Página 6 dos resultados de 45945 itens digitais encontrados em 0.057 segundos

‣ Galectin-1 Induces Reversible Phosphatidylserine Exposure at the Plasma Membrane

STOWELL, Sean R.; KARMAKAR, Sougata; ARTHUR, Connie M.; JU, Tongzhong; RODRIGUES, Lilian C.; RIUL, Thalita B.; DIAS-BARUFFI, Marcelo; MINER, Jonathan; MCEVER, Rodger P.; CUMMINGS, Richard D.
Fonte: AMER SOC CELL BIOLOGY Publicador: AMER SOC CELL BIOLOGY
Tipo: Artigo de Revista Científica
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Cells normally undergo physiological turnover through the induction of apoptosis and phagocytic removal, partly through exposure of cell surface phosphatidylserine (PS). In contrast, neutrophils appear to possess apoptosis-independent mechanisms of removal. Here we show that Galectin-1 (Gal-1) induces PS exposure independent of alterations in mitochondrial potential, caspase activation, or cell death. Furthermore, Gal-1-induced PS exposure reverts after Gal-1 removal without altering cell viability. Gal-1-induced PS exposure is uniquely microdomain restricted, yet cells exposing PS do not display evident alterations in membrane morphology nor do they exhibit bleb formation, typically seen in apoptotic cells. Long-term exposure to Gal-1 prolongs PS exposure with no alteration in cell cycle progression or cell growth. These results demonstrate that Gal-1-induced PS exposure and subsequent phagocytic removal of living cells represents a new paradigm in cellular turnover.; National Institutes of Health (NIH)[HL085607]

‣ Regulation of Endoplasmic Reticulum Stress-induced Cell Death by ATF4 in Neuroectodermal Tumor Cells*

Armstrong, Jane L.; Flockhart, Ross; Veal, Gareth J.; Lovat, Penny E.; Redfern, Christopher P. F.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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The neuroectodermal tumors neuroblastoma and melanoma represent biologically aggressive and chemoresistant cancers. The chemotherapeutic agents fenretinide and bortezomib induce apoptosis through endoplasmic reticulum (ER) stress in these tumor types. The aim of this study was to test the hypothesis that the early events of ER stress signaling and response pathways induced by fenretinide and bortezomib are mediated by the eukaryotic initiation factor 2α (eIF2α)-ATF4 signaling pathway. Treatment of neuroblastoma and melanoma cell lines with fenretinide, bortezomib, or thapsigargin resulted in induction of eIF2α signaling, characterized by increased expression of phosphorylated eIF2α, ATF4, ATF3, and GADD34. These events correlated with induction of the pro-apoptotic protein Noxa. The cytotoxic response, characterized by up-regulation of Noxa and cell death, was dependent on ATF4, but not the ER-related pro-death signaling pathways involving GADD153 or IRE1. Although PERK-dependent phosphorylation of eIF2α enhanced ATF4 protein levels during ER stress, cell death in response to fenretinide, bortezomib, or thapsigargin was not abrogated by inhibition of eIF2α phosphorylation through PERK knockdown or overexpression of wild-type eIF2α. Furthermore...

‣ A Novel LZAP-binding Protein, NLBP, Inhibits Cell Invasion*

Kwon, Junhye; Cho, Hyun Jung; Han, Seung Hun; No, Jin Gu; Kwon, Jae Young; Kim, Hongtae
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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LXXLL/leucine zipper-containing alternative reading frame (ARF)-binding protein (LZAP) was recently shown to function as a tumor suppressor through inhibition of the NF-κB signaling pathway. LZAP is also known as a negative regulator of cell invasion, and its expression was demonstrated to be reduced in several tumor tissues. However, the molecular mechanism of the negative effect of LZAP on cell invasion is unclear. In this study, we identify NLBP as a novel LZAP-binding protein using tandem affinity purification. We demonstrate the negative effects of NLBP on cell invasion and the NF-κB signaling pathway. NLBP expression was not detected in hepatocellular carcinoma cells with strong invasive activity, whereas its expression was detected in a hepatocellular carcinoma cell line with no invasive activity. We also demonstrate that these two proteins mutually affect the stability of each other by inhibiting ubiquitination of the other protein. Based on these results, we suggest that NLBP may act as a novel tumor suppressor by inhibiting cell invasion, blocking NF-κB signaling, and increasing stability of the LZAP protein.

