Activating mutations in fibroblast growth factor receptor 3 (FGFR3) account for the most prevalent form of dwarfism in humans, the achondroplasia. Excessive activation of FGFR3 causes premature chondrocyte growth arrest, abnormal extracellular matrix homeostasis, and altered chondrocyte differentiation, resulting in achondroplasia. Although achondroplasia is considered a curable condition, no treatment is available to date. This is mostly due to our ingnorance of the basic mechanisms governing the FGFR3 signal transduction. Prolonged activation of Erk MAP kinase, mediated by Frs2 adapter protein, is a major effector of FGFR3 activation in achondroplasia. The molecular mechanisms underlying this phenotype are not known. The dynamics of the Frs2 interaction with FGFR3, Erk and other associated proteins holds a key to understanding of pathology of achondroplasia, and will be addressed in this proposal. Via systematic mapping of interactions among FGFR3, Frs2, Erk and other proteins, we aim to identify novel treatment opportunities for achondroplasia.

Aktivující mutace v FGFR3 receptorové tyrozinové kináze způsobují nejrozšířenější formu trpasličího vzrůstu u lidí, achondroplázii. Nadměrná aktivace FGFR3 způsobuje předčasné ukončení růstu chondrocytů, abnormální homeostázu extracelulární matrix a abnormální diferenciaci chondrocytů, vedoucí k achondroplázii. I když je achondroplázie považovaná za léčitelné onemocnění, v současnosti žádná léčba neexistuje. To je především dáno naší neznalostí základních mechanismů, kterými se řídí FGFR3 signální transdukce v chrupavce. Nekontrolovatelná aktivace Erk MAP kinázy, zprostředkovaná signálním adaptérovým proteinem Frs2, představuje hlavní mediátor FGFR3 aktivace u achondroplázie. Molekulární mechanizmy tohoto fenotypu nejsou známy. Dynamika interakce Frs2 s FGFR3, Erk a dalších proteinů představují klíč k pochopení patologie achondroplázie a budou řešena v tomto návrhu. Prostřednictvím systematického mapování interakcí mezi FGFR3, Frs2, Erk a ostaních proteinů budou identifikovány nové možnosti léčby achondroplázie.

• MeSH
• Achondroplasia therapy   MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Chondrocytes MeSH
• Cartilage MeSH
• Extracellular Signal-Regulated MAP Kinases MeSH
• Phosphoric Monoester Hydrolases MeSH
• MAP Kinase Signaling System MeSH
• Receptor, Fibroblast Growth Factor, Type 3 MeSH
• Transforming Growth Factors MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• osteologie
• genetika, lékařská genetika
• molekulární biologie, molekulární medicína
• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.

• MeSH
• NIH 3T3 Cells MeSH
• Cilia metabolism   MeSH
• CRISPR-Cas Systems MeSH
• Fibroblast Growth Factors metabolism   MeSH
• Phosphorylation MeSH
• HEK293 Cells MeSH
• Protein Interaction Domains and Motifs MeSH
• Humans MeSH
• Models, Animal MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   MeSH
• Hedgehog Proteins metabolism   MeSH
• Proteomics MeSH
• Receptor, Fibroblast Growth Factor, Type 1 metabolism   MeSH
• Receptor, Fibroblast Growth Factor, Type 3 genetics   metabolism   MeSH
• Receptor, Fibroblast Growth Factor, Type 4 metabolism   MeSH
• Receptors, Fibroblast Growth Factor genetics   metabolism   MeSH
• Signal Transduction MeSH
• Molecular Docking Simulation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the known human RTKs.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• NIH 3T3 Cells MeSH
• Cyclin-Dependent Kinases genetics   metabolism   MeSH
• Peptide Elongation Factor 1 genetics   metabolism   MeSH
• Endosomal Sorting Complexes Required for Transport genetics   metabolism   MeSH
• Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases genetics   metabolism   MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Phosphorylation MeSH
• HEK293 Cells MeSH
• Homeodomain Proteins genetics   metabolism   MeSH
• Humans MeSH
• Protein Interaction Mapping MeSH
• Mice MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   MeSH
• Proteomics methods   MeSH
• Receptor, Fibroblast Growth Factor, Type 3 genetics   metabolism   MeSH
• Gene Expression Regulation * MeSH
• Signal Transduction genetics   MeSH
• Gene Expression Profiling MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Studies have suggested a role for the mammalian (or mechanistic) target of rapamycin (mTOR) in skeletal development and homeostasis, yet there is no evidence connecting mTOR with the key signaling pathways that regulate skeletogenesis. We identified a parathyroid hormone (PTH)/PTH-related peptide (PTHrP)-salt-inducible kinase 3 (SIK3)-mTOR signaling cascade essential for skeletogenesis. While investigating a new skeletal dysplasia caused by a homozygous mutation in the catalytic domain of SIK3, we observed decreased activity of mTOR complex 1 (mTORC1) and mTORC2 due to accumulation of DEPTOR, a negative regulator of both mTOR complexes. This SIK3 syndrome shared skeletal features with Jansen metaphyseal chondrodysplasia (JMC), a disorder caused by constitutive activation of the PTH/PTHrP receptor. JMC-derived chondrocytes showed reduced SIK3 activity, elevated DEPTOR, and decreased mTORC1 and mTORC2 activity, indicating a common mechanism of disease. The data demonstrate that SIK3 is an essential positive regulator of mTOR signaling that functions by triggering DEPTOR degradation in response to PTH/PTHrP signaling during skeletogenesis.

