Review
The regulation of MDM2 by multisite phosphorylation—Opportunities for molecular-based intervention to target tumours?

https://doi.org/10.1016/j.semcancer.2009.10.005Get rights and content

Abstract

The p53 tumour suppressor is a tightly controlled transcription factor that coordinates a broad programme of gene expression in response to various cellular stresses leading to the outcomes of growth arrest, senescence, or apoptosis. MDM2 is an E3 ubiquitin ligase that plays a key role in maintaining p53 at critical physiological levels by targeting it for proteasome-mediated degradation. Expression of the MDM2 gene is p53-dependent and thus p53 and MDM2 operate within a negative feedback loop in which p53 controls the levels of its own regulator. Induction and activation of p53 involves mainly the uncoupling of p53 from its negative regulators, principally MDM2 and MDMX, an MDM2-related and -interacting protein that inhibits p53 transactivation function. MDM2 is tightly regulated through various mechanisms including gene expression, protein turnover (mediated by auto-ubiquitylation), protein–protein interaction with key regulators, and post-translational modification, mainly, but not exclusively, by multisite phosphorylation. The purpose of the present article is to review our current knowledge of the signalling mechanisms that focus on MDM2, and indeed MDMX, through both phosphorylation mechanisms and peptide-docking events and to consider the wider implications of these regulatory events in the context of coordinated regulation of the p53 response. This analysis also provides an opportunity to consider the signalling pathways regulating MDM2 as potential targets for non-genotoxic therapies aimed at restoring p53 function in tumour cells.

Introduction

The p53 tumour suppressor is a potent transcription factor that is maintained at low levels through ubiquitylation and proteasome-dependent degradation in normal unstressed cells [1], [2]. Activation of p53 occurs in response to a variety of stresses including DNA damage, hyper-proliferation and hypoxia, and is mediated by a complex series of events that are determined by the type and intensity of the inducing stress signal and the cell type [3], [4]. Induction of p53 leads to changes in the balance of gene expression by up-regulating genes that promote growth arrest, apoptosis and repair and repressing genes involved in proliferation [5]. p53 can also mediate its apoptotic effects, at least in part, by regulating the interaction of key BCL2 family members. Additionally, p53 is critically involved in normal cellular processes such as glycolysis, mitochondrial respiration, differentiation, angiogenesis and ageing [1]. Inactivation of p53 through mutation of the TP53 gene or disruption of the p53 pathway eliminates p53 function in most, if not all, cancers.

Several E3 ubiquitin ligases are known to promote p53 degradation, including MDM2, COP1, PirH2, TOPORS and ARF-BP1 [6]. Three of these, MDM2, COP1 and PirH2 are established transcriptional targets of p53, thereby ensuring a balance between the levels of p53 and its regulators under non-stressed conditions. Of this group, MDM2 is the most extensively studied and the observation that mice lacking MDM2 die of massive p53-dependent apoptosis during the early stages of development provides strong evidence underpinning a critical role for MDM2 in p53 homeostasis [7], [8]. Elevated expression or amplification of MDM2 is a common event in several types of cancer. While this is thought principally to suppress p53 function during tumorigenesis, various lines of evidence suggest that MDM2 contributes to tumour development through additional p53-independent mechanisms [9]. In addition to p53, MDM2 has a growing list of substrates (including MDMX [see below], Rb, androgen receptor, E2F1, JMY and others) and interacts with a host of cellular factors that can influence its function [10]. Interaction of MDM2 with one or more of these proteins may contribute to tumorigenesis. Additionally, small nucleotide polymorhisms in the MDM2 promoter can have a profound effect on cancer susceptibility [11]. MDM2 itself, and the p53/MDM2 interaction, has therefore been under significant scrutiny from the perspective of developing small molecules that will alleviate its bearing on tumour development [12].

MDM2 comprises several functional domains including: (i) a p53 binding domain at its N-terminus; (ii) a region containing nuclear import and export sequences that mediate nucleo-cytoplasmic shuttling; this region also contains several important phosphorylation sites; (iii) a highly phosphorylated acidic domain that is critical for ubiquitylation and degradation of p53 and of MDM2 itself; (iv) a zinc finger that is thought to play a role in interacting with ribosomal proteins; and (v) a RING finger domain that mediates ubiquitylation of substrates (Fig. 1). Mechanistically, MDM2 and p53 interact strongly through their respective N-termini. Current evidence suggests that, following this interaction, a second point of contact becomes available in which the acidic domain of MDM2 can act as a key ubiquitylation signal that interacts with the core (DNA binding) domain of p53, thus permitting ubiquitylation of the C-terminus of p53 by the RING finger [13], [14], [15], [16].

