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1.
Cell Rep ; 40(13): 111428, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36170818

RESUMO

Proteasome machinery is a major proteostasis control system in human cells, actively compensated upon its inhibition. To understand this compensation, we compared global protein landscapes upon the proteasome inhibition with carfilzomib, in normal fibroblasts, cells of multiple myeloma, and cancers of lung, colon, and pancreas. Molecular chaperones, autophagy, and endocytosis-related proteins are the most prominent vulnerabilities in combination with carfilzomib, while targeting of the HSP70 family chaperones HSPA1A/B most specifically sensitizes cancer cells to the proteasome inhibition. This suggests a central role of HSP70 in the suppression of the proteasome downregulation, allowing to identify pathways impinging on HSP70 upon the proteasome inhibition. HSPA1A/B indeed controls proteasome-inhibition-induced autophagy, unfolded protein response, and endocytic flux, and directly chaperones the proteasome machinery. However, it does not control the NRF1/2-driven proteasome subunit transcriptional bounce-back. Consequently, targeting of NRF1 proves effective in decreasing the viability of cancer cells with the inhibited proteasome and HSP70.


Assuntos
Proteínas de Choque Térmico HSP70 , Neoplasias , Complexo de Endopeptidases do Proteassoma , Humanos , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP70/metabolismo , Neoplasias/genética , Fator 1 Relacionado a NF-E2/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteostase
2.
Mol Cancer Res ; 20(3): 446-455, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34782372

RESUMO

AXL, a receptor tyrosine kinase from the TAM (TYRO3 AXL and MER) subfamily, and its ligand growth arrest-specific 6 (GAS6) are implicated in pathogenesis of a wide array of cancers, acquisition of resistance to diverse anticancer therapies and cellular entry of viruses. The continuous development of AXL inhibitors for treatment of patients with cancer and COVID-19 underscores the need to better characterize the cellular effects of AXL targeting.In the present study, we compared the cellular phenotypes of CRISPR-Cas9-induced depletion of AXL and its pharmacological inhibition with bemcentinib, LDC1267 and gilteritinib. Specifically, we evaluated GAS6-AXL signaling, cell viability and invasion, the endo-lysosomal system and autophagy in glioblastoma cells. We showed that depletion of AXL but not of TYRO3 inhibited GAS6-induced phosphorylation of downstream signaling effectors, AKT and ERK1/2, indicating that AXL is a primary receptor for GAS6. AXL was also specifically required for GAS6-dependent increase in cell viability but was dispensable for viability of cells grown without exogenous addition of GAS6. Furthermore, we revealed that LDC1267 is the most potent and specific inhibitor of AXL activation among the tested compounds. Finally, we found that, in contrast to AXL depletion and its inhibition with LDC1267, cell treatment with bemcentinib and gilteritinib impaired the endo-lysosomal and autophagy systems in an AXL-independent manner. IMPLICATIONS: Altogether, our findings are of high clinical importance as we discovered that two clinically advanced AXL inhibitors, bemcentinib and gilteritinib, may display AXL-independent cellular effects and toxicity.


Assuntos
Compostos de Anilina/uso terapêutico , Benzocicloeptenos/uso terapêutico , Lisossomos/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirazinas/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Triazóis/uso terapêutico , Compostos de Anilina/farmacologia , Autofagia , Benzocicloeptenos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , Transdução de Sinais , Transfecção , Triazóis/farmacologia , Receptor Tirosina Quinase Axl
3.
J Biomed Sci ; 28(1): 69, 2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34635096

