Nutlin-3 downregulates p53 phosphorylation on serine392 and induces apoptosis in hepatocellular carcinoma cells

Drug-resistance and imbalance of apoptotic regulation limit chemotherapy clinical application for the human hepatocellular carcinoma (HCC) treatment. The reactivation of p53 is an attractive therapeutic strategy in cancer with disrupted-p53 function. Nutlin-3, a MDM2 antagonist, has antitumor activity in various cancers. The post-translational modifications of p53 are a hot topic, but there are some controversy ideas about the function of phospho-Ser³⁹²-p53 protein in cancer cell lines in response to Nutlin-3. Therefore, we investigated the relationship between Nutlin-3 and phospho-Ser³⁹²-p53 protein expression levels in SMMC-7721 (wild-type TP53) and HuH-7 cells (mutant TP53). We demonstrated that Nutlin-3 induced apoptosis through down-regulation phospho-Ser³⁹²-p53 in two HCC cells. The result suggests that inhibition of p53 phosphorylation on Ser³⁹² presents an alternative for HCC chemotherapy. [BMB Reports 2014; 47(4): 221-226]     

Serine 312 phosphorylation is dispensable for wild-type p53 functions in vivo

Cellular stimulation results in phosphorylation of the tumor suppressor p53 on multiple residues, though the functional relevance is not always clear. It is noteworthy that the serine (S) 315 residue is unique, as it has been suggested to be phosphorylated not only by genotoxic signals, but also during cell-cycle progression and by endoplasmic-reticulum stress. However, in vitro data have been conflicting as phosphorylation at this site was shown to both positively and negatively regulate p53 functions. We have thus generated knock-in mice expressing an unphosphorylable S312 (equivalent to human S315), by substitution with an alanine (A) residue, to clarify the conflicting observations and to evaluate its functional relevance in vivo. Born at Mendelian ratios, the p53^S312A/S312A mice show no anomalies during development and adulthood. p53 activation, stability, localization and ability to induce apoptosis, cell-cycle arrest and prevent centrosome amplification are not compromised in p53^S312A/S312A cells. p53^S312A/S312A mice are unable to rescue mdm2^−/− lethality, and tumorigenesis – both spontaneous and irradiation/oncogene-induced – is not accentuated. Taken together, the results show that the S312 phosphorylation site is not in itself necessary for efficient p53 function, and advocates the possibility that it is neither relevant in the mouse context nor important for p53 functions in vivo.     

The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser359 Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity

The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr³¹³-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδ''s Tyr³¹³-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδ''s enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser³⁵⁹. We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser^359. In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser³⁵⁹ phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr³¹³ docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity.     

