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Functions of Cyclin A1 in the Cell Cycle and Its Interactions with Transcription Factor E2F-1 and the Rb Family of Proteins

Yang, Rong; Müller, Carsten; Huynh, Vong; Fung, Yuen K.; Yee, Amy S.; Koeffler, H. Phillip
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/1999 Português
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857.02625%
Human cyclin A1, a newly discovered cyclin, is expressed in testis and is thought to function in the meiotic cell cycle. Here, we show that the expression of human cyclin A1 and cyclin A1-associated kinase activities was regulated during the mitotic cell cycle. In the osteosarcoma cell line MG63, cyclin A1 mRNA and protein were present at very low levels in cells at the G0 phase. They increased during the progression of the cell cycle and reached the highest levels in the S and G2/M phases. Furthermore, the cyclin A1-associated histone H1 kinase activity peaked at the G2/M phase. We report that cyclin A1 could bind to important cell cycle regulators: the Rb family of proteins, the transcription factor E2F-1, and the p21 family of proteins. The in vitro interaction of cyclin A1 with E2F-1 was greatly enhanced when cyclin A1 was complexed with CDK2. Associations of cyclin A1 with Rb and E2F-1 were observed in vivo in several cell lines. When cyclin A1 was coexpressed with CDK2 in sf9 insect cells, the CDK2-cyclin A1 complex had kinase activities for histone H1, E2F-1, and the Rb family of proteins. Our results suggest that the Rb family of proteins and E2F-1 may be important targets for phosphorylation by the cyclin A1-associated kinase. Cyclin A1 may function in the mitotic cell cycle in certain cells.

Induced Focal Adhesion Kinase (FAK) Expression in FAK-Null Cells Enhances Cell Spreading and Migration Requiring Both Auto- and Activation Loop Phosphorylation Sites and Inhibits Adhesion-Dependent Tyrosine Phosphorylation of Pyk2

Owen, James D.; Ruest, Paul J.; Fry, David W.; Hanks, Steven K.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/1999 Português
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856.774%
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration...

Distinct Pathways of Cell Migration and Antiapoptotic Response to Epithelial Injury: Structure-Function Analysis of Human Intestinal Trefoil Factor

Kinoshita, Koichi; Taupin, Douglas R.; Itoh, Hiroshi; Podolsky, Daniel K.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2000 Português
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860.50695%
The trefoil peptide intestinal trefoil factor (ITF) plays a critical role in the protection of colonic mucosa and is essential to restitution after epithelial damage. These functional properties are accomplished through coordinated promotion of cell migration and inhibition of apoptosis. ITF contains a unique three-looped trefoil motif formed by intrachain disulfide bonds among six conserved cysteine residues, which is thought to contribute to its marked protease resistance. ITF also has a seventh cysteine residue, which permits homodimer formation. A series of cysteine-to-serine substitutions and a C-terminally truncated ITF were made by PCR site-directed mutagenesis. Any alteration of the trefoil motif or truncation resulted in loss of protease resistance. However, neither an intact trefoil domain nor dimerization was required to promote cell migration. This pro-restitution activity correlated with the ability of the ITF mutants to activate mitogen-activated protein (MAP) kinase independent of phosphorylation of the epidermal growth factor (EGF) receptor. In contrast, only intact ITF retained both phosphatidylinositol 3-kinase and the EGF receptor-dependent antiapoptotic effect in HCT116 and IEC-6 cells. The inability to block apoptosis correlated with a loss of trefoil peptide-induced transactivation of the EGF receptor or Akt kinase in HT-29 cells. In addition to defining structural requirements for the functional properties of ITF...

