The Wnt/β-catenin/Tcf and IκB/NF-κB cascades are independent pathways involved in cell cycle control, cellular differentiation, and inflammation. Constitutive Wnt/β-catenin signaling occurs in ...certain cancers from mutation of components of the pathway and from activating growth factor receptors, including RON and MET. The resulting accumulation of cytoplasmic and nuclear β-catenin interacts with the Tcf/LEF transcription factors to induce target genes. The IκB kinase complex (IKK) that phosphorylates IκB contains IKKα, IKKβ, and IKKγ. Here we show that the cyclin D1 gene functions as a point of convergence between the Wnt/β-catenin and IκB pathways in mitogenic signaling. Mitogenic induction of G
1
-S phase progression and cyclin D1 expression was PI3K dependent, and cyclin D1
−/−
cells showed reduced PI3K-dependent S-phase entry. PI3K-dependent induction of cyclin D1 was blocked by inhibitors of PI3K/Akt/IκB/IKKα or β-catenin signaling. A single Tcf site in the cyclin D1 promoter was required for induction by PI3K or IKKα. In IKKα
−/−
cells, mitogen-induced DNA synthesis, and expression of Tcf-responsive genes was reduced. Reintroduction of IKKα restored normal mitogen induction of cyclin D1 through a Tcf site. In IKKα
−/−
cells, β-catenin phosphorylation was decreased and purified IKKα was sufficient for phosphorylation of β-catenin through its N-terminus in vitro. Because IKKα but not IKKβ induced cyclin D1 expression through Tcf activity, these studies indicate that the relative levels of IKKα and IKKβ may alter their substrate and signaling specificities to regulate mitogen-induced DNA synthesis through distinct mechanisms.
Regulation of β-Catenin Function by the IκB Kinases Lamberti, Carmela; Lin, Keng-Mean; Yamamoto, Yumi ...
Journal of biological chemistry/The Journal of biological chemistry,
11/2001, Letnik:
276, Številka:
45
Journal Article
Recenzirano
Odprti dostop
Both the β-catenin and the nuclear factor κB (NF-κB) proteins are important regulators of gene expression and cellular proliferation. Two kinases, IKKα and IKKβ, are critical activators of the NF-κB ...pathway. Here we present evidence that these kinases are also important in the regulation of β-catenin function. IKKα- and IKKβ-deficient mouse embryo fibroblasts exhibited different patterns of β-catenin cellular localization. IKKβ decreases β-catenin-dependent transcriptional activation, while IKKα increases β-catenin-dependent transcriptional activity. IKKα and IKKβ interact with and phosphorylate β-catenin using both in vitro and in vivo assays. Our results suggest that differential interactions of β-catenin with IKKα and IKKβ may in part be responsible for regulating β-catenin protein levels and cellular localization and integrating signaling events between the NF-κB and Wingless pathways.
The HSV-1 genome is composed of two unique regions (ULand US) flanked by inverted repeats. During the course of DNA replication the two unique regions ULand USinvert relative to one another. In this ...report we present evidence that cleavage is not necessary for genomic inversion to occur. We isolated and characterized a UL6::lacZinsertion mutant (hr74) that produces wild-type levels of replicating viral DNA but fails to cleave and package DNA. We demonstrate that this virus is still able to undergo genomic inversion. Furthermore we confirm that replicating DNA from cells infected with wild-type virus contains specific ULtermini but not UStermini, whereas cells infected with the mutanthr74do not contain either USor ULtermini. This demonstrates that the specific ULends found in replicating DNA are the result of the cleavage/packaging process.
Both the β-catenin and the nuclear factor κB (NF-κB) proteins are important regulators of gene expression and cellular proliferation.
Two kinases, IKKα and IKKβ, are critical activators of the ...NF-κB pathway. Here we present evidence that these kinases are
also important in the regulation of β-catenin function. IKKα- and IKKβ-deficient mouse embryo fibroblasts exhibited different
patterns of β-catenin cellular localization. IKKβ decreases β-catenin-dependent transcriptional activation, while IKKα increases
β-catenin-dependent transcriptional activity. IKKα and IKKβ interact with and phosphorylate β-catenin using both in vitro and in vivo assays. Our results suggest that differential interactions of β-catenin with IKKα and IKKβ may in part be responsible for
regulating β-catenin protein levels and cellular localization and integrating signaling events between the NF-κB and Wingless
pathways.