Plk1-mediated Phosphorylation of Topors Regulates p53 Stability

Polo-like kinase 1 (Plk1) overexpression is associated with tumorigenesis by an unknown mechanism. Likewise, Plk1 was suggested to act as a negative regulator of tumor suppressor p53, but the mechanism remains to be determined. Herein, we have identified topoisomerase I-binding protein (Topors), a p53-binding protein, as a Plk1 target. We show that Plk1 phosphorylates Topors on Ser⁷¹⁸ in vivo. Significantly, expression of a Plk1-unphosphorylatable Topors mutant (S718A) leads to a dramatic accumulation of p53 through inhibition of p53 degradation. Topors is an ubiquitin and small ubiquitin-like modifier ubiquitin-protein isopeptide ligase (SUMO E3) ligase. Plk1-mediated phosphorylation of Topors inhibits Topors-mediated sumoylation of p53, whereas p53 ubiquitination is enhanced, leading to p53 degradation. These results demonstrate that Plk1 modulates Topors activity in suppressing p53 function and identify a likely mechanism for the tumorigenic potential of Plk1.Polo-like kinase-1 (Plk1)³ has multiple functions required for cell cycle progression, and overexpression of Plk1 is observed in various types of human tumors (1, 2). Thus, Plk1 has been proposed as a novel diagnostic marker for cancers. Accumulating evidence suggests that Plk1 negatively regulates the function of the tumor suppressor p53, whose loss-of-function mutations have been observed in nearly 50% of human tumors (1). In our earlier studies, we were the first to demonstrate that Plk1 depletion results in increased p53 level in HeLa cells (3) and that human cells with different levels of p53 respond to Plk1 depletion differently (4). Subsequently, it was shown that Plk1 directly binds to the DNA-binding domain of p53 through its N-terminal kinase domain and inhibits the transactivation as well as the proapoptotic function of p53 (5). Although it has been suggested that Plk1 might regulate p53 through direct phosphorylation (5), our repeated efforts to prove p53 as a direct target of Plk1 have been unsuccessful.Topors was discovered in a screen searching for proteins that bind to DNA topoisomerase I (6) and was also identified as a p53-binding protein (7). Although Topors is widely expressed in normal human tissues, its expression is decreased or undetectable in colon, lung, and brain adenocarcinomas, indicating that it might function as a tumor suppressor (8). Topors contains an N-terminal C3HC4-type RING domain that is closely related in sequence to the RING domains of known E3 ligases (see Fig. 1A) and is the first example of a protein that has both ubiquitin and SUMO-1 E3 ligase activity. Topors functions as an E3 ubiquitin ligase for p53 and NKX3.1, and Topors-mediated ubiquitination leads to the degradation of these proteins (9, 10). Substrates of the SUMO-1 E3 ligase activity of Topors include DNA topoisomerase I and p53 (11, 12). In contrast to ubiquitination-induced protein degradation, Topors-induced p53 sumoylation is accompanied by an increase in the level of p53 protein (11). Taken together, these studies indicate that Topors functions both as an ubiquitin and as a SUMO-1 E3 ligase for p53. Therefore, it is likely that the effects of Topors on p53 depend on cellular context (10).Open in a separate windowFIGURE 1.Plk1 phosphorylates Topors at Ser⁷¹⁸in vitro and in vivo. A, schematic representation of the domain structure of Topors. Two separate regions encoding putative p53-binding domains are aa 456–731 and 854–916. Amino acid residues in the putative Ring finger motif are shown in a black box. PEST, sequences rich in Pro, Glu, Ser, and Thr; RS domain, Arg- and Ser-rich domain; NLS, nuclear localization sequence; NB, nuclear bodies. B, purified Plk1 was incubated with purified GST-Topors (aa 1–510) or GST-Topors (aa 511–1045) for 30 min at 30 °C in the presence of [γ-³²P]ATP (³²P). Reaction mixtures were resolved by SDS-PAGE followed by autoradiography. Coom., Coomassie Blue. C and D, Plk1 phosphorylates Topors (aa 679–760). Purified Plk1 was incubated with purified GST-Topors fragments (aa 1–250, 251–510, 511–760, 756–1045, 511–596, 597–678, and 679–760). Kinase assays were performed as described in B. E, Ser⁷¹⁸ of Topors is a Plk1 phosphorylation site in vitro. Purified Plk1 was incubated with the indicated serine to alanine Topors (aa 679–760) mutants and analyzed as in B. F, Topors is phosphorylated in vivo at Ser⁷¹⁸ by Plk1. HEK293T cells were transfected with WT-Topors-Myc (lanes 1 and 3) or S718A-Topors-Myc (lane 2) and depleted of Plk1 by using double-stranded RNA targeting Plk1 (lane 3). After overnight incubation, cells were treated with nocodazole for 10 h and metabolically labeled with [³²P]orthophosphate. Phosphoproteins were immunoprecipitated with anti-Myc antibodies, resolved by SDS-PAGE, and subjected to autoradiography. Relative ³²P (Rel. ³²P) incorporations of Topors are indicated on the bottom.In this study, we provide evidence that Plk1 phosphorylates Topors on Ser⁷¹⁸. Significantly, we demonstrate that the Plk1-mediated phosphorylation of Topors results in reduced sumoylation of p53, whereas the ubiquitination activity toward p53 is increased, thereby facilitating p53 degradation.     

