Borek Vojtesek

Novel phosphorylation sites of human tumour suppressor protein p53 at Ser20 and Thr18 that disrupt the binding of mdm2 (mouse double minute 2) protein are modified in human cancers

The ability to separate the isoforms of human tumour suppressor protein p53 expressed in insect cells using heparin-Sepharose correlates with differences in the isoelectric point of p53, demonstrating that p53 can be heterogeneously modified and providing support for the use of insect cells as a model system for identifying novel signalling pathways that target p53. One p53 isoform that was reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its binding to DO-1 by prior incubation with protein phosphatases, suggesting the presence of a previously unidentified N-terminal phosphorylation site capable of masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53 containing phosphate at Ser(20) inhibited DO-1 binding, thus identifying the phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies overlapping the DO-1 epitope were developed that are specific for phospho-Thr(18) (adjacent to the DO-1 epitope) and phospho-Ser(20) (within the DO-1 epitope) to determine whether direct evidence could be obtained for novel phosphorylation sites in human p53. A monoclonal antibody highly specific for phospho-Ser(20) detected significant phosphorylation of human p53 expressed in insect cells, whereas the relative proportion of p53 modified at Thr(18) was substantially lower. The relevance of these two novel phosphorylation sites to p53 regulation in human cells was made evident by the extensive phosphorylation of human p53 at Thr(18) and Ser(20) in a panel of human breast cancers with a wild-type p53 status. Phospho-Ser(20) or phospho-Thr(18) containing p53 peptides are as effective as the phospho-Ser(15) peptide at reducing mdm2 (mouse double minute 2) protein binding, indicating that the functional effects of these phosphorylation events might be to regulate the binding of heterologous proteins to p53. These results provide evidence in vivo for two novel phosphorylation sites within p53 at Ser(20) and Thr(18) that can affect p53 protein-protein interactions and indicate that some human cancers might have amplified one or more Ser(20) and Thr(18) kinase signalling cascades to modulate p53 activity.

Stoichiometric phosphorylation of human p53 at Ser315 stimulates p53-dependent transcription

p53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression. A phospho-specific monoclonal antibody to the C-terminal Ser315phospho-epitope was used to determine whether phosphorylation of endogenous p53 at Ser315 can be detected in vivo, whether steady-state Ser315 phosphorylation increases or decreases in an irradiated cell, and whether this phosphorylation event activates or inhibits p53 in vivo. A native phospho-specific IgG binding assay was developed for quantitating the extent of p53 phosphorylation at Ser315 where one, two, three, or four phosphates/tetramer could be defined after in vitro phosphorylation by cyclin-dependent protein kinases. Using this assay, near-stoichiometric Ser315 phosphorylation of endogenous p53 protein was detected in vivo after UV irradiation of MCF7 and A375 cells, coinciding with elevated p53-dependent transcription. Transfection of the p53 gene with an alanine mutation at the Ser315 site into Saos-2 cells gave rise to a form of p53 protein with a substantially reduced specific activity as a transcription factor. The treatment of cells with the cyclin-dependent protein kinase inhibitor Roscovitine promoted a reduction in the specific activity of endogenous p53 or ectopically expressed p53. These results indicate that the majority of p53 protein has been phosphorylated at Ser315 after irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.

Hsp90 Is Essential for Restoring Cellular Functions of Temperature-sensitive p53 Mutant Protein but Not for Stabilization and Activation of Wild-type p53 IMPLICATIONS FOR CANCER THERAPY

Several signaling pathways that monitor the dynamic state of the cell converge on the tumor suppressor p53. The ability of p53 to process these signals and exert a dynamic downstream response in the form of cell cycle arrest and/or apoptosis is crucial for preventing tumor development. This p53 function is abrogated by p53 gene mutations leading to alteration of protein conformation. Hsp90 has been implicated in regulating both wild-type and mutant p53 conformations, and Hsp90 antagonists are effective for the therapy of some human tumors. Using cell lines that contain human tumor-derived temperature-sensitive p53 mutants we show that Hsp90 is required for both stabilization and reactivation of mutated p53 at the permissive temperature. A temperature decrease to 32 °C causes conversion to a protein conformation that is capable of inducing expression of MDM2, leading to reduction of reactivated p53 levels by negative feedback. Mutant reactivation is enhanced by simultaneous treatment with agents that stabilize the reactivated protein and is blocked by geldanamycin, a specific inhibitor of Hsp90 activity, indicating that Hsp90 antagonist therapy and therapies that act to reactivate mutant p53 will be incompatible. In contrast, Hsp90 is not required for maintaining wild-type p53 or for stabilizing wild-type p53 after treatment with chemotherapeutic agents, indicating that Hsp90 therapy might synergize with conventional therapies in patients with wild-type p53. Our data demonstrate the importance of the precise characterization of the interaction between p53 mutants and stress proteins, which may shed valuable information for fighting cancer via the p53 tumor suppressor pathway.

