Biomedical subjects

George R Stark

Publications and source records attributed to George R Stark.

34 records · Page 2Linked to original sources

PubMed2004

Identification of a human NF-kappaB-activating protein, TAB3.

The NF-kappaB pathway plays a critical role in regulating cellular processes such as immune responses, stress responses, apoptosis, proliferation and differentiation, whereas dysfunction of this pathway has been associated with numerous cancer and immune disorders. We have applied our Random Activation of Gene Expression technology to an NF-kappaB reporter cell line to facilitate the discovery of positive regulators of NF-kappaB activation. A small protein expression library, corresponding to approximately 0.1x genome coverage, was generated and screened for clones exhibiting constitutive activation of NF-kappaB. After isolation of cellular clones displaying the relevant phenotypes, we identified two known components of the NF-kappaB pathway and a hypothetical gene that we have designated the human ortholog of Xenopus TAK1-binding protein 3 (TAB3). Overexpression of human TAB3 was found to activate both NF-kappaB and AP-1 transcription factors. Furthermore, the activation of NF-kappaB by TAB3 was blocked by the NF-kappaB inhibitor, SN50, and by expression of dominant-negative forms of tumor necrosis factor alpha-associated factor 6 and transforming growth factor beta-activated kinase. Taken together, these data demonstrate that TAB3 transforming growth factor is a constituent of the NF-kappaB pathway functioning upstream of tumor necrosis factor alpha-associated factor 6/transforming growth factor beta-activated kinase. Interestingly, increased expression of TAB3 was found in some cancer tissues, and its overexpression in NIH 3T3 cells resulted in cellular transformation, thus establishing a causative link between elevated TAB3 expression, constitutive NF-kappaB activation, and oncogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Analyzing the G2/M checkpoint.

The G2 checkpoint prevents cells from entering mitosis when DNA is damaged, providing an opportunity for repair and stopping the proliferation of damaged cells. Because the G2 checkpoint helps to maintain genomic stability, it is an important focus in understanding the molecular causes of cancer. Many different methods have been used to investigate the G2 checkpoint and uncover some of the underlying mechanisms. Because cell-cycle controls are highly conserved, a remarkable synergy between the genetic power of model organisms and biochemical analyses is possible and has uncovered control mechanisms that operate in many diverse species, including humans. Cdc2, the cyclin-dependent kinase that normally drives cells into mitosis, is an important target of pathways that mediate rapid arrest in G2 in response to DNA damage. Additional pathways ensure that the arrest is stably maintained. When mammalian cells contain damaged DNA, the p53 tumor suppressor and the Rb family of transcriptional repressors work together to downregulate a large number of genes that encode proteins required for G2 and M. Elimination of these essential cell cycle proteins helps to keep the cells arrested in G2.

Animals↗

PubMed2003

Mutant human cells with constitutive activation of NF-kappaB.

We have used a genetic approach to generate eight different mutant human cell lines in which NF-kappaB is constitutively activated. These independent clones have different phenotypes and belong to several different genetic complementation groups. In one clone inhibitor of kappaB(IkappaB) kinase is constitutively active, but in the seven others it is not, despite the fact that IkappaB is degraded in all eight clones. Thus, IkappaB kinase-independent mechanisms of IkappaB degradation and NF-kappaB activation are predominant in these mutants. Biochemical analyses of the mutants revealed that they fall into at least five different categories, differing in the sets of upstream kinases that are activated, confirming multiple mechanisms of NF-kappaB activation. By introducing a retroviral cDNA library into the Ras C6 cell line, with constitutively active NF-kappaB, followed by selection for functional complementation, we isolated a cDNA encoding a C-terminal fragment of enolase 1 and identified it as negative regulator of NF-kappaB.

Cell Line↗

PubMed2003

SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling.

The Toll-like receptor-interleukin 1 receptor signaling (TLR-IL-1R) receptor superfamily is important in differentially recognizing pathogen products and eliciting appropriate immune responses. These receptors alter gene expression, mainly through the activation of nuclear factor-kappaB and activating protein 1. SIGIRR (single immunoglobulin IL-1R-related molecule), a member of this family that does not activate these factors, instead negatively modulates immune responses. Inflammation is enhanced in SIGIRR-deficient mice, as shown by their enhanced chemokine induction after IL-1 injection and reduced threshold for lethal endotoxin challenge. Cells from SIGIRR-deficient mice showed enhanced activation in response to either IL-1 or certain Toll ligands. Finally, biochemical analysis indicated that SIGIRR binds to the TLR-IL-1R signaling components in a ligand-dependent way. Our data show that SIGIRR functions as a biologically important modulator of TLR-IL-1R signaling.

