Detection of Post-translationally Modified p53 by Western Blotting.

The p53 tumor suppressor has a central role in many key cellular processes including the DNA damage response, aging, stem cell differentiation, and fertility. p53 undergoes extensive regulatory post-translational modification through events such as phosphorylation, acetylation, methylation, and ubiquitylation. Here, we describe western blotting-based methodology for the detection and relative quantification of individual phosphorylation events in p53.

Author Correction: The responses of cancer cells to PLK1 inhibitors reveal a novel protective role for p53 in maintaining centrosome separation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The responses of cancer cells to PLK1 inhibitors reveal a novel protective role for p53 in maintaining centrosome separation.

Polo-like kinase-1 (PLK1) plays a major role in driving mitotic events, including centrosome disjunction and separation, and is frequently over-expressed in human cancers. PLK1 inhibition is a promising therapeutic strategy and works by arresting cells in mitosis due to monopolar spindles. The p53 tumour suppressor protein is a short-lived transcription factor that can inhibit the growth, or stimulate the death, of developing cancer cells.

p53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells.

Cancer genome sequencing has implicated the cytosine deaminase activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes as an important source of mutations in diverse cancers, with APOBEC3B (A3B) expression especially correlated with such cancer mutations. To better understand the processes directing A3B over-expression in cancer, and possible therapeutic avenues for targeting A3B, we have investigated the regulation of A3B gene expression. Here, we show that A3B expression is inversely related to p53 status in different cancer types and demonstrate that this is due to a direct and pivotal role for p53 in repressing A3B expression.

A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity.

The PIM1 serine/threonine protein kinase mediates growth factor and survival signalling, and cooperates potently with c-MYC during tumorigenesis. PIM1 is overexpressed in many human cancers and is a promising target for drug development. PIM1 levels are regulated mainly through cytokine-induced transcription and protein degradation, but mechanisms regulating its activity and levels remain largely unexplored.

Regulation of the p53 response and its relationship to cancer.

p53 has been studied intensively as a major tumour suppressor that detects oncogenic events in cancer cells and eliminates them through senescence (a permanent non-proliferative state) or apoptosis. Consistent with this role, p53 activity is compromised in a high proportion of all cancer types, either through mutation of the TP53 gene (encoding p53) or changes in the status of p53 modulators. p53 has additional roles, which may overlap with its tumour-suppressive capacity, in processes including the DNA damage response, metabolism, aging, stem cell differentiation and fertility.

MAGE-A Cancer/Testis Antigens Inhibit MDM2 Ubiquitylation Function and Promote Increased Levels of MDM4.

Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2.

Critical role for p53-serine 15 phosphorylation in stimulating transactivation at p53-responsive promoters.

The p53 tumour suppressor is induced by various stress stimuli and coordinates an adaptive gene expression programme leading to growth arrest or cell death. Some stimuli, such as DNA damage, lead to rapid and substantial multisite phosphorylation of p53, nucleated initially through phosphorylation of serine 15. Other stimuli, such as hyper-proliferation, do not stimulate p53-phosphorylation, raising questions regarding the physiological role for phosphorylation.

The RNA helicase/transcriptional co-regulator, p68 (DDX5), stimulates expression of oncogenic protein kinase, Polo-like kinase-1 (PLK1), and is associated with elevated PLK1 levels in human breast cancers.

p68 (DDX5) acts both as an ATP-dependent RNA helicase and as a transcriptional co-activator of several cancer-associated transcription factors, including the p53 tumor suppressor. p68 is aberrantly expressed in a high proportion of cancers, but the oncogenic drive for, or the consequences of, these expression changes remain unclear. Here we show that elevated p68 expression in a cohort of human breast cancers is associated significantly with elevated levels of the oncogenic protein kinase, Polo-like kinase-1 (PLK1).

MAGE-A antigens as targets in tumour therapy.

MAGE-A proteins constitute a sub-family of Cancer-Testis Antigens which are expressed mainly, but not exclusively, in germ cells. They are also expressed in various human cancers where they are associated with, and may drive, malignancy. MAGE-A proteins are highly immunogenic and are considered as potential targets for cancer vaccines and/or immuno-therapy.

Immunohistochemical detection of Polo-like kinase-1 (PLK1) in primary breast cancer is associated with TP53 mutation and poor clinical outcom.

Introduction: Polo-like kinase-1 (PLK1) is a crucial driver of cell cycle progression and its down-regulation plays an important checkpoint role in response to DNA damage. Mechanistically, this is mediated by p53 which represses PLK1 expression through chromatin remodelling. Consistent with this model, cultured cells lacking p53 fail to repress PLK1 expression.

Induction and activation of the p53 pathway: a role for the protein kinase CK2?

Protein kinase CK2 has many established in vitro substrates, but it is only within the past few years that we have begun to ascertain which of these are its real physiological targets, how their phosphorylation may contribute towards regulating normal cell physiology, and how phosphorylation of these proteins might influence the development of diseases such as cancer. One of the well-characterised in vitro substrates for CK2 is the tumour suppressor protein, p53. However, the physiological nature of this interaction has never been fully established.

Mage-A cancer/testis antigens inhibit p53 function by blocking its interaction with chromatin.