RESUMO

BACKGROUND: Overexpression of FGFR1 is observed in numerous tumors and therefore this receptor constitutes an attractive molecular target for selective cancer treatment with cytotoxic conjugates. The success of cancer therapy with cytotoxic conjugates largely relies on the precise recognition of a cancer-specific marker by a targeting molecule within the conjugate and its subsequent cellular internalization by receptor mediated endocytosis. We have recently demonstrated that efficiency and mechanism of FGFR1 internalization are governed by spatial distribution of the receptor in the plasma membrane, where clustering of FGFR1 into larger oligomers stimulated fast and highly efficient uptake of the receptor by simultaneous engagement of multiple endocytic routes. Based on these findings we aimed to develop a modular, self-assembly system for generation of oligomeric cytotoxic conjugates, capable of FGFR1 clustering, for targeting FGFR1-overproducing cancer cells. METHODS: Engineered FGF1 was used as FGFR1-recognition molecule and tailored for enhanced stability and site-specific attachment of the cytotoxic drug. Modified streptavidin, allowing for controlled oligomerization of FGF1 variant was used for self-assembly of well-defined FGF1 oligomers of different valency and oligomeric cytotoxic conjugate. Protein biochemistry methods were applied to obtain highly pure FGF1 oligomers and the oligomeric cytotoxic conjugate. Diverse biophysical, biochemical and cell biology tests were used to evaluate FGFR1 binding, internalization and the cytotoxicity of obtained oligomers. RESULTS: Developed multivalent FGF1 complexes are characterized by well-defined architecture, enhanced FGFR1 binding and improved cellular uptake. This successful strategy was applied to construct tetrameric cytotoxic conjugate targeting FGFR1-producing cancer cells. We have shown that enhanced affinity for the receptor and improved internalization result in a superior cytotoxicity of the tetrameric conjugate compared to the monomeric one. CONCLUSIONS: Our data implicate that oligomerization of the targeting molecules constitutes an attractive strategy for improvement of the cytotoxicity of conjugates recognizing cancer-specific biomarkers. Importantly, the presented approach can be easily adapted for other tumor markers.


Assuntos
Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , Ligação Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo
4.
Proc Natl Acad Sci U S A ; 118(28)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34244439

RESUMO

AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand, GAS6, are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion turnover, cell spreading, and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL, but not TYRO3, and downstream activation of PI3K and RAC1. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements that contribute to cancer-cell invasion.


Assuntos
Actinas/metabolismo , Extensões da Superfície Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Pinocitose , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Citoesqueleto de Actina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Glioblastoma/patologia , Glutamina/farmacologia , Células HEK293 , Humanos , Modelos Biológicos , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas rac1 de Ligação ao GTP/metabolismo , Receptor Tirosina Quinase Axl
5.
Mol Med ; 27(1): 46, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33962559

RESUMO

BACKGROUND: Antibody drug conjugates (ADCs) represent one of the most promising approaches in the current immuno-oncology research. The precise delivery of cytotoxic drugs to the cancer cells using ADCs specific for tumor-associated antigens enables sparing the healthy cells and thereby reduces unwanted side effects. Overexpression of fibroblast growth factor receptor 1 (FGFR1) has been demonstrated in numerous tumors and thereby constitutes a convenient molecular target for selective cancer treatment. We have recently engineered tetravalent anti-FGFR1 antibody, T-Fc, and have demonstrated that it displays extremely efficient internalization into FGFR1 producing cells, a feature highly desirable in the ADC approach. We have revealed that T-Fc mediates clustering of FGFR1, largely enhancing the uptake of FGFR1-T-Fc complexes by induction of clathrin-independent endocytic routes. The aim of this study was to obtain highly internalizing cytotoxic conjugate of the T-Fc for specific delivery of drugs into FGFR1-positive cancer cells. METHODS: Conjugation of the T-Fc to a cytotoxic payload, vcMMAE, was carried out via maleimide chemistry, yielding the T-Fc-vcMMAE. The specific binding of the T-Fc-vcMMAE conjugate to FGFR1 was confirmed in vitro with BLI technique. Confocal microscopy and flow cytometry were applied to determine FGFR1-dependence of the T-Fc-vcMMAE internalization. Western blot analyses of FGFR1-dependent signaling were conducted to assess the impact of the T-Fc-vcMMAE on FGFR1 activation and initiation of downstream signaling cascades. Finally, using FGFR1-negative and FGFR1-possitive cell lines, the cytotoxic potential of the T-Fc-vcMMAE was evaluated. RESULTS: We have performed the efficient conjugation of the tetravalent engineered antibody with a cytotoxic drug and generated FGFR1-specific ADC molecule, T-Fc-vcMMAE. We have demonstrated that T-Fc-vcMMAE conjugate exhibits high selectivity and affinity for FGFR1, similarly to T-Fc. Furthermore, we have shown that T-Fc constitutes an effective drug delivery vehicle as T-Fc-vcMMAE was efficiently and selectively internalized by FGFR1-producing cells leading to their death. Interestingly, we show that the efficiency of the uptake of T-Fc-vcMMAE corresponds well with the cytotoxicity of the conjugate, but doesn't correlate with the FGFR1expression level. CONCLUSION: Our results show that T-Fc-vcMMAE fulfills the key criteria for the successful cytotoxic drug carrier in a targeted approach against FGFR1-positive cancer cells. Furthermore, our data implicate that not solely expression level of the receptor, but rather its cellular trafficking should be taken into account for selection of suitable molecular targets and cancer models for successful ADC approach.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Imunoconjugados/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antineoplásicos Imunológicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunofluorescência , Expressão Gênica , Engenharia Genética , Humanos , Imunoconjugados/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo
6.
Int J Biol Macromol ; 180: 470-483, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33745974