A Divergent Substrate-Binding Loop within the Pro-oncogenic Protein Anterior Gradient-2 Forms a Docking Site for Reptin

Anterior gradient-2 (AGR2) functions in a range of biological systems, including goblet cell formation, limb regeneration, inhibition of p53, and metastasis. There are no well-validated binding proteins for AGR2 protein despite the wealth of data implicating an important cellular function in vertebrates. The yeast two-hybrid system was used to isolate the ATP binding protein Reptin as an AGR2-interacting protein. AGR2 formed a stable complex in human cell lysates with Reptin, thus validating Reptin as an AGR2 binding protein in cells. Reptin was also shown to be overproduced in a panel of primary breast cancer biopsy specimens, relative to normal adjacent tissue from the same patient, suggesting a role in cancer growth in vivo. Mutations were made at the two ATP binding motifs in Reptin to evaluate the effects of ATP on Reptin-AGR2 complex stability. Loss-of-ATP binding mutations at the Walker A motif (K83A) or gain-of-ATP binding mutations at the Walker B motif (D299N) resulted in Reptin mutants with altered oligomerization, thermostability, and AGR2 binding properties. These data indicate that the two ATP binding motifs of Reptin play a role in regulating the stability of the AGR2-Reptin complex. The minimal region of AGR2 interacting with Reptin was localized using overlapping peptide libraries derived from the AGR2 protein sequence. The Reptin docking site was mapped to a divergent octapeptide loop in the AGR2 superfamily between amino acids 104 and 111. Mutations at codon Y104 or F111 in full-length AGR2 destabilized the binding of Reptin. These data highlight the existence of a protein docking motif on AGR2 and an ATP-regulated peptide-binding activity for Reptin. This knowledge has implications for isolating other AGR2-interacting proteins, for developing assays to isolate small molecules that target the Reptin ATP binding site, and for measuring the effects of the Reptin-AGR2 complex in cancer cell growth.

Stress-dependent changes in the properties of p53 complexes by the alternative translation product p53/47

P53 plays a key role in the cellular response to damage exposure and in preventing the development of human cancers. Activation of p53 results in changes in the expression of a large number of gene products. However, relatively little is still known how p53 activation differentiates between different types of damages in different types of tissues or how this triggers either an apoptotic response or cell cycle arrest and DNA repair. The p53 message is translated into two products with distinct activities and stabilities through alternative mechanisms of initiation. P53/47 is initiated 40 codons down stream of the full length p53 and does not include the binding site for the E3 ubiquitin ligase Mdm2 or the transactivation domain I but retains the capacity to form p53 hetero- and homo-oligomers. Here we report that p53/47 controls the folding, the oligomerisation and the post-translational modification of p53 complexes and that it diversifies p53 properties in a cell stress-dependent fashion. P21 expression, for example, is under normal conditions not affected by p53/47 but is induced 18-fold after treatment of cells with the DNA damaging drug doxorubicin. This is accompanied by the recruitment of p53/47 to the p21 promoter.

The Development and Use of Phospho-Specific Antibodies to Study Protein Phosphorylation

The reversible phosphorylation of proteins is a key mechanism whereby signalling cascades involved in the response to extracellular stimuli bring about changes in cellular function. These proteins include the kinases/phosphatases that form such signaling pathways as well as the transcription factors involved in inducible changes in gene expression (1). Phosphorylation induces changes in the function of these proteins either by induction of allosteric conformational changes in the protein itself or in the regulation of its interaction with other cellular factors.

Cross-talk between HIF and p53 as mediators of molecular responses to physiological and genotoxic stresses

Abnormal rates of growth together with metastatic potential and lack of susceptibility to cellular signals leading to apoptosis are widely investigated characteristics of tumors that develop via genetic or epigenetic mechanisms. Moreover, in the growing tumor, cells are exposed to insufficient nutrient supply, low oxygen availability (hypoxia) and/or reactive oxygen species. These physiological stresses force them to switch into more adaptable and aggressive phenotypes. This paper summarizes the role of two key mediators of cellular stress responses, namely p53 and HIF, which significantly affect cancer progression and compromise treatment outcomes. Furthermore, it describes cross-talk between these factors.