‣ The Transmembrane Domains of L-selectin and CD44 Regulate Receptor Cell Surface Positioning and Leukocyte Adhesion under Flow*

Buscher, Konrad; Riese, Sebastian B.; Shakibaei, Mehdi; Reich, Christian; Dernedde, Jens; Tauber, Rudolf; Ley, Klaus
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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During inflammation and immune surveillance, initial contacts (tethering) between free-flowing leukocytes and the endothelium are vitally dependent on the presentation of the adhesion receptor L-selectin on leukocyte microvilli. Determinants that regulate receptor targeting to microvilli are, however, largely elusive. Therefore, we systematically swapped the extracellular (EC), transmembrane (TM), and intracellular (IC) domains of L-selectin and CD44, a hyaluronan receptor expressed on the cell body and excluded from microvilli. Electron microscopy of transfected human myeloid K562 cells showed that the highly conserved TM domains are responsible for surface positioning. The TM segment of L-selectin forced chimeric molecules to microvilli, and the CD44 TM domain evoked expression on the cell body, whereas the IC and EC domains hardly influenced surface localization. Transfectants with microvillus-based chimeras showed a significantly higher adhesion rate under flow but not under static conditions compared with cells with cell body-expressed receptors. Substitution of the IC domain of L-selectin caused diminished tethering but no change in surface distribution, indicating that both microvillus positioning and cytoskeletal anchoring contribute to leukocyte tethering. These findings demonstrate that TM domains of L-selectin and CD44 play a crucial role in cell adhesion under flow by targeting receptors to microvilli or the cell body...

‣ CADM1 Interacts with Tiam1 and Promotes Invasive Phenotype of Human T-cell Leukemia Virus Type I-transformed Cells and Adult T-cell Leukemia Cells*

Masuda, Mari; Maruyama, Tomoko; Ohta, Tsutomu; Ito, Akihiko; Hayashi, Tomayoshi; Tsukasaki, Kunihiko; Kamihira, Shimeru; Yamaoka, Shoji; Hoshino, Hiroo; Yoshida, Teruhiko; Watanabe, Toshiki; Stanbridge, Eric J.; Murakami, Yoshinori
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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CADM1 encodes a multifunctional immunoglobulin-like cell adhesion molecule whose cytoplasmic domain contains a type II PSD95/Dlg/ZO-1 (PDZ)-binding motif (BM) for associating with other intracellular proteins. Although CADM1 lacks expression in T lymphocytes of healthy individuals, it is overexpressed in adult T-cell leukemia-lymphoma (ATL) cells. It has been suggested that the expression of CADM1 protein promotes infiltration of leukemic cells into various organs and tissues, which is one of the frequent clinical manifestations of ATL. Amino acid sequence alignment revealed that Tiam1 (T-lymphoma invasion and metastasis 1), a Rac-specific guanine nucleotide exchange factor, has a type II PDZ domain similar to those of membrane-associated guanylate kinase homologs (MAGUKs) that are known to bind to the PDZ-BM of CADM1. In this study, we demonstrated that the cytoplasmic domain of CADM1 directly interacted with the PDZ domain of Tiam1 and induced formation of lamellipodia through Rac activation in HTLV-I-transformed cell lines as well as ATL cell lines. Our results indicate that Tiam1 integrates signals from CADM1 to regulate the actin cytoskeleton through Rac activation, which may lead to tissue infiltration of leukemic cells in ATL patients.

‣ ZF21 Protein Regulates Cell Adhesion and Motility*

Nagano, Makoto; Hoshino, Daisuke; Sakamoto, Takeharu; Kawasaki, Noritaka; Koshikawa, Naohiko; Seiki, Motoharu
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Cell migration on an extracellular matrix (ECM) requires continuous formation and turnover of focal adhesions (FAs) along the direction of cell movement. However, our knowledge of the components of FAs and the mechanism of their regulation remains limited. Here, we identify ZF21, a member of a protein family characterized by the presence of a phosphatidylinositol 3-phosphate-binding FYVE domain, to be a new regulator of FAs and cell movement. Knockdown of ZF21 expression in cells increased the number of FAs and suppressed cell migration. Knockdown of ZF21 expression also led to a significant delay in FA disassembly following induction of synchronous disassembly of FAs by nocodazole treatment. ZF21 bound to focal adhesion kinase, localized to FAs, and was necessary for dephosphorylation of FAK at Tyr397, which is important for disassembly of FAs. Thus, ZF21 represents a new component of FAs, mediates disassembly of FAs, and thereby regulates cell motility.

