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T R Hupp

Publications and source records attributed to T R Hupp.

At least 37 records · Page 2Linked to original sources
PubMed1999

Dephosphorylation of p53 at Ser20 after cellular exposure to low levels of non-ionizing radiation.

Induction of the transactivation function of p53 after cellular irradiation was studied under conditions in which upstream signaling events modulating p53 activation were uncoupled from those regulating stabilization. This investigation prompted the discovery of a novel radiation-responsive kinase pathway targeting Ser20 that results in the masking of the DO-1 epitope in undamaged cells. Unmasking of the DO-1 epitope via dephosphorylation occurs in response to low doses of non-ionizing radiation. Our data show that phosphorylation at Ser20 reduces binding of the mdm2 protein, suggesting that a function of the Ser20-kinase pathway may be to produce a stable pool of inactive p53 in undamaged cells which can be readily activated after cellular injury. Phospho-specific monoclonal antibodies were used to determine whether the Ser20 signaling pathway is coupled to the Ser15 and Ser392 radiation-responsive kinase pathways. These results demonstrated that: (1) dephosphorylation at Ser20 is co-ordinated with an increased steady-state phosphorylation at Ser392 after irradiation, without p53 protein stabilization, and (2) stabilization of p53 protein can occur without Ser15 phosphorylation at higher doses of radiation. These data show that the Ser20 and Ser392 phosphorylation sites are both targeted by an integrated network of signaling pathways which is acutely sensitive to radiation injury.

Antibodies, Monoclonal↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

Regulation of p53 protein function through alterations in protein-folding pathways.

The tumour suppressor protein p53 is a stress-activated transcription factor whose activity is required for regulating the cellular response to stress and damage. The biochemical activity of p53 as a transcription factor can be regulated by partner proteins affecting stability, nuclear transport, signalling pathways modulating phosphorylation and interactions with components of the transcriptional machinery. The key structural determinants of p53 protein that drive sequence-specific DNA binding include the core specific DNA-binding domain and the tetramerization domain. Flanking these domains are more evolutionarily divergent carboxy- and amino-terminal regulatory motifs that further modulate tetramerization and sequence-specific transactivation. This review will mainly focus on the mechanisms whereby the tetramerization domain modulates sequence-specific DNA binding and how missense point mutations in p53 protein and the activity of molecular chaperones may lead to unfolding of mutant p53 tetramers in human tumours.

Antigens, Viral, Tumor↗
PubMed1998

DNA damage triggers DRB-resistant phosphorylation of human p53 at the CK2 site.

The sequence-specific DNA binding activity of p53 is negatively regulated by a C-terminal domain whose phosphorylation in vitro can activate the latent DNA binding function of the protein. The DNA binding activity of p53 is a core component of its stress-activated transcription function, yet it is not yet clear whether phosphorylation within the C-terminal domain plays a role in the p53 damage response in vivo. As the casein kinase 2 (CK2) site at serine 392 is the C-terminal phosphorylation motif that exhibits the most pronounced conservation at the primary amino acid level, we have focused on determining whether the CK2 site is modified in vivo and whether radiation effects the extent of that phosphorylation. Using antibodies that can detect serine 392-phosphorylation of p53, we demonstrate that UV radiation can trigger extensive phosphorylation at the CK2 site. The CK2 inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), can partially inhibit the UV-induced phosphorylation at serine 392, suggesting that CK2 is one of the major serine 392-kinases. However, a striking increase in UV-induced serine 392 phosphorylation and p53 transactivation function at higher levels of DRB suggests that a DRB-resistant/stress-activated pathway may target serine 392 in vivo. These data demonstrate that radiation-induced phosphorylation of p53 can occur in vivo at serine 392 and implicate a CK2-independent signal cascade that can function to modulate serine 392 phosphorylation in cells.

Binding Sites↗
PubMed1998

Characterization of p53 oligomerization domain mutations isolated from Li-Fraumeni and Li-Fraumeni like family members.

p53 is a tumour suppressor gene which functions as a transcription factor to upregulate genes for growth arrest and apoptosis following DNA damage. p53 mutations are associated with Li-Fraumeni and Li-Fraumeni like syndromes. Recently mutations of the oligomerization domain have been isolated from an LFS and an LFL family affecting respectively codon 344 (Leu to Pro) and 337 (Arg to Cys). The present study was designed to determine the affect of these mutations on the function of p53 protein. p53 344 Leu to Pro existed only in a monomeric form and could not bind to DNA. It was inactive at inducing apoptosis, transactivating luciferase from a bax promoter and inhibiting cell growth. In contrast, p53 337 Arg to Cys could form tetramers and could bind to DNA. However, p53 337 Arg to Cys was not fully active and could only induce apoptosis, transactivate luciferase from a bax promoter and inhibit cell growth with approximately 60% of the ability of wild-type p53. Both mutant proteins had reduced ability to bind to MDM2, p53 337 Arg to Cys being more reduced than p53 344 Leu to Pro. These results indicate that point mutations in the oligomerization domain can disrupt p53 function. In addition, the value of LFS and LFL families for the further understanding of the biological and biochemical properties of p53 is demonstrated.

Apoptosis↗
PubMed1997

HSP70 binding sites in the tumor suppressor protein p53.