Dyrk1A Phosphorylates p53 and Inhibits Proliferation of Embryonic Neuronal Cells

Down syndrome (DS) is associated with many neural defects, including reduced brain size and impaired neuronal proliferation, highly contributing to the mental retardation. Those typical characteristics of DS are closely associated with a specific gene group “Down syndrome critical region” (DSCR) on human chromosome 21. Here we investigated the molecular mechanisms underlying impaired neuronal proliferation in DS and, more specifically, a regulatory role for dual-specificity tyrosine-(Y) phosphorylation-regulated kinase 1A (Dyrk1A), a DSCR gene product, in embryonic neuronal cell proliferation. We found that Dyrk1A phosphorylates p53 at Ser-15 in vitro and in immortalized rat embryonic hippocampal progenitor H19-7 cells. In addition, Dyrk1A-induced p53 phosphorylation at Ser-15 led to a robust induction of p53 target genes (e.g. p21^CIP1) and impaired G₁/G₀-S phase transition, resulting in attenuated proliferation of H19-7 cells and human embryonic stem cell-derived neural precursor cells. Moreover, the point mutation of p53-Ser-15 to alanine rescued the inhibitory effect of Dyrk1A on neuronal proliferation. Accordingly, brains from embryonic DYRK1A transgenic mice exhibited elevated levels of Dyrk1A, Ser-15 (mouse Ser-18)-phosphorylated p53, and p21^CIP1 as well as impaired neuronal proliferation. These findings suggest that up-regulation of Dyrk1A contributes to altered neuronal proliferation in DS through specific phosphorylation of p53 at Ser-15 and subsequent p21^CIP1 induction.     

The Effects of Phosphomimetic Lid Mutation on the Thermostability of the N-terminal Domain of MDM2

The multidomain E3 ubiquitin ligase MDM2 catalyzes p53 ubiquitination by a “dual-site” docking mechanism whereby MDM2 binding to at least two distinct peptide motifs on p53 promotes ubiquitination. One protein-protein interaction occurs between the N-terminal hydrophobic pocket of MDM2 and the transactivation motif of p53, and the second interaction occurs between the acidic domain of MDM2 and a motif in the DNA-binding domain of p53. A flexible N-terminal pseudo-substrate or “lid” adjacent to the N-terminal hydrophobic pocket of MDM2 has a phosphorylation site, and there are distinct models proposed on how the phosphorylated lid could affect MDM2 function. Biochemical studies have predicted that phosphomimetic mutation will stabilize the lid on the surface of MDM2 and will “open” the hydrophobic pocket and stabilize the MDM2-p53 complex, while NMR studies proposed that phosphomimetic mutation “closes” the lid over the MDM2 pocket and inhibits MDM2-p53 complex formation. To resolve these discrepancies, we utilized a quantitative fluorescence-based dye binding assay to measure the thermal unfolding of wild-type (wt), ΔLid, and S17D N-terminal domains of MDM2 as a function of increasing ligand concentration. Our data reveal that S17D lid mutation increases, rather than decreases, the thermostability of the N-terminal domain of MDM2 in the absence or in the presence of ligand. ΔLid mutation, by contrast, increases MDM2 thermoinstability. This is consistent with biochemical data, using full-length MDM2, showing that the S17D mutation stabilizes the MDM2-p53 complex and increases the specific activity of the E3 ubiquitin ligase function of MDM2. These data indicate that phosphomimetic lid mutation results in an “opening,” rather than a “closing,” of the pocket of MDM2 and highlight the ability of small intrinsically disordered or unstructured peptide motifs to regulate the specific activity of a protein.     

