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. This occurs through the induction of p21 (CDKN1A) and the recruitment of the repressive DREAM complex to the A3B gene promoter, such that loss of p53 through mutation, or human papilloma virus-mediated inhibition, prevents recruitment of the complex, thereby causing elevated A3B expression and cytosine deaminase activity in cancer cells. As p53 is frequently mutated in cancer, our findings provide a mechanism by which p53 loss can promote cancer mutagenesis.

LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner.

Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53.

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. Moreover, many mutant p53 proteins, termed 'gain-of-function' (GOF), acquire new activities that help drive cancer aggression. p53 is regulated mainly through protein turnover and operates within a negative-feedback loop with its transcriptional target, MDM2 (murine double minute 2), an E3 ubiquitin ligase which mediates the ubiquitylation and proteasomal degradation of p53. Induction of p53 is achieved largely through uncoupling the p53-MDM2 interaction, leading to elevated p53 levels. Various stress stimuli acting on p53 (such as hyperproliferation and DNA damage) use different, but overlapping, mechanisms to achieve this. Additionally, p53 activity is regulated through critical context-specific or fine-tuning events, mediated primarily through post-translational mechanisms, particularly multi-site phosphorylation and acetylation. In the present review, I broadly examine these events, highlighting their regulatory contributions, their ability to integrate signals from cellular events towards providing most appropriate response to stress conditions and their importance for tumour suppression. These are fascinating aspects of molecular oncology that hold the key to understanding the molecular pathology of cancer and the routes by which it may be tackled therapeutically.

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. Here, we show that a basal level of Ser15 phosphorylation occurs in both unstimulated cells and cells stimulated pharmacologically to induce p53. p53 in which Ser15 is substituted by alanine (S15A) fails to mediate p53-dependent transcription or growth arrest but can be rescued by substitution with aspartate (S15D: a phospho-mimic). Chromatin immunoprecipitation (ChIP) analyses show that, while wt- and S15A-p53 are detectable on the CDKN1A (p21) promoter (as a representative p53-responsive promoter), S15A-p53 does not stimulate histone acetylation (a measure of chromatin relaxation), nor is its recruitment stimulated, in response to a DNA damage or pharmacological stimulus. These data demonstrate that Ser15 phosphorylation is required for p53 function in the physiological context of p53-responsive promoters and suggest a key and possibly universal role even for low levels of this modification in promoting p53-transcription function.

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. This study examined PLK1 expression, p53 mutation and clinical outcome in breast cancer. METHODS: Immunohistochemistry was performed using antibodies to PLK1, MDM2 and Ki67 on Tissue Micro-Array (TMA) slides of a cohort of 215 primary breast cancers. The TP53 gene (encoding p53) was sequenced in all tumour samples. Protein expression scored using the "Quickscore" method was compared with clinical and pathological data, including survival. RESULTS: Staining of PLK1 was observed in 11% of primary breast tumours and was significantly associated with the presence of TP53 mutation (P = 0.0063). Moreover, patients with both PLK1 expression and TP53 mutation showed a significantly worse survival than those with either PLK1 expression or TP53 mutation alone. There was also a close association of elevated PLK1 with triple negative tumours, considered to be poor prognosis breast cancers that generally harbour TP53 mutation. Further association was observed between elevated PLK1 levels and the major p53 negative regulator, MDM2. CONCLUSIONS: The significant association between elevated PLK1 and TP53 mutation in women with breast cancer is consistent with escape from repression of PLK1 expression by mutant p53. Tumours expressing elevated PLK1, but lacking functional p53, may be potential targets for novel anti-PLK1-targeted drugs.

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. 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.

