The ribosomal protein S26 regulates p53 activity in response to DNA damage

Abstract

Ribosomal proteins have emerged as novel regulators of the Mdm2-p53 feedback loop, especially in the context of ribosomal stress. RPS26 is a recently identified Diamond-Blackfan Anemia-related ribosomal protein and its role in p53 activation has not been previously explored. In this study we found knockdown of RPS26 induced p53 stabilization and activation via a RPL11-dependent mechanism, resulting in p53-dependent cell growth inhibition. Moreover, RPS26 has the ability to interact with Mdm2 and inhibits Mdm2-mediated p53 ubiquitination that leads to p53 stabilization upon overexpression. Importantly, we discovered that RPS26 knockdown impaired p53's ability to transcriptionally activate its target genes in response to DNA damage, without affecting its stability. Accordingly, the cells lost the ability to induce G2/M cell cycle arrest. We further found that upon RPS26 knockdown, the DNA damage induced recruitment of p53 to the promoters of its target genes and p53 acetylation were both greatly reduced. In addition, RPS26 can interact with p53 independent of Mdm2 and coexist in a complex with p53 and p300. These data establish a role of RPS26 in DNA damage response by directly influencing p53 transcriptional activity, and suggest that RPS26 acts distinctively in different scenarios of p53 activation. Our finding also implicates p53 transcriptional activity control as an important mechanism of p53 regulation by ribosomal proteins.

A Novel Role for Pyruvate Kinase M2 as a Corepressor for P53 during the DNA Damage Response in Human Tumor Cells

“…Real-time RT-PCR was performed and data were analyzed according to our previous report (42). Specific oligonucleotide primers for P21 (43), GADD45 (44), MDM2 (43), and 14-3-3 (29) were used and listed below. ␤-Actin cDNA was amplified as internal positive controls (sense, 5Ј-CATCCTCACCCTGAAGTACCC-3Ј, antisense, 5Ј-AGCCTGGATAGCAACGTACATG-3Ј).…”

“…G, GST pull-down analysis of GST-P53 with PKM2 in the presence of the escalating concentrations (10/100/1,000 M) of FBP, PEP or ADP. H, GST pull-down analysis of GST or GST-P53 with PKM2 tetramer (fraction 38), dimer (43), and monomer (47) isolated by size exclusion chromatography. I, GST pull-down analysis of interaction between P53 and PKM2-WT or PKM2-R399E mutant, followed by immunoblotting with indicated antibodies.…”

Bclaf1 is an important NF-κB signaling transducer and C/EBPβ regulator in DNA damage-induced senescence

“…The siRNA target sequences of Atm, Atr, p53, Nemo, p65, c-Rel, and C/EBPβ have been previously reported. [41][42][43][44][45][46][47] Real-time PCR. Total RNA was extracted using TRIzol (Invitrogen) following the manufacturer's protocol.…”

“…Subsequent procedures were performed as previously described. 43 SA-β-gal and growth curves. HCT116 or MCF-7 cells were transfected with indicated siRNAs and cultured for 24 h. Subsequently, each group of cells were reseeded in 12-well plates and subjected to different treatments.…”

“…Immunofluorescence was performed as previously described. 43 For immunohistochemistry, xenografts were fixed in formalin, embedded in paraffin and then sliced. Antigens were retrieved using citric acid and steam and then stained with a Ki67 antibody (1 : 400, Cell Signaling Technology) and hematoxylin.…”