Destabilizing missense mutations in the tumour suppressor protein p53 enhance its ubiquitination in vitro and in vivo

p53 ubiquitination catalysed by MDM2 (murine double minute clone 2 oncoprotein) provides a biochemical assay to dissect stages in E3-ubiquitin-ligase-catalysed ubiquitination of a conformationally flexible protein. A mutant form of p53 (p53(F270A)) containing a mutation in the second MDM2-docking site in the DNA-binding domain of p53 (F270A) is susceptible to modification of long-lived and high-molecular-mass covalent adducts in vivo. Mutant F270A is hyperubiquitinated in cells as defined by immunoprecipitation and immunoblotting with an anti-ubiquitin antibody. Transfection of His-tagged ubiquitin along with p53(R175H) or p53(F270A) also results in selective hyperubiquitination in cells under conditions where wild-type p53 is refractory to covalent modification. The extent of mutant p53(R175H) or p53(F270A) unfolding in cells as defined by exposure of the DO-12 epitope correlates with the extent of hyperubiquitination, suggesting a link between substrate conformation and E3 ligase function. The p53(F270A:6KR) chimaeric mutant (where 6KR refers to the simultaneous mutation of lysine residues at positions 370, 372, 373, 381, 382 and 386 to arginine) maintains the high-molecular-mass covalent adducts and is modified in an MDM2-dependent manner. Using an in vitro ubiquitination system, mutant p53(F270A) and the p53(F270A:6KR) chimaeric mutant is also subject to hyperubiquitination outwith the C-terminal domain, indicating direct recognition of the mutant p53 conformation by (a) factor(s) in the cell-free ubiquitination system. These data identify an in vitro and in vivo assay with which to dissect how oligomeric protein conformational alterations are linked to substrate ubiquitination in cells. This has implications for understanding the recognition of misfolded proteins during aging and in human diseases such as cancer.     

Regulation of Human Cytidine Triphosphate Synthetase 2 by Phosphorylation

Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the K_m and IC₅₀ values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. Metabolic labeling of hCTPS2 with [³²P]H₃PO₄ demonstrated that Ser⁵⁶⁸ and Ser⁵⁷¹ were two major phosphorylation sites, and additional studies demonstrated that Ser⁵⁶⁸ was phosphorylated by casein kinase 1 both in vitro and in vivo. Interestingly, mutation of Ser⁵⁶⁸ (S568A) but not Ser⁵⁷¹ significantly increased hCTPS2 activity, demonstrating that Ser⁵⁶⁸ is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the V_max of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser⁵⁶⁸ as a major site of CTPS2 regulation by phosphorylation.     

A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation

The neuron-specific cation chloride cotransporter KCC2 plays a crucial role in hyperpolarizing synaptic inhibition. Transporter dysfunction is associated with various neurological disorders, raising interest in regulatory mechanisms. Phosphorylation has been identified as a key regulatory process. Here, we retrieved experimentally observed phosphorylation sites of KCC2 from public databases and report on the systematic analysis of six phosphorylated serines, Ser²⁵, Ser²⁶, Ser⁹³⁷, Ser¹⁰²², Ser¹⁰²⁵, and Ser¹⁰²⁶. Alanine or aspartate substitutions of these residues were analyzed in HEK-293 cells. All mutants were expressed in a pattern similar to wild-type KCC2 (KCC2^WT). Tl⁺ flux measurements demonstrated unchanged transport activity for Ser²⁵, Ser²⁶, Ser¹⁰²², Ser¹⁰²⁵, and Ser¹⁰²⁶ mutants. In contrast, KCC2^S937D, mimicking phosphorylation, resulted in a significant up-regulation of transport activity. Aspartate substitution of Thr⁹³⁴, a neighboring putative phosphorylation site, resulted in a comparable increase in KCC2 transport activity. Both KCC2^T934D and KCC2^S937D mutants were inhibited by the kinase inhibitor staurosporine and by N-ethylmaleimide, whereas KCC2^WT, KCC2^T934A, and KCC2^S937A were activated. The inverse staurosporine effect on aspartate versus alanine substitutions reveals a cross-talk between different phosphorylation sites of KCC2. Immunoblot and cell surface labeling experiments detected no alterations in total abundance or surface expression of KCC2^T934D and KCC2^S937D compared with KCC2^WT. These data reveal kinetic regulation of transport activity by these residues. In summary, our data identify a novel key regulatory phosphorylation site of KCC2 and a functional interaction between different conformation-changing post-translational modifications. The action of pharmacological agents aimed to modulate KCC2 activity for therapeutic benefit might therefore be highly context-specific.     