FRL, a Novel Formin-Related Protein, Binds to Rac and Regulates Cell Motility and Survival of Macrophages

Yayoshi-Yamamoto, Shinri; Taniuchi, Ichiro; Watanabe, Takeshi
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/2000 Português
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857.0895%
We have isolated a cDNA, frl (formin-related gene in leukocytes), a novel mammalian member of the formin gene family. The frl cDNA encodes a 160-kDa protein, FRL, that possesses FH1, FH2, and FH3 domains that are well conserved among other Formin-related proteins. An FRL protein is mainly localized in the cytosol and is highly expressed in spleen, lymph node, and bone marrow cells. Formin-related genes and proteins have been reported to play crucial roles in morphogenesis, cell polarity, and cytokinesis through interaction with Rho family small GTPases. FRL binds to Rac at its N-terminal region including the FH3 domain and associates with profilin at the FH1 domain. In a macrophage cell line, P388D1, overexpression of a truncated form of FRL containing only the FH3 domain (FH3-FRL) strongly inhibited cell adhesion to fibronectin and migration upon stimulation with a chemokine. Moreover, expression of the truncated FH3-FRL protein resulted in apoptotic cell death of P388D1 cells, suggesting that the truncated FH3-FRL protein may interfere with signals of FRL. Overexpression in the P388D1 cells of full-length FRL or of the truncated protein containing the FH3 and FH1 domains, with simultaneous expression of the truncated FH3-FRL protein...

Negative Regulation of Protein Translation by Mitogen-Activated Protein Kinase-Interacting Kinases 1 and 2

Knauf, Ursula; Tschopp, Claude; Gram, Hermann
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2001 Português
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858.1149%
Eukaryotic initiation factor 4E (eIF4E) is a key component of the translational machinery and an important modulator of cell growth and proliferation. The activity of eIF4E is thought to be regulated by interaction with inhibitory binding proteins (4E-BPs) and phosphorylation by mitogen-activated protein (MAP) kinase-interacting kinase (MNK) on Ser209 in response to mitogens and cellular stress. Here we demonstrate that phosphorylation of eIF4E via MNK1 is mediated via the activation of either the Erk or p38 pathway. We further show that expression of active mutants of MNK1 and MNK2 in 293 cells diminishes cap-dependent translation relative to cap-independent translation in a transient reporter assay. The same effect on cap-dependent translation was observed when MNK1 was activated by the Erk or p38 pathway. In line with these findings, addition of recombinant active MNK1 to rabbit reticulocyte lysate resulted in a reduced protein synthesis in vitro, and overexpression of MNK2 caused a decreased rate of protein synthesis in 293 cells. By using CGP 57380, a novel low-molecular-weight kinase inhibitor of MNK1, we demonstrate that eIF4E phosphorylation is not crucial to the formation of the initiation complex, mitogen-stimulated increase in cap-dependent translation...

E2F1 and E2F2 Determine Thresholds for Antigen-Induced T-Cell Proliferation and Suppress Tumorigenesis

Zhu, Jing W.; Field, Seth J.; Gore, Lia; Thompson, Margaret; Yang, Haidi; Fujiwara, Yuko; Cardiff, Robert D.; Greenberg, Michael; Orkin, Stuart H.; DeGregori, James
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2001 Português
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860.0382%
E2F activity is critical for the control of the G1 to S phase transition. We show that the combined loss of E2F1 and E2F2 results in profound effects on hematopoietic cell proliferation and differentiation, as well as increased tumorigenesis and decreased lymphocyte tolerance. The loss of E2F1 and E2F2 impedes B-cell differentiation, and hematopoietic progenitor cells in the bone marrow of mice lacking E2F1 and E2F2 exhibit increased cell cycling. Importantly, we show that E2F1 and E2F2 double-knockout T cells exhibit more rapid entry into S phase following antigenic stimulation. Furthermore, T cells lacking E2F1 and E2F2 proliferate much more extensively in response to subthreshold antigenic stimulation. Consistent with these observations, E2F1/E2F2 mutant mice are highly predisposed to the development of tumors, and some mice exhibit signs of autoimmunity.

