Dataset Information

CSA and CSB proteins interact with p53 and regulate its Mdm2-dependent ubiquitination.

ABSTRACT: Mutations in Cockayne syndrome (CS) A and B genes (CSA and CSB) result in a rare genetic disease that affects the development and homeostasis of a wide range of tissues and organs. We previously correlated the degenerative phenotype of patients to the enhanced apoptotic response, exhibited by CS cells, which is associated with the exceptional induction of p53 protein, upon a variety of stress stimuli. Here we showed that the elevated and persistent levels of p53 displayed by CS cells are due to the insufficient ubiquitination of the p53 protein. We further demonstrated that CSA and CSB proteins associate in a unique complex with p53 and Mdm2; this interaction greatly stimulates the ubiquitination of p53 in an Mdm2-dependent manner. Tandem affinity purification and immunoprecipitations combined with mass spectrometry studies indicate that CSA and CSB associate within a Cullin Ring Ubiquitin Ligase complex responsible, under certain circumstances, for p53 ubiquitination. This study identifies CSA and CSB as the key elements of a regulatory mechanism that equilibrate beneficial and detrimental effects of p53 activity upon cellular stress. The deregulation of p53, in absence of either of the CS proteins, can potentially explain the early onset degeneration of tissues and organs observed in CS patients.

SUBMITTER: Latini P

PROVIDER: S-EPMC6245570 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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CSA and CSB proteins interact with p53 and regulate its Mdm2-dependent ubiquitination.

Latini Paolo P Frontini Mattia M Caputo Manuela M Gregan Juraj J Cipak Lubos L Filippi Silvia S Kumar Vivek V Vélez-Cruz Renier R Stefanini Miria M Proietti-De-Santis Luca L

Cell cycle (Georgetown, Tex.) 20111101 21

Mutations in Cockayne syndrome (CS) A and B genes (CSA and CSB) result in a rare genetic disease that affects the development and homeostasis of a wide range of tissues and organs. We previously correlated the degenerative phenotype of patients to the enhanced apoptotic response, exhibited by CS cells, which is associated with the exceptional induction of p53 protein, upon a variety of stress stimuli. Here we showed that the elevated and persistent levels of p53 displayed by CS cells are due to ...[more]

PMID: 22032989

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Project description:MDM2 is the principal antagonist of the tumor suppressor p53. p53 binds to its cognate DNA element within promoters and activates the transcription of adjacent genes. These target genes include MDM2. Upon induction by p53, the MDM2 protein binds and ubiquitinates p53, triggering its proteasomal degradation and providing negative feedback. This raises the question whether MDM2 can also remove p53 from its target promoters, and whether this also involves ubiquitination. In the present paper, we employ the MDM2-targeted small molecule Nutlin-3a (Nutlin) to disrupt the interaction of MDM2 and p53 in three different cancer cell lines: SJSA-1 (osteosarcoma), 93T449 (liposarcoma; both carrying amplified MDM2), and MCF7 (breast adenocarcinoma). Remarkably, removing Nutlin from the culture medium for less than five minutes not only triggered p53 ubiquitination, but also dissociated most p53 from its chromatin binding sites, as revealed by chromatin immunoprecipitation. This also resulted in reduced p53-responsive transcription, and it occurred much earlier than the degradation of p53 by the proteasome, arguing that MDM2 removes p53 from promoters prior to and thus independent of degradation. Accordingly, the short-term pharmacological inhibition of the proteasome did not alter the removal of p53 from promoters by Nutlin washout. However, when the proteasome inhibitor was applied for several hours, depleting non-conjugated ubiquitin prior to eliminating Nutlin, this compromised the removal of DNA-bound p53, as did an E1 ubiquitin ligase inhibitor. This suggests that the ubiquitination of p53 by MDM2 is necessary for its clearance from promoters. Depleting the MDM2 cofactor MDM4 in SJSA cells did not alter the velocity by that p53 was removed from promoters upon Nutlin washout. We conclude that MDM2 antagonizes p53 not only by covering its transactivation domain and by destabilization, but also by the rapid, ubiquitin-dependent termination of p53-chromatin interactions.

Dataset Information

CSA and CSB proteins interact with p53 and regulate its Mdm2-dependent ubiquitination.

Publications

CSA and CSB proteins interact with p53 and regulate its Mdm2-dependent ubiquitination.

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