RESUMO

Fibroblast growth factor receptors (FGFRs) are integral membrane proteins involved in various biological processes including proliferation, migration and apoptosis. There are a number of regulatory mechanisms of FGFR signaling, which tightly control the specificity and duration of transmitted signals. The effect of the FGFRs spatial distribution in the plasma membrane on receptor-dependent functions is still largely unknown. We have demonstrated that oligomerization of FGF1 with coiled-coil motifs largely improves FGF1 affinity for FGFRs and heparin. Set of developed FGF1 oligomers evoked prolonged activation of FGFR1 and receptor-downstream signaling pathways, as compared to the wild type FGF1. The majority of obtained oligomeric FGF1 variants showed increased stability, enhanced mitogenic activity and largely improved internalization via FGFR1-dependent endocytosis. Importantly, FGF1 oligomers with the highest oligomeric state exhibited reduced ability to stimulate FGFR-dependent glucose uptake, while at the same time remained hyperactive in the induction of cell proliferation. Our data implicate that oligomerization of FGF1 alters the biological activity of the FGF/GFR1 signaling system. Furthermore, developed FGF1 oligomers, due to improved stability and proliferative potential, can be applied in the regenerative medicine or as drug delivery vehicles in the ADC approach against FGFR1-overproducing cancers.


Assuntos
Proliferação de Células , Fator 1 de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Células 3T3-L1 , Animais , Ligação Competitiva , Linhagem Celular Tumoral , Fator 1 de Crescimento de Fibroblastos/química , Heparina/metabolismo , Humanos , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Ligação Proteica , Multimerização Proteica
7.
Elife ; 92020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32795391

RESUMO

Intracellular transport undergoes remodeling upon cell differentiation, which involves cell type-specific regulators. Bone morphogenetic protein 2-inducible kinase (BMP2K) has been potentially implicated in endocytosis and cell differentiation but its molecular functions remained unknown. We discovered that its longer (L) and shorter (S) splicing variants regulate erythroid differentiation in a manner unexplainable by their involvement in AP-2 adaptor phosphorylation and endocytosis. However, both variants interact with SEC16A and could localize to the juxtanuclear secretory compartment. Variant-specific depletion approach showed that BMP2K isoforms constitute a BMP2K-L/S regulatory system that controls the distribution of SEC16A and SEC24B as well as SEC31A abundance at COPII assemblies. Finally, we found L to promote and S to restrict autophagic degradation and erythroid differentiation. Hence, we propose that BMP2K-L and BMP2K-S differentially regulate abundance and distribution of COPII assemblies as well as autophagy, possibly thereby fine-tuning erythroid differentiation.


Assuntos
Processamento Alternativo/genética , Autofagia/fisiologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Proteínas Serina-Treonina Quinases/genética , Animais , Diferenciação Celular/genética , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo
8.
Mol Oncol ; 14(9): 1998-2021, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32511887