Influence of Cell Cycle and Oncogene Activity upon Topoisomerase IIα Expression and Drug Toxicity

Stacey, Dennis W.; Hitomi, Masahiro; Chen, Guan
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2000 Português
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856.9405%
The cell cycle, oncogenic signaling, and topoisomerase (topo) IIα levels all influence sensitivity to anti-topo II drugs. Because the cell cycle and oncogenic signaling influence each other as well as topo IIα levels, it is difficult to assess the importance of any one of these factors independently of the others during drug treatment. Such information, however, is vital to an understanding of the cellular basis of drug toxicity. We, therefore, developed a series of analytical procedures to individually assess the role of each of these factors during treatment with the anti-topo II drug etoposide. All studies were performed with asynchronously proliferating cultures by the use of time-lapse and quantitative fluorescence staining procedures. To our surprise, we found that neither oncogene action nor the cell cycle altered topo IIα protein levels in actively cycling cells. Only a minor population of slowly cycling cells within these cultures responded to constitutively active oncogenes by elevating topo IIα production. Thus, it was possible to study the effects of the cell cycle and oncogene action on drug-treated cells while topo IIα levels remained constant. Toxicity analyses were performed with two consecutive time-lapse observations separated by a brief drug treatment. The cell cycle phase was determined from the first observation...

Inhibition of PrKX, a Novel Protein Kinase, and the Cyclic AMP-Dependent Protein Kinase PKA by the Regulatory Proteins of Adeno-Associated Virus Type 2

Chiorini, John A.; Zimmermann, Bastian; Yang, Linda; Smith, Richard H.; Ahearn, Aaron; Herberg, Friedrich; Kotin, Robert M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/1998 Português
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855.9852%
Adeno-associated virus encodes four nonstructural proteins, which are known as Rep78, Rep68, Rep52, and Rep40. Expression of these nonstructural proteins affects cell growth and gene expression through processes that have not yet been characterized. Using a yeast two-hybrid screen, we have demonstrated that a stable interaction occurs between the viral proteins Rep78 and Rep52 and the putative protein kinase PrKX, which is encoded on the X chromosome. The stability and specificity of the Rep-PrKX interaction were confirmed by coimmunoprecipitation of complexes assembled in vitro and in vivo. Overexpressed PrKX, which was purified from cos cells, was shown to phosphorylate a synthetic protein kinase A (PKA) substrate. However, this activity was dramatically inhibited by stoichiometric amounts of Rep52 and weakly inhibited with Rep68, which lacks the carboxy-terminal sequence contained in Rep52. Similarly, a stable interaction was observed with Rep78, which also contains the carboxy-terminal sequence of Rep52. A stable interaction and inhibition were also observed between Rep52 and the catalytic subunit of PKA. By using surface plasmon resonance and kinetic studies, Kis of approximately 300 and 167 nM were calculated for Rep52 with PKA and with PrKX...

ASAP1, a Phospholipid-Dependent Arf GTPase-Activating Protein That Associates with and Is Phosphorylated by Src

Brown, Megan T.; Andrade, Josefa; Radhakrishna, Harish; Donaldson, Julie G.; Cooper, Jonathan A.; Randazzo, Paul A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/1998 Português
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858.3452%
Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4,5-biphosphate (PIP2)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP2-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization...

Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

Ueki, Kohjiro; Fruman, David A.; Brachmann, Saskia M.; Tseng, Yu-Hua; Cantley, Lewis C.; Kahn, C. Ronald
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2002 Português
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861.1641%
Class Ia phosphoinositide (PI) 3-kinase is a central component in growth factor signaling and is comprised of a p110 catalytic subunit and a regulatory subunit, the most common family of which is derived from the p85α gene (Pik3r1). Optimal signaling through the PI 3-kinase pathway depends on a critical molecular balance between the regulatory and catalytic subunits. In wild-type cells, the p85 subunit is more abundant than p110, leading to competition between the p85 monomer and the p85-p110 dimer and ineffective signaling. Heterozygous disruption of Pik3r1 results in increased Akt activity and decreased apoptosis by insulin-like growth factor 1 (IGF-1) through up-regulated phosphatidylinositol (3,4,5)-triphosphate production. Complete depletion of p85α, on the other hand, results in significantly increased apoptosis due to reduced PI 3-kinase-dependent signaling. Thus, a reduction in p85α represents a novel therapeutic target for enhancing IGF-1/insulin signaling, prolongation of cell survival, and protection against apoptosis.

