving 4 amino acids (E286, M318, I360, and D381) that form hydrogen bonds with ponatinib. We therefore introduced mutations in each of these residues, either preserving or altering their physicochemical properties. We found that E286, M318, I360, and D381 are dispensable for ABL and BCR-ABL protein stability but are critical for preserving catalytic activity. Indeed, only a "conservative" I360T substitution retained kinase proficiency and transforming potential. Molecular dynamics simulations of BCR-ABLI360T revealed differences in both helix αC dynamics and protein-correlated motions, consistent with a modified ATP-binding pocket. Nevertheless, this mutant remained sensitive to ponatinib, imatinib, and dasatinib. These results suggest that changes in the 4 BCR-ABL residues described here would be selected against by a lack of kinase activity or by maintained responsiveness to TKIs. Notably, amino acids equivalent to those identified in BCR-ABL are conserved in 51% of human tyrosine kinases. Hence, these residues may represent an appealing target for the design of pharmacological compounds that would inhibit additional oncogenic tyrosine kinases while avoiding the emergence of resistance due to point mutations.This work was supported by an investigator grant to P.V. from Associazione Italiana per la Ricerca sul Cancro (AIRC) and by funding from the Biotechnology and Biological Sciences Research Council (BB/I023291/1 and BB/H018409/1 to AP and FF). P.B. is the recipient of an AIRC - Marie Curie fellowship

Differential responses to kinase inhibition in FGFR2-addicted triple negative breast cancer cells: a quantitative phosphoproteomics study

Fibroblast Growth Factor (FGF) dependent signalling is frequently activated in cancer by a variety of different mechanisms. However, the downstream signal transduction pathways involved are poorly characterised. Here a quantitative differential phosphoproteomics approach, SILAC, is applied to identify FGF-regulated phosphorylation events in two triple- negative breast tumour cell lines, MFM223 and SUM52, that exhibit amplified expression of FGF receptor 2 (FGFR2) and are dependent on continued FGFR2 signalling for cell viability. Comparative Gene Ontology proteome analysis revealed that SUM52 cells were enriched in proteins associated with cell metabolism and MFM223 cells enriched in proteins associated with cell adhesion and migration. FGFR2 inhibition by SU5402 impacts a significant fraction of the observed phosphoproteome of these cells. This study expands the known landscape of FGF signalling and identifies many new targets for functional investigation. FGF signalling pathways are found to be flexible in architecture as both shared, and divergent, responses to inhibition of FGFR2 kinase activity in the canonical RAF/MAPK/ERK/RSK and PI3K/AKT/PDK/mTOR/S6K pathways are identified. Inhibition of phosphorylation-dependent negative-feedback pathways is observed, defining mechanisms of intrinsic resistance to FGFR2 inhibition. These findings have implications for the therapeutic application of FGFR inhibitors as they identify both common and divergent responses in cells harbouring the same genetic lesion and pathways of drug resistance

Regulation and Kinase Activity of the Trk Family of Receptor Tyrosine Kinases

The tropomyosin-related kinase (Trk) family consists of three receptor tyrosine kinases (RTKs) called TrkA, TrkB, and TrkC. These RTKs are regulated by the neurotrophins, a class of secreted growth factors responsible for the development and function of neurons. Given the high homology between the Trks and their use of overlapping signaling pathways, the key question to be addressed is how the activation of the different Trks can lead to distinct cellular outcomes. To this end, I first sought to determine the mechanism of autoregulation for the Trk tyrosine kinase domain (TKD). The Trk TKDs are members of the insulin receptor kinase (IRK) superfamily and recent data suggest that the IRK family displays a wide array of autoinhibitory mechanisms. To determine where TrkA and the closely related Ror2 TKD (from an unconventional Wnt receptor) lie in this spectrum, we determined the crystal structures of the kinase domains of these RTKs. In both cases, the conformation of the activation loop resembles the IRK activation loop conformation, with subtle but notable differences in the case of Ror2. These findings aid in understanding the range of autoinhibitory mechanisms of the IRK family, in addition to providing a foundation for deciphering consequences of TKD mutations in this family. I also observed crystallographic dimers of the inactive TrkA TKD that resemble those seen for other RTK TKDs - which may aid in understanding the reported pre-formed inactive TrkA dimers observed in cells. To understand the molecular basis for differences in signaling specificity of the Trk receptors, I investigated whether the TrkA and TrkB TKDs differ in their intrinsic kinase activities. I show that the TrkA TKD autophosphorylates itself faster than its TrkB counterpart. However, this difference of autophosphorylation is not due to a difference in kinase activity per se. Rather, my data indicate that the difference in autophosphorylation may arise because of self-association of the TrkA TKD that does not occur with TrkB. My work sheds light on potential differences between TrkA and TrkB signaling, as well as providing a quantitative understanding of Trk TKD activation, which is useful for effective and selective inhibitor design

Functional dissection of the "Drosophila melanogaster" fibroblast growth factor signalling pathway in branching morphogenesis of the developing tracheal system