Animals↗

PubMed2003

The cell death regulator GRIM-19 is an inhibitor of signal transducer and activator of transcription 3.

GRIM-19 (gene associated with retinoid-IFN-induced mortality 19), isolated as a cell death activator in a genetic screen used to define mechanisms involved in IFN-beta- and retinoic acid-induced cell death, codes for a approximately 16-kDa protein that induces apoptosis in a number of cell lines. Antisense ablation of GRIM-19 caused resistance to cell death induced by IFN plus retinoic acid and conferred a growth advantage to cells. To understand the molecular bases for its cell death regulatory activity, we used a yeast two-hybrid screen and identified that the transcription factor STAT3 (signal transducer and activator of transcription 3) binds to GRIM-19. GRIM-19 inhibits transcription driven by activation of STAT3, but not STAT1. It neither inhibits the ligand-induced activation of STAT3 nor blocks its ability to bind to DNA. Mutational analysis indicates that the transactivation domain of STAT3, especially residue S727, is required for GRIM-19 binding. Because GRIM-19 does not bind significantly to other STATs, our studies identify a specific inhibitor of STAT3. Because constitutively active STAT3 up-regulates antiapoptotic genes to promote tumor survival, its inhibition by GRIM-19 also demonstrates an antioncogenic effect exerted by biological therapeutics.

Animals↗

PubMed2003

G2 arrest in response to topoisomerase II inhibitors: the role of p53.

We have previously found that the overexpression of p53 causes G(2) arrest and represses the synthesis of cyclin-dependent kinase 1 and cyclin B1, two proteins required for cells to traverse from G(2) into M. G(2) arrest occurs in response to DNA damage caused by a variety of agents and treatments. Here, we investigate the role of p53 in the G(2) arrest that occurs in response to the topoisomerase inhibitors etoposide and merbarone. In HT1080 cells expressing a dominant-negative form of p53, treatment with etoposide still caused G(2) arrest, but the arrest could be overcome by additional treatment with caffeine, which inhibits the damage-responsive kinases ataxia telangiectasia mutated (ATM) and atm and rad3-related (ATR). However, caffeine could not overcome etoposide-induced G(2) arrest in HT1080 cells with functional p53. We conclude that etoposide activates two pathways, one of which depends on p53 and the other of which is sensitive to caffeine, and that either pathway is sufficient to activate G(2) arrest. Etoposide inhibits topoisomerase II by trapping the enzyme in a complex with cleaved DNA. Inhibition of topoisomerase II with merbarone, which does not stabilize a cleavage complex, causes G(2) arrest by a checkpoint that monitors the decatenation of chromatin. We find that caffeine can abrogate merbarone-induced G(2) arrest even in cells with functional p53, indicating that p53 does not contribute to the decatenation-sensitive response. Thus, p53 has a differential role in effecting G(2) arrest in response to topoisomerase II inhibitors, depending upon the mechanisms of action of the inhibitors tested.

CDC2 Protein Kinase↗

PubMed2003

IRAK-dependent phosphorylation of Stat1 on serine 727 in response to interleukin-1 and effects on gene expression.

Interleukin-1 (IL-1) induces the phosphorylation of Stat1 on serine 727 but not on tyrosine 701. Analyses of mutant I1A cells, which lack the IL-1 receptor-associated kinase (IRAK), and of I1A cells reconstituted with deletion mutants of IRAK show that the IL-1-mediated phosphorylation of Stat1 on serine requires the IRAK protein but not its kinase activity and does not involve phosphatidylinositol-3'-kinase (PI3K) or the mitogen-activated protein (MAP) kinases p38 or ERK. IRAK and Stat1 interact in vivo, and this interaction is increased in response to IL-1, suggesting that IRAK may serve to recruit the as yet unknown IL-1-induced Stat1 serine kinase. Chemical inhibitors or dominant-negative forms of signaling components required to activate NF-kappa B, ATF, or AP-1 in response to IL-1 do not affect the phosphorylation of Stat1 on serine. IL-1 and tumor necrosis factor (TNF) enhance the serine phosphorylation of Stat1 that occurs in response to interferon-gamma (IFN-gamma) and potentiate IFN-gamma-mediated, Stat1-driven gene expression, thus contributing to the synergistic activities of these proinflammatory cytokines.