The p53 tumor suppressor plays a major protective role in tumor prevention by coordinating changes in gene expression that lead to the elimination of cancer cells. Mage-A proteins comprise a family of metastasis-associated transcriptional regulators that potently inhibit p53 function. Here, we show that Mage-A interacts with 3 distinct peptides each of which is located within the DNA binding surface of the core domain of p53 and encompasses amino acids that are critical for site-specific DNA binding.

p53-dependent repression of polo-like kinase-1 (PLK1).

PLK1 is a critical mediator of G₂/M cell cycle transition that is inactivated and depleted as part of the DNA damage-induced G₂/M checkpoint. Here we show that downregulation of PLK1 expression occurs through a transcriptional repression mechanism and that p53 is both necessary and sufficient to mediate this effect. Repression of PLK1 by p53 occurs independently of p21 and of arrest at G₁/S where PLK1 levels are normally repressed in a cell cycle-dependent manner through a CDE/CHR element.

Posttranslational modification of p53: cooperative integrators of function.

The p53 protein is modified by as many as 50 individual posttranslational modifications. Many of these occur in response to genotoxic or nongenotoxic stresses and show interdependence, such that one or more modifications can nucleate subsequent events. This interdependent nature suggests a pathway that operates through multiple cooperative events as opposed to distinct functions for individual, isolated modifications.

Phosphorylation of serine 392 in p53 is a common and integral event during p53 induction by diverse stimuli.

Post-translational modifications play important roles during the stabilisation and activation of p53 by various genotoxic and non-genotoxic stresses. Ser392 has been reported to be a major UV-stimulated phosphorylation site that is modified through the p38 MAPK pathway in a manner that may involve recruitment of CK2. Here we show that phosphorylation of Ser392 is an integral event that occurs not only in response to UV, but also during the induction of p53 by a range of stimuli including treatment of cells with the MDM2 inhibitor, Nutlin 3a.

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

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.

Polo-like kinase-1 phosphorylates MDM2 at Ser260 and stimulates MDM2-mediated p53 turnover.

The E3 ubiqutin ligase, murne double-minute clone 2 (MDM2), promotes the degradation of p53 under normal homeostatic conditions. Several serine residues within the acidic domain of MDM2 are phosphorylated to maintain its activity but become hypo-phosphorylated following DNA damage, leading to inactivation of MDM2 and induction of p53. However, the signalling pathways that mediate these phosphorylation events are not fully understood.

CK1alpha plays a central role in mediating MDM2 control of p53 and E2F-1 protein stability.

The ubiquitin ligase murine double minute clone 2 (MDM2) mediates ubiquitination and degradation of the tumor suppressor p53. The activation and stabilization of p53 by contrast is maintained by enzymes catalyzing p53 phosphorylation and acetylation. Casein kinase 1 (CK1) is one such enzyme; it stimulates p53 after transforming growth factor-beta treatment, irradiation, or DNA virus infection.

Tumour suppression by p53: a role for the DNA damage response?

Loss of p53 function occurs during the development of most, if not all, tumour types. This paves the way for genomic instability, tumour-associated changes in metabolism, insensitivity to apoptotic signals, invasiveness and motility. However, the nature of the causal link between early tumorigenic events and the induction of the p53-mediated checkpoints that constitute a barrier to tumour progression remains uncertain.

Nitrogen fertilizer effects on soil carbon balances in midwestern U.S. agricultural systems.

A single ecosystem dominates the Midwestern United States, occupying 26 million hectares in five states alone: the corn-soybean agroecosystem [Zea mays L.-Glycine max (L.) Merr.

Heat shock factor-1 modulates p53 activity in the transcriptional response to DNA damage.

Here we define an important role for heat shock factor 1 (HSF1) in the cellular response to genotoxic agents. We demonstrate for the first time that HSF1 can complex with nuclear p53 and that both proteins are co-operatively recruited to p53-responsive genes such as p21. Analysis of natural and synthetic cis elements demonstrates that HSF1 can enhance p53-mediated transcription, whilst depletion of HSF1 reduces the expression of p53-responsive transcripts.

14-3-3 Binding to Pim-phosphorylated Ser166 and Ser186 of human Mdm2--Potential interplay with the PKB/Akt pathway and p14(ARF).

Here we show that 14-3-3 proteins bind to Pim kinase-phosphorylated Ser166 and Ser186 on the human E3 ubiquitin ligase mouse double minute 2 (Mdm2), but not protein kinase B (PKB)/Akt-phosphorylated Ser166 and Ser188. Pim-mediated phosphorylation of Ser186 blocks phosphorylation of Ser188 by PKB, indicating potential interplay between the Pim and PKB signaling pathways in regulating Mdm2. In cells, expression of Pim kinases promoted phosphorylation of Ser166 and Ser186, interaction of Mdm2 with endogenous 14-3-3s and p14(ARF), and also increased the amount of Mdm2 protein by a mechanism that does not require Pim kinase activities.

A central role for CK1 in catalyzing phosphorylation of the p53 transactivation domain at serine 20 after HHV-6B viral infection.

The tumor suppressor protein p53 is activated by distinct cellular stresses including radiation, hypoxia, type I interferon, and DNA/RNA virus infection. The transactivation domain of p53 contains a phosphorylation site at Ser20 whose modification stabilizes the binding of the transcriptional co-activator p300 and whose mutation in murine transgenics induces B-cell lymphoma. Although the checkpoint kinase CHK2 is implicated in promoting Ser20 site phosphorylation after irradiation, the enzyme that triggers this phosphorylation after DNA viral infection is undefined.