The Wnt/␤-catenin/Tcf and IB/NF-B cascades are independent pathways involved in cell cycle control, cellular differentiation, and inflammation. Constitutive Wnt/␤-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 ␤-catenin interacts with the Tcf/LEF transcription factors to induce target genes. The IB kinase complex (IKK) that phosphorylates IB contains IKK␣, IKK␤, and IKK␥. Here we show that the cyclin D1 gene functions as a point of convergence between the Wnt/␤-catenin and IB pathways in mitogenic signaling. Mitogenic induction of G 1 -S phase progression and cyclin D1 expression was PI3K dependent, and cyclin D1 Ϫ/Ϫ cells showed reduced PI3K-dependent S-phase entry. PI3K-dependent induction of cyclin D1 was blocked by inhibitors of PI3K/Akt/IB/IKK␣ or ␤-catenin signaling. A single Tcf site in the cyclin D1 promoter was required for induction by PI3K or IKK␣. In IKK␣ Ϫ/Ϫ cells, mitogen-induced DNA synthesis, and expression of Tcf-responsive genes was reduced. Reintroduction of IKK␣ restored normal mitogen induction of cyclin D1 through a Tcf site. In IKK␣ Ϫ/Ϫ cells, ␤-catenin phosphorylation was decreased and purified IKK␣ was sufficient for phosphorylation of ␤-catenin through its N-terminus in vitro. Because IKK␣ but not IKK␤ induced cyclin D1 expression through Tcf activity, these studies indicate that the relative levels of IKK␣ and IKK␤ may alter their substrate and signaling specificities to regulate mitogeninduced DNA synthesis through distinct mechanisms. INTRODUCTIONThe Wingless/Wnt pathway plays a crucial role in development and cell cycle control (Cadigan and Nusse, 1997;Huelsken and Behrens, 2000). Dysregulation of the Wingless/ (Wnt)/␤-catenin/Tcf pathway has been implicated in tumorigenesis of diverse types (Polakis, 2000a). Axin/Conductin, together with APC, promote ␤-catenin degradation through serine-threonine phosphorylation of the ␤-catenin N-terminus by GSK3␤, which targets ␤-catenin for ubiquitination by a SCF ␤-TRCP (␤-transducin repeat-containing protein) ubiquitin ligase complex (Fuchs et al., 1999;Winston et al., 1999) and its degradation by the proteasome. On induction of Wnt signaling by extracellular ligands, the Frizzled receptors are activated. The activity of GSK3␤ and its effect on ␤-catenin is antagonized by Dishevelled, a downstream target of Frizzled, thus preventing the degradation of ␤-cate- nin by the proteasome. The resulting accumulation of ␤-catenin leads to its nuclear translocation and binding to Tcf/Lef transcription factors to induce target genes including cyclin D1 and c-Myc (He et al., 1998;Shtutman et al., 1999;Huelsken and Behrens, 2000). In addition to components in the Wnt signaling pathway, several other pathways can regulate ␤-catenin/Tcf signaling and gene expression and confer aberrant cellular growth. The protein encoded by Gas6, a growth factor of the vitamin K-dependent family, which binds member...