Anterior Gradient-3: A novel biomarker for ovarian cancer that mediates cisplatin resistance in xenograft models

The Anterior Gradient (AGR) genes AGR2 and AGR3 are part of the Protein Disulfide Isomerase (PDI) family and harbour core thioredoxin folds (CxxS motifs) that have the potential to regulate protein folding and maturation. A number of proteomics and transcriptomics screens in the fields of limb regeneration, cancer cell metastasis, pro-oncogenic oestrogen-signalling, and p53 regulation have identified AGR2 as a novel component of these signalling pathways. Curiously, despite the fact that the AGR2 and AGR3 genes are contiguous on chromosome 7p21.1-3, the AGR3 protein has rarely been identified in such OMICs screens along with AGR2 protein. Therefore there is little information on how AGR3 protein is expressed in normal and diseased states. A panel of three monoclonal antibodies was generated towards AGR3 protein for identifying novel clinical models that can be used to define whether AGR3 protein could play a positive or negative role in human cancer development. One monoclonal antibody was AGR3-specific and bound a linear epitope that could be defined using both pep-scan and phage-peptide library screening. Using this monoclonal antibody, endogenous AGR3 protein expression was shown to be cytosolic in four human ovarian cancer subtypes; serous, endometrioid, clear cell, and mucinous. Mucinous ovarian cancers produced the highest number of AGR3 positive cells. AGR3 expression is coupled to AGR2 expression only in mucinous ovarian cancers, whereas AGR3 and AGR2 expressions are uncoupled in the other three types of ovarian cancer. AGR3 expression in ovarian cancer is independent of oestrogen-receptor expression, which is distinct from the oestrogen-receptor dependent expression of AGR3 in breast cancers. Isogenic cancer cell models were created that over-express AGR3 and these demonstrated that AGR3 mediates cisplatin-resistance in mouse xenografts. These data indicate that AGR3 is over-expressed by a hormone (oestrogen-receptor α)-independent mechanism and identify a novel protein-folding associated pathway that could mediate resistance to DNA-damaging agents in human cancers.

The calcium‐binding domain of the stress protein SEP53 is required for survival in response to deoxycholic acid‐mediated injury

Stress protein responses have evolved in part as a mechanism to protect cells from the toxic effects of environmental damaging agents. Oesophageal squamous epithelial cells have evolved an atypical stress response that results in the synthesis of a 53 kDa protein of undefined function named squamous epithelial‐induced stress protein of 53 kDa (SEP53). Given the role of deoxycholic acid (DCA) as a potential damaging agent in squamous epithelium, we developed assays measuring the effects of DCA on SEP53‐mediated responses to damage. To achieve this, we cloned the human SEP53 gene, developed a panel of monoclonal antibodies to the protein, and showed that SEP53 expression is predominantly confined to squamous epithelium. Clonogenic assays were used to show that SEP53 can function as a survival factor in mammalian cell lines, can attenuate DCA‐induced apoptotic cell death, and can attenuate DCA‐mediated increases in intracellular free calcium. Deletion of the highly conserved EF‐hand calcium‐binding domain in SEP53 neutralizes the colony survival activity of the protein, neutralizes the protective effects of SEP53 after DCA exposure, and permits calcium elevation in response to DCA challenge. These data indicate that the squamous cell‐stress protein SEP53 can function as a modifier of the DCA‐mediated calcium influx and identify a novel survival pathway whose study may shed light on mechanisms relating to squamous cell injury and associated cancer development.

Identification of a second Nutlin-3 responsive interaction site in the N-terminal domain of MDM2 using hydrogen/deuterium exchange mass spectrometry

MDM2 is a multidomain protein that functions as an E3 ubiquitin ligase, transcription repressor, mRNA‐binding protein, translation factor, and molecular chaperone. The small molecule Nutlin‐3 has been engineered to bind to the N‐terminal hydrophobic pocket domain of MDM2. This binding of Nutlin‐3 has two consequences: (i) antagonistic effects through competitive disruption of the MDM2‐p53 complex and (ii) agonist effects that allosterically stabilize MDM2 protein–protein interactions that increase p53 ubiquitination as well as nucleophosmin deoligomerization. We present a methodology using a hydrogen/deuterium (H/D) exchange platform that measures Nutlin‐3 binding to the N‐terminal domain of MDM2 (MDM21–126) in order to begin to develop dynamic assays that evaluate MDM2 allostery. In order to localize the regions in MDM2 being suppressed by Nutlin‐3, MDM2 was incubated with the ligand and H/D amide exchange was measured after pepsin digestion. One dynamic segment containing amino acids 55–60 exhibited slower deuterium exchange after Nutlin‐3 binding, reflecting ligand binding within the hydrophobic pocket. However, another dominant suppression of H/D exchange was observed in a motif from amino acids 103–107 that reflects surface hydrophobic residues surrounding the hydrophobic pocket of MDM2. In order to explore the consequences of this latter Nutlin‐3 interaction site on MDM2, the Y104G and L107G mutant series was constructed. The MDM2Y104G and MDM2L107G mutants were fully active in p53 binding. However, the authentic p53‐derived peptide:MDM2Y104G complex exhibited partial resistance to Nutlin‐3 inhibition, while the p53‐mimetic 12.1 peptide:MDM2Y104G complex retained normal Nutlin‐3 responsiveness. These data reveal the existence of a second functional Nutlin‐3‐binding site in a surface hydrophobic patch of MDM2, flanking the hydrophobic pocket. This reveals two modes of peptide binding by MDM2 and highlights the utility of H/D exchange as an assay for measuring allosteric effects in MDM2.