Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2

Abstract

NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator.

Np9, a cellular protein of retroviral ancestry restricted to human, chimpanzee and gorilla, binds and regulates ubiquitin ligase MDM2

“…Tera-1 cells were grown at 37°C and in a 5% CO 2 atmosphere in McCoy's 5a medium, while The proteins were separated by SDS-PAGE, immobilized on PVDF membrane (Immobilon P, Millipore) and detected by the indicated antibodies 65 .…”

“…GST pull-down analyses were performed by immobilizing equal amounts of GST, GST-MDM2, GST-MDM2 deletion mutants, or GST-Np9, respectively, on Gluthation-Sepharose beads (GE-Healthcare), following the protocol published by us recently 65 …”

“…pcDNA3-HA-ubiquitin was purchased from Addgene. pcmdm2, pCMV-p53, p21-luc, MG15-luc and PG13-luc have been used by us before64,65 .Epoxomicin was purchased from Enzo (Farmingdale, USA), and MG132 from Sigma, as were the -actin monoclonal antibody AC-15, the peroxidase-conjugated secondary anti-mouse and anti-rabbit antibodies, and the monoclonal anti-glutathione-s-transferase antibody (clone GST-2). The secondary anti-rat antibody was provided by one of us (EK).…”

Tandem Affinity Purification and Nano HPLC-ESI-MS/MS Reveal Binding of Vitamin D Receptor to p53 and other New Interaction Partners in HEK 293T Cells

“…Moreover, mutant p53 is able to convert 1,25-D 3 from a pro-to an antiapoptotic agent (16 Considering the high physiological relevance of wild-type p53 as major barrier to cancer progression, our new finding will encourage future studies to elucidate the significance of the association of both proteins. Remarkably, our study identified new nuclear VDRbinding partners that control p53-mediated biological effects, including the novel inhibitor of histone acetyl transferases (INHAT) repressor NIR, which modulates the transcriptional activity of p53 and of its relative p63 (6,17,18) as well as nuclease-sensitive element-binding protein 1 (19), ELAV-like protein 1 (20)(21)(22) and nucleophosmin (23). Our MS data that indicate VDR binding to nuclear located mediator of RNA polymerase II (Table I) are in line with the results of Rachez and coworkers.…”

“…), as well as for transactivation via binding of NCoAs. It has been reported that VDR-NCoAs interfaces correspond to helices H3 and H12 (the latter representing the activation function-2 domain), and an area immediately Nterminal of the zinc fingers (residues [18][19][20][21][22]. Binding of human VDR to basal transcription factors such as transcription factor II B (TFIIB) (near the N-terminus of VDR), as well as with transcriptional corepressors such as the hairless (Hr) gene product, which associates with the VDR hinge and H3, have also been shown before (26).…”