Animals↗

PubMed2002

Requirement of phosphoinositide 3-kinase and Akt for interferon-beta-mediated induction of the beta-R1 (SCYB11) gene.

We previously reported (Rani, M. R., Asthagiri, A. R., Singh, A., Sizemore, N., Sathe, S. S., Li, X., DiDonato, J. D., Stark, G. R., and Ransohoff, R. M. (2001) J. Biol. Chem. 276, 44365-44368) that IFN-beta induction of beta-R1 in fibrosarcoma cells required transcription factors ISGF-3 and NF-kappa B. IFN-beta treatment did not augment the abundance of NF-kappa B, but led to phosphorylation of the NF-kappa B subunit p65 and induced a nuclear activity capable of phosphorylating a p65-GST fusion construct in the carboxy-terminal transactivation domain (TAD), residues 354-551. We now present evidence for the involvement of phosphoinositide 3-kinase (PI3K) in this pathway. Pretreatment of HT1080-derived fibrosarcoma cells with pharmacological inhibitors of PI3K (wortmannin or LY294002) selectively inhibited IFN-beta-induced beta-R1 mRNA accumulation in a dose-dependent manner. In stably transfected cell lines, bovine p85, the regulatory subunit of PI3K, functioned as a dominant-negative inhibitor of interferon (IFN) signaling via PI3K and selectively suppressed IFN-beta-mediated induction of beta-R1. Overexpression of PTEN (phosphatase and tensin homologue mutated on chromosome ten), an antagonist of PI3K, blocked induction of a beta-R1 promoter-reporter construct. Studies with PTEN mutants suggested that the lipid kinase activity of PI3K was essential for IFN-beta-induced transcription of beta-R1. Consistent with this finding, a dominant-negative mutant of the serine-threonine kinase Akt, a downstream effector of PI3K, selectively blocked IFN-beta-mediated induction of the beta-R1 promoter reporter. Furthermore, IFN-beta-mediated phosphorylation of GST-p65 was blocked by pretreatment with LY294002. These data suggest that IFN-beta acts through PI3K to enhance the transactivation competence of NF-kappa B complexes through phosphorylation of p65 within the TAD. The results provide novel insight into the role of PI3K in the transcriptional response to IFN-beta.

Animals↗

PubMed2002

IRF-3-dependent, NFkappa B- and JNK-independent activation of the 561 and IFN-beta genes in response to double-stranded RNA.

Double-stranded (ds) RNA induces transcription of the 561 gene by activating IFN regulatory factor (IRF) transcription factors, whereas similar induction of the IFN-beta gene is thought to require additional activation of NFkappaB and AP-1. In mutant P2.1 cells, dsRNA failed to activate NFkappaB, IRF-3, p38, or c-Jun N-terminal kinase, and transcription of neither 561 mRNA nor IFN-beta mRNA was induced. The defect in the IRF-3 pathway was traced to a low cellular level of this protein because of its higher rate of degradation in P2.1 cells. As anticipated, in several clonal derivatives of P2.1 cells expressing different levels of transfected IRF-3, activation of IRF-3 and induction of 561 mRNA by dsRNA was restored fully, although the defects in other responses to dsRNA persisted. Surprisingly, IFN-beta mRNA also was induced strongly in these cells in response to dsRNA, demonstrating that the activation of NFkappaB and AP-1 is not required. This conclusion was confirmed in wild-type cells overexpressing IRF-3 by blocking NFkappaB activation with the IkappaB superrepressor and AP-1 activation with a p38 inhibitor. Therefore, IRF-3 activation by dsRNA is sufficient to induce the transcription of genes with simple promoters such as 561 as well as complex promoters such as IFN-beta.

Actins↗

PubMed2002

NFkappaB-dependent signaling pathways.

The transcription factor NFkappaB is activated by numerous stimuli. Once NFkappaB is fully activated, it participates in the regulation of various target genes in different cells to exert its biological functions. NFkappaB has often been referred to as a central mediator of the immune response, since a large variety of bacteria and viruses can lead to the activation of NFkappaB, which in turn controls the expression of many inflammatory cytokines, chemokines, immune receptors, and cell surface adhesion molecules. Recent studies have shown that NFkappaB may function more generally as a central regulator of stress responses, since different stressful conditions, including physical stress, oxidative stress, and exposure to certain chemicals, also lead to NFkappaB activation. Furthermore, NFkappaB blocks cell apoptosis in several cell types. Taken together, these findings make it clear that NFkappaB plays an important role in cell proliferation and differentiation. It is the intention of this review to cover the various NFkappaB-dependent signaling pathways, thereby to achieve a better understanding of the mechanisms of NFkappaB activation and the physiological functions of activated NFkappaB.

