The hypermorphic PLCγ2 S707Y variant dysregulates microglial cell function - Insight into PLCγ2 activation in brain health and disease, and opportunities for therapeutic modulation.

Phospholipase C-gamma 2 (PLCγ2) is highly expressed in hematopoietic and immune cells, where it is a key signalling node enabling diverse cellular functions. Within the periphery, gain-of-function (GOF) PLCγ2 variants, such as the strongly hypermorphic S707Y, cause severe immune dysregulation. The milder hypermorphic mutation PLCγ2 P522R increases longevity and confers protection in central nervous system (CNS) neurodegenerative disorders, implicating PLCγ2 as a novel therapeutic target for treating these CNS indications. Currently, nothing is known about what consequences strong PLCγ2 GOF has on CNS functionality, and more precisely on the specific biological functions of microglia. Using the PLCγ2 S707Y variant as a model of chronic activation we investigated the functional consequences of strong PLCγ2 GOF on human microglia. PLCγ2 S707Y expressing human inducible pluripotent stem cells (hiPSC)-derived microglia exhibited hypermorphic enzymatic activity under both basal and stimulated conditions, compared to PLCγ2 wild type. Despite the increase in PLCγ2 enzymatic activity, the PLCγ2 S707Y hiPSC-derived microglia display diminished functionality for key microglial processes including phagocytosis and cytokine secretion upon inflammatory challenge. RNA sequencing revealed a downregulation of genes related to innate immunity and response, providing molecular support for the phenotype observed. Our data suggests that chronic activation of PLCγ2 elicits a detrimental phenotype that is contributing to unfavourable CNS functions, and informs on the therapeutic window for targeting PLCγ2 in the CNS. Drug candidates targeting PLCγ2 will need to precisely mimic the effects of the PLCγ2 P522R variant on microglial function, but not those of the PLCγ2 S707Y variant.

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells.

BACKGROUND: Development of adult T-cell leukemia/lymphoma (ATL) involves human T-cell leukemia virus type 1 (HTLV-1) infection and accumulation of somatic mutations. The most frequently mutated gene in ATL (36 % of cases) is phospholipase C gamma1 (PLCG1). PLCG1 is also frequently mutated in other T-cell lymphomas. However, the functional consequences of the PLCG1 mutations in cancer cells have not been characterized. METHODS: We compared the activity of the wild-type PLCγ1 with that of a mutant carrying a hot-spot mutation of PLCγ1 (S345F) observed in ATL, both in cells and in cell-free assays. To analyse the impact of the mutation on cellular properties, we quantified cellular proliferation, aggregation, chemotaxis and apoptosis by live cell-imaging in an S345F+ ATL-derived cell line (KK1) and a KK1 cell line in which we reverted the mutation to the wild-type sequence using CRISPR/Cas9 and homology-directed repair. FINDINGS: The PLCγ1 S345F mutation results in an increase of basal PLC activity in vitro and in different cell types. This higher basal activity is further enhanced by upstream signalling. Reversion of the S345F mutation in the KK1 cell line resulted in reduction of the PLC activity, lower rates of proliferation and aggregation, and a marked reduction in chemotaxis towards CCL22. The PLCγ1-pathway inhibitors ibrutinib and ritonavir reduced both the PLC activity and the tested functions of KK1 cells. INTERPRETATION: Consistent with observations from clinical studies, our data provide direct evidence that activated variants of the PLCγ1 enzyme contribute to the properties of the malignant T-cell clone in ATL. FUNDING: MRC (UK) Project Grant (P028160).

Characterization of the membrane interactions of phospholipase Cγ reveals key features of the active enzyme.

PLCγ enzymes are autoinhibited in resting cells and form key components of intracellular signaling that are also linked to disease development. Insights into physiological and aberrant activation of PLCγ require understanding of an active, membrane-bound form, which can hydrolyze inositol-lipid substrates. Here, we demonstrate that PLCγ1 cannot bind membranes unless the autoinhibition is disrupted. Through extensive molecular dynamics simulations and experimental evidence, we characterize membrane binding by the catalytic core domains and reveal previously unknown sites of lipid interaction. The identified sites act in synergy, overlap with autoinhibitory interfaces, and are shown to be critical for the phospholipase activity in cells. This work provides direct evidence that PLCγ1 is inhibited through obstruction of its membrane-binding surfaces by the regulatory region and that activation must shift PLCγ1 to a conformation competent for membrane binding. Knowledge of the critical sites of membrane interaction extends the mechanistic framework for activation, dysregulation, and therapeutic intervention.

