RELA

Abstract: The nuclear expression and action of the nuclear factor kappa B (NF-κB) transcription factor requires signal-coupled phosphorylation and degradation of the IκB inhibitors, which normally bind and sequester this pleiotropically active factor in the cytoplasm. The subsequent molecular events that regulate the termination of nuclear NF-κB action remain poorly defined, although the activation of de novo IκBα gene expression by NF-κB likely plays a key role. Our studies now demonstrate that the RelA subunit of NF-κB is subject to inducible acetylation and that acetylated forms of RelA interact weakly, if at all, with IκBα. Acetylated RelA is subsequently deacetylated through a specific interaction with histone deacetylase 3 (HDAC3). This deacetylation reaction promotes effective binding to IκBα and leads in turn to IκBα-dependent nuclear export of the complex through a chromosomal region maintenance-1 (CRM-1)–dependent pathway. Deacetylation of RelA by HDAC3 thus acts as an intranuclear molecular switch that both controls the duration of the NF-κB transcriptional response and contributes to the replenishment of the depleted cytoplasmic pool of latent NF-κB–IκBα complexes.

Abstract: The nuclear function of the heterodimeric NF-κB transcription factor is regulated in part through reversible acetylation of its RelA subunit. We now demonstrate that the p300 and CBP acetyltransferases play a major role in the in vivo acetylation of RelA, principally targeting lysines 218, 221 and 310 for modification. Analysis of the functional properties of hypoacetylated RelA mutants containing lysine-to-arginine substitutions at these sites and of wild-type RelA co-expressed in the presence of a dominantly interfering mutant of p300 reveals that acetylation at lysine 221 in RelA enhances DNA binding and impairs assembly with IκBα. Conversely, acetylation of lysine 310 is required for full transcriptional activity of RelA in the absence of effects on DNA binding and IκBα assembly. Together, these findings highlight how site-specific acetylation of RelA differentially regulates distinct biological activities of the NF-κB transcription factor complex.

Abstract: The work of Reddy et al. (S. A. Reddy, J. A. Huang, and W. S. Liao, J. Biol. Chem. 272:29167–29173, 1997) reveals that phosphatidylinositol 3-kinase (PI3K) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear factor κB (NF-κB), but the underlying mechanism remains to be determined. We have found that IL-1 stimulates interaction of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-κB-dependent gene expression but have no effect on the IL-1-stimulated degradation of IκBα, the nuclear translocation of NF-κB, or the ability of NF-κB to bind to DNA. In contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-κB itself, especially the p65/RelA subunit. Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, we found that overexpression of the p110 catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-κB, a process quite distinct from the liberation of NF-κB from its cytoplasmic inhibitor IκB.

Abstract: Pancreatic adenocarcinoma is a leading cause of adult cancer mortality in the United States. Recent studies have revealed that point mutation of the K-ras oncogene is a common event in pancreatic cancer, and oncogenesis mediated by Ras may also involve activation of Rel/nuclear factor (NF)-kappa B transcription factors. Furthermore, the c-rel member of Rel/NF-kappa B transcription factor family was first identified as a cellular homologue of the v-rel oncogene, suggesting that other members of the Rel/NF-kappa B family are potentially oncogenes. We therefore investigated the possibility that Rel/NF-kappa B transcription factors are activated in pancreatic cancer. Immunohistochemical analysis, Western blot and Northern blot analysis, electrophoretic mobility shift assays, and chloramphenicol acetyltransferase assays were performed to determine RelA activity in human pancreatic adenocarcinomas and normal tissues and nontumorigenic or tumorigenic cell lines. RelA, the p65 subunit of NF-kappa B, was constitutively activated in approximately 67% (16 of 24) of pancreatic adenocarcinomas but not in normal pancreatic tissues. Constitutive RelA activity was also detected in 9 of 11 human pancreatic tumor cell lines but not in nontumorigenic Syrian golden hamster cell lines. I kappa B alpha, a previously identified NF-kappa B-inducible gene, was overexpressed in human pancreatic tumor tissues and cell lines, and RelA activation could be inhibited by curcumin and dominant-negative mutants of I kappa B alpha, raf, and MEKK1. This is the first report demonstrating constitutive activation of RelA in nonlymphoid human cancer. These data are consistent with the possibility that RelA is constitutively activated by the upstream signaling pathway involving Ras and mitogen-activated protein kinases in pancreatic tumor cells. Constitutive RelA activity may play a key role in pancreatic tumorigenesis through activation of its downstream target genes.