RESUMO
Annexin A10 is the latest identified member of the annexin family of Ca(2+)- and phospholipid-binding proteins. In previous studies, downregulation of annexin A10 was correlated with dedifferentiation, invasion, and tumor progression, pointing to a possible tumor suppressor role. However, the biochemical characteristics and functions of annexin A10 remain unknown. We show that annexin A10 displays biochemical characteristics atypical for an annexin, indicating a Ca(2+)- and membrane-binding-independent function. Annexin A10 co-localizes with the mRNA-binding proteins SFPQ and PSPC1 at paraspeckles, an only recently discovered nuclear body, and decreases paraspeckle numbers when overexpressed in HeLa cells. In addition, annexin A10 relocates to dark perinucleolar caps upon transcriptional inhibition of RNA polymerase II. We mapped the cap-binding function of annexin A10 to the proximal part of the core domain, which is missing in the short isoform of annexin A10, and show its independence from the remaining functional type II Ca(2+)-binding site. In contrast to this, paraspeckle recruitment required additional core regions and was negatively affected by the mutation of the last type II Ca(2+)-binding site. Additionally, we show that overexpression of annexin A10 in HeLa cells increases their sensitivity to apoptosis and reduces colony formation. The identification of unique nuclear and biochemical characteristics of annexin A10 points towards its membrane-independent role in paraspeckle-associated mRNA regulation or processing.
Assuntos
Anexinas/metabolismo , Núcleo Celular/metabolismo , Animais , Anexinas/análise , Anexinas/genética , Antibióticos Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Núcleo Celular/ultraestrutura , Cães , Doxorrubicina/toxicidade , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Proteínas Nucleares/metabolismo , Fator de Processamento Associado a PTB , Isoformas de Proteínas/metabolismo , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Endocytosis of activated growth factor receptors regulates spatio-temporal cellular signaling. In the case of the EGF receptor, sorting into multivesicular bodies (MVBs) controls signal termination and subsequently leads to receptor degradation in lysosomes. Annexin A1, a Ca(2+)-regulated membrane binding protein often deregulated in human cancers, interacts with the EGF receptor and is phosphorylated by internalized EGF receptor on endosomes. Most relevant for EGF receptor signal termination, annexin A1 is required for the formation of internal vesicles in MVBs that sequester ligand-bound EGF receptor away from the limiting membrane. To elucidate the mechanism underlying annexin A1-dependent EGF receptor trafficking we employed an N-terminally truncated annexin A1 mutant that lacks the EGF receptor phosphorylation site and the site for interaction with its protein ligand S100A11. Overexpression of this dominant-negative mutant induces a delay in EGF-induced EGF receptor transport to the LAMP1-positive late endosomal/lysosomal compartment and impairs ligand-induced EGF receptor degradation. Consistent with these findings, EGF-stimulated EGF receptor and MAP kinase pathway signaling is prolonged. Importantly, depletion of S100A11 also results in a delayed EGF receptor transport and prolonged MAP kinase signaling comparable to the trafficking defect observed in cells expressing the N-terminally truncated annexin A1 mutant. These results strongly suggest that the function of annexin A1 as a regulator of EGF receptor trafficking, degradation and signaling is critically mediated through an N-terminal interaction with S100A11 in the endosomal compartment. This interaction appears to be essential for lysosomal targeting of the EGF receptor, possibly by providing a physical scaffold supporting inward vesiculation in MVBs. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.
Assuntos
Anexina A1/metabolismo , Movimento Celular , Proliferação de Células , Receptores ErbB/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas S100/metabolismo , Anexina A1/antagonistas & inibidores , Anexina A1/genética , Compartimento Celular , Ensaio de Unidades Formadoras de Colônias , Endocitose/fisiologia , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Células HeLa , Humanos , Técnicas Imunoenzimáticas , Proteínas de Membrana Lisossomal/genética , Lisossomos/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Transporte Proteico , Proteínas S100/antagonistas & inibidores , Proteínas S100/genética , Ressonância de Plasmônio de SuperfícieRESUMO
Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2), an enzyme mediating 3-O-sulfation of heparan sulfate (HS), is silenced by hypermethylation in breast cancer. As HS has an important co-receptor function for numerous signal transduction pathways, the phenotypical changes due to HS3ST2 reexpression were investigated in vitro using high and low invasive breast cancer cell lines. Compared to controls, highly invasive HS3ST2-expressing MDA-MB-231 cells showed enhanced Matrigel invasiveness, transendothelial migration and motility. Affymetrix screening and confirmatory real-time PCR and Western blotting analysis revealed increased expression of several matrix metalloproteinases, cadherin-11, E-cadherin and CEACAM-1, while protease inhibitor and annexin A10 expression were decreased. Low invasive HS3ST2 -expressing MCF-7 cells became even less invasive, with no change in gelatinolytic MMP activity. HS3ST2 expression increased HS-dependent basal and FGF2-specific signaling through the constitutively active p44/42 MAPK pathway in MDA-MB-231 cells. Increased MAPK activation was accompanied by upregulation of ß-catenin in MDA-MB-231, and of the transcription factor Tcf4 in both cell lines. Dysregulation of Tcf4-regulated ion transporters and increased cytosolic acidification were observed in HS3ST2-expressing MDA-MB-231 cells, which is a possible underlying cause of increased chemosensitivity towards doxorubicine and paclitaxel in these cells. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion and chemosensitivity.