• MeSH
• HEK293 Cells MeSH
• Homozygote MeSH
• Intracellular Signaling Peptides and Proteins metabolism   MeSH
• Humans MeSH
• Mechanistic Target of Rapamycin Complex 1 metabolism   MeSH
• Mechanistic Target of Rapamycin Complex 2 metabolism   MeSH
• Mutation, Missense genetics   MeSH
• Mutant Proteins chemistry   metabolism   MeSH
• Osteogenesis * MeSH
• Parathyroid Hormone metabolism   MeSH
• Parathyroid Hormone-Related Protein metabolism   MeSH
• Protein Kinases chemistry   deficiency   genetics   metabolism   MeSH
• Proteolysis MeSH
• Growth Plate metabolism   MeSH
• Amino Acid Sequence MeSH
• Signal Transduction * MeSH
• TOR Serine-Threonine Kinases metabolism   MeSH
• Inheritance Patterns genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Sustained activation of extracellular signal-regulated kinase (ERK) drives pathologies caused by mutations in fibroblast growth factor receptors (FGFRs). We previously identified the inositol phosphatase SHIP2 (also known as INPPL1) as an FGFR-interacting protein and a target of the tyrosine kinase activities of FGFR1, FGFR3, and FGFR4. We report that loss of SHIP2 converted FGF-mediated sustained ERK activation into a transient signal and rescued cell phenotypes triggered by pathologic FGFR-ERK signaling. Mutant forms of SHIP2 lacking phosphoinositide phosphatase activity still associated with FGFRs and did not prevent FGF-induced sustained ERK activation, demonstrating that the adaptor rather than the catalytic activity of SHIP2 was required. SHIP2 recruited Src family kinases to the FGFRs, which promoted FGFR-mediated phosphorylation and assembly of protein complexes that relayed signaling to ERK. SHIP2 interacted with FGFRs, was phosphorylated by active FGFRs, and promoted FGFR-ERK signaling at the level of phosphorylation of the adaptor FRS2 and recruitment of the tyrosine phosphatase PTPN11. Thus, SHIP2 is an essential component of canonical FGF-FGFR signal transduction and a potential therapeutic target in FGFR-related disorders.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Enzyme Activation MeSH
• Extracellular Signal-Regulated MAP Kinases genetics   metabolism   MeSH
• Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases genetics   metabolism   MeSH
• Phosphorylation MeSH
• HEK293 Cells MeSH
• Humans MeSH
• MAP Kinase Signaling System * MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Receptors, Fibroblast Growth Factor genetics   metabolism   MeSH
• src-Family Kinases genetics   metabolism   MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 11 genetics   metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

The blocking of specific protein-protein interactions using nanoparticles is an emerging alternative to small molecule-based therapeutic interventions. However, the nanoparticles designed as "artificial proteins" generally require modification of their surface with (bio)organic molecules and/or polymers to ensure their selectivity and specificity of action. Here, we show that nanosized diamond crystals (nanodiamonds, NDs) without any synthetically installed (bio)organic interface enable the specific and efficient targeting of the family of extracellular signalling molecules known as fibroblast growth factors (FGFs). We found that low nanomolar solutions of detonation NDs with positive ζ-potential strongly associate with multiple FGF ligands present at sub-nanomolar concentrations and effectively neutralize the effects of FGF signalling in cells without interfering with other growth factor systems and serum proteins unrelated to FGFs. We identified an evolutionarily conserved FGF recognition motif, ∼17 amino acids long, that contributes to the selectivity of the ND-FGF interaction. In addition, we inserted this motif into a de novo constructed chimeric protein, which significantly improved its interaction with NDs. We demonstrated that the interaction of NDs, as purely inorganic nanoparticles, with proteins can mitigate pathological FGF signalling and promote the restoration of cartilage growth in a mouse limb explant model. Based on our observations, we foresee that NDs may potentially be applied as nanotherapeutics to neutralize disease-related activities of FGFs in vivo.

