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1.
J Cell Sci ; 136(12)2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37232246

RESUMO

Endocytic recycling controls the return of internalised cargoes to the plasma membrane to coordinate their positioning, availability and downstream signalling. The Rab4 and Rab11 small GTPase families regulate distinct recycling routes, broadly classified as fast recycling from early endosomes (Rab4) and slow recycling from perinuclear recycling endosomes (Rab11), and both routes handle a broad range of overlapping cargoes to regulate cell behaviour. We adopted a proximity labelling approach, BioID, to identify and compare the protein complexes recruited by Rab4a, Rab11a and Rab25 (a Rab11 family member implicated in cancer aggressiveness), revealing statistically robust protein-protein interaction networks of both new and well-characterised cargoes and trafficking machinery in migratory cancer cells. Gene ontological analysis of these interconnected networks revealed that these endocytic recycling pathways are intrinsically connected to cell motility and cell adhesion. Using a knock-sideways relocalisation approach, we were further able to confirm novel links between Rab11, Rab25 and the ESCPE-1 and retromer multiprotein sorting complexes, and identify new endocytic recycling machinery associated with Rab4, Rab11 and Rab25 that regulates cancer cell migration in the 3D matrix.


Assuntos
Proteínas rab de Ligação ao GTP , Proteínas rab4 de Ligação ao GTP , Humanos , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismo , Transporte Biológico , Transporte Proteico/fisiologia , Endossomos/metabolismo
2.
Cell ; 139(7): 1327-41, 2009 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-20064378

RESUMO

p53 is a tumor suppressor protein whose function is frequently lost in cancers through missense mutations within the Tp53 gene. This results in the expression of point-mutated p53 proteins that have both lost wild-type tumor suppressor activity and show gain of functions that contribute to transformation and metastasis. Here, we show that mutant p53 expression can promote invasion, loss of directionality of migration, and metastatic behavior. These activities of p53 reflect enhanced integrin and epidermal growth factor receptor (EGFR) trafficking, which depends on Rab-coupling protein (RCP) and results in constitutive activation of EGFR/integrin signaling. We provide evidence that mutant p53 promotes cell invasion via the inhibition of TAp63, and simultaneous loss of p53 and TAp63 recapitulates the phenotype of mutant p53 in cells. These findings open the possibility that blocking alpha5/beta1-integrin and/or the EGF receptor will have therapeutic benefit in mutant p53-expressing cancers.


Assuntos
Movimento Celular , Integrina alfa5beta1/metabolismo , Metástase Neoplásica , Proteína Supressora de Tumor p53/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Mutação , Pseudópodes/metabolismo , Proteína Supressora de Tumor p53/genética
3.
Nat Rev Mol Cell Biol ; 10(12): 843-53, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19904298

RESUMO

Since it has become clear that adhesion receptors are trafficked through the endosomal pathway and that this can influence their function, much effort has been invested in obtaining detailed descriptions of the molecular machinery responsible for internalizing and recycling integrins. New findings indicate that integrin trafficking dictates the nature of Rho GTPase signalling during cytokinesis and cell migration. Furthermore, integrins can exert control over the trafficking of other receptors in a way that drives cancer cell invasion and tumour angiogenesis.


Assuntos
Endocitose , Integrinas/metabolismo , Animais , Matriz Extracelular/metabolismo , Humanos , Transporte Proteico , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo
4.
Nucleic Acids Res ; 47(9): 4375-4392, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30927008

RESUMO

Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate.


Assuntos
Neoplasias/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Fosforotioatos/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas rab5 de Ligação ao GTP/genética , Endossomos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HT29 , Humanos , Neoplasias/patologia , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Fosforotioatos/química , Oligonucleotídeos Fosforotioatos/farmacologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética
5.
J Cell Sci ; 130(4): 697-711, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28062852

RESUMO

We have previously shown that Rab17, a small GTPase associated with epithelial polarity, is specifically suppressed by ERK2 (also known as MAPK1) signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this, are unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17- and Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption that accompanies the transition between DCIS and a more invasive phenotype.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Endossomos/metabolismo , Proteômica/métodos , Aminoácidos/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Membranas Intracelulares/metabolismo , Marcação por Isótopo , Modelos Biológicos , Gradação de Tumores , Invasividade Neoplásica , Neuropilina-2/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas R-SNARE/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Estrogênio/metabolismo , Proteínas SNARE/metabolismo , Análise de Sobrevida , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
6.
PLoS Genet ; 10(3): e1004262, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24676055

RESUMO

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours.