Animals↗

PubMed2002

Stat1-dependent and -independent pathways in IFN-gamma-dependent signaling.

The paradigm that emerged from studies during the past decade established a central role for Jak-Stat (signal transducer and activator of transcription) signaling pathways in promoting the diverse cellular responses induced by interferon gamma (IFN-gamma). However, recent studies have shown that the IFN-gamma receptor activates additional signaling pathways and can regulate gene expression by Stat1-independent pathways. The diversity of gene-expression patterns mediated by Stat1-dependent and -independent mechanisms and the balance between these two pathways play an important role in the biological response to IFN-gamma.

Animals↗

PubMed2002

Resistance to interferons in melanoma cells does not correlate with the expression or activation of signal transducer and activator of transcription 1 (Stat1).

Defects in expression or activation signal transducer and activator of transcription-1 (Stat1) in response to interferon-alpha2 (IFN-alpha2) have been implicated as a mechanism for IFN resistance in melanoma cells. To further determine the significance of this observation, 17 melanoma cell lines sensitive or resistant to the antiproliferative effects of IFN-alpha2 and IFN-beta, as well as 30 melanoma patient samples, were analyzed for Stat1 levels by either Western blot analysis or immunohistochemistry. Although the expression level varied between samples, all the cell lines except one and all melanoma biopsy specimens expressed Stat1. IFN-stimulated levels of Stat1 and Stat2, which constitute the transcriptional activation complexes, such as, gamma activated factor (GAF) and IFN-stimulated gene factor 3 (ISGF3), for IFN-stimulated gene (ISG) induction were assessed in melanoma cell lines. Both IFN-alpha2 and INF-beta induced equivalent amounts of Stat1 and Stat2 proteins in cell lines, although compared with IFN-alpha2, IFN-beta had greater antiproliferative effects. No significant differences were observed in tyrosine or serine phosphorylation of Stat1 or the formation of GAF or ISGF3 complexes following IFN-alpha2 or IFN-beta treatment of IFN-resistant or IFN-sensitive cell lines. Comparable induction of two ISGs, ISG54 and IFN regulatory factor-1 (IRF-1), was observed in both sensitive WM9 and resistant A375 cells. Therefore, we report that defects in expression or activation of Stat1 or Stat2 were infrequent in melanoma cell lines and tumor samples and did not correlate with IFN resistance. Cellular resistance to IFNs likely results from defective quantitative or qualitative expression of specific ISGs.

Cell Division↗

PubMed2002

Stat1-dependent, p53-independent expression of p21(waf1) modulates oxysterol-induced apoptosis.

7-Ketocholesterol (7kchol) is prominent in atherosclerotic lesions where apoptosis occurs. Using mouse fibroblasts lacking p53, p21(waf1), or Stat1, we found that optimal 7kchol-induced apoptosis requires p21(waf1) and Stat1 but not p53. Findings were analogous in a human cell system. Apoptosis was restored in Stat1-null human cells when wild-type Stat1 was restored. Phosphorylation of Stat1 on Ser(727) but not Tyr(701) was essential for optimum apoptosis. A neutralizing antibody against beta interferon (IFN-beta) blunted Ser(727) phosphorylation and apoptosis after 7kchol treatment; cells deficient in an IFN-beta receptor subunit exhibited blunted apoptosis. IFN-beta alone did not induce apoptosis; thus, 7kchol-induced release of IFN-beta was necessary but not sufficient for optimal apoptosis. In Stat1-null cells, expression of p21(waf1) was much less than in wild-type cells; introducing transient expression of p21(waf1) restored apoptosis. Stat1 and p21(waf1) were essential for downstream apoptotic events, including cytochrome c release from mitochondria and activation of caspases 9 and 3. Our data reveal key elements of the cellular pathway through which an important oxysterol induces apoptosis. Identification of the essential signaling events that may pertain in vivo could suggest targets for therapeutic intervention.

