Nucleolar protein, Myb-binding protein 1A, specifically binds to nonacetylated p53 and efficiently promotes transcriptional activation

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Highlights

  • MYBBPA interacts with p53 via lysine residues in the CRD region of p53.

  • MYBBP1A binds specifically to non-acetylated p53 and regulates the activation of p53.

  • Our p53-activating model explains how MYBBP1A cycle efficiently enhances p53 activation.

Abstract

Nucleolar dynamics are important for cellular stress response. We previously demonstrated that nucleolar stress induces nucleolar protein Myb-binding protein 1A (MYBBP1A) translocation from the nucleolus to the nucleoplasm and enhances p53 activity. However, the underlying molecular mechanism is understood to a lesser extent. Here we demonstrate that MYBBP1A interacts with lysine residues in the C-terminal regulatory domain region of p53. MYBBP1A specifically interacts with nonacetylated p53 and induces p53 acetylation. We propose that MYBBP1A dissociates from acetylated p53 because MYBBP1A did not interact with acetylated p53 and because MYBBP1A was not recruited to the p53 target promoter. Therefore, once p53 is acetylated, MYBBP1A dissociates from p53 and interacts with nonacetylated p53, which enables another cycle of p53 activation. Based on our observations, this MYBBP1A–p53 binding property can account for efficient p53-activation by MYBBP1A under nucleolar stress. Our results support the idea that MYBBP1A plays catalytic roles in p53 acetylation and activation.

Introduction

The tumor suppressor p53 is a critical mediator of cellular stress response, which maintains genomic integrity and prevents oncogenic transformation [1]. The protein p53 regulates many target genes that induces cell cycle arrest or apoptosis [2], [3].

DNA damage is known to activate p53 as a transcription factor through post-translational modifications [4], [5] such as phosphorylation, ubiquitination, and acetylation, which are critical in regulating p53 function [6], [7], [8], [9], [10], [11], [12]. The p300/CBP protein that possesses histone acetyltransferase activity and acetylates p53, acts as a coactivator of p53 and augments p53 transcriptional activity [13], [14], [15]. p53 acetylation occurs at multiple lysine residues in the C-terminal regulatory domain (CRD) of p53 (residues 370, 372, 373, 381, 382, and 386) in response to DNA-damaging agents [16], [17], [18]. p53 acetylation also correlates with its sequence-specific DNA binding [19], and augments recruitment of transcriptional activators to p53 [20]. Based on these observations, p53 acetylation is considered to play a vital role in p53 activation [21], [22].

DNA damage induces repression of rRNA transcription by RNA polymerase I, resulting in disruption of nucleolar structure [23], [24]. Low concentrations of actinomycin D (ActD) specifically inhibit RNA polymerase I-driven transcription, but do not affect RNA polymerase II-driven transcription [25], [26]. Therefore, ActD treatment also induces nucleolar disruption [27].

We previously reported that nucleolar disruption induces acetylation and accumulation of p53 without phosphorylation. The nucleolar protein Myb-binding protein 1A (MYBBP1A) binds to p53 and facilitates p53 acetylation to enhance p53-mediated transcription by enhancing the p53–p300 interaction [28]. However, the mechanism by which MYBBP1A enhances p53 acetylation and induces p53 activity is still ambiguous.

Here we demonstrate that MYBBP1A specifically recognizes nonacetylated lysine residues of p53, promotes its acetylation, and dissociates from acetylated p53. The dissociated MYBBP1A may recognize nonacetylated p53 for another cycle of p53 activation. We predict that this MYBBP1A–p53 binding property may explain the effective p53-activating function of MYBBP1A.

Section snippets

Cell culture and treatments

MCF-7 human breast cancer cells and H1299 p53-deficient human lung cancer cells were maintained in DMEM (Sigma). All media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution (Nacalai Tesque). Cells were maintained at 37 °C in an atmosphere containing 5% CO2 and 100% humidity. To induce nucleolar stress, cells were exposed to ActD (5 nM).

siRNA and plasmid DNA transfection

For siRNA transfection, cells at 30–50% confluency were transfected using Lipofectamine RNAi MAX (Invitrogen, Carlsbad,

Lysine residues in the CRD region of p53 play an important role in MYBBP1A–p53 interaction

We have previously demonstrated that cellular stresses induce nucleolar disruption, which leads to MYBBP1A translocation from the nucleolus to the nucleoplasm. Subsequently, MYBBP1A binds to p53 and promotes acetylation by enhancing p53–p300/CBP association, thereby activating the p53 function [28]. However, determination of the MYBBP1A–p53 binding properties remains. To investigate this, we attempted to dissect the mechanism of p53–MYBBP1A interaction. Co-immunoprecipitation experiments

Acknowledgment

This work was supported by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS).

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      Further, NCL associates with both p53 and MDM2, and depending on post-translational regulation can either antagonize their interaction or promote p53 degradation [121,158,159]. Finally, p53 transcriptional activity is mediated by acetylation by its coactivator p300/CBP; this is facilitated by neddylated RPL11 and MYBBP1A, which is sequestered in the nucleolus and released upon nucleolar stress [160,161,151,162]. In addition to sequestration of factors that regulate p53, the nucleolus may play a direct role in p53 transport and its degradation [163].

    • The nucleolar protein Myb-binding protein 1A (MYBBP1A) enhances p53 tetramerization and acetylation in response to nucleolar disruption

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      Taken together, these results suggest that MYBBP1A binds to the p53 dimer via two distinct MYBBP1A sites. We previously reported that MYBBP1A-WT did not bind to the p53–6KA mutant, which bears simultaneous lysine substitutions to alanines at positions 370, 372, 373, 381, 382, and 386 within the p53-CDR region (57). Thus, we investigated whether these lysine residues within p53-CRD regions were critical in the p53-MYBBP1A interaction via the middle (a.a. 643–1150) and C-terminal (a.a. 1151–1328) regions of MYBBP1A.

    1

    These authors contributed equally to this work.

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