NF-κB Activates Prostate-Specific Antigen Expression and Is Upregulated in Androgen-Independent Prostate Cancer

Chen, Charlie D.; Sawyers, Charles L.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2002 Português
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860.3684%
The transcription factor NF-κB regulates gene expression involved in cell growth and survival and has been implicated in progression of hormone-independent breast cancer. By expressing a dominant-active form of mitogen-activated protein kinase kinase kinase 1, by exposure to tumor necrosis factor alpha, or by overexpression of p50/p65, we show that NF-κB activates a transcription regulatory element of the prostate-specific antigen (PSA)-encoding gene, a marker for prostate cancer development, treatment, and progression. By DNase I footprinting, we identified four NF-κB binding sites in the PSA core enhancer. We also demonstrate that androgen-independent prostate cancer xenografts have higher constitutive NF-κB binding activity than their androgen-dependent counterparts. These results suggest a role of NF-κB in prostate cancer progression.

The Immunosuppressant Rapamycin Mimics a Starvation-Like Signal Distinct from Amino Acid and Glucose Deprivation

Peng, Tao; Golub, Todd R.; Sabatini, David M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2002 Português
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856.05484%
RAFT1/FRAP/mTOR is a key regulator of cell growth and division and the mammalian target of rapamycin, an immunosuppressive and anticancer drug. Rapamycin deprivation and nutrient deprivation have similar effects on the activity of S6 kinase 1 (S6K1) and 4E-BP1, two downstream effectors of RAFT1, but the relationship between nutrient- and rapamycin-sensitive pathways is unknown. Using transcriptional profiling, we show that, in human BJAB B-lymphoma cells and murine CTLL-2 T lymphocytes, rapamycin treatment affects the expression of many genes involved in nutrient and protein metabolism. The rapamycin-induced transcriptional profile is distinct from those induced by glucose, glutamine, or leucine deprivation but is most similar to that induced by amino acid deprivation. In particular, rapamycin treatment and amino acid deprivation up-regulate genes involved in nutrient catabolism and energy production and down-regulate genes participating in lipid and nucleotide synthesis and in protein synthesis, turnover, and folding. Surprisingly, however, rapamycin had effects opposite from those of amino acid starvation on the expression of a large group of genes involved in the synthesis, transport, and use of amino acids. Supported by measurements of nutrient use...

Inactivation of the Retinoblastoma Protein Family Can Bypass the HCF-1 Defect in tsBN67 Cell Proliferation and Cytokinesis

Reilly, Patrick T.; Wysocka, Joanna; Herr, Winship
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2002 Português
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857.09766%
Owing to a single missense mutation in the cell proliferation factor HCF-1, the temperature-sensitive tsBN67 hamster cell line arrests proliferation at nonpermissive temperatures, primarily in a G0/G1 state, and displays temperature-sensitive cytokinesis defects. The HCF-1 mutation in tsBN67 cells also causes a temperature-sensitive dissociation of HCF-1 from chromatin prior to cell proliferation arrest, suggesting that HCF-1-chromatin association is important for mammalian-cell proliferation. Here, we report that the simian virus 40 (SV40) early region, in particular, large T antigen (Tag), and the adenovirus oncoprotein E1A can rescue the tsBN67 cell proliferation defect at nonpermissive temperatures. The SV40 early region rescues the tsBN67 cell proliferation defect without restoring the HCF-1-chromatin association, indicating that these oncoproteins bypass a requirement for HCF-1 function. The SV40 early region also rescues the tsBN67 cytokinesis defect, suggesting that the roles of HCF-1 in cell proliferation and proper cytokinesis are intimately linked. The ability of SV40 Tag and adenovirus E1A to inactivate members of the pRb protein family—pRb, p107, and p130—is important for the bypass of HCF-1 function. These results suggest that HCF-1 regulates mammalian-cell proliferation and cytokinesis...

