Inhibition of Nuclear Factor-κB-mediated Transcription by Association with the Amino-terminal Enhancer of Split, a Groucho-related Protein Lacking WD40 Repeats

Abstract

The amino-terminal enhancer of split (AES) encodes a 197-amino acid protein that is homologous to the NH 2 -terminal domain of the Drosophila Groucho protein but lacks COOH-terminal WD40 repeats. Although the Drosophila Groucho protein and its mammalian homologs, transducin-like enhancer of split proteins, are known to act as non-DNA binding corepressors, the role of the AES protein remains unclarified. Using the yeast twohybrid system, we have identified the protein-protein interaction between AES and the p65 (RelA) subunit of the transcription factor nuclear factor B (NF-B), which activates various target genes involved in inflammation, apoptosis, and embryonic development. The interaction between AES and p65 was confirmed by in vitro glutathione S-transferase pull down assay and by in vivo co-immunoprecipitation study. In transient transfection assays, AES repressed p65-driven gene expression. AES also inhibited NF-B-dependent gene expression induced by tumor necrosis factor ␣, interleukin-1␤, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1, which is an upstream kinase for NF-B activation. These data indicate that AES acts as a corepressor for NF-B and suggest that AES may play a pivotal role in the regulation of NF-B target genes.Nuclear factor B (NF-B) 1 is an inducible cellular transcription factor that regulates a wide variety of cellular and viral genes including several cytokines, cell adhesion molecules, and human immunodeficiency virus (HIV) (1-4). The members of the NF-B family in mammalian cells include the proto-oncogene c-Rel, Rel A (p65), Rel B, NF B1 (p50/105), and NF B2 (p52/p100). These proteins share a conserved 300-amino acids region known as the Rel homology domain, which is responsible for DNA binding, dimerization, and nuclear translocation of NF-B (1-4). In most cells, Rel family members form heteroand homodimers with distinct specificities in various combinations. p65, Rel B, and c-Rel are transcriptionally active members of the NF-B family, whereas p50 and p52 primarily serve as mere DNA binding subunits (1-4). The transactivation domains of p65, Rel B, and c-Rel have been mapped in their unique COOH-terminal regions. p65 was shown to contain at least two independent transactivation domains within its COOH-terminal 120 amino acids (5-8). One p65 activation domain, TA1, is confined to the COOH-terminal 30 amino acids of p65. The second domain TA2 is contained within the NH 2 -terminally adjacent 90 amino acids.A common feature of the regulation of NF-B family is their sequestration in the cytoplasm as inactive complexes with a class of inhibitory molecules known as I Bs (1-4). Treatment of cells with a variety of inducers such as phorbol esters, interleukin-1, and tumor necrosis factor (TNF) results in phosphorylation, ubiquitination, and degradation of the I B proteins (4, 9, 10). The degradation of I B proteins exposes the nuclear localization sequence in the remaining NF-B dimers, leading to nuclear translocation and subsequent binding of NF...

Cdc25C interacts with PCNA at G2/M transition

“…Yeast Y190 cells were co-transformed with expression vectors encoding various Cdc25C proteins fused to Gal4 DNA-binding domain and pACT2-PCNA expressing PCNA fused to Gal4 activation domain. These transformants were streaked on selective medium lacking leucine and tryptophan, allowed to grow for 2 days at 308C and tested for b-galactosidase activity using X-gal colony ®lter assay as described previously (Yang et al, 1999;Tetsuka et al, 2000). Hatched box (1 ± 40) indicates the minimal PCNA interaction region of Cdc25C obtained by this analysis.`+', positive for b-galactosidase activity;`itself', auto-activation of Lac Z reporter gene in Y190;`ND', not determined Thus, we further examined the speci®city of interaction by creating various Cdc25C deletion mutants and tested the interaction with PCNA by yeast two-hybrid system.…”

“…Human cDNA library from an osteosarcoma cell line for the yeast two-hybrid screening was obtained commercially (Matchmaker cDNA Library, Clontech). Screening was performed according to the supplier's protocol using yeast strain Y190 as described previously (Yang et al, 1999;Tetsuka et al, 2000;Uranishi et al, 2001). For the subsequent two-hybrid assays to examine the protein ± protein interaction in the yeast, the pAS2-1 plasmids expressing various portions of Cdc25C (as described in Figure 1) were transfected into the yeast together with pACT2 ± PCNA, expressing PCNA fused to Gal4 activation domain, and detected the expression of b-galactosidase enzyme activity to con®rm the speci®c interaction.…”

The role of TLE1 in synovial sarcoma

“…11,12 Gro/TLEs inhibit NF-kB signaling, a pathway for inflammation and proliferation of inflammatory cells. 13 Although most of these related studies reported that the Groucho/TLE family of corepressors inhibits gene expression and the signaling pathway, it also enhances the gene expression including the macrophage colony stimulating factor receptor, 14 Notch ligand Jagged1, 15 and Wnt pathway mediator plakoglobin. 16 The Groucho/ TLE family of corepressors was also suggested to be an oncogene because the over-expression of Gro/TLE family members is associated with lung adenocarcinoma, 17 grade 1 astrocytomas, 18 higher-grade meningiomas, 19 pituitary adenomas, 20,21 and synovial sarcomas, 7,22 which is consistent with our results.…”

Growth defect in Grg5 null mice is associated with reduced Ihh signaling in growth plates

“…Members of the other subgroup, e.g., Grg5 in mice and AES in humans, contain the amino-terminal region of gro, including the Q-rich domain, but lack most of the variable region and the entire WD-40 repeat region. Mammalian gro homologues in the first group bind to transcription factors that target gene repression (Aronson et al, 1997;Roose et al, 1998;Eberhard et al, 2000;Tetsuka et al, 2000;Wang et al, 2000;Brantjes et al, 2001), whereas the short form of gro, such as XGrg5 in Xenopus, has been shown to enhance transcriptional activity (Roose et al, 1998;Brantjes et al, 2001). It has been speculated that Grg5 may act as a dominant negative molecule within a transcription complex.…”