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1.
Org Biomol Chem ; 20(21): 4342-4351, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35575175

RESUMO

Herein, we report emissive aminoquinoline derivatives (TFMAQ) containing alkylmorpholine and arylmorpholine groups and their photophysical properties, acid-responsiveness, and organelle targeting. The alkylmorpholine group is well-known to favour accumulation in lysosomes and be acid-responsive, but, counterintuitively, the TFMAQ derivatives containing ethylmorpholine groups showed limited accumulation in lysosomes and, instead, preferential accumulation in lipid droplets. The findings reported here will aid the development of organelle/tissue specific dyes for cell imaging and diagnosis.


Assuntos
Aminoquinolinas , Corantes Fluorescentes , Lisossomos , Imagem Óptica , Organelas
2.
J Biol Chem ; 286(50): 43537-48, 2011 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-22027834

RESUMO

Cell movement is an important cellular function not only in physiological but also in pathological conditions. Although numerous studies have been conducted to reveal the mechanism of cell movement, the full picture has yet to be depicted, likely due to the complex features of cell movement. We show here that the scaffold protein afadin dilute domain-interacting protein (ADIP), an afadin-binding protein, is involved in the regulation of cell movement. ADIP localized at the leading edge of moving cells in response to platelet-derived growth factor (PDGF) and was required for the formation of the leading edge and the promotion of cell movement. Impaired cell movement observed in ADIP knockdown cells was not rescued by expression of an ADIP mutant that is incapable of binding to afadin, leading to the notion that the function of ADIP in moving cells depends on its interaction with afadin. Knockdown of ADIP as well as knockdown of afadin inhibited the activation of the small G protein Rac, which is important for the formation of the leading edge and the promotion of cell movement. Furthermore, ADIP interacted with Vav2, a GDP/GTP exchange factor for Rac, in a Src phosphorylation-dependent manner, suggesting that ADIP mediates the activation of Rac through Vav2. These results indicate that ADIP plays an essential role in PDGF-induced cell movement by interacting with afadin and Vav2 and regulating the activation of Rac.


Assuntos
Proteínas de Transporte/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Western Blotting , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Movimento Celular/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Imunoprecipitação , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos , Células NIH 3T3 , Ligação Proteica , Proteínas Proto-Oncogênicas c-vav/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rac de Ligação ao GTP/genética
3.
J Biol Chem ; 285(7): 5003-12, 2010 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-20008323

RESUMO

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with beta- and alpha-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-DeltaPR1-2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Junções Íntimas/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Cães , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Microscopia de Fluorescência , Fosfoproteínas/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Junções Íntimas/genética , Proteína da Zônula de Oclusão-1
4.
J Cell Sci ; 121(Pt 6): 727-35, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18322269

RESUMO

The cadherin molecules at adherens junctions have multiple isoforms. Cadherin isoform switching (cadherin switching) occurs during normal developmental processes to allow cell types to segregate from one another. Tumor cells often recapitulate this activity and the result is an aggressive tumor cell that gains the ability to leave the site of the tumor and metastasize. At present, we understand some of the mechanisms that promote cadherin switching and some of the pathways downstream of this process that influence cell behavior. Specific cadherin family members influence growth-factor-receptor signaling and Rho GTPases to promote cell motility and invasion. In addition, p120-catenin probably plays multiple roles in cadherin switching, regulating Rho GTPases and stabilizing cadherins.


Assuntos
Caderinas/metabolismo , Neoplasias/metabolismo , Junções Aderentes/ultraestrutura , Animais , Caderinas/antagonistas & inibidores , Caderinas/genética , Sobrevivência Celular , Transformação Celular Neoplásica/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Transcrição Gênica , Proteínas rho de Ligação ao GTP/metabolismo
5.
J Cell Biol ; 180(6): 1277-89, 2008 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-18362184

RESUMO

Tumor cells undergo epithelial-to-mesenchymal transition (EMT) to convert from a benign to a malignant phenotype. Our recent focus has been signaling pathways that promote EMT in response to collagen. We have shown that human pancreatic cancer cells respond to collagen by up-regulating N-cadherin, which promotes tumor growth, invasion, and metastasis. Initial characterization showed that knocking down c-Jun NH2-terminal kinase prevented N-cadherin up-regulation and limited tumor growth and invasion in a mouse model for pancreatic cancer. The current study was designed to understand the pathway from collagen to N-cadherin up-regulation. Initiation of the signal requires two collagen receptors, alpha2beta1 integrin and discoidin domain receptor (DDR) 1. Each receptor propagates signals through separate pathways that converge to up-regulate N-cadherin. Focal adhesion kinase (FAK)-related protein tyrosine kinase (Pyk2) is downstream of DDR1, whereas FAK is downstream of alpha2beta1 integrin. Both receptor complexes rely on the p130 Crk-associated substrate scaffold. Interestingly, Rap1, but not Rho family guanosine triphosphatases, is required for the response to collagen I.