CDK9 Is Constitutively Expressed throughout the Cell Cycle, and Its Steady-State Expression Is Independent of SKP2

Garriga, Judit; Bhattacharya, Sabyasachi; Calbó, Joaquim; Marshall, Renée M.; Truongcao, May; Haines, Dale S.; Graña, Xavier
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2003 Português
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857.09766%
CDK9 is a CDC2-related kinase and the catalytic subunit of the positive-transcription elongation factor b and the Tat-activating kinase. It has recently been reported that CDK9 is a short-lived protein whose levels are regulated during the cell cycle by the SCFSKP2 ubiquitin ligase complex (R. E. Kiernan et al., Mol. Cell. Biol. 21:7956-7970, 2001). The results presented here are in contrast to those observations. CDK9 protein levels remained unchanged in human cells entering and progressing through the cell cycle from G0, despite dramatic changes in SKP2 expression. CDK9 levels also remained unchanged in cells exiting from mitosis and progressing through the next cell cycle. Similarly, the levels of CDK9 protein did not change as cells exited the cell cycle and differentiated along various lineages. In keeping with these observations, the kinase activity associated with CDK9 was found to not be regulated during the cell cycle. We have also found that endogenous CDK9 is a very stable protein with a half-life (t1/2) of 4 to 7 h, depending on the cell type. In contrast, when CDK9 is overexpressed, it is not stabilized and is rapidly degraded, with a t1/2 of less than 1 h, depending on the level of expression. Treatment of cells with proteasome inhibitors blocked the degradation of short-lived proteins...

ΔNp73 Facilitates Cell Immortalization and Cooperates with Oncogenic Ras in Cellular Transformation In Vivo

Petrenko, Oleksi; Zaika, Alexander; Moll, Ute M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2003 Português
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861.2105%
TP73, despite significant homology to TP53, is not a classic tumor suppressor gene, since it exhibits upregulation of nonmutated products in human tumors and lacks a tumor phenotype in p73-deficient mice. We recently reported that an N-terminally truncated isoform, ΔNp73, is upregulated in breast and gynecological cancers. We further showed that ΔNp73 is a potent transdominant inhibitor of wild-type p53 and TAp73 in cultured human tumor cells by efficiently counteracting their target gene transactivations, apoptosis, and growth suppression functions (A. I. Zaika et al., J. Exp. Med. 6:765-780, 2002). Although these data strongly suggest oncogenic properties of ΔNp73, this can only be directly shown in primary cells. We report here that ΔNp73 confers resistance to spontaneous replicative senescence of primary mouse embryo fibroblasts (MEFs) and immortalizes MEFs at a 1,000-fold-higher frequency than occurs spontaneously. ΔNp73 cooperates with cMyc and E1A in promoting primary cell proliferation and colony formation and compromises p53-dependent MEF apoptosis. Importantly, ΔNp73 rescues Ras-induced senescence. Moreover, ΔNp73 cooperates with oncogenic Ras in transforming primary fibroblasts in vitro and in inducing MEF-derived fibrosarcomas in vivo in nude mice. Wild-type p53 is likely a major target of ΔNp73 inhibition in primary fibroblasts since deletion of p53 or its requisite upstream activator ARF abrogates the growth-promoting effect of ΔNp73. Taken together...

