Posttranslational modification of the tumor suppressor p53 plays important roles in regulating its stability and activity. Six lysine residues at the p53 C terminus can be posttranslationally modified by various mechanisms, including acetylation, ubiquitination, neddylation, methylation, and sumoylation. Previous cell line transfection studies show that ubiquitination of these lysine residues is required for ubiquitin-dependent degradation of p53. In addition, biochemical and cell line studies suggested that p53 acetylation at the C terminus might stabilize p53 and activate its transcriptional activities. To investigate the physiological functional outcome of these C-terminal modifications in regulating p53 stability and activity, we introduced missense mutations (lysine to arginine) at the six lysine residues (K6R) into the endogenous p53 gene in mouse embryonic stem (ES) cells. The K6R mutation prevents all posttranslational modifications at these sites but conserves the structure of p53. In contrast to conclusions of previous studies, analysis of p53 stability in K6R ES cells, mouse embryonic fibroblasts, and thymocytes showed normal p53 stabilization in K6R cells both before and after DNA damage, indicating that ubiquitination of these lysine residues is not required for efficient p53 degradation. However, p53-dependent gene expression was impaired in K6R ES cells and thymocytes in a promoter-specific manner after DNA damage, indicating that the net outcome of the posttranslational modifications at the C terminus is to activate p53 transcriptional activities after DNA damage.

In this study, we have demonstrated that translocated in liposarcoma (TLS), also termed FUS, is an interacting molecule of the p65 (RelA) subunit of the transcription factor nuclear factor B (NF-B) using a yeast two-hybrid screen. We confirmed the interaction between TLS and p65 by the pull-down assay in vitro and by a coimmunoprecipitation experiment followed by Western blot of the cultured cell in vivo. TLS was originally identified as part of a fusion protein with CHOP arising from chromosomal translocation in human myxoid liposarcomas. TLS has been shown to be involved in TFIID complex formation and associated with RNA polymerase II. However, the role of TLS in transcriptional regulation has not yet been clearly elucidated. We found that TLS enhanced the NF-B-mediated transactivation induced by physiological stimuli such as tumor necrosis factor ␣, interleukin-1␤, and overexpression of NF-B-inducing kinase. TLS augmented NF-B-dependent promoter activity of the intercellular adhesion molecule-1 gene and interferon-␤ gene. These results suggest that TLS acts as a coactivator of NF-B and plays a pivotal role in the NF-B-mediated transactivation. Nuclear factor B (NF-B)1 is an inducible cellular transcription factor that regulates a wide variety of cellular and viral genes including cytokines, cell adhesion molecules and human immunodeficiency virus (1-5). The members of the NF-B family in mammalian cells include the proto-oncogene c-Rel, RelA (p65), RelB, NFkB1 (p50/105), and NFkB2 (p52/p100). These proteins share a conserved 300-amino acid region known as the Rel homology domain, which is responsible for DNA binding, dimerization, and nuclear translocation of NF-B (1, 2, 4, 5). In most cells, Rel family members form hetero-and homodimers with distinct specificities in various combinations. p65, RelB, and c-Rel are transcriptionally active members of the NF-B family, whereas p50 and p52 primarily serve as DNA binding subunits (1, 2, 4, 5). These proteins play fundamental roles in immune and inflammatory responses and in the control of cell proliferation (4, 6 -9). A common feature of the regulation of NF-B is the sequestration in the cytoplasm as an inactive complex with a class of inhibitory molecules known as IBs (2, 10). Treatment of cells with a variety of inducers such as phorbol esters, interleukin-1 (IL-1), and tumor necrosis factor ␣ (TNF-␣) results in phosphorylation, ubiquitination, and degradation of the IB proteins (5, 11, 12). The degradation of IB proteins exposes the nuclear localization sequence in the remaining NF-B dimers, followed by the rapid translocation of NF-B to the nucleus where it activates the target genes by binding to the DNA regulatory element (1, 2, 4, 5).The protein regions responsible for the transcriptional activation (called "transactivation domain") of p65, RelB, and c-Rel have been mapped in their unique C-terminal regions. p65 contains at least two independent transactivation domains within its C-terminal 120 amino acids (Fig. 1A) (13-16). One of these transactivation doma...

The cAMP-dependent protein kinase (PKA) signaling pathway plays a major role in a number of pathophysiological conditions. However, there have been conflicting evidences regarding the action of cAMP/PKA on nuclear factor-jB (NF-jB). In this study, we have explored the effect of cAMP/PKA on NF-jB activity and determined its molecular mechanism. PKA activating agents or expression of the catalytic subunit of PKA (PKAc) inhibited the NF-jBdependent reporter gene expression induced by tumor necrosis factor a (TNFa). PKA activators affected neither IjBa phosphorylation, IjBa degradation, nor the NF-jB/DNA binding. Expression of PKAc inhibited the transactivation potential of suggesting that the inhibitory action of PKA is through the C-terminal transactivation domain of p65 but not by phosphorylation of the consensus PKA recognition site containing serine at position 276. Overexpression of coactivators, CBP (CREB-binding protein) and p300, failed to reverse the PKA-mediated inhibition of p65 transactivation. Thus, the inhibitory action of the cAMP/PKA pathway on the transcriptional activity of NF-jB appears to be exhibited by modifying the C-terminal transactivation domain of p65, either directly or indirectly.Keywords: NF-jB; PKA; cAMP; signal transduction.Nuclear factor jB (NF-jB) 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][2][3][4]. Members of the NF-jB family in mammalian cells include the proto-oncogene c-Rel, Rel A (p65), Rel B, NF-kB1 (p50/105), and NF-kB2 (p52/p100) 1 . 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 [1][2][3][4]. In most cells, Rel family members form hetero-and homo-dimers with distinct specificities in various combinations. A common feature of the regulation of the NF-jB family is their sequestration in the cytoplasm as inactive complexes with a class of inhibitory molecules known as IjBs [1][2][3][4]. Treatment of cells with a variety of inducers such as phorbol ester, interleukin-1b (IL-1b) and tumor necrosis factor a (TNFa) results in phosphorylation, ubiquitination and degradation of the IjB proteins [3,5,6]. The degradation of IjB proteins exposes the nuclear localization sequence in the remaining NF-jB dimers, leading to nuclear translocation and subsequent binding of NF-jB to the DNA cis-regulatory element of target genes [1][2][3][4]. In addition to the nuclear translocation and DNA-binding of NF-jB, its transcriptional activity is regulated by coactivators, CBP (CREB-binding protein) and p300, that associate with the C-terminal transactivation domain of p65. It has been demonstrated that these coactivators physically interact with p65 and promote its transcriptional activity [7,8].On the other hand, elevation of intracellular cAMP induces the expression of numerous genes through the protein kinase A (PKA)-med...

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...