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. In the present article, we summarise a recent study from our laboratory showing that phosphorylation of p53 at Ser392, the sole site modified by CK2 in vitro, is regulated by a novel mechanism where the stoichiometry of phosphorylation is governed by the rate of turnover of the p53 protein. Such a model is entirely consistent with phosphorylation by a constitutively active protein kinase such as CK2. In contrast to this, while there is overwhelming evidence that CK2 phosphorylates p53 in vitro and is the only detectable Ser392 protein kinase in cell extracts, our data raise uncertainty as to whether this interaction truly reflects events underpinning Ser392 phosphorylation in vivo. We consider the possible role of CK2 in regulating the p53 response in a wider context and suggest key issues that should be addressed experimentally to provide a more cohesive picture of the relationship between this important protein kinase and a pivotal anti-cancer surveillance system in cells.

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. We find that HSF1 is required for optimal p21 expression and p53-mediated cell-cycle arrest in response to genotoxins while loss of HSF1 attenuates apoptosis in response to these agents. To explain these novel properties of HSF1 we show that HSF1 can complex with DNA damage kinases ATR and Chk1 to effect p53 phosphorylation in response to DNA damage. Our data reveal HSF1 as a key transcriptional regulator in response to genotoxic compounds widely used in the clinical setting, and suggest that HSF1 will contribute to the efficacy of these agents.

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. While we focus on well-established N-terminal modifications for the purpose of illustration, this approach can be used to investigate other post-translational modifications of the protein, drawing upon a broad range of commercially available modification-specific antibodies.

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. We analyzed whether CK1 regulates p53 protein stability in unstressed conditions. Depletion of CK1 using small interfering RNA or inhibition of CK1 using the kinase inhibitor (D4476) activated p53 and destabilized E2F-1, indicating that steady-state levels of these proteins are controlled by CK1. Co-immunoprecipitation of endogenous CK1 with MDM2 occurred in undamaged cells, indicating the existence of a stable multiprotein complex, and as such, we evaluated whether the MDM2 Nutlin had similar pharmacological properties to the CK1 inhibitor D4476. Indeed, D4476 or Nutlin treatments resulted in the same p53 and E2F-1 steady-state protein level changes, indicating that the MDM2 x CK1 complex is both a negative regulator of p53 and a positive regulator of E2F-1 in undamaged cells. Although the treatment of cells with D4476 resulted in a partial p53-dependent growth arrest, the induction of p53-independent apoptosis by D4476 suggested a critical role for the MDM2 x CK1 complex in maintaining E2F-1 anti-apoptotic signaling. These data highlighting a pharmacological similarity between MDM2 and CK1 small molecule inhibitors and the fact that CK1 and MDM2 form a stable complex suggest that the MDM2 x CK1 complex is a component of a genetic pathway that co-regulates the stability of the p53 and E2F-1 transcription factors.

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.]. Nitrogen (N) fertilization could influence the soil carbon (C) balance in this system because the corn phase is fertilized in 97-100% of farms, at an average rate of 135 kg N x ha(-1) x yr(-1). We evaluated the impacts on two major processes that determine the soil C balance, the rates of organic-carbon (OC) inputs and decay, at four levels of N fertilization, 0, 90, 180, and 270 kg/ha, in two long-term experimental sites in Mollisols in Iowa, USA. We compared the corn-soybean system with other experimental cropping systems fertilized with N in the corn phases only: continuous corn for grain; corn-corn-oats (Avena sativa L.)-alfalfa (Medicago sativa L.; corn-oats-alfalfa-alfalfa; and continuous soybean. In all systems, we estimated long-term OC inputs and decay rates over all phases of the rotations, based on long-term yield data, harvest indices (HI), and root:shoot data. For corn, we measured these two ratios in the four N treatments in a single year in each site; for other crops we used published ratios. Total OC inputs were calculated as aboveground plus belowground net primary production (NPP) minus harvested yield. For corn, measured total OC inputs increased with N fertilization (P < 0.05, both sites). Belowground NPP, comprising only 6-22% of total corn NPP, was not significantly influenced by N fertilization. When all phases of the crop rotations were evaluated over the long term, OC decay rates increased concomitantly with OC input rates in several systems. Increases in decay rates with N fertilization apparently offset gains in carbon inputs to the soil in such a way that soil C sequestration was virtually nil in 78% of the systems studied, despite up to 48 years of N additions. The quantity of belowground OC inputs was the best predictor of long-term soil C storage. This indicates that, in these systems, in comparison with increased N-fertilizer additions, selection of crops with high belowground NPP is a more effective management practice for increasing soil C sequestration.