Human Heterochromatin Protein 1 Isoforms HP1Hsα and HP1Hsβ Interfere with hTERT-Telomere Interactions and Correlate with Changes in Cell Growth and Response to Ionizing Radiation

Sharma, Girdhar G.; Hwang, Kyu-kye; Pandita, Raj K.; Gupta, Arun; Dhar, Sonu; Parenteau, Julie; Agarwal, Manjula; Worman, Howard J.; Wellinger, Raymund J.; Pandita, Tej K.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2003 Português
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860.6301%
Telomeres are associated with the nuclear matrix and are thought to be heterochromatic. We show here that in human cells the overexpression of green fluorescent protein-tagged heterochromatin protein 1 (GFP-HP1) or nontagged HP1 isoforms HP1Hsα or HP1Hsβ, but not HP1Hsγ, results in decreased association of a catalytic unit of telomerase (hTERT) with telomeres. However, reduction of the G overhangs and overall telomere sizes was found in cells overexpressing any of these three proteins. Cells overexpressing HP1Hsα or HP1Hsβ also display a higher frequency of chromosome end-to-end associations and spontaneous chromosomal damage than the parental cells. None of these effects were observed in cells expressing mutants of GFP-ΔHP1Hsα, GFP-ΔHP1Hsβ, or GFP-ΔHP1Hsγ that had their chromodomains deleted. An increase in the cell population doubling time and higher sensitivity to cell killing by ionizing radiation (IR) treatment was also observed for cells overexpressing HP1Hsα or HP1Hsβ. In contrast, cells expressing mutant GFP-ΔHP1Hsα or GFP-ΔHP1Hsβ showed a decrease in population doubling time and decreased sensitivity to IR compared to the parental cells. The effects on cell doubling times were paralleled by effects on tumorigenicity in mice: overexpression of HP1Hsα or HP1Hsβ suppressed tumorigenicity...

Ribosomal Protein L11 Negatively Regulates Oncoprotein MDM2 and Mediates a p53-Dependent Ribosomal-Stress Checkpoint Pathway

Zhang, Yanping; White Wolf, Gabrielle; Bhat, Krishna; Jin, Aiwen; Allio, Theresa; Burkhart, William A.; Xiong, Yue
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2003 Português
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858.04734%
The gene encoding p53 mediates a major tumor suppression pathway that is frequently altered in human cancers. p53 function is kept at a low level during normal cell growth and is activated in response to various cellular stresses. The MDM2 oncoprotein plays a key role in negatively regulating p53 activity by either direct repression of p53 transactivation activity in the nucleus or promotion of p53 degradation in the cytoplasm. DNA damage and oncogenic insults, the two best-characterized p53-dependent checkpoint pathways, both activate p53 through inhibition of MDM2. Here we report that the human homologue of MDM2, HDM2, binds to ribosomal protein L11. L11 binds a central region in HDM2 that is distinct from the ARF binding site. We show that the functional consequence of L11-HDM2 association, like that with ARF, results in the prevention of HDM2-mediated p53 ubiquitination and degradation, subsequently restoring p53-mediated transactivation, accumulating p21 protein levels, and inducing a p53-dependent cell cycle arrest by canceling the inhibitory function of HDM2. Interference with ribosomal biogenesis by a low concentration of actinomycin D is associated with an increased L11-HDM2 interaction and subsequent p53 stabilization. We suggest that L11 functions as a negative regulator of HDM2 and that there might exist in vivo an L11-HDM2-p53 pathway for monitoring ribosomal integrity.

