Abstract
The p53 tumor suppressor protein is a transcription factor that plays a prominent role in protecting cells from malignant transformation. Protein levels of p53 and its transcriptional activity are tightly regulated by the ubiquitin E3 ligase MDM2, the gene expression of which is transcriptionally regulated by p53 in a negative feedback loop. The p53 protein is transcriptionally active as a tetramer, and this oligomerization state is modulated by a complex formed by NEURL4 and the ubiquitin E3 ligase HERC2. Here, we report that MDM2 forms a complex with oligomeric p53, HERC2, and NEURL4. HERC2 knockdown results in a decline in MDM2 protein levels without affecting its protein stability, as it reduces its mRNA expression by inhibition of its promoter activation. DNA damage induced by bleomycin dissociates MDM2 from the p53/HERC2/NEURL4 complex and increases the phosphorylation and acetylation of oligomeric p53 bound to HERC2 and NEURL4. Moreover, the MDM2 promoter, which contains p53‐response elements, competes with HERC2 for binding of oligomeric, phosphorylated and acetylated p53. We integrate these findings in a model showing the pivotal role of HERC2 in p53‐MDM2 loop regulation. Altogether, these new insights in p53 pathway regulation are of great interest in cancer and may provide new therapeutic targets.
Highlights
The tumoral protein p53 (TP53) gene encodes the p53 tumor suppressor protein which is a master transcription regulator of an extensive number of genes involved in apoptosis, proliferation, senescence, and metabolism among other cellular processes
As it has been reported that homologous to the E6AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1) 2 (HERC2) binds p53 (Cubillos-Rojas et al, 2014, 2017), and given that mouse double minute (MDM2) is a well-known interactor with p53 (Moll and Petrenko, 2004; Wu et al, 1993), we decided to investigate whether these two events occur simultaneously
A simultaneous interaction of HERC2 with both endogenous p53 and MDM2 can be observed in immunoprecipitation experiments using specific anti-HERC2 antibody in U2OS cells (Fig. 1A)
Summary
The TP53 gene encodes the p53 tumor suppressor protein which is a master transcription regulator of an extensive number of genes involved in apoptosis, proliferation, senescence, and metabolism among other cellular processes. TP53 is the most frequently mutated gene in human cancer Inactivating mutations of this gene are common, being linked to poor patient prognosis. Abbreviations ATM, ataxia/telangiectasia-mutated; ATR, ataxia, telangiectasia and Rad3-related; Bleo, bleomycin; BRCA1, breast cancer 1; CDDP, cisdiamminedichloro platinum (II); CHC, clathrin heavy chain; CHX, cycloheximide; CPH, cullin 7, Parc, HERC2; DNA-PK, DNA-dependent protein kinase; FBXL5, F-box and leucine-rich repeat protein 5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; HERC2, HECT (homologous to the E6AP carboxyl terminus) and RCC1 (regulator of chromosome condensation 1) 2; IP, immunoprecipitation; MDM2, mouse double minute; mut, mutant; NEURL4, neuralized E3 ubiquitin protein ligase 4; NSCLC, non-small-cell lung cancer; NT, nontargeting; PAGE, polyacrylamide gel electrophoresis; PI, pre-immune serum; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene fluoride; RE, response element; RING, really interesting new gene; shRNA, short hairpin RNA; siRNA, small interfering RNA; TP53, tumoral protein p53; wt, wild-type; XPA, xeroderma pigmentosum antigen A
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