Cells of the human intestinal tract mapped across space and time.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Phospholipase C families: Common themes and versatility in physiology and pathology.

Phosphoinositide-specific phospholipase Cs (PLCs) are expressed in all mammalian cells and play critical roles in signal transduction. To obtain a comprehensive understanding of these enzymes in physiology and pathology, a detailed structural, biochemical, cell biological and genetic information is required. In this review, we cover all these aspects to summarize current knowledge of the entire superfamily. The families of PLCs have expanded from 13 enzymes to 16 with the identification of the atypical PLCs in the human genome. Recent structural insights highlight the common themes that cover not only the substrate catalysis but also the mechanisms of activation. This involves the release of autoinhibitory interactions that, in the absence of stimulation, maintain classical PLC enzymes in their inactive forms. Studies of individual PLCs provide a rich repertoire of PLC function in different physiologies. Furthermore, the genetic studies discovered numerous mutated and rare variants of PLC enzymes and their link to human disease development, greatly expanding our understanding of their roles in diverse pathologies. Notably, substantial evidence now supports involvement of different PLC isoforms in the development of specific cancer types, immune disorders and neurodegeneration. These advances will stimulate the generation of new drugs that target PLC enzymes, and will therefore open new possibilities for treatment of a number of diseases where current therapies remain ineffective.

Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane.

Phosphatidylinositol(4,5) bisphosphate (PI(4,5)P2) has become a major focus in biochemistry, cell biology and physiology owing to its diverse functions at the plasma membrane. As a result, the functions of PI(4,5)P2 can be explored in two separate and distinct roles - as a substrate for phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) and as a primary messenger, each having unique properties. Thus PI(4,5)P2 makes contributions in both signal transduction and cellular processes including actin cytoskeleton dynamics, membrane dynamics and ion channel regulation. Signalling through plasma membrane G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immune receptors all use PI(4,5)P2 as a substrate to make second messengers. Activation of PI3K generates PI(3,4,5)P3 (phosphatidylinositol(3,4,5)trisphosphate), a lipid that recruits a plethora of proteins with pleckstrin homology (PH) domains to the plasma membrane to regulate multiple aspects of cellular function. In contrast, PLC activation results in the hydrolysis of PI(4,5)P2 to generate the second messengers, diacylglycerol (DAG), an activator of protein kinase C and inositol(1,4,5)trisphosphate (IP3/I(1,4,5)P3) which facilitates an increase in intracellular Ca2+. Decreases in PI(4,5)P2 by PLC also impact on functions that are dependent on the intact lipid and therefore endocytosis, actin dynamics and ion channel regulation are subject to control. Spatial organisation of PI(4,5)P2 in nanodomains at the membrane allows for these multiple processes to occur concurrently.

Severe Autoinflammatory Manifestations and Antibody Deficiency Due to Novel Hypermorphic PLCG2 Mutations.

Autoinflammatory diseases (AIDs) were first described as clinical disorders characterized by recurrent episodes of seemingly unprovoked sterile inflammation. In the past few years, the identification of novel AIDs expanded their phenotypes toward more complex clinical pictures associating vasculopathy, autoimmunity, or immunodeficiency. Herein, we describe two unrelated patients suffering since the neonatal period from a complex disease mainly characterized by severe sterile inflammation, recurrent bacterial infections, and marked humoral immunodeficiency. Whole-exome sequencing detected a novel, de novo heterozygous PLCG2 variant in each patient (p.Ala708Pro and p.Leu845_Leu848del). A clear enhanced PLCγ2 activity for both variants was demonstrated by both ex vivo calcium responses of the patient's B cells to IgM stimulation and in vitro assessment of PLC activity. These data supported the autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) diagnosis in both patients. Immunological evaluation revealed a severe decrease of immunoglobulins and B cells, especially class-switched memory B cells, with normal T and NK cell counts. Analysis of bone marrow of one patient revealed a reduced immature B cell fraction compared with controls. Additional investigations showed that both PLCG2 variants activate the NLRP3-inflammasome through the alternative pathway instead of the canonical pathway. Collectively, the evidences here shown expand APLAID diversity toward more severe phenotypes than previously reported including dominantly inherited agammaglobulinemia, add novel data about its genetic basis, and implicate the alternative NLRP3-inflammasome activation pathway in the basis of sterile inflammation.