SWAP-70 Regulates c-kit-Induced Mast Cell Activation, Cell-Cell Adhesion, and Migration†

Sivalenka, Raja Rajeswari; Jessberger, Rolf
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2004 Português
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857.0601%
SWAP-70, an unusual phosphatidylinositol-3-kinase-dependent protein that interacts with the RhoGTPase Rac, is highly expressed in mast cells. Cultured bone marrow mast cells (BMMC) from SWAP-70−/− mice are reduced in FcɛRI-triggered degranulation. This report describes the hitherto-unknown role of SWAP-70 in c-kit receptor signaling, a key proliferation and differentiation pathway in mast cells. Consistent with the role of Rac in cell motility and regulation of the actin cytoskeleton, mutant cells show abnormal actin rearrangements and are deficient in migration in vitro and in vivo. SWAP-70−/− BMMC are impaired in calcium flux, in proper translocation and activity of Akt kinase (required for mast cell activation and survival), and in translocation of Rac1 and Rac2 upon c-kit stimulation. Adhesion to fibronectin is reduced, but homotypic cell association induced through c-kit is strongly increased in SWAP-70−/− BMMC. Homotypic association requires extracellular Ca2+ and depends on the integrin αLβ2 (LFA-1). ERK is hyperactivated upon c-kit signaling in adherent and dispersed mutant cells. Together, we suggest that SWAP-70 is an important regulator of specific effector pathways in c-kit signaling, including mast cell activation...

Brk Activates Rac1 and Promotes Cell Migration and Invasion by Phosphorylating Paxillin

Chen, Hsin-Yi; Shen, Che-Hung; Tsai, Yuh-Tyng; Lin, Feng-Chi; Huang, Yuan-Ping; Chen, Ruey-Hwa
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2004 Português
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859.7858%
Brk (for breast tumor kinase) is a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. Brk was originally identified from a human metastatic breast tumor, and its overexpression is frequently observed in breast cancer and several other cancer types. However, the molecular mechanism by which this kinase participates in tumorigenesis remains poorly characterized. In the present study, we not only identified paxillin as the binding partner and substrate of Brk but also discovered a novel signaling pathway by which Brk mediates epidermal growth factor (EGF)-induced paxillin phosphorylation. We show that EGF stimulation activates the catalytic activity of Brk, which in turn phosphorylates paxillin at Y31 and Y118. These phosphorylation events promote the activation of small GTPase Rac1 via the function of CrkII. Through this pathway, Brk is capable of promoting cell motility and invasion and functions as a mediator of EGF-induced migration and invasion. In accordance with these functional roles, Brk translocates to membrane ruffles, where it colocalizes with paxillin during cell migration. Together, our findings identify novel signaling and biological roles of Brk and indicate the first potential link between Brk and metastatic malignancy.

HMG-I/Y, a New c-Myc Target Gene and Potential Oncogene

Wood, Lisa J.; Mukherjee, Mita; Dolde, Christine E.; Xu, Yi; Maher, Joseph F.; Bunton, Tracie E.; Williams, John B.; Resar, Linda M. S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2000 Português
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859.6831%
The HMG-I/Y gene encodes the HMG-I and HMG-Y proteins, which function as architectural chromatin binding proteins important in the transcriptional regulation of several genes. Although increased expression of the HMG-I/Y proteins is associated with cellular proliferation, neoplastic transformation, and several human cancers, the role of these proteins in the pathogenesis of malignancy remains unclear. To better understand the role of these proteins in cell growth and transformation, we have been studying the regulation and function of HMG-I/Y. The HMG-I/Y promoter was cloned, sequenced, and subjected to mutagenesis analysis. A c-Myc–Max consensus DNA binding site was identified as an element important in the serum stimulation of HMG-I/Y. The oncoprotein c-Myc and its protein partner Max bind to this site in vitro and activate transcription in transfection experiments. HMG-I/Y expression is stimulated by c-Myc in a Myc-estradiol receptor cell line in the presence of the protein synthesis inhibitor cycloheximide, indicating that HMG-I/Y is a direct c-Myc target gene. HMG-I/Y induction is decreased in Myc-deficient fibroblasts. HMG-I/Y protein expression is also increased in Burkitt's lymphoma cell lines, which are known to have increased c-Myc protein. Like Myc...