Assuntos
Neoplasias da Mama/genética , Quinase 1 de Adesão Focal/genética , Sistema de Sinalização das MAP Quinases/genética , Receptores Proteína Tirosina Quinases/genética , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Drosophila melanogaster/genética , Células Epiteliais/metabolismo , Feminino , Quinase 1 de Adesão Focal/metabolismo , Humanos , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo
7.
Traffic ; 15(6): 665-83, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24589086

RESUMO

Fibroblast growth factor receptor 4 (FGFR4) plays important roles during development and in the adult to maintain tissue homeostasis. Moreover, overexpression of FGFR4 or activating mutations in FGFR4 has been identified as tumour-promoting events in several forms of cancer. Endocytosis is important for regulation of signalling receptors and we have previously shown that FGFR4 is mainly localized to transferrin-positive structures after ligand-induced endocytosis. Here, using a cell line with a defined pericentriolar endocytic recycling compartment, we show that FGFR4 accumulates in this compartment after endocytosis. Furthermore, using classical recycling assays and a new, photoactivatable FGFR4-PA-GFP fusion protein combined with live-cell imaging, we demonstrate that recycling of FGFR4 is dependent on Rab11. Upon Rab11b depletion, FGFR4 is trapped in the pericentriolar recycling compartment and the total levels of FGFR4 in cells are increased. Moreover, fibroblast growth factor 1 (FGF1)-induced autophosphorylation of FGFR4 as well as phosphorylation of phospholipase C (PLC)-γ is prolonged in cells depleted of Rab11. Interestingly, the activation of mitogen-activated protein kinase and AKT pathways were not prolonged but rather reduced in Rab11-depleted cells, indicating that recycling of FGFR4 is important for the nature of its signalling output. Thus, Rab11-dependent recycling of FGFR4 maintains proper levels of FGFR4 in cells and regulates FGF1-induced FGFR4 signalling.


Assuntos
Endocitose , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Endossomos/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Humanos , Fosfolipase C gama/metabolismo , Transporte Proteico , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genética
8.
J Cell Sci ; 127(Pt 18): 3893-901, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25015290

RESUMO

Chloride intracellular channel 3 (CLIC3) drives invasiveness of pancreatic and ovarian cancer by acting in concert with Rab25 to regulate the recycling of α5ß1 integrin from late endosomes to the plasma membrane. Here, we show that in two estrogen receptor (ER)-negative breast cancer cell lines, CLIC3 has little influence on integrin recycling, but controls trafficking of the pro-invasive matrix metalloproteinase MT1-MMP (also known as MMP14). In MDA-MB-231 cells, MT1-MMP and CLIC3 are localized primarily to late endosomal/lysosomal compartments located above the plane of adhesion and near the nucleus. MT1-MMP is transferred from these late endosomes to sites of cell-matrix adhesion in a CLIC3-dependent fashion. Correspondingly, CLIC3-knockdown opposes MT1-MMP-dependent invasive processes. These include the disruption of the basement membrane as acini formed from MCF10DCIS.com cells acquire invasive characteristics in 3D culture, and the invasion of MDA-MB-231 cells into Matrigel or organotypic plugs of type I collagen. Consistent with this, expression of CLIC3 predicts poor prognosis in ER-negative breast cancer. The identification of MT1-MMP as a cargo of a CLIC3-regulated pathway that drives invasion highlights the importance of late endosomal sorting and trafficking in breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Canais de Cloreto/metabolismo , Endossomos/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Movimento Celular , Canais de Cloreto/genética , Feminino , Humanos , Metaloproteinase 14 da Matriz/genética , Invasividade Neoplásica , Metástase Neoplásica , Transporte Proteico
9.
J Biol Chem ; 289(1): 122-32, 2014 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-24220032

