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1.
Mol Cell Proteomics ; 17(5): 850-870, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29371290

RESUMO

Recently, FGFR1 was found to be overexpressed in osteosarcoma and represents an important target for precision medicine. However, because targeted cancer therapy based on FGFR inhibitors has so far been less efficient than expected, a detailed understanding of the target is important. We have here applied proximity-dependent biotin labeling combined with label-free quantitative mass spectrometry to identify determinants of FGFR1 activity in an osteosarcoma cell line. Many known FGFR interactors were identified (e.g. FRS2, PLCG1, RSK2, SRC), but the data also suggested novel determinants. A strong hit in our screen was the tyrosine phosphatase PTPRG. We show that PTPRG and FGFR1 interact and colocalize at the plasma membrane where PTPRG directly dephosphorylates activated FGFR1. We further show that osteosarcoma cell lines depleted for PTPRG display increased FGFR activity and are hypersensitive to stimulation by FGF1. In addition, PTPRG depletion elevated cell growth and negatively affected the efficacy of FGFR kinase inhibitors. Thus, PTPRG may have future clinical relevance by being a predictor of outcome after FGFR inhibitor treatment.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Fatores de Crescimento de Fibroblastos/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Osteossarcoma/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteômica , Reprodutibilidade dos Testes
2.
Biochemistry ; 57(26): 3807-3816, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29812912

RESUMO

Fibroblast growth factor 1 (FGF1) binds to specific FGF receptors (FGFRs) at the surface of target cells to initiate intracellular signaling. While heparan sulfate proteoglycans (HSPGs) are well-described coreceptors, it is uncertain whether there are additional binding sites for FGF1 at the cell surface. To address this, we devised and tested a method to identify novel binding sites for FGF1 at the cell surface, which may also be applicable for other protein ligands. We constructed an APEX2-FGF1 fusion protein to perform proximal biotin labeling of proteins following binding of the fusion protein to the cell surface. After functional validation of the fusion protein by a signaling assay, we used this method to identify binding sites for FGF1 on cell surfaces of living cells. We confirmed the feasibility of our approach by detection of FGFR4, a well-known and specific receptor for FGF1. We subsequently screened for novel interactors using RPE1 cells and identified the proteoglycans CSPG4 (NG2) and CD44. We found that FGF1 binds CD44 through its heparin-binding moiety. Moreover, we found that FGF1 was colocalized with both CSPG4 and CD44 at the cell surface, suggesting that these receptors act as storage molecules that create a reservoir of FGF1. Importantly, our data demonstrate that recombinant ligand-APEX2 fusion proteins can be used to identify novel receptor interactions on the cell surface.


Assuntos
Proteoglicanas de Sulfatos de Condroitina/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Fator 1 de Crescimento de Fibroblastos/química , Receptores de Hialuronatos/química , Proteínas de Membrana/química , Proteínas Recombinantes de Fusão/química , Proteoglicanas de Sulfatos de Condroitina/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases , Fator 1 de Crescimento de Fibroblastos/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Proteínas de Membrana/metabolismo , Enzimas Multifuncionais , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem
3.
J Proteome Res ; 15(10): 3841-3855, 2016 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-27615514

RESUMO

The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells. Upon addition of biotin to these cells, proteins in proximity to the FGFR4-BirA* fusion protein became biotinylated and could be isolated and identified by quantitative mass spectrometry. We identified in total 291 proteins, including 80 proteins that were enriched in samples where the receptor was activated by the ligand (FGF1), among them several proteins previously found to be involved in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly, many of the identified proteins were implicated in endosomal transport, and by precise annotation we were able to trace the intracellular pathways of activated FGFR4. Validating the data by confocal and three-dimensional structured illumination microscopy analysis, we concluded that FGFR4 uses clathrin-mediated endocytosis for internalization and is further sorted from early endosomes to the recycling compartment and the trans-Golgi network. Depletion of cells for clathrin heavy chain led to accumulation of FGFR4 at the cell surface and increased levels of active FGFR4 and PLCγ, while AKT and ERK signaling was diminished, demonstrating that functional clathrin-mediated endocytosis is required for proper FGFR4 signaling. Thus, this study reveals proteins and pathways involved in FGFR4 transport and signaling that provide possible targets and opportunities for therapeutic intervention in FGFR4 aberrant cancer.