Identification of Multiple Phosphorylation Sites on Maize Endosperm Starch Branching Enzyme IIb,a Key Enzyme in Amylopectin Biosynthesis

Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser⁶⁴⁹, Ser²⁸⁶, and Ser²⁹⁷. Two Ca²⁺-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser⁶⁴⁹ and Ser²⁸⁶ phosphorylation, and K2, responsible for Ser⁶⁴⁹ and Ser²⁹⁷ phosphorylation. The Ser²⁸⁶ and Ser²⁹⁷ phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel β-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser²⁹⁷ forms a stable salt bridge with Arg⁶⁶⁵, part of a conserved Cys-containing domain in plant branching enzymes. Ser⁶⁴⁹ conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application.     

The conformationally flexible S9-S10 linker region in the core domain of p53 contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo

Although the N-terminal BOX-I domain of the tumor suppressor protein p53 contains the primary docking site for MDM2, previous studies demonstrated that RNA stabilizes the MDM2.p53 complex using a p53 mutant lacking the BOX-I motif. In vitro assays measuring the specific activity of MDM2 in the ligand-free and RNA-bound state identified a novel MDM2 interaction site in the core domain of p53. As defined using phage-peptide display, the RNA.MDM2 isoform exhibited a notable switch in peptide binding specificity, with enhanced affinity for novel peptide sequences in either p53 or small nuclear ribonucleoprotein-U (snRNP-U) and substantially reduced affinity for the primary p53 binding site in the BOX-I domain. The consensus binding site for the RNA.MDM2 complex within p53 is SGXLLGESXF, which links the S9-S10 beta-sheets flanking the BOX-IV and BOX-V motifs in the core domain and which is a site of reversible conformational flexibility in p53. Mutation of conserved amino acids in the linker at Ser(261) and Leu(264), which bridges the S9-S10 beta-sheets, stimulated p53 activity from reporter templates and increased MDM2-dependent ubiquitination of p53. Furthermore, mutation of the conserved Phe(270) within the S10 beta-sheet resulted in a mutant p53, which binds more stably to RNA.MDM2 complexes in vitro and which is strikingly hyper-ubiquitinated in vivo. Introducing an Ala(19) mutation into the p53(F270A) protein abolished both RNA.MDM2 complex binding and hyper-ubiquitination in vivo, thus indicating that p53(F270A) protein hyper-ubiquitination depends upon MDM2 binding to its primary site in the BOX-I domain. Together, these data identify a novel MDM2 binding interface within the S9-S10 beta-sheet region of p53 that plays a regulatory role in modulating the rate of MDM2-dependent ubiquitination of p53 in cells.     

Phosphorylation of Cdc42 Effector Protein-4 (CEP4) by Protein Kinase C Promotes Motility of Human Breast Cells

Cdc42 effector protein-4 (CEP4) was recently identified by our laboratory to be a substrate of multiple PKC isoforms in non-transformed MCF-10A human breast cells. The significance of phosphorylated CEP4 to PKC-stimulated motility of MCF-10A cells was evaluated. Single site mutants at Ser residues embedded in potential PKC consensus sites (Ser¹⁸, Ser⁷⁷, Ser⁸⁰, and Ser⁸⁶) were individually replaced with Asp residues to simulate phosphorylation. Following expression in weakly motile MCF-10A cells, the S18D and S80D mutants each promoted increased motility, and the double mutant (S18D/S80D) produced a stronger effect. MS/MS analysis verified that Ser¹⁸ and Ser⁸⁰ were directly phosphorylated by PKCα in vitro. Phosphorylation of CEP4 severely diminished its affinity for Cdc42 while promoting Rac activation and formation of filopodia (microspikes). In contrast, the phosphorylation-resistant double mutant S18A/S80A-CEP4 blocked CEP4 phosphorylation and inhibited motility of MCF-10A cells that had been stimulated with PKC activator diacylglycerol lactone. In view of the dissociation of phospho-CEP4 from Cdc42, intracellular binding partners were explored by expressing each CEP4 double mutant from a tandem affinity purification vector followed by affinity chromatography, SDS-PAGE, and identification of protein bands evident only with S18D/S80D-CEP4. One binding partner was identified as tumor endothelial marker-4 (TEM4; ARHGEF17), a guanine nucleotide exchange factor that is involved in migration. In motile cells expressing S18D/S80D-CEP4, knockdown of TEM4 inhibited both Rac activation and motility. These findings support a model in which PKC-mediated phosphorylation of CEP4 at Ser¹⁸ and Ser⁸⁰ causes its dissociation from Cdc42, thereby increasing its affinity for TEM4 and producing Rac activation, filopodium formation, and cell motility.     

Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code

Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein–DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.     

Effect of Y220C Mutation on p53 and Its Rescue Mechanism: A Computer Chemistry Approach

Mutation causes inactivation of ‘p53’ tumor suppressor protein in almost fifty percent of cancers in humans. Outside the DNA-binding surface of p53, Y220C is the most common cancerous mutation. Previous studies have shown that a surface cavity is created by this mutation which destabilizes p53. PhiKan083, a carbazole derivative capable of binding with that cavity, and slows down its thermal denaturation rate. We investigated, theoretically, on mechanisms of structural stability loss due to Y220C mutation and mechanisms of stability restoration by PhiKan083 at the atomic level. From this study it is found that in Tp53C, Tyr220 has five electrostatic interactions with residues Val 147, Prol51, Pro153 and Pro223 located on S3/S4 loop and S7/S8 loop. The S7/S8 loop is stabilized by these electrostatic interactions. Due to the Y220C mutation all these electrostatic interactions are lost. As a result the structural fluctuation occurs at S7/S8 loop, and the loop is displaced from its original position after 6 ns MD simulation. When PhiKan083 is present (inserted) at the mutation site it provides five electrostatic interactions with Pro155, Glu221 and Thr230, and two hydrogen bonds with Leu145 and Asp228, respectively. These interactions provided by Pkikan083 stabilized the S7/S8 loop, and as a result it couldn’t be displaced. Our results showed that due to Y220C mutation p53 became destabilized through structural fluctuations surrounding the mutation site. When PhiKan083 is present at the Y220C mutation site (in 2vuk), it provides electrostatic and hydrogen bonding interactions among residue-220, its neighboring residues and PhiKan08. These interactions give additional stability to Y220C mutant p53, thus Y220C mutant p53 doesn’t destabilize.     

A novel Aurora-A-mediated phosphorylation of p53 inhibits its interaction with MDM2

Purpose: Crosstalk between Aurora-A kinase and p53 has been proposed. While the genetic amplification of Aurora-A has been observed in many human cancers, how p53 is regulated by Aurora-A remains ambiguous. In this study, Aurora-A-mediated phosphorylation of p53 was analyzed by mass spectrometry in order to identify a new phosphorylation site. Subsequently, the functional consequences of such phosphorylation were examined. Experimental design: In vitro phosphorylation of p53 by Aurora-A was performed and the phosphorylated protein was then digested with trypsin and enriched for phosphopeptides by immobilized metal affinity chromatography. Subsequently, a combination of β-elimination and Michael addition was applied to the phosphopeptides in order to facilitate the identification of phosphorylation sites by MS. The functional consequences of the novel phosphorylation of p53 on the protein–protein interactions, protein stability and transactivation activity were then examined using co-immunoprecipitation, Western blotting and reporter assays. Results: Ser-106 of p53 was identified as a novel site phosphorylated by Aurora-A. A serine-to-alanine mutation at this site was found to attenuate Aurora-A-mediated phosphorylation in vitro. In addition, phosphate-sensitive Phos-tag SDS-PAGE was used to confirm that the Ser-106 of p53 is in vivo phosphorylated by Aurora-A. Finally, co-immunoprecipitation studies suggested that Ser-106 phosphorylation of p53 decreases its interaction with MDM2 and prolongs the half-life of p53. Conclusions: The inhibition of the interaction between p53 and MDM2 by a novel Aurora-A-mediated p53 phosphorylation was identified in this study and this provides important information for further investigations into the interaction between p53 and Aurora-A in terms of cancer biology.