4C, E). facilitates p53 degradation and thereby negatively regulates p53 target gene expression provides insight into an oncogenic role of TRAIL-R2 in tumorigenesis that particularly manifests in p53 wild-type tumors. Benzoylmesaconitine transcriptional target of Benzoylmesaconitine p53, the most important tumor suppressor protein frequently inactivated in human cancers. P53 plays a central role in coordinating cellular responses to various intrinsic and extrinsic stress factors to maintain genomic stability. Depending on the stress level, p53 induces cell survival or cell death signaling pathways leading to transient or permanent cell cycle arrest (senescence) or to cell death. P53 is an unstable protein with a short half-life . At physiological conditions p53 is kept at a low steady-state level and a broad network of interacting proteins regulate its Benzoylmesaconitine stability and activity. An important negative regulator is the E3 ubiquitin ligase murine double minute 2 (MDM2). MDM2 interacts with p53, influences its cellular distribution and initiates its proteasomal degradation via ubiquitination [20, 21]. At the same time p53 transcriptionally regulates representing a negative feedback loop initiated by p53. Consequently, the p53-MDM2 feedback loop keeps p53 at a low level under unstressed conditions . The promyelocytic leukemia protein (PML) also plays a key role in regulating the rate of p53 protein turnover. PML modulates the p53CMDM2 interaction in the nucleus thereby reducing p53 degradation [23, 24]. Most tumors escape p53 tumor suppressor functions by developing mutations or inactivating mechanisms . Nevertheless, some tumors express functional p53 and treatment with chemo- or radiotherapy in these tumors activate a p53-mediated stress response. Since TRAIL-R2 is a transcriptional target of p53 , anti-tumor therapy has also aimed at potentiating cell death in wild-type p53-expressing malignant cells by enhancing TRAIL-R2 expression at the plasma membrane [27C29]. While regulation of TRAIL-R2 expression by p53 has been well established, the recent discovery Smad1 of pro-tumoral functions of Benzoylmesaconitine endogenous level expression of plasma membrane TRAIL-R2 [8C11] and nTRAIL-R2  prompted us to investigate a potential negative feedback regulation of p53 by TRAIL-R2. Here, we show that both proteins interact in the nucleus and that TRAIL-R2 functions as a novel negative regulator of p53. Results Nuclear TRAIL-R2 co-localizes with p53 Since both, TRAIL-R2 and p53 are present in the nucleus and each of them can interact with the chromatin and with the microprocessor complex, we asked whether both proteins may interact with each other within the nuclear compartment. First, we studied the intracellular distribution of TRAIL-R2 and p53 in wild-type p53-expressing HCT116 colon carcinoma cells by indirect immunofluorescence staining followed by confocal laser scanning microscopy (LSM). LSM analyses demonstrated co-localization of a subset of both proteins in a distinct compartment of the nucleus (Fig. ?(Fig.1A).1A). ImageStream high-throughput microscopy showed also a nuclear co-localization of TRAIL-R2 and p53 in 62% of the analyzed HCT116 cells (Fig. ?(Fig.1B).1B). Similar results were obtained for A549 lung cancer cells (Fig. S2). Open in a separate window Fig. 1 TRAIL-R2 interacts with p53 in the nucleus.Intracellular distribution of TRAIL-R2 and p53 in HCT116 cells was analyzed by indirect immunofluorescence followed by (A) confocal LSM and (B) ImageStream high-throughput microscopy. Scale bar 20?m. The respective antibody controls are shown in Supplementary Fig. 1. C TRAIL-R1 and TRAIL-R2 were precipitated from nuclear fractions of HCT116 p53 WT and p53 KO cells by receptor-specific antibodies (MapaMapatumumab, anti-TRAIL-R1 antibody, lane 1 and 3; LexaLexatumumab, anti-TRAIL-R2 antibody, lane 2 and 4). As controls, antibodies alone were analyzed in parallel (lane 5, 6). Nuclear lysates and precipitated protein complexes were examined by western blotting (anti-p53 antibody DO-1). As gel loading control the levels of nuclear protein hnRNPA1 was analyzed in parallel. D AsPC-1 p53 null cells were stable transfected with a temperature-sensitive p53-mutant. These cells express mutant-p53 (p53 MT) at 37?C and wild-type p53 (p53 WT) at 32?C. Whole-cell lysates of AsPC-1 cells cultured for 24?h at 37?C or 32?C were analyzed for the presence of TRAIL-R2, p53 (anti-p53 antibody DO-1) and p21 by western blotting. The level of -Actin was determined in parallel and served as loading control. E TRAIL-R1 and TRAIL-R2 were precipitated from nuclear fractions of AsPC-1 cells cultured for 24?h at 37?C or 32?C. As controls, antibodies alone.