The Wnt/beta-catenin/Tcf and IkappaB/NF-kappaB cascades are independent pathways involved in cell cycle control, cellular differentiation, and inflammation. Constitutive Wnt/beta-catenin signaling ...occurs in certain cancers from mutation of components of the pathway and from activating growth factor receptors, including RON and MET. The resulting accumulation of cytoplasmic and nuclear beta-catenin interacts with the Tcf/LEF transcription factors to induce target genes. The IkappaB kinase complex (IKK) that phosphorylates IkappaB contains IKKalpha, IKKbeta, and IKKgamma. Here we show that the cyclin D1 gene functions as a point of convergence between the Wnt/beta-catenin and IkappaB pathways in mitogenic signaling. Mitogenic induction of G(1)-S phase progression and cyclin D1 expression was PI3K dependent, and cyclin D1(-/-) cells showed reduced PI3K-dependent S-phase entry. PI3K-dependent induction of cyclin D1 was blocked by inhibitors of PI3K/Akt/IkappaB/IKKalpha or beta-catenin signaling. A single Tcf site in the cyclin D1 promoter was required for induction by PI3K or IKKalpha. In IKKalpha(-/-) cells, mitogen-induced DNA synthesis, and expression of Tcf-responsive genes was reduced. Reintroduction of IKKalpha restored normal mitogen induction of cyclin D1 through a Tcf site. In IKKalpha(-/-) cells, beta-catenin phosphorylation was decreased and purified IKKalpha was sufficient for phosphorylation of beta-catenin through its N-terminus in vitro. Because IKKalpha but not IKKbeta induced cyclin D1 expression through Tcf activity, these studies indicate that the relative levels of IKKalpha and IKKbeta may alter their substrate and signaling specificities to regulate mitogen-induced DNA synthesis through distinct mechanisms.
The introduction of the bovine (BPV) or human papillomavirus E6 gene into susceptible cells can result in their transformation, but there are few clues to the mechanism of action of the E6 gene. The ...characteristic features of E6 proteins are their small size (approximately 150 amino acids) and the potential to form two large zinc fingers. To determine if E6 can function as a transcription factor, the BPV E6 gene was fused to the sequence specific DNA binding peptide encoded by the BPV E2 gene. This chimeric E6‐E2 protein trans‐activated promoters that incorporated E2 binding elements in both rodent cells and Saccharomyces cerevisiae. In the absence of E6‐E2 localization to the target promoter, trans‐activation did not occur. Alteration of the cysteine residues at the base of each finger abrogated the transcriptional activity of the E6‐E2 hybrids. These data demonstrated that the BPV E6 gene encodes a transcription activation domain and imply that a specific structure of the protein, most likely the zinc fingers, is critical for this function. Since these cysteine mutants are also transformation defective, E6 transcriptional functions may be required for its oncogenic activity.
These experiments were designed to further characterize the differential phenotypic constellation of the Naples High- (NHE) and Naples Low-Excitability (NLE) lines. In order to determine possible ...differences between NHE and NLE rats in activity and circadian rhythms, besides reactivity to novelty (selection trait), adult male rats of both strains were tested during two 10-min exposures to a Làt-maze. They were then kept in activity cages continuously for 3 days. Moreover, nociceptive thresholds were measured with the hot-plate and the tail-flick test, to probe the possibility that these rats could be differentially sensitive to nociceptive stimuli. Further, the integrity of the nigro-striatal and mesolimbic system was investigated by measuring tyrosine-hydroxylase activity in the striatum and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the striatum as well as in the nucleus accumbens. In addition, TH activity was measured in the adrenals to probe the sympathetic section of the neurovegetative system. The results indicate that NHE and NLE rats differ by a factor of two in their phasic activity in a Làt-maze. In contrast, no differences in 24-h activity during the dark or light phase could be observed in the activity cages. However, NHE rats anticipated the light-on stimulus in the morning by reducing their activity 1 h earlier than NLE rats. Further, no difference could be found with the hot-plate and the tail-flick test. Finally, biochemical analyses revealed no difference in the NHE and NLE rats in the main terminal zone of mesolimbic system (n. accumbens) nor of nigrostriatal system (striatum) nor in the adrenal glands. In conclusion, since the only consistent difference between NHE and NLE rats appears to be reactivity to spatial novelty, an hippocampus-dependent behavioral trait (selection trait), independent of altered activity in the sympathetic system or dopaminergic activity in the major dopaminergic brain systems, the usefulness of these strains as genetic model to test current hypotheses of spatial processor device(s) in the mammalian brain is supported.
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