RESUMO

Fibroblast growth factor receptor 1 (FGFR1) transmits signals through the plasma membrane regulating essential cellular processes like division, motility, metabolism, and death. Overexpression of FGFR1 is observed in numerous tumors and thus constitutes an attractive molecular target for selective cancer treatment. Targeted anti-cancer therapies aim for the precise delivery of drugs into cancer cells, sparing the healthy ones and thus limiting unwanted side effects. One of the key steps in targeted drug delivery is receptor-mediated endocytosis. Here, we show that the efficiency and the mechanism of FGFR1 internalization are governed by the spatial distribution of the receptor in the plasma membrane. Using engineered antibodies of different valency, we demonstrate that dimerization of FGFR1 with bivalent antibody triggers clathrin-mediated endocytosis (CME) of the receptor. Clustering of FGFR1 into larger oligomers with tetravalent antibody stimulates fast and highly efficient uptake of the receptor that occurs via two distinct mechanisms: CME and dynamin-dependent clathrin-independent endocytic routes. Furthermore, we show that all endocytic pathways engaged in FGFR1 internalization do not require receptor activation. Our data provide novel insights into the mechanisms of intracellular trafficking of FGFR1 and constitute guidelines for development of highly internalizing antibody-based drug carriers for targeted therapy of FGFR1-overproducing cancers.


Assuntos
Anticorpos/metabolismo , Endocitose , Engenharia de Proteínas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Afinidade de Anticorpos/imunologia , Células CHO , Linhagem Celular Tumoral , Clatrina/metabolismo , Análise por Conglomerados , Cricetulus , Dinaminas/metabolismo , Humanos , Camundongos , Modelos Biológicos , Células NIH 3T3 , Ligação Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Receptores Fc/metabolismo
9.
J Cell Sci ; 131(22)2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30333141

RESUMO

Cytokine receptors, such as tumor necrosis factor receptor I (TNFRI, also known as TNFRSF1A) and lymphotoxin ß receptor (LTßR), activate inflammatory nuclear factor (NF)-κB signaling upon stimulation. We have previously demonstrated that depletion of ESCRT components leads to endosomal accumulation of TNFRI and LTßR, and their ligand-independent signaling to NF-κB. Here, we studied whether other perturbations of the endolysosomal system could trigger intracellular accumulation and signaling of ligand-free LTßR. While depletion of the CORVET components had no effect, knockdown of Rab7a or HOPS components, or pharmacological inhibition of lysosomal degradation, caused endosomal accumulation of LTßR and increased its interaction with the TRAF2 and TRAF3 signaling adaptors. However, the NF-κB pathway was not activated under these conditions. We found that knockdown of Rab7a or HOPS components led to sequestration of LTßR in intraluminal vesicles of endosomes, thus precluding NF-κB signaling. This was in contrast to the LTßR localization on the outer endosomal membrane that was seen after ESCRT depletion and was permissive for signaling. We propose that the inflammatory response induced by intracellular accumulation of endocytosed cytokine receptors critically depends on the precise receptor topology within endosomal compartments.


Assuntos
Receptor beta de Linfotoxina/metabolismo , NF-kappa B/metabolismo , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Transporte Proteico , Transdução de Sinais , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
10.
J Cell Sci ; 130(3): 577-589, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27980069

RESUMO

Platelet-derived growth factor receptor ß (PDGFRß) is a receptor tyrosine kinase which upon activation by PDGF-BB stimulates cell proliferation, migration and angiogenesis. Ligand binding induces intracellular signaling cascades but also internalization of the receptor, eventually resulting in its lysosomal degradation. However, endocytic trafficking of receptors often modulates their downstream signaling. We previously reported that internalization of PDGFRß occurs via dynamin-dependent and -independent pathways but their further molecular determinants remained unknown. Here we show that, in human fibroblasts expressing endogenous PDGFRß and stimulated with 50 ng/ml PDGF-BB, ligand-receptor uptake proceeds via the parallel routes of clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE). CME involves the canonical AP2 complex as a clathrin adaptor, while CIE requires RhoA-ROCK, Cdc42 and galectin-3, the latter indicating lectin-mediated internalization via clathrin-independent carriers (CLICs). Although different uptake routes appear to be partly interdependent, they cannot fully substitute for each other. Strikingly, inhibition of any internalization mechanism impaired activation of STAT3 but not of other downstream effectors of PDGFRß. Our data indicate that multiple routes of internalization of PDGFRß contribute to a transcriptional and mitogenic response of cells to PDGF.


