Akinori Ishimoto

Casein kinase I phosphorylates the Armadillo protein and induces its degradation in Drosophila.

Casein kinase I (CKI) was recently reported as a positive regulator of Wnt signaling in vertebrates and Caenorhabditis elegans. To elucidate the function of Drosophila CKI in the wingless (Wg) pathway, we have disrupted its function by double‐stranded RNA‐mediated interference (RNAi). While previous findings were mainly based on CKI overexpression, this is the first convincing loss‐of‐function analysis of CKI. Surprisingly, CKIα‐ or CKIϵ‐RNAi markedly elevated the Armadillo (Arm) protein levels in Drosophila Schneider S2R+ cells, without affecting its mRNA levels. Pulse–chase analysis showed that CKI‐RNAi stabilizes Arm protein. Moreover, Drosophila embryos injected with CKIα double‐stranded RNA showed a naked cuticle phenotype, which is associated with activation of Wg signaling. These results indicate that CKI functions as a negative regulator of Wg/Arm signaling. Overexpression of CKIα induced hyper‐phosphorylation of both Arm and Dishevelled in S2R+ cells and, conversely, CKIα‐RNAi reduced the amount of hyper‐modified forms. His‐tagged Arm was phosphorylated by CKIα in vitro on a set of serine and threonine residues that are also phosphorylated by Zeste‐white 3. Thus, we propose that CKI phosphorylates Arm and stimulates its degradation.

Characterization of Mouse Dishevelled (Dvl) Proteins in Wnt/Wingless Signaling Pathway

The dishevelled (dsh) gene family encodes cytoplasmic proteins that have been implicated in Wnt/Wingless (Wg) signaling. To demonstrate functional conservation of Dsh family proteins, two mouse homologs of Drosophila Dsh, Dvl-1 and Dvl-2, were biochemically characterized in mouse andDrosophila cell culture systems. We found that treatment with a soluble Wnt-3A leads to hyperphosphorylation of Dvl proteins and a concomitant elevation of the cytoplasmic β-catenin levels in mouse NIH3T3, L, and C57MG cells. This coincides well with our finding in aDrosophila wing disc cell line, clone-8, that Wg treatment induced hyperphosphorylation of Dsh (Yanagawa, S., van Leeuwen, F., Wodarz, A., Klingensmith, J., and Nusse, R. (1995) Genes Dev. 9, 1087–1097). Furthermore, we showed that mouse Dvl proteins affect downstream components of Drosophila Wg signaling as Dsh does; overexpression of Dvl proteins in clone-8 cells results in elevation of Armadillo (Drosophila homolog of β-catenin) and Drosophila E-cadherin levels, hyperphosphorylation of Dvl proteins themselves, and inhibition of Zeste-White3 kinase-mediated phosphorylation of a microtubule-binding protein, Tau. In addition, casein kinase II was shown to coimmunoprecipitate with Dvl proteins, and Dvl proteins were phosphorylated in these immune complexes. These results are direct evidence that Dsh family proteins mediate a set of conserved biochemical processes in the Wnt/Wg signaling pathway.

IDENTIFICATION AND CHARACTERIZATION OF A NOVEL LINE OF DROSOPHILA SCHNEIDER S2 CELLS THAT RESPOND TO WINGLESS SIGNALING

Wingless (Wg) treatment of Drosophilawing disc clone 8 cells leads to Armadillo (Arm) protein elevation, and this effect has been used as the basis of in vitro assays for Wg protein. Previously analyzed stocks of DrosophilaSchneider S2 cells could not respond to added Wg, because they lack the Wg receptor, Dfrizzled-2. However, we found that a line of S2 cells obtained from another source express Dfrizzled-2 and Dfrizzled-1. Thus, we designated this cell line as S2R+ (S2 receptor plus). S2R+ cells respond to addition of extracellular Wg by elevating Arm and DE-cadherin protein levels and by hyperphosphorylating Dsh, just as clone 8 cells do. Moreover, overexpression of Wg in S2R+, but not in S2 cells, induced the same changes in Dsh, Arm, and DE-cadherin proteins as induced in clone 8 cells, indicating that these events are common effects of Wg signaling, which occurs in cells expressing functional Wg receptors. In addition, unphosphorylated Dsh protein in S2 cells was phosphorylated as a consequence of expression of Dfrizzled-2 or mouse Frizzled-6, suggesting that basal structures common to various frizzled family proteins trigger this phosphorylation of Dsh. S2R+ cells are as sensitive to Wg as are clone 8 cells but can grow in simpler medium. Therefore, the S2R+ cell line is likely to prove highly useful for in vitro analyses of Wg signaling.

Mechanisms of Human Papillomavirus E2-Mediated Repression of Viral Oncogene Expression and Cervical Cancer Cell Growth Inhibition

ABSTRACT The papillomavirus E2 gene product plays a pivotal role in viral replication. E2 has multiple functions, including (i) transcriptional activation and repression of viral promoters and (ii) the enhancement of viral DNA replication. It was previously reported that E2 suppressed the growth of papillomavirus-positive cervical carcinoma cell lines. In the present study, we investigated the mechanisms of E2 growth inhibition. We found that the transcriptional activation function of E2 is required for inhibition of the growth of HeLa cells as well as for transcriptional repression of the viralE6/E7 promoter. It had been previously postulated that transcriptional repression of the E6/E7 promoter results from E2 binding its cognate sites proximal to the E6/E7promoter and displacing other cellular transcriptional factors. In this study, we report a requirement for the transcription activation function for the binding of E2 to transcriptionally active templates.