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.

p53 represses cyclin D1 transcription through down regulation of Bcl-3 and inducing increased association of the p52 NF-kappaB subunit with histone deacetylase 1.

The p53 and NF-kappaB transcription factor families are important, multifunctional regulators of the cellular response to stress. Here we have investigated the regulatory mechanisms controlling p53-dependent cell cycle arrest and cross talk with NF-kappaB. Upon induction of p53 in H1299 or U-2 OS cells, we observed specific repression of cyclin D1 promoter activity, correlating with a decrease in cyclin D1 protein and mRNA levels. This repression was dependent on the proximal NF-kappaB binding site of the cyclin D1 promoter, which has been shown to bind the p52 NF-kappaB subunit. p53 inhibited the expression of Bcl-3 protein, a member of the IkappaB family that functions as a transcriptional coactivator for p52 NF-kappaB and also reduced p52/Bcl-3 complex levels. Concomitant with this, p53 induced a significant increase in the association of p52 and histone deacetylase 1 (HDAC1). Importantly, p53-mediated suppression of the cyclin D1 promoter was reversed by coexpression of Bcl-3 and inhibition of p52 or deacetylase activity. p53 therefore induces a transcriptional switch in which p52/Bcl-3 activator complexes are replaced by p52/HDAC1 repressor complexes, resulting in active repression of cyclin D1 transcription. These results reveal a unique mechanism by which p53 regulates NF-kappaB function and cell cycle progression.

Hypophosphorylation of Mdm2 augments p53 stability.

The Mdm2 protein mediates ubiquitylation and degradation of p53 and is a key regulator of this tumor suppressor. More recently, it has been shown that Mdm2 is highly phosphorylated within its central acidic domain. In order to address the issue of how these modifications might regulate Mdm2 function, putative phosphorylation sites within this domain were substituted, individually or in pairs, with alanine residues. Mutants with serine-to-alanine substitutions between residues 244 and 260 abolished or at least reduced the capacity of Mdm2 to promote p53 degradation. In each case, loss of degradation function was independent of the ability to bind to p53 or p14ARF. Moreover, each of the Mdm2 mutants completely retained the capacity to act as a ubiquitin ligase in vivo. Thus, ubiquitylation and degradation can be uncoupled. Two-dimensional phosphopeptide mapping coupled with the use of phospho-specific antibodies revealed that Mdm2 is phosphorylated physiologically at several sites within this region, consistent with the idea that phosphorylation is important for Mdm2 activity. Strikingly, treatment of cells with ionizing radiation resulted in a significant decrease in the phosphorylation of residues that are important for p53 turnover. This hypophosphorylation preceded p53 accumulation. These findings indicate that Mdm2 contributes an additional function toward the degradation of p53 that is distinct from its ubiquitin ligase activity and is regulated by phosphorylation. Our model suggests that hypophosphorylation of Mdm2 in response to ionizing irradiation inactivates this novel function, thereby contributing to p53 stabilization.

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. Here, we show that PIM1 is modified in vitro and in cultured cells by the Small ubiquitin-like modifier (SUMO) on two independent sites: K169, within a consensus SUMOylation motif (IK169DE171) in the active site of PIM1, and also at a second promiscuous site. Alanine substitution of E171 (within the consensus motif) abolished SUMOylation, significantly increased the half-life of PIM1, and markedly reduced its ubiquitylation. Mechanistically, SUMOylation promoted ubiquitin-mediated degradation of PIM1 via recruitment of the SUMO-targeted ubiquitin ligase, RNF4. Additionally, SUMOylated PIM1 showed enhanced protein kinase activity in vitro. Interestingly, the E171A mutant was active in vitro but displayed altered substrate specificity in cultured cells, consistent with the idea that SUMOylation may govern PIM1 substrate specificity under certain contexts. Taken together, these data demonstrate that the protein kinase activity and levels of PIM1 can be regulated by a covalent post-translational modification.