Both the ␤-catenin and the nuclear factor B (NF-B) proteins are important regulators of gene expression and cellular proliferation. Two kinases, IKK␣ and IKK␤, are critical activators of the NF-B pathway. Here we present evidence that these kinases are also important in the regulation of ␤-catenin function. IKK␣-and IKK␤-deficient mouse embryo fibroblasts exhibited different patterns of ␤-catenin cellular localization. IKK␤ decreases ␤-catenin-dependent transcriptional activation, while IKK␣ increases ␤-catenin-dependent transcriptional activity. IKK␣ and IKK␤ interact with and phosphorylate ␤-catenin using both in vitro and in vivo assays. Our results suggest that differential interactions of ␤-catenin with IKK␣ and IKK␤ may in part be responsible for regulating ␤-catenin protein levels and cellular localization and integrating signaling events between the NF-B and Wingless pathways.␤-Catenin, the mammalian homologue of the Drosophila armadillo protein, is a ubiquitously expressed protein that has at least two distinct roles in the cell. First, it participates in cell-cell adhesion by mediating the association of E-cadherin with the cytoskeleton (1, 2). Second, it is a critical downstream component of the Wnt 1 or Wingless signal transduction pathway (3-5). The Wnt family of secretory glycoproteins plays an important role in embryonic development, in the induction of cell polarity, and in the determination of cell fate. Deregulation of Wnt signaling disrupts axis formation in embryos (5-8) and is associated with multiple human malignancies (9).The current model of Wnt signaling indicates that the binding of the Wnt proteins to their receptor, frizzled, stabilizes ␤-catenin by inhibiting the activity of a serine/threonine kinase glycogen synthase kinase-3 or GSK-3␤ (9). GSK-3␤ is associated with ␤-catenin in a multiprotein complex that includes the adenomatous polyposis coli tumor suppressor protein (APC), axin or conductin, protein phosphatase 2A, and dishevelled. GSK-3␤ phosphorylation of residues in the amino terminus of ␤-catenin results in APC-mediated ␤-catenin degradation via the ubiquitin-proteosome pathway (10, 11). Increased levels of ␤-catenin are frequently found in colon cancer due to mutations in either the APC gene (12)(13)(14) or at residues in the amino terminus of ␤-catenin that are phosphorylated by . In the nucleus, ␤-catenin forms a complex with members of the T-cell factor (TCF)/lymphocyte-enhancer factor (LEF) family and activates gene expression of a variety of target genes (18 -23) including c-myc (24) and cyclin D1 (25, 26).NF-B comprises a family of transcription factors which are critical in activating the expression of genes involved in the immune and inflammatory response and in the regulation of cellular apoptosis (27, 28). NF-B is sequestered in the cytoplasm by a family of inhibitory proteins known as IB. Upon stimulation of this pathway by a variety of agents including IL-1 and TNF␣, the kinases IKK␣ and IKK␤ (29 -33) in conjunction with the scaffold protein IKK␥/NEMO (34 -36) l...

The HSV-1 genome is composed of two unique regions (UL and US) flanked by inverted repeats. During the course of DNA replication the two unique regions UL and US invert 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::lacZ insertion 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 UL terminal but not US termini, whereas cells infected with the mutant hr74 do not contain either US or UL termini. This demonstrates that the specific UL ends found in replicating DNA are the result of the cleavage/packaging process.

Six genes, including UL32, have been implicated in the cleavage and packaging of herpesvirus DNA into preassembled capsids. We have isolated a UL32 insertion mutant which is capable of near-wild-type levels of viral DNA synthesis; however, the mutant virus is unable to cleave and package viral DNA, consistent with the phenotype of a previously isolated temperature-sensitive herpes simplex virus type 1 mutant, tsN20 (P. A. Schaffer, G. M. Aron, N. Biswal, and M. Benyesh-Melnick, Virology 52:57–71, 1973). A polyclonal antibody which recognizes UL32 was previously used by Chang et al. (Y. E. Chang, A. P. Poon, and B. Roizman, J. Virol. 70:3938–3946, 1996) to demonstrate that UL32 accumulates predominantly in the cytoplasm of infected cells. In this report, a functional epitope-tagged version of UL32 showed that while UL32 is predominantly cytoplasmic, some nuclear staining which colocalizes with the major DNA binding protein (ICP8, UL29) in replication compartments can be detected. We have also used a monoclonal antibody (5C) specific for the hexon form of major capsid protein VP5 to study the distribution of capsids during infection. In cells infected with wild-type KOS (6 and 8 h postinfection), 5C staining patterns indicate that capsids are present in nuclei within replication compartments. These results suggest that cleavage and packaging occur in replication compartments at least at 6 and 8 h postinfection. Cells infected with the UL32 mutant exhibit a hexon staining pattern which is more diffusely distributed throughout the nucleus and which is not restricted to replication compartments. We propose that UL32 may play a role in “bringing” preassembled capsids to the sites of DNA packaging and that the failure to localize to replication compartments may explain the cleavage/packaging defect exhibited by this mutant. These results suggest that the UL32 protein is required at a step distinct from those at which other cleavage and packaging proteins are required and may be involved in the correct localization of capsids within infected cells.

The adenovirus type 12 mutants in700 and pm700 carry site-specific mutations within the reading frame encoding the E1B 19-kilodalton protein (19K protein) which prevent the production of the intact 19K protein. In cultures of human A549 cells, these mutants grow just as well as the wild-type virus does, but they display a large-plaque (Ip), cytocidal (cyt) phenotype. DNA in these infected cells is not degraded, but at late times in human KB cells infected by the mutants, the mutants display a DNA degradation (deg) phenotype. The transformation phenotype of these mutants is also host range. Although the mutants are defective for transformation of the 3Y1 rat cell line, they transform rat and mouse primary kidney cells in vitro at wild-type efficiency and are capable of inducing tumors in rats. These results support the view that the type 12 E1B 19K protein is not obligatory for oncogenic transformation.