• MeSH
• Amino Acid Motifs MeSH
• Cell Line MeSH
• Cartilage physiology   MeSH
• Embryo, Mammalian MeSH
• Fibroblast Growth Factors metabolism   MeSH
• Humans MeSH
• Ligands MeSH
• Mice MeSH
• Nanodiamonds chemistry   MeSH
• Cell Proliferation MeSH
• Receptors, Fibroblast Growth Factor metabolism   MeSH
• Signal Transduction MeSH
• Tissue Culture Techniques MeSH
• Tibia physiology   MeSH
• Protein Binding MeSH
• Cell Survival MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Conventional biophysical and chemical biology approaches for delineating relationships between the structure and biological function of nucleic acids (NAs) abstract NAs from their native biological context. However, cumulative experimental observations have revealed that the structure, dynamics and interactions of NAs might be strongly influenced by a broad spectrum of specific and nonspecific physical-chemical environmental factors. This consideration has recently sparked interest in the development of novel tools for structural characterization of NAs in the native cellular context. Here, we review the individual methods currently being employed for structural characterization of NA structure in a native cellular environment with a focus on recent advances and developments in the emerging fields of in-cell NMR and electron paramagnetic resonance spectroscopy and in-cell single-molecule FRET of NAs.

• MeSH
• Single-Cell Analysis MeSH
• Cells chemistry   MeSH
• Electron Spin Resonance Spectroscopy MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• Magnetic Resonance Spectroscopy MeSH
• Models, Molecular MeSH
• Nucleic Acids chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Cilia project from almost every cell integrating extracellular cues with signaling pathways. Constitutive activation of FGFR3 signaling produces the skeletal disorders achondroplasia (ACH) and thanatophoric dysplasia (TD), but many of the molecular mechanisms underlying these phenotypes remain unresolved. Here, we report in vivo evidence for significantly shortened primary cilia in ACH and TD cartilage growth plates. Using in vivo and in vitro methodologies, our data demonstrate that transient versus sustained activation of FGF signaling correlated with different cilia consequences. Transient FGF pathway activation elongated cilia, while sustained activity shortened cilia. FGF signaling extended primary cilia via ERK MAP kinase and mTORC2 signaling, but not through mTORC1. Employing a GFP-tagged IFT20 construct to measure intraflagellar (IFT) speed in cilia, we showed that FGF signaling affected IFT velocities, as well as modulating cilia-based Hedgehog signaling. Our data integrate primary cilia into canonical FGF signal transduction and uncover a FGF-cilia pathway that needs consideration when elucidating the mechanisms of physiological and pathological FGFR function, or in the development of FGFR therapeutics.

• MeSH
• Achondroplasia genetics   physiopathology   MeSH
• NIH 3T3 Cells MeSH
• Chondrocytes metabolism   MeSH
• Cartilage metabolism   MeSH
• Cilia pathology   physiology   MeSH
• Ciliopathies genetics   physiopathology   MeSH
• Phenotype MeSH
• Fibroblast Growth Factors metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Primary Cell Culture MeSH
• Receptor, Fibroblast Growth Factor, Type 3 genetics   metabolism   MeSH
• Growth Plate metabolism   MeSH
• Signal Transduction physiology   MeSH
• Thanatophoric Dysplasia genetics   physiopathology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.

• MeSH
• Point Mutation MeSH
• Computer-Aided Design * MeSH
• Embryonic Stem Cells cytology   metabolism   MeSH
• Fibroblast Growth Factor 2 chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Computer Simulation MeSH
• Protein Engineering * MeSH
• Directed Molecular Evolution MeSH
• Protein Folding MeSH
• Amino Acid Sequence MeSH
• Protein Stability * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Many tyrosine kinase inhibitors (TKIs) have failed to reach human use due to insufficient activity in clinical trials. However, the failed TKIs may still benefit patients if their other kinase targets are identified by providing treatment focused on syndromes driven by these kinases. Here, we searched for novel targets of AZD1480, an inhibitor of JAK2 kinase that recently failed phase two cancer clinical trials due to a lack of activity. Twenty seven human receptor tyrosine kinases (RTKs) and 153 of their disease-associated mutants were in-cell profiled for activity in the presence of AZD1480 using a newly developed RTK plasmid library. We demonstrate that AZD1480 inhibits ALK, LTK, FGFR1-3, RET and TRKA-C kinases and uncover a physical basis of this specificity. The RTK activity profiling described here facilitates inhibitor repurposing by enabling rapid and efficient identification of novel TKI targets in cells.

• Publication type
• Journal Article MeSH