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Sulfotransferases/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Caderinas/genética , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/genética , Invasividade Neoplásica , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Sulfotransferases/genética , Fator de Transcrição 4 , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Fatores de Transcrição/genética , Células Tumorais Cultivadas , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Ca(2+)-binding proteins of the S100 family participate in intracellular Ca(2+) signaling by binding to and regulating specific cellular targets in their Ca(2+)-loaded conformation. Because the information on specific cellular targets of different S100 proteins is still limited, we developed an affinity approach that selects for protein targets only binding to the physiologically active dimer of an S100 protein. Using this approach, we here identify IQGAP1 as a novel and dimer-specific target of S100P, a member of the S100 family enriched in the cortical cytoskeleton. The interaction between S100P and IQGAP1 is strictly Ca(2+)-dependent and characterized by a dissociation constant of 0.2 µM. Binding occurs primarily through the IQ domain of IQGAP1 and the first EF hand loop of S100P, thus representing a novel structural principle of S100-target protein interactions. Upon cell stimulation, S100P and IQGAP1 co-localize at or in close proximity to the plasma membrane, and complex formation can be linked to altered signal transduction properties of IQGAP1. Specifically, the EGF-induced tyrosine phosphorylation of IQGAP1 that is thought to function in assembling signaling intermediates at IQGAP1 scaffolds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing an S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occurring downstream of IQGAP1 in EGF-triggered signaling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca(2+)-dependent regulator of IQGAP1 that can down-regulate the function of IQGAP1 as a signaling intermediate by direct interaction.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Neoplasias/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Calmodulina/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Dimerização , Células HeLa , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fosforilação/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
microRNAs are small endogenous noncoding RNAs, which post-transcriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan-1, a predicted target of the oncomiR miR-10b, correlates with poor clinical outcome. To investigate the potential functional relationship of miR-10b and syndecan-1, MDA-MB-231 and MCF-7 breast cancer cells were transiently transfected with pre-miR-10b, syndecan-1 siRNA or control reagents, respectively. Altered cell behavior was monitored by proliferation, migration and invasion chamber assays, and time-lapse video microscopy. miR-10b overexpression induced post-transcriptional downregulation of syndecan-1, as demonstrated by quantitative real-time PCR (qPCR), flow cytometry, and 3'UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan-1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation was increased. Syndecan-1 coimmunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan-1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan-1-deficient cells revealed upregulation of ATF-2, COX-2, cadherin-11, vinculin, actin γ 2, MYL9, transgelin-1, RhoA/C, matrix metalloproteinase 2 (MMP2) and heparanase, and downregulation of AML1/RUNX1, E-cadherin, CLDN1, p21WAF/CIP, cyclin-dependent kinase 6, TLR-4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin-2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase-dependent motility of syndecan-1-depleted cells, which displayed increased filopodia formation. We conclude that syndecan-1 is a novel target of the oncomiR miR-10b. Rho-GTPase-dependent modulation of cytoskeletal function and downregulation of E-cadherin expression are identified as relevant effectors of the miR-10b-syndecan-1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer.
Assuntos
Neoplasias da Mama/patologia , Caderinas/fisiologia , Movimento Celular , MicroRNAs/fisiologia , Sindecana-1/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/fisiologia , Fator 2 Ativador da Transcrição/genética , Comunicação Celular , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Progressão da Doença , Feminino , Humanos , Invasividade Neoplásica , Fosforilação , RNA Mensageiro/análise , RNA Interferente Pequeno/genética , Sindecana-1/fisiologiaRESUMO
To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63.
Assuntos
Anexinas/metabolismo , Adesão Celular/imunologia , Células Endoteliais/imunologia , Leucócitos/imunologia , Modelos Imunológicos , Tetraspanina 30/metabolismo , Corpos de Weibel-Palade/metabolismo , Análise de Variância , Animais , Anticorpos Monoclonais , Southern Blotting , Western Blotting , Primers do DNA/genética , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucócitos/metabolismo , Camundongos , Microscopia de Força Atômica , Selectina-P/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. Late endosomes, organelles of the degradative lysosomal route, seem to require associated actin filaments for proper localization and function. We show here that the F-actin and phospholipid binding protein annexin A8 is associated specifically with late endosomes. Altering intracellular annexin A8 levels drastically affected the morphology and intracellular distribution of late endosomes. Trafficking through the degradative pathway was delayed in the absence of annexin A8, resulting in attenuated ligand-induced degradation of the epidermal growth factor receptor and prolonged epidermal growth factor-induced activation of mitogen-activated protein kinase. Depletion of annexin A8 reduced the association of late endosomal membranes with actin filaments. These results indicate that the defective cargo transport through the late endocytic pathway and the imbalanced signaling of activated receptors observed in the absence of annexin A8 results from the disturbed association of late endosomal membranes with the actin network, resulting in impaired actin-based late endosome motility.