p53 is localized to a sub-nucleolar compartment after proteasomal inhibition in an energy-dependent manner

Abstract

process as reduction of ATP levels prevents nucleolar localization. In addition, p53 sub-nucleolar accumulation is abolished when cells are subjected to various types of genotoxic stress. Furthermore, we show that monoubiquitination of p53, which causes it to localize to the cytoplasm and nucleoplasm, does not prevent the association of p53 with the nucleolus after MG132 treatment. Importantly, we demonstrate that p53 nucleolar association occurs in lung and bladder carcinomas. Journal of Cell Science ATP-dependent p53 sub-nucleolar localizationThe nucleolus is the site of rRNA transcription, processing and ribosome assembly (Andersen et al., 2005; Carmo-Fonseca et al., 2000;Lam et al., 2005;Lamond and Sleeman, 2003;Leung et al., 2003;Olson and Dundr, 2005). As diagrammed in Fig. 1A the nucleolus consists of at least three morphologically distinct regions: the fibrillar center (FC), a dense fibrillar center (DFC) and a granular component (GC) (Scheer and Hock, 1999). Although, still not fully described, several studies indicate that the FC or the FC-DFC boundaries are the sites of pre-rRNA transcription (reviewed by Boisvert et al., 2007;Lamond and Sleeman, 2003). Ribosomal proteins and assembly factors are found in the GCs (Huang, 2002;Koberna et al., 2002;Lamond and Sleeman, 2003). FCs are particularly enriched in Pol I, topoisomerase I and the upstream binding factor (UBF, an activator of Pol I), whereas most processing molecules localize to the DFC and/or GC including nucleolin, nucleophosmin and fibrillarin [Casafont et al. (Casafont et al., 2007) and references therein].In a previous study we showed that p53 accumulation in the nucleolus, after proteasome inhibition, requires two regions within the carboxyl terminus of p53 (Karni-Schmidt et al., 2007). We show now that after proteasome inhibition, p53 can associate with a discrete sub-nucleolar component, the FC. We demonstrate that p53 association with the FC is ATP dependent and it is abrogated by genotoxic stress. Our results also suggest that monoubiquitination of p53 does not preclude its association with nucleoli. Perhaps most relevantly we have found that p53 associates with nucleoli in human bladder and lung carcinomas. Resultsp53 associates with a discrete sub-nucleolar compartment after MG132 treatment To extend our observations and those of others that the treatment of cells with a proteasome inhibitor causes nucleolar localization of p53 (Karni-Schmidt et al., 2007; Klibanov et al., 2001;Latonen et al., 2003;Pokrovskaja et al., 2001) we examined the precise location of ectopic p53 within the nucleolus. H1299 cells were transfected with a construct expressing wild-type p53 and after 24 hours, cells were treated with different concentrations of MG132 for various times or left untreated. The levels of transfected p53 either did not change or were modestly reduced by treatment with H1299 cells were transfected with wild-type p53 and after 24 hours were treated with different doses (5 μM, 10 μM and 20 μM for 6-10 hours and 1 μM, 5 μM and 10 μM f...

The PML nuclear bodies-associated protein TTRAP regulates ribosome biogenesis in nucleolar cavities upon proteasome inhibition

“…TTRAP was previously shown to interact with PML in Y2H and to localize within PML-NBs in transfected cells. 7 As proteins associated with PML-NBs accumulate in the nucleolus upon treatment with high concentration of proteasome inhibitors, 11,12 we proved that TTRAP nucleolar granules were PML-NBs by To confirm that the nucleolar TTRAP is a part of PML-NBs, we tested its physical interaction with PML-IV and PML-NBs component DAXX. Both proteins (Figures 2d and e) were detectable in TTRAP-bound fractions but not in negative controls.…”

“…In particular, PML itself, Sp100, SUMO-1 and p53 localize to subnucleolar regions in response to high doses of MG132. [11][12][13] We previously showed that treatment of SH-SY5Y neuroblastoma cells with the low concentrations of proteasome inhibitors triggered TTRAP relocalization to the cytoplasm, to form juxtanuclear aggresome-like structures. 8 We thus investigated the cellular localization of PML-NBs-associated protein TTRAP in the cells treated with increasing concentrations of drug.…”

Firefly luciferase mutants as sensors of proteome stress

“…Moreover, many cellular proteins such as p53 (Karni-Schmidt et al, 2008;Klibanov et al, 2001), promyelocytic leukemia gene product (PML) (Mattsson et al, 2001), cyclins, cyclin dependent kinases (CDKs) and several transcription factors such as Sp1 and Sp3 (Latonen et al, 2011) have been shown to accumulate in the nucleolus upon proteasome inhibition.…”

Proteasome-independent p53 degradation

“…This is reminiscent of the action of the nucleolar protein, p14 ARF, that binds and sequesters Mdm2 in the nucleolus in response to oncogenic stress. Elevated levels of p53 protein in nucleoli are observed in response to proteasome inhibition with MG132 [10,11]. The function of p53 in the nucleolus is uncertain but nucleolar localization may facilitate the ability of p53 to repress RNA polymerase 1 activity which is required for synthesis npg of 47S precursor rRNA transcripts that are processed to form 18S, 5.8S and 28S rRNAs [11].…”

“…Elevated levels of p53 protein in nucleoli are observed in response to proteasome inhibition with MG132 [10,11]. The function of p53 in the nucleolus is uncertain but nucleolar localization may facilitate the ability of p53 to repress RNA polymerase 1 activity which is required for synthesis npg of 47S precursor rRNA transcripts that are processed to form 18S, 5.8S and 28S rRNAs [11]. The importance of def during embryonic development in fish suggests that nucleolar p53 needs to be tightly regulated so that it is available to regulate ribosome biogenesis at times of stress without interfering with normal embryonic development under normal stress-free conditions.…”