RESUMO

The control and processing of microRNAs (miRs) is critical in the regulation of all cellular responses. Previous studies have suggested that a reduction in the expression of certain miRs, or an overall decrease in miR processing through the partial depletion of Dicer, can promote enhanced metastatic potential. We show here that Dicer depletion can promote the invasive behavior of cells that is reflected in enhanced recycling and activation of the growth factor receptors Met and EGF receptor. These responses are also seen in response to the expression of tumor-derived mutant p53s, and we show that mutant p53 can down-regulate Dicer expression through both direct inhibition of the TAp63-mediated transcriptional activation of Dicer and a TAp63-independent control of Dicer protein expression. Our results delineate a clear relationship between mutant p53, TAp63, and Dicer that might contribute to the metastatic function of mutant p53 but, interestingly, also reveal TAp63-independent functions of mutant p53 in controlling Dicer activity.


Assuntos
RNA Helicases DEAD-box/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Mutação , Neoplasias/metabolismo , Ribonuclease III/biossíntese , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/genética , Neoplasias/patologia , Ribonuclease III/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética
10.
Bioessays ; 35(6): 523-32, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23605698

RESUMO

Recently it has become clear that trafficking of integrins to late endosomes is key to the regulation of integrin expression and function during cell migration. Here we discuss the molecular machinery that dictates whether integrins are sorted to recycling endosomes or are targeted to late endosomes and lysosomes. Integrins and other receptors that are sorted to late endosomes are not necessarily degraded and, under certain circumstances, can be spared destruction and returned to the cell surface to drive cell migration and invasion. We will discuss how the exchange of adhesion receptors and other key regulators of cell migration between late endosomes/lysosomes and the plasma membrane can promote dynamic turnover of adhesions during cell migration.


Assuntos
Movimento Celular , Endossomos/metabolismo , Integrinas/fisiologia , Lisossomos/metabolismo , Neoplasias/patologia , Animais , Humanos , Invasividade Neoplásica , Transporte Proteico
11.
J Cell Sci ; 125(Pt 6): 1465-77, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22328529

RESUMO

Upregulation of the extracellular signal-regulated kinase (ERK) pathway has been shown to contribute to tumour invasion and progression. Because the two predominant ERK isoforms (ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively) are highly homologous and have indistinguishable kinase activities in vitro, both enzymes were believed to be redundant and interchangeable. To challenge this view, we show that ERK2 silencing inhibits invasive migration of MDA-MB-231 cells, and re-expression of ERK2 but not ERK1 restores the normal invasive phenotype. A detailed quantitative analysis of cell movement on 3D matrices indicates that ERK2 knockdown impairs cellular motility by decreasing the migration velocity as well as increasing the time that cells spend not moving. Using gene expression arrays we found that the expression of the genes for Rab17 and liprin-ß2 was increased by knockdown of ERK2 and restored to normal levels following re-expression of ERK2, but not ERK1. Both play inhibitory roles in the invasive behaviour of three independent cancer cell lines. Importantly, knockdown of either Rab17 or liprin-ß2 restores invasiveness of ERK2-depleted cells, indicating that ERK2 drives invasion of MDA-MB-231 cells by suppressing expression of these genes.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/antagonistas & inibidores , Movimento Celular/fisiologia , Proteínas de Membrana/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Microambiente Tumoral/fisiologia , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Neoplasias da Mama/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab de Ligação ao GTP/genética
12.
Neuro Oncol ; 26(4): 625-639, 2024 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-37936324