Modulation of the cell division cycle by human papillomavirus type 18 E4

ABSTRACT The life cycle of human papillomaviruses (HPVs) is tightly coupled to the differentiation program of their host epithelial cells. HPV E4 gene expression is first observed in the parabasal layers of squamous epithelia, suggesting that the E4 gene product contributes to the mechanism of differentiation-dependent virus replication, although its biological function remains unclear. We analyzed the effect of HPV type 18 E4 on cell proliferation and found that E4 expression induced cell cycle arrest at the G2/M boundary. The functional region of E4 necessary for the growth arrest activity was located in the central portion of the molecule, and this activity was independent of the E4-mediated collapse of cytokeratin intermediate filament structures.

Accumulation of Armadillo Induced by Wingless, Dishevelled, and Dominant-negative Zeste-white 3 Leads to Elevated DE-cadherin inDrosophila Clone 8 Wing Disc Cells

Drosophila genetic studies suggest that in the Wingless (Wg) signaling pathway, the segment polarity gene products, Dishevelled (Dsh), Zeste-white 3 (ZW-3), and Armadillo (Arm), work sequentially; wg and dsh negatively regulate zw-3, which in turn down-regulatesarm. To biochemically analyze interactions between the Wg pathway and Drosophila E-cadherin (DE-cadherin) which bind to Arm, we overexpressed Dsh, ZW-3, and Arm, in theDrosophila wing disc cell line, clone 8, which responds to Wg signal. Dsh overexpression led to accumulation of Arm primarily in the cytosol and elevation of DE-cadherin at cell junctions. Overexpression of wild-type and dominant-negative forms of ZW-3 decreased and increased Arm levels, respectively, indicating that modulation in zw-3 activity negatively regulates Arm levels. Overexpression of an Arm mutant with an amino-terminal deletion elevated DE-cadherin levels, suggesting that Dsh-induced DE-cadherin elevation is caused by the Arm accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative ZW-3-, and truncated Arm-induced accumulation of DE-cadherin protein was accompanied by a marked increase in the steady-state levels of DE-cadherin mRNA, suggesting that transcription of DE-cadherin is activated by Wg signaling. In addition, overexpression of DE-cadherin elevated Arm levels by stabilizing Arm at cell-cell junctions.

Growth ability of human immunodeficiency virus type 1 auxiliary gene mutants in primary blood macrophage cultures.

A strain of human immunodeficiency virus type 1 that is strictly tropic for primary human blood cell cultures was constructed in vitro. Mutational studies on the vif, vpr, vpu and nef genes of this virus were performed to evaluate their biological functions in natural target cells. For this purpose, replication properties of mutant viruses in peripheral blood mononuclear cells (PBMCs) and macrophages (PBMPs) were determined. Three phenotypes with respect to virus replication were noticed: normal or mildly retarded growth (nef and vpr mutants), impaired growth (vpu mutant), and no growth (vif mutant). These results suggest that the Vif and Vpu proteins are more important than the Nef and Vpr proteins for virus replication in PBMCs and PBMPs.

Rapid emergence of mink cell focus-forming (MCF) virus in various mice infected with NB-tropic friend virus

Abstract Mink cell focus forming (MCF) virus was always detected in the enlarged spleens of NFS, BALB/c, SL, and C 3 H mice infected with NB-tropic ecotropic Friend virus. The MCF virus emerged from the spleens as early as 7 days postinfection. The MCF viruses isolated from various mouse strains were NB-tropic dualtropic and highly oncogenic to newborn NFS and BALB/c mice. When cells infected with MCF virus were overlaid with XC cells without uv irradiation, the viruses were XC positive. Interference and host-range properties suggested that the MCF viruses were recombinants between exogenous NB-tropic ecotropic Friend virus and endogenous xenotropic virus.

Downregulation of CD4 is required for maintenance of viral infectivity of HIV-1

Downregulation of virus receptors on the cell surface is considered to be important in preventing superinfection. HIV-1 encodes multiple gene products, Env, Vpu, and Nef, involved in downregulation of CD4, a major HIV-1 receptor. We found that simultaneous mutations in both vpu and nef severely impaired virus replication. We examined the involvement of CD4 downregulation mediated by Vpu and Nef in the modification of virus infectivity. The mutation in vpu increased CD4 incorporation into virions without affecting the Env content in it, inhibiting the attachment step of virions to the CD4-positive cell surface. Although a single mutation in nef suppresses virus infectivity via a CD4-independent mechanism, it could augment CD4 incorporation in virions in combination with a vpu mutation. These results indicated that CD4 downregulation was necessary for maintenance of Env function in the virion.

Identification of Notch1 as a Frequent Target for Provirus Insertional Mutagenesis in T-Cell Lymphomas Induced by Leukemogenic Mutants of Mouse Mammary Tumor Virus

ABSTRACT In contrast to wild-type mouse mammary tumor virus (MMTV), the MMTV mutants with specific deletions in the U3 region of their long terminal repeats cause T-cell lymphomas. In 30% of T-cell lymphomas arising in BALB/c mice infected with MLA-MMTV, a leukemogenic MMTV mutant, we have found that MMTV proviruses were integrated into a short region of theNotch1 genome, so that truncated Notch1transcripts encoding the transmembrane and the cytoplasmic domains of Notch1 protein could be expressed. Thus, Notch1 is a major target of provirus insertional mutagenesis in these T-cell lymphomas.