Animals↗

PubMed2001

Distinct roles of the Ikappa B kinase alpha and beta subunits in liberating nuclear factor kappa B (NF-kappa B) from Ikappa B and in phosphorylating the p65 subunit of NF-kappa B.

Phosphatidylinositol 3'-kinase (PI3K) and the serine/threonine kinase AKT have critical roles in phosphorylating and transactivating the p65 subunit of nuclear factor kappaB (NF-kappaB) in response to the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF). Mouse embryo fibroblasts (MEFs) lacking either the alpha or beta subunit of IkappaB kinase (IKK) were deficient in NF-kappaB-dependent transcription following treatment with IL-1 or TNF. However, in contrast to IKKbeta-null MEFs, IKKalpha-null MEFs were not substantially defective in the cytokine-stimulated degradation of Ikappabetaalpha or in the nuclear translocation of NF-kappaB. The IKK complexes from IKKalpha- or IKKbeta-null MEFs were both deficient in PI3K-mediated phosphorylation of the transactivation domain of the p65 subunit of NF-kappaB in response to IL-1 and TNF, and constitutively activated forms of PI3K or AKT did not potentiate cytokine-stimulated activation of NF-kappaB in either IKKalpha- or IKKbeta-null MEFs. Collectively, these data indicate that, in contrast to IKKbeta, which is required for both NF-kappaB liberation and p65 phosphorylation, IKKalpha is required solely for the cytokine-induced phosphorylation and activation of the p65 subunit of NF-kappaB that are mediated by the PI3K/AKT pathway.

Animals↗

PubMed2001

Regulation of the accumulation and function of p53 by phosphorylation of two residues within the domain that binds to Mdm2.

The function and stability of the tumor suppressor p53 are tightly controlled by the negative regulator mouse double minute 2 (Mdm2), which binds to p53, blocking DNA binding and targeting p53 for proteosome-mediated degradation. Following DNA damage or cellular stress, p53 is phosphorylated within the Mdm2 binding domain on threonine 18 and serine 20. To analyze the roles of these phosphorylation events, residues 18 and 20 were mutated to alanines. Transient transfection into p53-null cells demonstrated that the T18A protein can be expressed stably, but the S20A protein is very unstable, precluding further analysis. When expressed stably at low basal levels in p53-null human fibroblasts or fibrosarcoma cells, the T18A mutant accumulated 5-10-fold less well than wild-type p53 following exposure to UV. Analysis of p53-dependent transcription following UV revealed that the phosphorylation of threonine 18 is required for transactivation of the p21, Hdm2 (the human ortholog of Mdm2), and GADD45 genes. The phosphorylation of serine 33, another early event following DNA damage, is not required for p53 accumulation or p53-dependent transactivation following UV.

Base Sequence↗

PubMed2001

Interferon signaling is dependent on specific tyrosines located within the intracellular domain of IFNAR2c. Expression of IFNAR2c tyrosine mutants in U5A cells.

Type I interferons (IFNs) are cytokines that play a central role in mediating antiviral, antiproliferative, and immunomodulatory activities in virtually all cells. These activities are entirely dependent on the interaction of IFNs with their particular cell surface receptor. In this report, we identify two specific tyrosine residues located within the cytoplasmic domain of IFNAR2c that are obligatory for IFN-dependent signaling. Various IFNAR2c tyrosine mutants were expressed in a human lung fibroscarcoma cell line lacking IFNAR2c (U5A). Stable clones expressing these mutants were analyzed for their ability to induce STAT1 and STAT2 activation, ISGF3 transcriptional complex formation, gene expression, and cell growth regulation in response to stimulation with type I IFNs. The replacement of all seven cytoplasmic tyrosine residues of IFNAR2c with phenylalanine resulted in a receptor unable to respond to IFN stimulation. Substitution of single tyrosines at amino acid residue 269, 316, 318, 337, or 512 with phenylalanine had no effect on IFN-dependent signaling, suggesting that no single tyrosine is essential for IFN receptor-mediated signaling. In addition, IFNAR2c retaining five proximal tyrosines residues (269, 306, 316, 318, and 337) or either two distal tyrosine residues (411 or 512) continued to be responsive to IFN stimulation. Surprisingly, the presence of only a single tyrosine at either position 337 or 512 was sufficient to restore a complete IFN response. These results indicate that IFN-dependent signaling proceeds through the redundant usage of two tyrosine residues in the cytoplasmic domain of IFNAR2c.

Animals↗