SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus

Activating transcription factor 3, a stress sensor, activates p53 by blocking its ubiquitination

Activating transcription factor 3 (ATF3) is rapidly induced by diverse environmental insults including genotoxic stress. We report herein that its interaction with p53, enhanced by genotoxic stress, stabilizes the tumor suppressor thereby augmenting functions of the latter. Overexpression of ATF3 (but not a mutated ATF3 protein (Δ102–139) devoid of its p53-binding region) prevents p53 from MDM2-mediated degradation and leads to increased transcription from p53-regulated promoters. ATF3, but not the Δ102–139 protein, binds the p53 carboxy-terminus and diminishes its ubiquitination and nuclear export. Genotoxic-stressed ATF3-null mouse embryonic fibroblasts, or cells in which ATF3 was reduced by small interference RNA, show inefficient p53 induction and impaired apoptosis compared with wild-type cells. ATF3-null cells (but not wild-type cells), which poorly accumulate p53, are transformed by oncogenic Ras. Thus, ATF3 is a novel stress-activated regulator of p53 protein stability/function providing the cell with a means of responding to a wide range of environmental insult, thus maintaining DNA integrity and protecting against cell transformation

The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells

Data on the relationship between ribosome biogenesis and p53 function indicate that the tumour suppressor can be activated by either nucleolar disruption or ribosomal protein defects. However, there is increasing evidence that the induction of p53 does not always require these severe cellular changes, and data are still lacking on a possible role of ribosome biogenesis in the downregulation of p53. Here, we studied the effect of the up-and downregulation of the rRNA transcription rate on p53 induction in mammalian cells. We found that a downregulation of rRNA synthesis, induced by silencing the POLR1A gene coding for the RNA polymerase I catalytic subunit, stabilised p53 without altering the nucleolar integrity in human cancer cells. p53 stabilisation was due to the inactivation of the MDM2-mediated p53 degradation by the binding of ribosomal proteins no longer used for ribosome building. p53 stabilisation did not occur when rRNA synthesis downregulation was associated with a contemporary reduction of protein synthesis. Furthermore, we demonstrated that in three different experimental models characterised by an upregulation of rRNA synthesis, cancer cells treated with insulin or exposed to the insulin-like growth factor 1, rat liver stimulated by cortisol and regenerating rat liver after partial hepatectomy, the p53 protein level was reduced due to a lowered ribosomal protein availability for MDM2 binding. It is worth noting that the upregulation of rRNA synthesis was responsible for a decreased p53-mediated response to cytotoxic stresses. These findings demonstrated that the balance between rRNA and ribosomal protein synthesis controls the function of p53 in mammalian cells, that p53 can be induced without the occurrence of severe changes of the cellular components controlling ribosome biogenesis, and that conditions characterised by an upregulated rRNA synthesis are associated with a reduced p53 response

AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: Implications for targeting mTOR during malignancy

The phosphatidylinositol 3-kinase (PI3K)/AKT and RAS oncogenic signalling modules are frequently mutated in sporadic human cancer. Although each of these pathways has been shown to play critical roles in driving tumour growth and proliferation, their activation in normal human cells can also promote cell senescence. Although the mechanisms mediating RAS-induced senescence have been well characterised, those controlling PI3K/AKT-induced senescence are poorly understood. Here we show that PI3K/AKT pathway activation in response to phosphatase and tensin homolog (PTEN) knockdown, mutant PI3K, catalytic, α polypeptide (PIK3CA) or activated AKT expression, promotes accumulation of p53 and p21, increases cell size and induces senescence-associated β-galactosidase activity. We demonstrate that AKT-induced senescence is p53-dependent and is characterised by mTORC1-dependent regulation of p53 translation and stabilisation of p53 protein following nucleolar localisation and inactivation of MDM2. The underlying mechanisms of RAS and AKT-induced senescence appear to be distinct, demonstrating that different mediators of senescence may be deregulated during transformation by specific oncogenes. Unlike RAS, AKT promotes rapid proliferative arrest in the absence of a hyperproliferative phase or DNA damage, indicating that inactivation of the senescence response is critical at the early stages of PI3K/AKT-driven tumourigenesis. Furthermore, our data imply that chronic activation of AKT signalling provides selective pressure for the loss of p53 function, consistent with observations that PTEN or PIK3CA mutations are significantly associated with p53 mutation in a number of human tumour types. Importantly, the demonstration that mTORC1 is an essential mediator of AKT-induced senescence raises the possibility that targeting mTORC1 in tumours with activated PI3K/AKT signalling may exert unexpected detrimental effects due to inactivation of a senescence brake on potential cancer-initiating cells