RESUMO

BACKGROUND: Glioblastomas have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist that counter glioblastoma invasion. Here, we report that inhibition of ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of glioblastoma cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking. METHODS: Glioblastoma motility and invasion were assessed in vitro and in vivo in response to ATR inhibition (ATRi) and ATR overexpression using time-lapse microscopy, two orthotopic glioblastoma models, and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high-throughput, super-resolution and intravital imaging. RESULTS: High ATR expression was associated with significantly poorer survival in clinical datasets while histological, protein expression, and spatial transcriptomics using glioblastoma tumor specimens revealed higher ATR expression at infiltrative margins. Pharmacological inhibition with two different compounds and RNAi targeting of ATR opposed the invasion of glioblastoma, whereas overexpression of ATR drove migration. Subsequent investigation revealed that cytoskeletal dysregulation reduced macropinocytotic internalization of integrins at growth-cone-like structures, resulting in a tumor microtube retraction defect. The biological relevance and translational potential of these findings were confirmed using two orthotopic in vivo models of glioblastoma and intravital imaging. CONCLUSIONS: We demonstrate a novel role for ATR in determining invasion in glioblastoma cells and propose that pharmacological targeting of ATR could have far-reaching clinical benefits beyond radiosensitization.


Assuntos
Glioblastoma , Humanos , Glioblastoma/patologia , Integrinas/metabolismo , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Invasividade Neoplásica , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo
13.
J Neurosci ; 32(30): 10352-64, 2012 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-22836268

RESUMO

Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of α9 integrin promotes axon regeneration, so we have investigated α9ß1 trafficking and transport in axons and at the growth cone. We have previously found that α9 and ß1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied α9 and ß1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of ß1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Transporte Axonal/fisiologia , Gânglios Espinais/metabolismo , Integrinas/metabolismo , Neurônios/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Animais , Axônios/metabolismo , Células Cultivadas , Endocitose/fisiologia , Gânglios Espinais/citologia , Cones de Crescimento/metabolismo , Masculino , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Vesículas Sinápticas/genética , Vesículas Sinápticas/metabolismo
14.
Curr Opin Cell Biol ; 18(5): 549-57, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16904305

RESUMO

The past five years have seen a steady accumulation of data reinforcing the view that Rab-regulated recycling pathways contribute to cell migration. In particular, detailed descriptions have emerged of the mechanisms that recruit integrins and growth factor receptors to Rab4- and Rab11-driven pathways. Recent work provides new insight into the importance of particular recycling events in cell migration within a variety of physiological contexts.


Assuntos
Membrana Celular/metabolismo , Movimento Celular/fisiologia , Endocitose/fisiologia , Transdução de Sinais/fisiologia , Fatores de Ribosilação do ADP/metabolismo , Animais , Exocitose/fisiologia , Integrinas/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
15.
J Am Soc Nephrol ; 23(9): 1506-17, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22859853

RESUMO

The aquaporin 2 (AQP2) water channel, expressed in kidney collecting ducts, contributes critically to water homeostasis in mammals. Animals lacking or having significantly reduced levels of AQP2, however, have not only urinary concentrating abnormalities but also renal tubular defects that lead to neonatal mortality from renal failure. Here, we show that AQP2 is not only a water channel but also an integrin-binding membrane protein that promotes cell migration and epithelial morphogenesis. AQP2 expression modulates the trafficking and internalization of integrin ß1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin ß1 by mutating the RGD motif led to reduced endocytosis, retention of integrin ß1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin ß1 at the basolateral membrane and had tubular abnormalities. In summary, these data suggest that the water channel AQP2 interacts with integrins to promote renal epithelial cell migration, contributing to the structural and functional integrity of the mammalian kidney.


Assuntos
Aquaporina 2/fisiologia , Movimento Celular/fisiologia , Células Epiteliais/citologia , Rim/citologia , Morfogênese/fisiologia , Animais , Aquaporina 2/deficiência , Aquaporina 2/genética , Linhagem Celular , Permeabilidade da Membrana Celular/fisiologia , Cães , Endocitose/fisiologia , Células Epiteliais/fisiologia , Técnicas In Vitro , Integrina beta1/fisiologia , Rim/crescimento & desenvolvimento , Rim/fisiologia , Camundongos , Camundongos Knockout , Modelos Animais , Mutação/genética , Oligopeptídeos/genética , Oligopeptídeos/fisiologia , Suínos , Transfecção
16.
Dev Cell ; 13(4): 496-510, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17925226