‣ Essential Role of p400/mDomino Chromatin-remodeling ATPase in Bone Marrow Hematopoiesis and Cell-cycle Progression*

Fujii(藤井, Toshihiro, 俊裕); Ueda(上, Takeshi, 田健); Nagata(長田, Shigekazu, 重一); Fukunaga(福永理, Rikiro, 己郎)
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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p400/mDomino is an ATP-dependent chromatin-remodeling protein that catalyzes the deposition of histone variant H2A.Z into nucleosomes to regulate gene expression. We previously showed that p400/mDomino is essential for embryonic development and primitive hematopoiesis. Here we generated a conditional knock-out mouse for the p400/mDomino gene and investigated the role of p400/mDomino in adult bone marrow hematopoiesis and in the cell-cycle progression of embryonic fibroblasts. The Mx1-Cre- mediated deletion of p400/mDomino resulted in an acute loss of nucleated cells in the bone marrow, including committed myeloid and erythroid cells as well as hematopoietic progenitor and stem cells. A hematopoietic colony assay revealed a drastic reduction in colony-forming activity after the deletion of p400/mDomino. Moreover, the loss of p400/mDomino in mouse embryonic fibroblasts (MEFs) resulted in strong growth inhibition. Cell-cycle analysis revealed that the mDomino-deficient MEFs exhibited a pleiotropic cell-cycle defect at the S and G2/M phases, and polyploid and multi-nucleated cells with micronuclei emerged. DNA microarray analysis revealed that the p400/mDomino deletion from MEFs caused the impaired expression of many cell-cycle-regulatory genes...

‣ Maspin Regulates Endothelial Cell Adhesion and Migration through an Integrin Signaling Pathway*

Qin, Li; Zhang, Ming
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Maspin has been identified as a potent angiogenesis inhibitor. However, the molecular mechanism responsible for its anti-angiogenic property is unclear. In this study, we examined the effect of maspin on endothelial cell (EC) adhesion and migration in a cell culture system. We found that maspin was expressed in blood vessels ECs and human umbilical vein endothelial cells (HUVECs). Maspin significantly enhanced HUVEC cell adhesion to various matrix proteins. This effect was dependent on the activation of integrin β1, which subsequently led to distribution pattern changes of vinculin and F-actin. These results indicated that maspin affects cell adhesion and cytoskeleton reorganization through an integrin signal transduction pathway. Analysis of HUVECs following maspin treatment revealed increased integrin-linked kinase activities and phosphorylated FAK levels, consistent with increased cell adhesion. Interestingly, when HUVECs were induced to migrate by migration stimulatory factor bFGF, active Rac1 and cdc42 small GTPase levels were decreased dramatically at 30 min following maspin treatment. Using phosphorylated FAK at Tyr397 as an indicator of focal adhesion disassembly, maspin-treated HUVECs had elevated FAK phosphorylation compared with the mock treated control. The results were a reduction in focal adhesion disassembly and the retardation in EC migration. This study uncovers a mechanism by which maspin exerts its effect on EC adhesion and migration through an integrin signal transduction pathway.

‣ Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation*

Vogel, Sabine; Wottawa, Marieke; Farhat, Katja; Zieseniss, Anke; Schnelle, Moritz; Le-Huu, Sinja; von Ahlen, Melanie; Malz, Cordula; Camenisch, Gieri; Katschinski, Dörthe M.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.

‣ Pax6 Controls the Expression of Critical Genes Involved in Pancreatic α Cell Differentiation and Function*

Gosmain, Yvan; Marthinet, Eric; Cheyssac, Claire; Guérardel, Audrey; Mamin, Aline; Katz, Liora S.; Bouzakri, Karim; Philippe, Jacques
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased β and δ cells and absent α cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat α cells. To study the transcriptional network of Pax6 in adult and differentiated α cells, we generated Pax6-deficient primary rat α cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat α cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and α cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on α cell differentiation is independent of those of Arx and Foxa2...