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. Curiously, although p53 normally acts in an anti-cancer capacity, it can offer significant protection against inhibitors of PLK1, but the events underpinning this effect are not known. Here, we show that functional p53 reduces the sensitivity to PLK1 inhibitors by permitting centrosome separation to occur, allowing cells to traverse mitosis and re-enter cycle with a normal complement of 2N chromosomes. Protection entails the activation of p53 through the DNA damage-response enzymes, ATM and ATR, and requires the phosphorylation of p53 at the key regulatory site, Ser15. These data highlight a previously unrecognised link between p53, PLK1 and centrosome separation that has therapeutic implications for the use of PLK1 inhibitors in the clinic.

The p53 response to DNA damage.

The p53 tumour suppressor protein is a highly potent transcription factor which, under normal circumstances, is maintained at low levels through the action of MDM2, an E3 ubiquitin ligase which directs p53 ubiquitylation and degradation. Expression of the mdm2 gene is stimulated by p53 and this reciprocal relationship forms the basis of a negative feedback loop. Both genotoxic and non-genotoxic stresses that induce p53 focus principally on interruption of the p53-MDM2 loop with the consequence that p53 becomes stabilised, leading to changes in the expression of p53-responsive genes. The biological outcome of inducing this pathway can be either growth arrest or apoptosis: factors affecting the functioning of the loop, the biochemical activity of p53 itself and the cellular environment govern the choice between these outcomes in a cell type- and stress-specific manner.

Posttranslational modification of MDM2.

The functions of the MDM2 protein, in particular its E3 ubiquitin ligase activity and its ability to interact with a number of cellular proteins intimately involved in growth regulation, are modulated by sumoylation and multisite phosphorylation. These posttranslational mechanisms not only regulate the intrinsic activity of MDM2 in response to cellular stresses, but also govern its subcellular localization, differentiate between MDM2-mediated ubiquitination of p53 and autoubiquitination, integrate the stress response with mechanisms that mediate cell survival, and modulate the interaction of MDM2 with cellular and viral proteins. In this review, we summarize our current knowledge of the role of posttranslational modifications of MDM2 and their functional relevance.

Centrosomal anchoring of the protein kinase CK1delta mediated by attachment to the large, coiled-coil scaffolding protein CG-NAP/AKAP450.

Protein kinase CK1 (formerly termed casein kinase I) is ubiquitous in eukaryotic cells and comprises a family of as many as 14 isoforms (including splice variants) in mammalian cells. Mammalian CK1delta and CK1epsilon, which are highly related to each other, are enriched at the centrosomes in interphase cells and at the spindle during mitosis. In the present study we have isolated, using the yeast two-hybrid system, a 182 amino acid residue fragment of the centrosomal and golgi N-kinase anchoring protein (CG-NAP, also known as AKAP450), which specifically interacts with CK1delta and CK1epsilon, but not with other CK1 isoforms. The 182 amino acid residue CG-NAP fragment, or full length CG-NAP, co-immunoprecipitates with CK1delta and CK1epsilon from mammalian cells. Consistent with this association, endogenous CG-NAP/AKAP450 and CK1delta co-localize in cells. Moreover, when expressed in the presence of CK1delta the 182 amino acid residue CG-NAP fragment adopts the same sub-cellular localization as CK1delta. Strikingly, attachment of the CG-NAP fragment to the plasma membrane is sufficient to re-localize a significant level of CK1delta to the membrane. These findings support a model in which sub-cellular localization of CK1delta/epsilon molecules at the centrosome is mediated, at least in part, through the action of CG-NAP/AKAP450 and provide a potential mechanism by which the contribution to cell cycle progression by CK1delta/epsilon may be regulated.

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. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically.