Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses

p53 protects against cancer through its capacity to induce cell cycle arrest or apoptosis under a large variety of cellular stresses. It is not known how such diversity of signals can be integrated by a single molecule. However, the literature reveals that a common denominator in all p53-inducing stresses is nucleolar disruption. We thus postulated that the impairment of nucleolar function might stabilize p53 by preventing its degradation. Using micropore irradiation, we demonstrate that large amounts of nuclear DNA damage fail to stabilize p53 unless the nucleolus is also disrupted. Forcing nucleolar disruption by anti-upstream binding factor (UBF) microinjection (in the absence of DNA damage) also causes p53 stabilization. We propose that the nucleolus is a stress sensor responsible for maintenance of low levels of p53, which are automatically elevated as soon as nucleolar function is impaired in response to stress. Our model integrates all known p53-inducing agents and also explains cell cycle-related variations in p53 levels which correlate with established phases of nucleolar assembly/disassembly through the cell cycle.     

E2F1 and E2F3 activate ATM through distinct mechanisms to promote E1A-induced apoptosis

Deregulation of the Rb-E2F pathway occurs in many cancers and results in aberrant cell proliferation as well as an increased propensity to undergo apoptosis. In most cases, apoptosis in response to Rb inactivation involves the activation of p53 but the molecular details of the signaling pathway connecting Rb loss to p53 are poorly understood. Here we demonstrate that the E1A oncoprotein, which binds and inhibits Rb family members, induces the accumulation and phosphorylation of p53 through the DNA damage-responsive ATM kinase. As a result, E1A-induced apoptosis is significantly impaired in cells lacking ATM. In contrast, inactivation of ARF, which is widely believed to activate p53 in response to oncogenic stress, has no effect on p53 induction and only a modest effect on apoptosis in response to E1A. Both E2F1 and E2F3 contribute to ATM-dependent phosphorylation of p53 and apoptosis in cells expressing E1A. However, deregulated E2F3 activity is implicated in the DNA damage caused by E1A while E2F1 stimulates ATM- and NBS1-dependent p53 phosphorylation and apoptosis through a mechanism that does not involve DNA damage.     

Perturbation of RNA Polymerase I transcription machinery by ablation of HEATR1 triggers the RPL5/RPL11-MDM2-p53 ribosome biogenesis stress checkpoint pathway in human cells

Ribosome biogenesis is an energy consuming process which takes place mainly in the nucleolus. By producing ribosomes to fuel protein synthesis, it is tightly connected with cell growth and cell cycle control. Perturbation of ribosome biogenesis leads to the activation of p53 tumor suppressor protein promoting processes like cell cycle arrest, apoptosis or senescence. This ribosome biogenesis stress pathway activates p53 through sequestration of MDM2 by a subset of ribosomal proteins (RPs), thereby stabilizing p53. Here, we identify human HEATR1, as a nucleolar protein which positively regulates ribosomal RNA (rRNA) synthesis. Downregulation of HEATR1 resulted in cell cycle arrest in a manner dependent on p53. Moreover, depletion of HEATR1 also caused disruption of nucleolar structure and activated the ribosomal biogenesis stress pathway – RPL5 / RPL11 dependent stabilization and activation of p53. These findings reveal an important role for HEATR1 in ribosome biogenesis and further support the concept that perturbation of ribosome biosynthesis results in p53-dependent cell cycle checkpoint activation, with implications for human pathologies including cancer.     

Loss of Peter Pan protein is associated with cell cycle defects and apoptotic events

The nucleolus is a subnuclear compartment, which governs ribosome biogenesis. Moreover, it functions as hub in the stress response by orchestrating a variety of processes, such as regulation of cell cycle progression, senescence and apoptosis. Emerging evidence links the nucleolus also to the control of genomic stability and the development of human malignancies. Peter Pan (PPAN) is an essential ribosome biogenesis factor localized to nucleoli and mitochondria. We earlier showed that PPAN depletion triggers p53-independent nucleolar stress and apoptosis. In this study we investigated the precise localization of nucleolar PPAN during cell cycle and its function in cell cycle regulation. We show that PPAN knockdown impairs cell proliferation and induces G0/G1 as well as later G2/M cell cycle arrest in cancer cells. Although PPAN knockdown stabilizes the tumor suppressor p53 and induces CDKN1A/p21, the proliferation defects occur largely in a p53/p21-independent manner. We noticed a reduced number of knockdown cells entering cytokinesis and an elevation of binucleation. PPAN knockdown is also associated with increased H2A.X phosphorylation (γH2A.X) in cancer cells. We evaluated a potential signaling axis through the DNA damage response kinases ATM and ATR and alternatively apoptosis as a potent driver of γH2A.X. Interestingly, PPAN knockdown does not involve activation of ATM/ATR. Instead, γH2A.X is generated as a consequence of apoptosis induction in cancer cells. Strikingly, PPAN depletion in human fibroblasts did neither provoke apoptosis nor H2A.X phosphorylation, but recapitulated p53 stabilization. In summary, our data underline the notion that the PPAN-mediated, p53-independent nucleolar stress response has multiple facets.     