‣ Identification and Characterization of the Integrin α2β1 Binding Motif in Chondroadherin Mediating Cell Attachment*

Haglund, Lisbet; Tillgren, Viveka; Addis, Laura; Wenglén, Christina; Recklies, Anneliese; Heinegård, Dick
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α2β1 and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same α2β1 receptor. We identified a cell binding motif, CQLRGLRRWLEAK318 by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK318 remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the α2β1 integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC326, mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK318. Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus...

‣ Endogenous Expression of Matriptase in Neural Progenitor Cells Promotes Cell Migration and Neuron Differentiation*

Fang, Jung-Da; Chou, Hsiao-Chin; Tung, Hsiu-Hui; Huang, Pao-Yi; Lee, Sheau-Ling
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted basement membrane, whereas overexpression of MTP promoted it. The NP cell mobilization induced by either vascular endothelial growth factor or hepatocyte growth factor was also impaired by knocking down MTP expression. MTP acts upstream of matrix metalloproteinase 2 in promoting NP cell mobility. In embryonic stem cell differentiation to neural cells, MTP knockdown had no effect on entry of embryonic stem cells into the neural lineage. High MTP expression or activity, however, shifts the population dynamics from NP cells toward neurons to favor neuronal differentiation. This is the first report to demonstrate the direct involvement of type II transmembrane serine protease in NP cell function.

‣ Immunoinhibitory Adapter Protein Src Homology Domain 3 Lymphocyte Protein 2 (SLy2) Regulates Actin Dynamics and B Cell Spreading*

von Holleben, Max; Gohla, Antje; Janssen, Klaus-Peter; Iritani, Brian M.; Beer-Hammer, Sandra
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Appropriate B cell activation is essential for adaptive immunity. In contrast to the molecular mechanisms that regulate positive signaling in immune responses, the counterbalancing negative regulatory pathways remain insufficiently understood. The Src homology domain 3 (SH3)-containing adapter protein SH3 lymphocyte protein 2 (SLy2, also known as hematopoietic adapter-containing SH3 and sterile α-motif (SAM) domains 1; HACS1) is strongly up-regulated upon B cell activation and functions as an endogenous immunoinhibitor in vivo, but the underlying molecular mechanisms of SLy2 function have been elusive. We have generated transgenic mice overexpressing SLy2 in B and T cells and have studied the biological effects of elevated SLy2 levels in Jurkat and HeLa cells. Our results demonstrate that SLy2 induces Rac1-dependent membrane ruffle formation and regulates cell spreading and polarization and that the SLy2 SH3 domain is essential for these effects. Using immunoprecipitation and confocal microscopy, we provide evidence that the actin nucleation-promoting factor cortactin is an SH3 domain-directed interaction partner of SLy2. Consistent with an important role of SLy2 for actin cytoskeletal reorganization, we further show that SLy2-transgenic B cells are severely defective in cell spreading. Together...

‣ Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion*

McCluskey, Jane T.; Hamid, Muhajir; Guo-Parke, Hong; McClenaghan, Neville H.; Gomis, Ramon; Flatt, Peter R.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca2+ channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K+ were all able to increase [Ca2+]i in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells...

‣ Down-modulation of the G-protein-coupled Estrogen Receptor, GPER, from the Cell Surface Occurs via a trans-Golgi-Proteasome Pathway*

Cheng, Shi-Bin; Quinn, Jeffrey A.; Graeber, Carl T.; Filardo, Edward J.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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GPER is a Gs-coupled seven-transmembrane receptor that has been linked to specific estrogen binding and signaling activities that are manifested by plasma membrane-associated enzymes. However, in many cell types, GPER is predominately localized to the endoplasmic reticulum (ER), and only minor amounts of receptor are detectable at the cell surface, an observation that has caused controversy regarding its role as a plasma membrane estrogen receptor. Here, we show that GPER constitutively buds intracellularly into EEA-1+ endosomes from clathrin-coated pits. Nonvisual arrestins-2/-3 do not co-localize with GPER, and expression of arrestin-2 dominant-negative mutants lacking clathrin- or β-adaptin interaction sites fails to block GPER internalization suggesting that arrestins are not involved in GPER endocytosis. Like β1AR, which recycles to the plasma membrane, GPER co-traffics with transferrin+, Rab11+ recycling endosomes. However, endocytosed GPER does not recycle to the cell surface, but instead returns to the trans-Golgi network (TGN) and does not re-enter the ER. GPER is ubiquitinated at the cell surface, exhibits a short half-life (t½ <1 h), and is protected from degradation by the proteasome inhibitor, MG132. Disruption of the TGN by brefeldin A induces the accumulation of endocytosed GPER in Rab11+ perinuclear endosomes and prevents GPER degradation. Our results provide an explanation as to why GPER is not readily detected on the cell surface in some cell types and further suggest that TGN serves as the checkpoint for degradation of endocytosed GPER.

‣ Met Receptors Induce Sam68-dependent Cell Migration by Activation of Alternate Extracellular Signal-regulated Kinase Family Members*

Locatelli, Alessia; Lange, Carol A.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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The hepatocyte growth factor (HGF)/Met receptor signaling pathway is deregulated in diverse human malignancies and plays a central role in oncogenesis, tumor progression, and invasive cancer growth. Similarly, altered expression and splicing (i.e. inclusion of variant exon 5, “v5”) of the cell adhesion marker, CD44, is associated with advanced cancer phenotypes. We sought to further understand how HGF regulates CD44v5 expression. Immortalized nontumorigenic keratinocyte (HaCaT) cells abundantly express both Met receptors and CD44v5 transmembrane glycoproteins. HGF stimulated CD44v5 protein expression and HaCaT cell migration; these events required activation of the ERK1/2 MAPK module and Sam68, a protein involved in RNA processing, splicing, and v5 inclusion. Similar to HaCaT cells, highly migratory MDA-MB-231 breast cancer cells also required Sam68 expression for HGF-induced migration. However, MDA-MB-231 cell migration occurred independently of ERK1/2 and CD44v5 expression and instead required ERK5 signaling to Sam68. Phospho-mutant, but not WT-Sam68, blocked HGF-induced cell migration in both cell types; MDA-MB-435 cells behaved similarly. These results suggest that Sam68 acts as a convergence point for ERK signaling to cell migration; blockade of phospho-Sam68 may provide a new avenue for therapeutic inhibition of metastatic cancers.

‣ Molecular Insight into How HIV-1 Vpr Protein Impairs Cell Growth through Two Genetically Distinct Pathways

Maudet, Claire; Bertrand, Matthieu; Le Rouzic, Erwann; Lahouassa, Hichem; Ayinde, Diana; Nisole, Sébastien; Goujon, Caroline; Cimarelli, Andrea; Margottin-Goguet, Florence; Transy, Catherine
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Vpr, a small HIV auxiliary protein, hijacks the CUL4 ubiquitin ligase through DCAF1 to inactivate an unknown cellular target, leading to cell cycle arrest at the G2 phase and cell death. Here we first sought to delineate the Vpr determinants involved in the binding to DCAF1 and to the target. On the one hand, the three α-helices of Vpr are necessary and sufficient for binding to DCAF1; on the other hand, nonlinear determinants in Vpr are required for binding to the target, as shown by using protein chimeras. We also underscore that a SRIG motif conserved in the C-terminal tail of Vpr proteins from HIV-1/SIVcpz and HIV-2/SIVsmm lineages is critical for G2 arrest. Our results suggest that this motif may be predictive of the ability of Vpr proteins from other SIV lineages to mediate G2 arrest. We took advantage of the characterization of a subset of G2 arrest-defective, but DCAF1 binding-proficient mutants, to investigate whether Vpr interferes with cell viability independently of its ability to induce G2 arrest. These mutants inhibited cell colony formation in HeLa cells and are cytotoxic in lymphocytes, unmasking a G2 arrest-independent cytopathic effect of Vpr. Furthermore these mutants do not block cell cycle progression at the G1 or S phases but trigger apoptosis through caspase 3. Disruption of DCAF1 binding restored efficiency of colony formation. However...