Targeting the Src Pathway Enhances the Efficacy of Selective FGFR Inhibitors in Urothelial Cancers with FGFR3 Alterations.

Selective FGFR inhibitors such as infigratinib (BGJ398) and erdafitinib (JNJ-42756493) have been evaluated in clinical trials for cancers with FGFR3 molecular alterations, particularly in urothelial carcinoma patients. However, a substantial proportion of these patients (up to 50%) display intrinsic resistance to these drugs and receive minimal clinical benefit. There is thus an unmet need for alternative therapeutic strategies to overcome primary resistance to selective FGFR inhibitors. In this study, we demonstrate that cells expressing cancer-associated activating FGFR3 mutants and the FGFR3-TACC3 fusion showed primary resistance to infigratinib in long-term colony formation assays in both NIH-3T3 and urothelial carcinoma models. We find that expression of these FGFR3 molecular alterations resulted in elevated constitutive Src activation compared to wildtype FGFR3 and that cells co-opted this pathway as a means to achieve intrinsic resistance to infigratinib. Targeting the Src pathway with low doses of the kinase inhibitor dasatinib synergistically sensitized multiple urothelial carcinoma lines harbouring endogenous FGFR3 alterations to infigratinib. Our data provide preclinical rationale that supports the use of dasatinib in combination with selective FGFR inhibitors as a means to overcome intrinsic drug resistance in the salvage therapy setting in urothelial cancer patients with FGFR3 molecular alterations.

Structural insights and activating mutations in diverse pathologies define mechanisms of deregulation for phospholipase C gamma enzymes.

BACKGROUND: PLCγ enzymes are key nodes in cellular signal transduction and their mutated and rare variants have been recently implicated in development of a range of diseases with unmet need including cancer, complex immune disorders, inflammation and neurodegenerative diseases. However, molecular nature of activation and the impact and dysregulation mechanisms by mutations, remain unclear; both are critically dependent on comprehensive characterization of the intact PLCγ enzymes. METHODS: For structural studies we applied cryo-EM, cross-linking mass spectrometry and hydrogen-deuterium exchange mass spectrometry. In parallel, we compiled mutations linked to main pathologies, established their distribution and assessed their impact in cells and in vitro. FINDINGS: We define structure of a complex containing an intact, autoinhibited PLCγ1 and the intracellular part of FGFR1 and show that the interaction is centred on the nSH2 domain of PLCγ1. We define the architecture of PLCγ1 where an autoinhibitory interface involves the cSH2, spPH, TIM-barrel and C2 domains; this relative orientation occludes PLCγ1 access to its substrate. Based on this framework and functional characterization, the mechanism leading to an increase in PLCγ1 activity for the largest group of mutations is consistent with the major, direct impact on the autoinhibitory interface. INTERPRETATION: We reveal features of PLCγ enzymes that are important for determining their activation status. Targeting such features, as an alternative to targeting the PLC active site that has so far not been achieved for any PLC, could provide new routes for clinical interventions related to various pathologies driven by PLCγ deregulation. FUND: CR UK, MRC and AstaZeneca.

Frequent and Persistent PLCG1 Mutations in Sézary Cells Directly Enhance PLCγ1 Activity and Stimulate NFκB, AP-1, and NFAT Signaling.

Phospholipase C Gamma 1 (PLCG1) is frequently mutated in primary cutaneous T-cell lymphoma (CTCL). This study functionally interrogated nine PLCG1 mutations (p.R48W, p.S312L, p.D342N, p.S345F, p.S520F, p.R1158H, p.E1163K, p.D1165H, and the in-frame indel p.VYEEDM1161V) identified in Sézary Syndrome, the leukemic variant of CTCL. The mutations were demonstrated in diagnostic samples and persisted in multiple tumor compartments over time, except in patients who achieved a complete clinical remission. In basal conditions, the majority of the mutations confer PLCγ1 gain-of-function activity through increased inositol phosphate production and the downstream activation of NFκB, AP-1, and NFAT transcriptional activity. Phosphorylation of the p.Y783 residue is essential for the proximal activity of wild-type PLCγ1, but we provide evidence that activating mutations do not require p.Y783 phosphorylation to stimulate downstream NFκB, NFAT, and AP-1 transcriptional activity. Finally, the gain-of-function effects associated with the p.VYEEDM1161V indel suggest that the C2 domain may have a role in regulating PLCγ1 activity. These data provide compelling evidence to support the development of therapeutic strategies targeting mutant PLCγ1.