Assuntos
Endocitose/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Complexo 2 de Proteínas Adaptadoras/metabolismo , Clatrina/metabolismo , DNA/biossíntese , Dinaminas/metabolismo , Endocitose/genética , Galectina 3/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/metabolismo , Masculino , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
11.
Sci Signal ; 9(411): ra8, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26787452

RESUMO

Because signaling mediated by the transcription factor nuclear factor κB (NF-κB) is initiated by ligands and receptors that can undergo internalization, we investigated how endocytic trafficking regulated this key physiological pathway. We depleted all of the ESCRT (endosomal sorting complexes required for transport) subunits, which mediate receptor trafficking and degradation, and found that the components Tsg101, Vps28, UBAP1, and CHMP4B were essential to restrict constitutive NF-κB signaling in human embryonic kidney 293 cells. In the absence of exogenous cytokines, depletion of these proteins led to the activation of both canonical and noncanonical NF-κB signaling, as well as the induction of NF-κB-dependent transcriptional responses in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin ß receptor (LTßR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTßR induced its ligand-independent oligomerization and signaling through the adaptors TNFR-associated factor 2 (TRAF2) and TRAF3. These data suggest that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling, which may represent a general mechanism to prevent spurious activation of NF-κB.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , NF-kappa B/metabolismo , Receptores de Citocinas/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Linhagem Celular Tumoral , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HEK293 , Humanos , NF-kappa B/genética , Transporte Proteico/fisiologia , Receptores de Citocinas/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
12.
PLoS One ; 10(6): e0130818, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26110841

RESUMO

Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of ß-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of ß-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of ß-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive ß-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Via de Sinalização Wnt/genética , Animais , Carcinogênese/genética , Desenvolvimento Embrionário/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transporte Proteico/fisiologia , Peixe-Zebra , beta Catenina/metabolismo
13.
Methods Enzymol ; 535: 167-77, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24377924

RESUMO

Microscopical analyses of endocytic trafficking require tools for efficient detection of internalized cargo. Due to the lack of suitable reagents and limitations related to its biological properties, visualization of platelet-derived growth factor (PDGF) by microscopy remained a challenge. To overcome these restrictions, we generated a biologically active PDGF labeled with up to five biotins on cleavable linkers. Subsequently, we stimulated cells with such ligand followed by removal of extracellular biotins. PDGF captured in endocytic vesicles was successfully detected with antibiotin antibodies with parallel detection of PDGF receptor, as well as other markers of endocytic compartments. Labeled PDGF was successfully validated and can be utilized in various microscopical techniques.


Assuntos
Endocitose , Fator de Crescimento Derivado de Plaquetas/metabolismo , Biotinilação , Linhagem Celular , Humanos , Microscopia de Fluorescência , Fator de Crescimento Derivado de Plaquetas/química , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Coloração e Rotulagem
14.
Traffic ; 14(6): 725-36, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23425318

RESUMO

Platelet-derived growth factor (PDGF) isoforms regulate cell proliferation, migration and differentiation both in embryonic development and adult tissue remodeling. At the cellular level, growth-factor signaling is often modulated by endocytosis. Despite important functions of PDGF, its endocytosis remains poorly studied, mainly for lack of tools to track internalized ligand by microscopy. Here, we developed such a tool and quantitatively analyzed internalization and endosomal trafficking of PDGF-BB in human fibroblasts. We further show that PDGF can be internalized in the presence of dynamin inhibitors, arguing that both dynamin-dependent and dynamin-independent pathways can mediate PDGF uptake. Although these routes operate with somewhat different kinetics, they both ultimately lead to lysosomal degradation of PDGF. Although acute inhibition of dynamin activity only moderately affects PDGF endocytosis, it specifically decreases downstream signaling of PDGF via signal transducer and activator of transcription 3 (STAT3). This correlates with reduced expression of MYC and impaired cell entry into S-phase, indicating that dynamin activity is required for PDGF-induced mitogenesis. Our data support a general view that the components governing endocytic trafficking may selectively regulate certain signaling effectors activated by a growth factor.


Assuntos
Dinaminas/antagonistas & inibidores , Endocitose , Sistema de Sinalização das MAP Quinases , Fator de Crescimento Derivado de Plaquetas/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Humanos , Lisossomos/metabolismo , Transporte Proteico , Proteólise , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fase S/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Transcrição Gênica
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