‣ Phosphorylation of Histone H2B Serine 32 Is Linked to Cell Transformation*

Lau, Andy T. Y.; Lee, Sung-Young; Xu, Yan-Ming; Zheng, Duo; Cho, Yong-Yeon; Zhu, Feng; Kim, Hong-Gyum; Li, Sheng-Qing; Zhang, Zhiguo; Bode, Ann M.; Dong, Zigang
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Various types of post-translational modifications of the histone tails have been revealed, but a few modifications have been found within the histone core sequences. Histone core post-translational modifications have the potential to modulate nucleosome structure and DNA accessibility. Here, we studied the histone H2B core domain and found that phosphorylation of H2B serine 32 occurs in normal cycling and mitogen-stimulated cells. Notably, this phosphorylation is elevated in skin cancer cell lines and tissues compared with normal counterparts. The JB6 Cl41 mouse skin epidermal cell line is a well established model for tumor promoter-induced cell transformation and was used to study the function of H2B during EGF-induced carcinogenesis. Remarkably, cells overexpressing a nonphosphorylatable H2BS32A mutant exhibited suppressed growth and EGF-induced cell transformation, possibly because of decreased activation of activator protein-1, compared with control cells overexpressing wild type H2B. We identified ribosomal S6 kinase 2 (RSK2) as the kinase responsible for H2BS32 phosphorylation. Serum-starved JB6 cells contain very little endogenous H2BS32 phosphorylation, and EGF treatment induced this phosphorylation. The phosphorylation was attenuated in RSK2 knock-out MEFs and RSK2 knockdown JB6 cells. Taken together...

‣ The Interactome of the Human Respiratory Syncytial Virus NS1 Protein Highlights Multiple Effects on Host Cell Biology

Wu, Weining; Tran, Kim C.; Teng, Michael N.; Heesom, Kate J.; Matthews, David A.; Barr, John N.; Hiscox, Julian A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2012 Português
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Viral proteins can have multiple effects on host cell biology. Human respiratory syncytial virus (HRSV) nonstructural protein 1 (NS1) is a good example of this. During the virus life cycle, NS1 can act as an antagonist of host type I and III interferon production and signaling, inhibit apoptosis, suppress dendritic cell maturation, control protein stability, and regulate transcription of host cell mRNAs, among other functions. It is likely that NS1 performs these different roles through interactions with multiple host cell proteins. To investigate this and identify cellular proteins that could interact with NS1, we used quantitative proteomics in combination with green fluorescent protein (GFP)-trap immunoprecipitation and bioinformatic analysis. This analysis identified 221 proteins that were potentially part of complexes that could interact with NS1, with many of these associated with transcriptional regulation as part of the mediator complex, cell cycle regulation, and other functions previously assigned to NS1. Specific immunoprecipitation using the GFP trap was used to confirm the ability of selected cellular proteins to interact individually with NS1. Infection of A549 cells with recombinant viruses deficient in the expression of NS1 and overexpression analysis both demonstrated that NS1 was necessary and sufficient for the enrichment of cells in the G1 phase of the cell cycle.

‣ Rgf1p Is a Specific Rho1-GEF That Coordinates Cell Polarization with Cell Wall Biogenesis in Fission Yeast

García Rodríguez, Patricia; Tajadura, Virginia; García, Ignacio; Sánchez Martín, Yolanda
Fonte: American Society for Cell Biology Publicador: American Society for Cell Biology
Tipo: Artículo Formato: 22195 bytes; application/pdf
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Copyright © by American Society for Cell Biology.-- Final full-text version of the paper available at: http://www.molbiolcell.org/content/vol17/issue4/; Rho1p regulates cell integrity by controlling the actin cytoskeleton and cell wall synthesis. We have identified a new GEF, designated Rgf1p, which specifically regulates Rho1p during polarized growth. The phenotype of rgf1 null cells was very similar to that seen after depletion of Rho1p, 30% of cells being lysed. In addition, rgf1+ deletion caused hypersensitivity to the antifungal drug Caspofungin and defects in the establishment of bipolar growth. rho1+, but none of the other GTPases of the Rho-family, suppressed the rgf1Δ phenotypes. Moreover, deletion of rgf1+ suppressed the severe growth defect in rga1+ null mutants (a Rho1-GAP, negative regulator). Rgf1p and Rho1p coimmunoprecipitated and overexpression of rgf1+ specifically increased the GTP-bound Rho1p; it caused changes in cell morphology, and a large increase in β(1,3)-glucan synthase activity. These effects were similar to those elicited when the hyperactive rho1-G15V allele was expressed. A genetic relationship was observed between Rgf1p, Bgs4p (β[1,3]-glucan synthase), and Pck1p (protein kinase C [PKC] homologue); Bgs4p and Pck1p suppressed the hypersensitivity to Caspofungin in rgf1Δ mutants. Rgf1p localized to the growing ends and the septum...