RESUMO

Here, we report a direct interaction between the beta1 integrin cytoplasmic tail and Rab25, a GTPase that has been linked to tumor aggressiveness and metastasis. Rab25 promotes a mode of migration on 3D matrices that is characterized by the extension of long pseudopodia, and the association of the GTPase with alpha5beta1 promotes localization of vesicles that deliver integrin to the plasma membrane at pseudopodial tips as well as the retention of a pool of cycling alpha5beta1 at the cell front. Furthermore, Rab25-driven tumor-cell invasion into a 3D extracellular matrix environment is strongly dependent on ligation of fibronectin by alpha5beta1 integrin and the capacity of Rab25 to interact with beta1 integrin. These data indicate that Rab25 contributes to tumor progression by directing the localization of integrin-recycling vesicles and thereby enhancing the ability of tumor cells to invade the extracellular matrix.


Assuntos
Movimento Celular/fisiologia , Matriz Extracelular/metabolismo , Integrina alfa5beta1/fisiologia , Invasividade Neoplásica , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Adesão Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Colágeno , Combinação de Medicamentos , Humanos , Integrina alfa5beta1/metabolismo , Laminina , Camundongos , Transporte Proteico , Proteoglicanas , Pseudópodes/metabolismo , Ratos
17.
J Cell Biol ; 177(3): 515-25, 2007 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-17485491

RESUMO

Accumulating evidence suggests that integrin recycling regulates cell migration. However, the lack of reagents to selectively target the trafficking of individual heterodimers, as opposed to endocytic transport as a whole, has made it difficult to define the contribution made by particular recycling pathways to directional cell movement. We show that autophosphorylation of protein kinase D1 (PKD1) at Ser(916) is necessary for its association with alphavbeta3 integrin. Expression of PKD1(916A) or the use of mutants of beta3 that do not bind to PKD1 selectively inhibits short-loop, Rab4-dependent recycling of alphavbeta3, and this suppresses the persistence of fibroblast migration. However, we report that short-loop recycling does not directly contribute to fibroblast migration by moving alphavbeta3 to the cell front, but by antagonizing alpha5beta1 recycling, which, in turn, influences the cell's decision to migrate with persistence or to move randomly.


Assuntos
Movimento Celular , Fibroblastos/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Células COS , Movimento Celular/genética , Chlorocebus aethiops , Endocitose/genética , Fibroblastos/citologia , Expressão Gênica , Integrina alfa5beta1/genética , Integrina alfaVbeta3/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Mutação de Sentido Incorreto , Células NIH 3T3 , Fosforilação , Ligação Proteica/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico/genética , Transdução de Sinais/genética , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo , Quinases Associadas a rho
18.
PLoS Biol ; 7(1): e25, 2009 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-19175293

RESUMO

Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes alpha5beta1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with alpha5beta1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active alpha5beta1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with alpha5beta1, and the associated motor myosin VI (Myo6) support active alpha5beta1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active alpha5beta1. Nrp1 modulation of alpha5beta1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice.


Assuntos
Proteínas de Transporte/metabolismo , Endotélio Vascular/metabolismo , Integrina alfa5beta1/metabolismo , Neuropeptídeos/metabolismo , Neuropilina-1/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Adesão Celular , Endotélio Vascular/citologia , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Integrina alfa5beta1/genética , Camundongos , Camundongos Knockout , Neovascularização Fisiológica , Neuropeptídeos/genética , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/genética , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Artérias Umbilicais/citologia , Artérias Umbilicais/metabolismo
19.
RSC Med Chem ; 13(2): 150-155, 2022 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-35308027

RESUMO

Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein-protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket ('WF-binding pocket') via a conserved tryptophan-phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets.

20.
Cell Death Differ ; 29(10): 2089-2104, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35473984

RESUMO

Glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. We investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti-apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non-malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti-apoptotic BCL-2 family members. High anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein-targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3-mimetics.


Assuntos
Glioblastoma , Adulto , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X
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