Assuntos
Caderinas/metabolismo , Transformação Celular Neoplásica/metabolismo , Colágeno Tipo I/metabolismo , Integrina alfa2beta1/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Mitogênicos/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Proteína Substrato Associada a Crk/metabolismo , Receptores com Domínio Discoidina , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Quinase 2 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Mesoderma/metabolismo , Mesoderma/patologia , Invasividade Neoplásica/patologia , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , Proteínas rap1 de Ligação ao GTP/metabolismo
6.
Exp Cell Res ; 314(1): 52-67, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17719574

RESUMO

In contrast to growth factor-stimulated tyrosine phosphorylation of p120, its relatively constitutive serine/threonine phosphorylation is not well understood. Here we examined the role of serine/threonine phosphorylation of p120 in cadherin function. Expression of cadherins in cadherin-null cells converted them to an epithelial phenotype, induced p120 phosphorylation and localized it to sites of cell contact. Detergent solubility and immunofluorescence confirmed that phosphorylated p120 was at the plasma membrane. E-cadherin constructs incapable of traveling to the plasma membrane did not induce serine/threonine phosphorylation of p120, nor did cadherins constructs incapable of binding p120. However, an E-cadherin cytoplasmic domain construct artificially targeted to the plasma membrane did induce serine/threonine phosphorylation of p120, suggesting phosphorylation occurs independently of signals from cadherin dimerization and trafficking through the ER/Golgi. Solubility assays following calcium switch showed that p120 isoform 3A was more effective at stabilizing E-cadherin at the plasma membrane relative to isoform 4A. Since the major phosphorylation domain of p120 is included in isoform 3A but not 4A, we tested p120 mutated in the known phosphorylation sites in this domain and found that it was even less effective at stabilizing E-cadherin. These data suggest that serine/threonine phosphorylation of p120 influences the dynamics of E-cadherin in junctions.


Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Fosfoproteínas/metabolismo , Animais , Sítios de Ligação/fisiologia , Caderinas/genética , Cateninas , Adesão Celular/fisiologia , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Membrana Celular/química , Membrana Celular/genética , Dimerização , Endocitose/fisiologia , Humanos , Junções Intercelulares/química , Junções Intercelulares/genética , Junções Intercelulares/metabolismo , Camundongos , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação , Ligação Proteica/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Serina/metabolismo , Solubilidade , Treonina/metabolismo , delta Catenina
7.
Int J Cancer ; 122(1): 71-7, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17721921

RESUMO

Pancreatic cancer is one of the most aggressive malignant diseases. We recently reported that N-cadherin plays a key role in tumor progression and metastasis in pancreatic cancer. For this study, we sought to determine if an N-cadherin-blocking peptide (ADH-1) could prevent N-cadherin-mediated tumor progression in a mouse model for pancreatic cancer. The effect of ADH-1 on N-cadherin-mediated cell scattering and migration on collagen I was examined using pancreatic cancer cells. We also examined the influence of ADH-1 on cell apoptosis. Furthermore, in vivo animal studies were performed using orthotopic injection of N-cadherin overexpressing BxPC-3 cells with or without ADH-1 treatment. BxPC-3 and Capan-1 cells exhibited increased expression of N-cadherin in response to collagen I. This increase in N-cadherin promoted cell scattering and migration in response to collagen I. ADH-1 prevented these changes, but did not inhibit upregulation of N-cadherin. TUNEL assays and immunoblots for caspase-3 showed that ADH-1 induced apoptosis in a concentration dependent and N-cadherin dependent manner in pancreatic cancer cells. ADH-1 treatment resulted in significant reductions in tumor growth and lung metastasis in a mouse model for pancreatic cancer. The N-cadherin antagonist, ADH-1 has significant antitumor activity against N-cadherin-expressing cells using in vitro assays and in an orthotopic mouse model for pancreatic cancer, raising the possibility that N-cadherin antagonists have therapeutic potential for the treatment of pancreatic cancer in humans.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Oligopeptídeos/farmacologia , Neoplasias Pancreáticas/prevenção & controle , Peptídeos Cíclicos/farmacologia , Animais , Antígenos CD/genética , Apoptose , Caderinas/antagonistas & inibidores , Caderinas/genética , Adesão Celular , Movimento Celular , Colágeno/metabolismo , Progressão da Doença , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , RNA Interferente Pequeno/farmacologia , Células Tumorais Cultivadas
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