Fibroblast Growth Factor (FGF) signalling is involved in numerous developmental processes ranging from cell determination to mitogenesis, and cell survival to cell migration. Interestingly, the same signalling pathway is used reiteratively throughout development and the question regarding the intracellular specificity is raised. Little is known about the intracellular signalling events of the FGF signalling pathway leading to specific cellular responses. Since the FGF signal is essential throughout embryonic and adult development and plays a role in many pathogenical processes, it is important to identify the factors, which determine the differential responses. We were interested to investigate the specificity of FGF signalling in a developmental context in which the signal induces directed cell migration, a cellular phenomenon that relies on changes of the cytoskeletal architecture. During gastrulation in early embryonic development, but also during the formation of organs in mammals and in Drosophila, FGFs have been shown to act as chemoattractants and guide cells toward their targets. In these contexts, FGF signalling has been shown to induce filopodia, which are long cellular extensions containing parallel actin bundles. Using Drosophila tracheal and mesodermal cell migration as model systems, we found that the intracellular domain of the two Drosophila FGF receptors (FGFRs) Breathless (Btl) and Heartless (Htl) can be replaced by the equivalent domains of Torso and EGFR, and yet these hybrid receptors will rescue cell migration in btl or htl mutant embryos, respectively. These chimeric receptors rescued cell migration even in the absence of Downstream-of-FGFR (Dof), a scaffolding protein that has been shown to be essential for FGF signalling in Drosophila. Thus, Dof acts specifically in the FGF signalling pathway. The functional characterization of Dof has demonstrated that Dof is indeed a FGFR specific phosphotarget and forms a complex with both FGFRs, but it is not a substrate of Torso. We performed a functional deletion analysis of the Btl receptor to define the interaction domains of Dof and other putative adapter proteins essential in the process of cell migration. Deletion of all putative interaction domains outside of the kinase domain did not affect the rescue capacity of the truncated Btl receptors in vivo suggesting that the kinase domain is sufficient for transmitting the signal. In line with this interpretation, results from S2 cell culture experiments revealed that Dof interacts with the kinase domain, and it does so independently of the activation state of the receptor. Surprisingly, in S2 cells, Btl receptors lacking the C-terminus did not auto-phosphorylate, as consequence we could not observe phosphorylation of Dof. We assume, that the short C-terminus is required for conformational changes of the kinase activation loop upon dimerization of the receptors to enable trans-phosphorylation. Dof belongs to a distinct family of adapter proteins than its functional homologue, the vertebrate FGFR adapter protein FRS2 that has been shown to constitutively interact with the juxtamembrane domain of the FGFRs. We could show that the human FGFR2, when expressed in the tracheal system, is only able to rescue cell migration defects effectively in the presence of Dof. These results suggest that Dof is able to interact with human FGFRs. At present, there is no evidence for a FRS2 homologue in Drosophila that might act as substitute for Dof. Upon receptor activation, Dof recruits the phosphatase Corkscrew (Csw), the Drosophila Shp2 homologue. Csw recruitment represents an essential step in FGF induced cell migration and transcriptional activation via the Ras/MAPK cascade. However, our results indicate that the activation of Ras is not sufficient to activate the migration machinery in tracheal and mesodermal cells. Ectopic activation of the Ras/MAPK cascade partially rescued tracheal cell migration in btl or dof mutant embryos. But high levels of sustained activation of Ras or the MAPK in wild-type tracheal cells did not disturb the migratory behaviour of the cells in contrast to ectopic activation of Branchless (Bnl), the Drosophila FGF homologue, which completely impaired primary branch outgrowth. In a wild-type tracheal system, MAPK activity is restricted to the tracheal tip cells. Single cell rescue experiments indicate that Bnl induces the migratory response exclusively in the tip cells of the outgrowing tracheal branches; the stalk cells are pulled forward by cell-cell adhesion contacts. The small GTPases of the Rho family have been shown to regulate cytoskeletal rearrangements. For tracheal development, Dcdc42 could function in collaboration with Drac in the regulation of actin dynamics according to our experiments. Additional proteins linking either Dof or Csw to the small GTPases have to be identified

The D647N mutation of FGFR1 induces ligand-independent receptor activation

The activation loop (A-loop) of kinases, a key regulatory region, is recurrently mutated in several kinase proteins in cancer resulting in dysregulated kinase activity and response to kinase inhibitors. FGFR1 receptor tyrosine kinase represents an important oncogene and therapeutic target for solid and hematological tumors. Here we investigate the biochemical and molecular effects of D647N mutation lying in the A-loop of FGFR1.When expressed in normal and tumoral in vitro cell models, FGFR1D647N is phosphorylated also in the absence of ligands, and this is accompanied by the activation of intracellular signaling. The expression of FGFR1D647N significantly increases single and collective migration of cancer cells in vitro and in vivo, when compared to FGFR1WT. FGFR1D647N expression exacerbates the aggressiveness of cancer cells, increasing their invasiveness in vitro and augmenting their pro-angiogenic capacity in vivo.Remarkably, the D647N mutation significantly increases the sensitivity of FGFR1 to the ATP-competitive inhibitor Erdafitinib suggesting the possibility that this mutation could become a specific target for the development of new inhibitors. Although further efforts are warranted for an exhaustive description of the activation mechanisms, for the identification of more specific inhibitors and for confirming the clinical significance of mutated FGFR1D647N, overall our data demonstrate that the D647N substitution of FGFR1 is a novel pro-oncogenic activating mutation of the receptor that, when found in cancer patients, may anticipate good response to erdafitinib treatment