Alzheimer's disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph.

BACKGROUND: Recent Genome Wide Association Studies (GWAS) have identified novel rare coding variants in immune genes associated with late onset Alzheimer's disease (LOAD). Amongst these, a polymorphism in phospholipase C-gamma 2 (PLCG2) P522R has been reported to be protective against LOAD. PLC enzymes are key elements in signal transmission networks and are potentially druggable targets. PLCG2 is highly expressed in the hematopoietic system. Hypermorphic mutations in PLCG2 in humans have been reported to cause autoinflammation and immune disorders, suggesting a key role for this enzyme in the regulation of immune cell function. METHODS: We assessed PLCG2 distribution in human and mouse brain tissue via immunohistochemistry and in situ hybridization. We transfected heterologous cell systems (COS7 and HEK293T cells) to determine the effect of the P522R AD-associated variant on enzymatic function using various orthogonal assays, including a radioactive assay, IP-One ELISA, and calcium assays. RESULTS: PLCG2 expression is restricted primarily to microglia and granule cells of the dentate gyrus. Plcg2 mRNA is maintained in plaque-associated microglia in the cerebral tissue of an AD mouse model. Functional analysis of the p.P522R variant demonstrated a small hypermorphic effect of the mutation on enzyme function. CONCLUSIONS: The PLCG2 P522R variant is protective against AD. We show that PLCG2 is expressed in brain microglia, and the p.P522R polymorphism weakly increases enzyme function. These data suggest that activation of PLCγ2 and not inhibition could be therapeutically beneficial in AD. PLCγ2 is therefore a potential target for modulating microglia function in AD, and a small molecule drug that weakly activates PLCγ2 may be one potential therapeutic approach.

Dynamic Allostery in PLCγ1 and Its Modulation by a Cancer Mutation Revealed by MD Simulation and NMR.

Phosphatidylinositol phospholipase Cγ (PLCγ) is an intracellular membrane-associated second-messenger signaling protein activated by tyrosine kinases such as fibroblast growth factor receptor 1. PLCγ contains the regulatory γ-specific array (γSA) comprising a tandem Src homology 2 (SH2) pair, an SH3 domain, and a split pleckstrin homology domain. Binding of an activated growth factor receptor to γSA leads to Tyr783 phosphorylation and consequent PLCγ activation. Several disease-relevant mutations in γSA have been identified; all lead to elevated phospholipase activity. In this work, we describe an allosteric mechanism that connects the Tyr783 phosphorylation site to the nSH2-cSH2 junction and involves dynamic interactions between the cSH2-SH3 linker and cSH2. Molecular dynamics simulations of the tandem SH2 protein suggest that Tyr783 phosphorylation is communicated to the nSH2-cSH2 junction by modulating cSH2 binding to sections of the cSH2-SH3 linker. NMR chemical shift perturbation analyses for designed tandem SH2 constructs reveal combined fast and slow dynamic processes that can be attributed to allosteric communication involving these regions of the protein, establishing an example in which complex N-site exchange can be directly inferred from 1H,15N-HSQC spectra. Furthermore, in tandem SH2 and γSA constructs, molecular dynamics and NMR results show that the Arg687Trp mutant in PLCγ1 (equivalent to the cancer mutation Arg665Trp in PLCγ2) perturbs the dynamic allosteric pathway. This combined experimental and computational study reveals a rare example of multistate kinetics involved in a dynamic allosteric process that is modulated in the context of a disease-relevant mutation. The allosteric influences and the weakened binding of the cSH2-SH3 linker to cSH2 should be taken into account in any more holistic investigation of PLCγ regulation.

NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 3 in apo state and in complex with inhibitor PD173074.

Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process, including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074. Analysis of chemical shift changes upon inhibitor binding highlights a characteristic pattern of allosteric network perturbations that is of relevance for future drug discovery activities aimed at development of conformationally-selective FGFR inhibitors.

Disease Variants of FGFR3 Reveal Molecular Basis for the Recognition and Additional Roles for Cdc37 in Hsp90 Chaperone System.