The mechanism of this multi-domain mediated ubiquitylation of p53 by MDM2 is highlighted in Fig. 2 with key elements including; (1) entry of the p53 BOX-I substrate with the N-terminal Hy domain of MDM2 [15]; (2) conformational changes that “open” the acidic central domain to the ubiquitylation signal in the DNA-binding domain of p53; and (3) an undefined interaction with E2 and the RING domain of MDM2 that drives p53 ubiquitylation after E2-mediated ubiquitin transfer [17], [18]. Key molecular evidence for this ubiquitylation model include the observations that: (a) Nutlin cannot block p53 ubiquitylation and can in fact stimulate ubiquitylation in trans [15]; (b) peptides that bind to the acidic domain derived from p53 itself, ARF, or Rb can block ubiquitylation [15], [19]; and (c) TAFII250 binding to MDM2 stabilizes the interaction between acidic domain of MDM2 and p53, driving p53 turnover [20]. Together, these data indicate the central role of the acidic domain of MDM2 is a key motif in driving MDM2 function and highlights the importance of understanding the kinase pathways that target this domain for regulating MDM2 activity or proteins like TAFII250 that appear to stimulate acidic domain functions. The role of other key proteins including MDMX in this core ubiquitylation model are undefined. Further structure–function studies will be required to fully reconstitute and understand this dynamic multi-protein and multi-interface ubiquitylation complex.

Nuclear export of p53 by MDM2 is important for its degradation by cytoplasmic proteasomes [21] (although various groups have shown that some degree of nuclear proteasomal degradation can occur [22] and references therein). Additionally, MDM2 can inhibit p53 transcriptional activity through binding to its N-terminal transactivation domain and sterically blocking interaction with transcription factors.

The MDMX protein (also known as MDM4) is structurally related to MDM2 and is also an essential non-redundant negative regulator of p53 (reviewed in [23], [24]). However, its role is mainly to regulate p53 transcriptional activity and is therefore distinct from that of MDM2 which regulates p53 levels [25]. The critical contribution of this function to p53 regulation is underscored by the observation that mice lacking MDMX, but retaining p53, die during the mid-gestation stages of development. MDMX expression is also elevated in a significant proportion of various human tumours that retain wild type p53, consistent with the idea that high levels of MDMX can alleviate the selection pressure for p53 mutation during tumorigenesis, particularly in diseases such as retinoblastoma [26], [27]. As with MDM2, MDMX can bind to the N-terminus of p53 and block its transcription function but, since it is a defective ubiquitin ligase, cannot directly mediate ubiquitylation. However, MDM2/MDMX heterodimerization, which occurs through their respective RING fingers, is thought to stimulate the intrinsic ligase function of MDM2 [28]. Expression of MDMX is not regulated by p53 but may be dependent on mitogenic signalling [29]. Additionally, like p53, MDMX is ubiquitylated and degraded in an MDM2-dependent manner. Under normal unstressed conditions, MDM2 is protected from auto-ubiquitylation through an interaction in which the adaptor protein, DAXX, recruits the de-ubiquitylating enzyme, HAUSP (Herpes virus-associated ubiquitin-specific protease). In response to stress signals, this complex is disrupted, leaving MDM2 free to preferentially mediate its own destruction and that of MDMX [30]. The mechanism of these events involves protein phosphorylation and is discussed below.

The fundamental event underlying the p53 induction process is the uncoupling of p53 from partner proteins that inhibit its activity and mediate its destruction, principally MDM2 and MDMX (reviewed in [31]). This can occur through various (overlapping) mechanisms and is dependent upon the type of stress. For example, UV radiation promotes phosphorylation of p53 and MDM2 (see below), and redistributes nucleophosmin to the nucleoplasm where it blocks the MDM2–p53 interaction [32]. In contrast, hyper-proliferation leads principally to the induction of the MDM2 negative regulatory partner, p14ARF (hereafter ARF), which inhibits MDM2 and sequesters it in the nucleolus (reviewed in [33]). In response to nucleolar stress, MDM2 function is inhibited through its interaction with key ribosomal proteins [34], [35], [36], [37], [38], [39]. Double strand breaks, however, acting mainly through the ATM (ataxia telangiectasia mutated) pathway, promote multiple post-translational events in p53, MDM2 and MDMX, that lead to the disruption of p53–MDM2–MDMX complexes and the ubiquitylation and destruction of MDM2 and MDMX (discussed below). Clinically important anti-cancer agents including ionizing radiation (IR) and a variety of genotoxic drugs induce p53 through these mechanisms and can stimulate apoptosis in cancer cells that retain wild type p53. However, given that these treatments act by damaging DNA, surviving cells can often harbour genetic changes that may lie beneath the longer term development of cancer. Consequently, non-genotoxic strategies aimed at inducing p53 in tumour cells are actively sought to replace these approaches. The signalling pathways that regulate p53 induction may provide potential avenues for such therapeutic consideration.

Section snippets

Phosphorylation of MDM2

The MDM2 protein is modified by multisite phosphorylation [40]. The various phosphorylation sites fall into essentially three clusters (Fig. 1). (a) A group of four sites (serines 157, 166, 186 and 188) lie adjacent to the nuclear export sequence and the two nuclear localization sequences. Each of these sites is modified through a pathway(s) that is responsive to growth factor/mitogenic signalling. (b) A group of up to eight sites are phosphorylated under normal unstressed conditions (serines

Phosphorylation of MDMX

No consideration of MDM2 phosphorylation, and its role in regulating the p53 response, would be complete without a discussion of its sister protein, MDMX, which plays an integral and coordinate role together with MDM2 in regulating p53 function. Like MDM2, MDMX is subject to multisite phosphorylation (summarised in Fig. 5) and much of our knowledge of the modification of this protein comes from analysis of its fate in response to DNA damage.