Assuntos
Endossomos/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Biotinilação , Linhagem Celular Tumoral , Clatrina/metabolismo , Endocitose , Humanos , Microscopia/métodos , Transporte Proteico , Transdução de Sinais , Coloração e Rotulagem , Rede trans-Golgi/metabolismo
4.
Sci Rep ; 13(1): 22982, 2023 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-38151514

RESUMO

The ability of cells to move and migrate is required during development, but also in the adult in processes such as wound healing and immune responses. In addition, cancer cells exploit the cells' ability to migrate and invade to spread into nearby tissue and eventually metastasize. The majority of cancer deaths are caused by metastasis and the process of cell migration is therefore intensively studied. A common way to study cell migration is to observe cells through an optical microscope and record their movements over time. However, segmenting and tracking moving cells in phase contrast time-lapse video sequences is a challenging task. Several tools to track the velocity of migrating cells have been developed. Unfortunately, most of the automated tools are made for fluorescence images even though unlabelled cells are often preferred to avoid phototoxicity. Consequently, researchers are constrained with laborious manual tracking tools using ImageJ or similar software. We have therefore developed a freely available, user-friendly, automated tracking tool called CellTraxx. This software makes it easy to measure the velocity and directness of migrating cells in phase contrast images. Here, we demonstrate that our tool efficiently recognizes and tracks unlabelled cells of different morphologies and sizes (HeLa, RPE1, MDA-MB-231, HT1080, U2OS, PC-3) in several types of cell migration assays (random migration, wound healing and cells embedded in collagen). We also provide a detailed protocol and download instructions for CellTraxx.


Assuntos
Software , Cicatrização , Adulto , Humanos , Movimento Celular/fisiologia , Células HeLa , Cicatrização/fisiologia , Ensaios de Migração Celular/métodos , Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos
5.
Biochem J ; 437(2): 199-213, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21711248

RESUMO

FGFs (fibroblast growth factors) and their receptors (FGFRs) play essential roles in tightly regulating cell proliferation, survival, migration and differentiation during development and adult life. Deregulation of FGFR signalling, on the other hand, has been associated with many developmental syndromes, and with human cancer. In cancer, FGFRs have been found to become overactivated by several mechanisms, including gene amplification, chromosomal translocation and mutations. FGFR alterations are detected in a variety of human cancers, such as breast, bladder, prostate, endometrial and lung cancers, as well as haematological malignancies. Accumulating evidence indicates that FGFs and FGFRs may act in an oncogenic fashion to promote multiple steps of cancer progression by inducing mitogenic and survival signals, as well as promoting epithelial-mesenchymal transition, invasion and tumour angiogenesis. Therapeutic strategies targeting FGFs and FGFRs in human cancer are therefore currently being explored. In the present review we will give an overview of FGF signalling, the main FGFR alterations found in human cancer to date, how they may contribute to specific cancer types and strategies for therapeutic intervention.


Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Neoplasias/fisiopatologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais/fisiologia , Animais , Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/fisiopatologia , Diferenciação Celular/genética , Proliferação de Células , Feminino , Fatores de Crescimento de Fibroblastos/genética , Humanos , Neoplasias Pulmonares/fisiopatologia , Masculino , Mieloma Múltiplo/fisiopatologia , Transtornos Mieloproliferativos/fisiopatologia , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/fisiopatologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/efeitos dos fármacos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Proteínas Recombinantes de Fusão , Rabdomiossarcoma/fisiopatologia , Transdução de Sinais/genética , Neoplasias da Bexiga Urinária/fisiopatologia
7.
Cells ; 10(6)2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071546

RESUMO

FGFR (fibroblast growth factor receptor) signaling controls fundamental processes in embryonic, fetal and adult human life. The magnitude, duration, and location of FGFR signaling must be strictly controlled in order to induce the correct biological response. Uncontrolled receptor signaling has been shown to lead to a variety of diseases, such as skeletal disorders and cancer. Here we review the numerous cellular mechanisms that regulate and turn off FGFR signaling, once the receptor is activated. These mechanisms include endocytosis and endocytic sorting, phosphatase activity, negative regulatory proteins and negative feedback phosphorylation events. The mechanisms act together simultaneously or sequentially, controlling the same or different steps in FGFR signaling. Although more work is needed to fully understand the regulation of FGFR signaling, it is clear that the cells in our body have evolved an extensive repertoire of mechanisms that together keep FGFR signaling tightly controlled and prevent excess FGFR signaling.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Humanos
8.
Cells ; 10(6)2021 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204611

RESUMO

Fibroblast growth factor receptors (FGFRs) have become an attractive target in cancer research and therapy due to their implication in several cancers. Limitations of current treatment options require a need for additional, more specific and potent strategies to overcome cancers driven by FGFRs. Photochemical internalization (PCI) is a light-controlled method for cytosolic delivery of drugs that are entrapped in endosomes and lysosomes. We here evaluated the efficacy and selectivity of PCI of FGF2-saporin (FGF-SAP) in cells overexpressing FGFR1. FGF-SAP is a conjugate of FGF2 and the highly cytotoxic ribosome-inactivating protein (RIP) saporin, which is used as payload to eliminate cancer cells. Evaluation of the targeting effect of PCI of FGF-SAP was done by comparing the cytotoxic response in osteosarcoma cells with very low levels of FGFR1 (U2OS) to cells overexpressing FGFR1 (U2OS-R1). We demonstrate that PCI greatly enhances cytotoxicity of the drug showing efficient cell killing at pM concentrations of the drug in U2OS-R1 cells. However, U2OS cells were also sensitive to the toxin after PCI. Binding experiments using confocal microscopy and Western blotting techniques indicate that FGF-SAP is taken up by cells through heparan sulfate proteoglycans (HSPGs) in U2OS cells. We further show that the cytotoxicity of FGF-SAP in U2OS cells was reduced when cells were co-treated with heparin to compete out binding to HSPG, demonstrating that the cytotoxic effect was due to internalization by HSPGs. We conclude that to prevent off-target effects of FGF-based toxins, it will be necessary to circumvent binding to HSPGs, for example by mutating the binding site of FGF2 to HSPGs.