Nucleolar Disruption Ensures Nuclear Accumulation of p21 upon DNA Damage

p21^cip1 is a protein with a dual function in oncogenesis depending mainly on its intracellular localization: tumor suppressor in the nucleus and oncogenic in the cytoplasm. After DNA damage, p21^cip1 increases and accumulates in the nucleus to ensure cell cycle arrest. We show here that the nuclear accumulation of p21^cip1 is not only a consequence of its increased levels but to a DNA damage cellular response, which is ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) and p53 independent. Furthermore, after DNA damage, p21^cip1 not only accumulates in the nucleoplasm but also in the disrupted nucleolus. Inside the nucleolus, it is found in spherical structures, which are not a protrusion of the nucleoplasm. The steady‐state distribution of p21^cip1 in the nucleolus resulted from a highly dynamic equilibrium between nucleoplasmic and nucleolar p21^cip1 and correlated with the inhibition of p21^cip1 nuclear export. Most interestingly, inhibition of ribosomal export after expressing a dominant‐negative mutant of nucleophosmin induced p21^cip1 accumulation in the nucleus and the nucleolus in the absence of DNA damage. This proved the existence of a nucleolar export route to the cytoplasm for p21^cip1 in control conditions that would be inhibited upon DNA damage leading to nuclear and nucleolar accumulation of p21^cip1.     

ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53

In response to DNA damage, ataxia-telangiectasia mutant and ataxia-telangiectasia and Rad-3 activate p53, resulting in either cell cycle arrest or apoptosis. We report here that DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ionizing irradiation (IR), and ultraviolet irradiation (UV) activate ERK1/2 (ERK) mitogen-activated protein kinase in primary (MEF and IMR90), immortalized (NIH3T3) and transformed (MCF-7) cells. ERK activation in response to ETOP was abolished in ATM-/- fibroblasts (GM05823) and was independent of p53. The MEK1 inhibitor PD98059 prevented ERK activation but not p53 stabilization. Maximal ERK activation in response to DNA damage was not attenuated in MEF(p53-/-). However, ERK activation contributes to either cell cycle arrest or apoptosis in response to low or high intensity DNA insults, respectively. Inhibition of ERK activation by PD98059 or U0126 attenuated p21(CIP1) induction, resulting in partial release of the G(2)/M cell cycle arrest induced by ETOP. Furthermore, PD98059 or U0126 also strongly attenuated apoptosis induced by high dose ETOP, ADR, or UV. Conversely, enforced activation of ERK by overexpression of MEK-1/Q56P sensitized cells to DNA damage-induced apoptosis. Taken together, these results indicate that DNA damage activates parallel ERK and p53 pathways in an ATM-dependent manner. These pathways might function cooperatively in cell cycle arrest and apoptosis.     

Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro

The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.     

The novel mouse Polo-like kinase 5 responds to DNA damage and localizes in the nucleolus

Polo-like kinases (Plk1-4) are emerging as an important class of proteins involved in many aspects of cell cycle regulation and response to DNA damage. Here, we report the cloning of a fifth member of the polo-like kinase family named Plk5. DNA and protein sequence analyses show that Plk5 shares more similarities with Plk2 and Plk3 than with Plk1 and Plk4. Consistent with this observation, we show that mouse Plk5 is a DNA damage inducible gene. Mouse Plk5 protein localizes predominantly to the nucleolus, and deletion of a putative nucleolus localization signal (NoLS) within its N-terminal moiety disrupts its nucleolar localization. Ectopic expression of Plk5 leads to cell cycle arrest in G1, decreased DNA synthesis, and to apoptosis, a characteristic it shares with Plk3. Interestingly, in contrast to mouse Plk5 gene, the sequence of human Plk5 contains a stop codon that produces a truncated protein lacking part of the kinase domain.