Agents that induce DNA strand breaks promote rapid

A role for modulation of MDM2 function?

It is widely accepted that understanding the mechanisms governing the activation of p53 in response to cellular stress, or down-regulation of the response through growth factor signalling or cancer-related hyper-proliferative mechanisms, will help identify possible targets for therapy in tumours that express wild type p53. In this respect several groups have developed small molecules aimed at activating or reactivating the p53 pathway. For example, compounds such as Nutlin-3a [96] and RITA [97]

Conflict of interest statement

None declared.

References (111)

  • C.L. Brooks et al.

    Mol Cell

    (2006)
  • G.L. Bond et al.

    Cell

    (2004)
  • R. Kulikov et al.

    J Biol Chem

    (2006)
  • H. Shimizu et al.

    J Biol Chem

    (2002)
  • M. Wallace et al.

    Mol Cell

    (2006)
  • M. Kostic et al.

    J Mol Biol

    (2006)
  • F. Toledo et al.

    Int J Biochem Cell Biol

    (2007)
  • F. Toledo et al.

    Cancer Cell

    (2006)
  • S. Kurki et al.

    Cancer Cell

    (2004)
  • D. Michael et al.

    Semin Cancer Biol

    (2003)
  • M.S. Dai et al.

    J Biol Chem

    (2004)
  • B. Guerra et al.

    FEBS Lett

    (1998)
  • T.J. Hay et al.

    FEBS Lett

    (2000)
  • X. Lu et al.

    Cancer Cell

    (2007)
  • S. Ries et al.

    Cell

    (2000)
  • J. Feng et al.

    J Biol Chem

    (2004)
  • D. Milne et al.

    FEBS Lett

    (2004)
  • Y. Ogawara et al.

    J Biol Chem

    (2002)
  • L.D. Mayo et al.

    J Biol Chem

    (2002)
  • L. Burch et al.

    J Mol Biol

    (2004)
  • M.W. Jackson et al.

    J Biol Chem

    (2006)
  • C. Hogan et al.

    J Biol Chem

    (2008)
  • Q. Zhu et al.

    J Biol Chem

    (2001)
  • B. Bothner et al.

    J Mol Biol

    (2001)
  • P. Sdek et al.

    J Biol Chem

    (2004)
  • L. Bozulic et al.

    Mol Cell

    (2008)
  • D. Baltzis et al.

    J Biol Chem

    (2007)
  • T. Leveillard et al.

    J Biol Chem

    (1997)
  • V. Lopez-Pajares et al.

    J Biol Chem

    (2008)
  • Y. Yang et al.

    Cancer Cell

    (2005)
  • V.J. Bykov et al.

    J Biol Chem

    (2005)
  • S. Lain et al.

    Cancer Cell

    (2008)
  • K.H. Vousden et al.

    Nat Rev

    (2007)
  • K.H. Vousden et al.

    Nat Rev Cancer

    (2002)
  • J.M. Espinosa

    Oncogene

    (2008)
  • F. Murray-Zmijewski et al.

    Nat Rev

    (2008)
  • T. Riley et al.

    Nat Rev

    (2008)
  • S.N. Jones et al.

    Nature

    (1995)
  • R. Montes de Oca Luna et al.

    Nature

    (1995)
  • K. Onel et al.

    Mol Cancer Res

    (2004)
  • G. Ganguli et al.

    Mol Cancer Res

    (2003)
  • W. Wang et al.

    Curr Opin Oncol

    (2008)
  • G.W. Yu et al.

    Proc Natl Acad Sci USA

    (2006)
  • K. Linke et al.

    Cell Death Differ

    (2008)
  • C.A. Midgley et al.

    Oncogene

    (2000)
  • N. Allende-Vega et al.

    Oncogene

    (2007)
  • J. Roth et al.

    EMBO J

    (1998)
  • J.M. Stommel et al.

    EMBO J

    (2004)
  • J.C. Marine et al.

    J Cell Sci

    (2007)
  • D. Danovi et al.

    Mol Cell Biol

    (2004)
  • Cited by (57)

    • TP53 and MDM2 genetic alterations in non-small cell lung cancer: Evaluating their prognostic and predictive value

      2016, Critical Reviews in Oncology/Hematology
      Citation Excerpt :

      MDM2 binds the N-terminal domain of p53, thereby inhibiting p53’s transcriptional activity and exporting p53 from the nucleus into the cytoplasm (Moll and Petrenko, 2003). In addition, MDM2 acts as an E3-ubiquitin ligase for p53 through its RING-finger domain, which leads to its proteasomal degradation (Meek and Hupp, 2010) (Fig. 2). However, in the presence of DNA damage the function of MDM2 is altered via several mechanisms.

    • Network regulation meets substrate modification chemistry

      2023, Journal of the Royal Society Interface
    View all citing articles on Scopus
    View full text