Assuntos
Portadores de Fármacos , Fator 2 de Crescimento de Fibroblastos , Terapia de Alvo Molecular/métodos , Fotoquimioterapia/métodos , Saporinas/administração & dosagem , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Humanos
9.
Cells ; 8(6)2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31146385

RESUMO

Tight regulation of signaling from receptor tyrosine kinases is required for normal cellular functions and uncontrolled signaling can lead to cancer. Fibroblast growth factor receptor 2 (FGFR2) is a receptor tyrosine kinase that induces proliferation and migration. Deregulation of FGFR2 contributes to tumor progression and activating mutations in FGFR2 are found in several types of cancer. Here, we identified a negative feedback loop regulating FGFR2 signaling. FGFR2 stimulates the Ras/MAPK signaling pathway consisting of Ras-Raf-MEK1/2-ERK1/2. Inhibition of this pathway using a MEK1/2 inhibitor increased FGFR2 signaling. The putative ERK1/2 phosphorylation site at serine 780 (S780) in FGFR2 corresponds to serine 777 in FGFR1 which is directly phosphorylated by ERK1/2. Substitution of S780 in FGFR2 to an alanine also increased signaling. Truncated forms of FGFR2 lacking the C-terminal tail, including S780, have been identified in cancer and S780 has been found mutated to leucine in bladder cancer. Substituting S780 in FGFR2 with leucine increased FGFR2 signaling. Importantly, cells expressing these mutated versions of S780 migrated faster than cells expressing wild-type FGFR2. Thus, ERK1/2-mediated phosphorylation of S780 in FGFR2 constitutes a negative feedback loop and inactivation of this feedback loop in cancer cells causes hyperactivation of FGFR2 signaling, which may result in increased invasive properties.


Assuntos
Retroalimentação Fisiológica , Sistema de Sinalização das MAP Quinases , Mutação/genética , Neoplasias/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , Progressão da Doença , Fator de Crescimento Epidérmico/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Fator 1 de Crescimento de Fibroblastos/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Serina/genética , Transdução de Sinais
10.
Anat Rec (Hoboken) ; 302(8): 1268-1275, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30950230

RESUMO

In the course of peripheral nerve regeneration, axons encounter different extracellular growth factors secreted by non-neuronal cells at the injury site and retrogradely transported after binding to neuronal membrane receptor tyrosine kinases. The present study reviews the role of receptor transport in peripheral axon outgrowth and provides novel data on trafficking of fibroblast growth factor receptor type 1 (FGFR1). Differences in receptor transport are determined by different numbers of lysine residues acting as ubiquitination sites in the intracellular receptor domain. We previously demonstrated that overexpression of mutant FGFR1-25R (25 out of 29 intracellular lysines replaced with arginine) results in enhanced receptor recycling as compared to wild-type FGFR1 followed by strong stimulation of elongative axon growth in vitro. Here, the effects of lysine-deficient FGFR1 (FGFR1-29R lacking all 29 cytoplasmic lysine residues) or of only 15 lysine mutations (FGFR1-15R) on axon outgrowth and concomitant changes in signal pathway activation were investigated by immunocytochemistry and morphometry of cultured primary neurons. Overexpression of FGFR1-15R in adult sensory neurons resulted in enhanced receptor recycling, which was accompanied by increased axon elongation without stimulating axon branching. By contrast, FGFR1-29R was neither endocytosed nor axon outgrowth affected. Although overexpression of FGFR1-15R or FGFR1-25Ra strongly promoted elongation, we did not detect increased signal pathway activation (ERK, AKT, PLC, or STAT3) in neurons expressing mutant FGFR1 as compared with wild-type neurons raising the possibility that other signaling pathways or signaling independent mechanisms may be involved in the axon outgrowth effects of recycled FGF receptors. Anat Rec, 302:1268-1275, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.


Assuntos
Axônios/fisiologia , Neurônios Dopaminérgicos/citologia , Endocitose , Neurogênese , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Células Receptoras Sensoriais/citologia , Animais , Neurônios Dopaminérgicos/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lisina/química , Lisina/genética , Mutação , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Células Receptoras Sensoriais/fisiologia , Transdução de Sinais
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