Receptor tyrosine kinase FGFR3 is involved in many signaling networks and is frequently mutated in developmental disorders and cancer. The Hsp90/Cdc37 chaperone system is essential for function of normal and neoplastic cells. Here we uncover the mechanistic inter-relationships between these proteins by combining approaches including NMR, HDX-MS, and SAXS. We show that several disease-linked mutations convert FGFR3 to a stronger client, where the determinant underpinning client strength involves an allosteric network through the N-lobe and at the lobe interface. We determine the architecture of the client kinase/Cdc37 complex and demonstrate, together with site-specific information, that binding of Cdc37 to unrelated kinases induces a common, extensive conformational remodeling of the kinase N-lobe, beyond localized changes and interactions within the binary complex. As further shown for FGFR3, this processing by Cdc37 deactivates the kinase and presents it, in a specific orientation established in the complex, for direct recognition by Hsp90.

Novel PLCG2 Mutation in a Patient With APLAID and Cutis Laxa.

Background: The auto-inflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) syndrome is a rare primary immunodeficiency caused by a gain-of-function mutation S707Y in the PLCG2 gene previously described in two patients from one family. The APLAID patients presented with early-onset blistering skin lesions, posterior uveitis, inflammatory bowel disease (IBD) and recurrent sinopulmonary infections caused by a humoral defect, but lacked circulating autoantibodies and had no cold-induced urticaria, contrary to the patients with the related PLAID syndrome. Case: We describe a new APLAID patient who presented with vesiculopustular rash in the 1st weeks of life, followed by IBD, posterior uveitis, recurrent chest infections, interstitial pneumonitis, and also had sensorineural deafness and cutis laxa. Her disease has been refractory to most treatments, including IL1 blockers and a trial with ruxolitinib has been attempted. Results: In this patient, we found a unique de novo heterozygous missense L848P mutation in the PLCG2 gene, predicted to affect the PLCγ2 structure. Similarly to S707Y, the L848P mutation led to the increased basal and EGF-stimulated PLCγ2 activity in vitro. Whole blood assays showed reduced production of IFN-γ and IL-17 in response to polyclonal T-cell stimulation and reduced production of IL-10 and IL-1ß after LPS stimulation. Reduced IL-1ß levels and the lack of clinical response to treatment with IL-1 blockers argue against NLRP3 inflammasome hyperactivation being the main mechanism mediating the APLAID pathogenesis. Conclusion: Our findings indicate that L848P is novel a gain-of-function mutation that leads to PLCγ2 activation and suggest cutis laxa as a possible clinical manifestations of the APLAID syndrome.

Unique signalling connectivity of FGFR3-TACC3 oncoprotein revealed by quantitative phosphoproteomics and differential network analysis.

The FGFR3-TACC3 fusion is an oncogenic driver in diverse malignancies, including bladder cancer, characterized by upregulated tyrosine kinase activity. To gain insights into distinct properties of FGFR3-TACC3 down-stream signalling, we utilised telomerase-immortalised normal human urothelial cell lines expressing either the fusion or wild-type FGFR3 (isoform IIIb) for subsequent quantitative proteomics and network analysis. Cellular lysates were chemically labelled with isobaric tandem mass tag reagents and, after phosphopeptide enrichment, liquid chromatography-high mass accuracy tandem mass spectrometry (LC-MS/MS) was used for peptide identification and quantification. Comparison of data from the two cell lines under non-stimulated and FGF1 stimulated conditions and of data representing physiological stimulation of FGFR3 identified about 200 regulated phosphosites. The identified phosphoproteins and quantified phosphosites were further analysed in the context of functional biological networks by inferring kinase-substrate interactions, mapping these to a comprehensive human signalling interaction network, filtering based on tissue-expression profiles and applying disease module detection and pathway enrichment methods. Analysis of our phosphoproteomics data using these bioinformatics methods combined into a new protocol-Disease Relevant Analysis of Genes On Networks (DRAGON)-allowed us to tease apart pathways differentially involved in FGFR3-TACC3 signalling in comparison to wild-type FGFR3 and to investigate their local phospho-signalling context. We highlight 9 pathways significantly regulated only in the cell line expressing FGFR3-TACC3 fusion and 5 pathways regulated only by stimulation of the wild-type FGFR3. Pathways differentially linked to FGFR3-TACC3 fusion include those related to chaperone activation and stress response and to regulation of TP53 expression and degradation that could contribute to development and maintenance of the cancer phenotype.

Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET.

Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.