Mutations within conserved regions of the tumor suppressor protein, p53, result in oncogenic forms of the protein with altered tertiary structures. In most cases, the mutant p53 proteins are selectively recognized and bound by members of the HSP70 family of molecular chaperones, but the binding site(s) in p53 for these chaperones have not been clearly defined. We have screened a library of overlapping biotinylated peptides, spanning the entire human p53 sequence, for binding to the HSP70 proteins, Hsc70 and DnaK. We show that most of the high affinity binding sites for these proteins map to secondary structure elements, particularly beta-strands, in the hydrophobic core of the central DNA binding domain, where the majority of oncogenic p53 mutations are found. Although peptides corresponding to the C-terminal region of p53 also contain potential binding sites, p53 proteins with C-terminal deletions are capable of binding to Hsc70, indicating that this region is not required for complex formation. We propose that mutations in the p53 protein alter the tertiary structure of the central DNA binding domain, thus exposing high affinity HSP70 binding sites that are cryptic in the wild-type molecule.

Amino Acid Sequence↗
PubMed1997

Tumor suppressor protein p53 binds preferentially to supercoiled DNA.

Wild type human tumor suppressor protein p53 (expressed in insect cells) binds strongly to negatively supercoiled (sc) plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of scDNA in agarose gels and imaging by scanning force microscopy (SFM). The binding occurs both in the presence and absence of the p53 consensus sequence. At relatively low p53/DNA ratios, binding of p53 to scDNA results in the appearance of several retarded DNA bands on the gels, similar to a conventional topoisomer ladder generated enzymatically. However, after removal of p53 by deproteination, the original mobility of the scDNA is recovered, indicating that the reduction of torsional stress accompanying p53 binding does not reflect changes in linking number. In DNA samples partially relaxed by topoisomerase I p53 binds preferentially to the scDNA molecules with the largest negative superhelix density. SFM imaging of the p53/scDNA complex reveals a partial or total relaxation of the compact scDNA, the degree of which increases with the number of bound p53 molecules. Competition assays with linear DNA reveal a preference of p53 for scDNA. In addition, scDNA induces dissociation of p53 from a preformed complex with a DNA fragment (474 bp) containing the consensus sequence. We conclude that the affinity of p53 for negatively supercoiled DNA is greater than that for the consensus sequence in linear fragments. However, thermally denatured linearized plasmid DNA is efficient in competing for the binding of p53 to scDNA, although the first retarded band (presumed to contain one bound p53 molecule) is retained in the case of the plasmid containing the consensus sequence. Thus, it appears that interactions involving both the core domain and the C-terminal domain regulate the binding of p53 to scDNA. The above results are not restricted to human p53; the wild type rat p53 protein also results in the retardation of scDNA on agarose gels. The biological implications of the novel DNA binding activities of p53 are discussed.

Animals↗
PubMed1997

Biochemical analysis of the stress protein response in human oesophageal epithelium.

BACKGROUND: The oesophageal epithelium is exposed routinely to noxious agents in the environment, including gastric acid, thermal stress, and chemical toxins. These epithelial cells have presumably evolved effective protective mechanisms to withstand tissue damage and repair injured cells. Heat shock protein or stress protein responses play a central role in protecting distinct cell types from different types of injury. AIM: To determine (i) whether biochemical analysis of stress protein responses in pinch biopsy specimens from human oesophageal epithelium is feasible; (ii) whether undue stresses are imposed on cells by the act of sample collection, thus precluding analysis of stress responses; and (iii) if amenable to experimentation, the type of heat shock protein (Hsp) response that operates in the human oesophageal epithelium. METHODS: Tissue from the human oesophagus comprised predominantly of squamous epithelium was acquired within two hours of biopsy and subjected to an in vitro heat shock. Soluble tissue cell lysates derived from untreated or heat shocked samples were examined using denaturing polyacrylamide gel electrophoresis for changes in: (i) the pattern of general protein synthesis by labelling epithelial cells with 35S-methionine and (ii) the levels of soluble Hsp70 protein and related isoforms using immunochemical protein blots. RESULTS: A single pinch biopsy specimen is sufficient to extract and analyse specific sets of polypeptides in the oesophageal epithelium. After ex vivo heat shock, a classic inhibition of general protein synthesis is observed and correlates with the increased synthesis of two major proteins of molecular weight of 60 and 70 kDa. Notably, cells from unheated controls exhibit a "stressed" biochemical state 22 hours after incubation at 37 degrees C, as shown by inhibition of general protein synthesis and increased synthesis of the 70 kDa protein. These data indicate that only freshly acquired specimens are suitable for studying stress responses ex vivo. No evidence was found that the two heat induced polypeptides are previously identified Hsp70 isoforms. In fact, heat shock results in a reduction in the steady state concentrations of Hsp70 protein in the oesophageal epithelium. CONCLUSION: Systematic and highly controlled studies on protein biochemistry are possible on epithelial biopsy specimens from the human oesophagus. These technical innovations have permitted the discovery of a novel heat shock response operating in the oesophageal epithelium. Notably, two polypeptides were synthesised after heat shock that seem to differ from Hsp70 protein. In addition, the striking reduction in steady state concentrations of Hsp70 protein after heat shock suggests that oesophageal epithelium has evolved an atypical biochemical response to thermal stress.

Biopsy↗
PubMed1996

The p53 response to ionising radiation in adult and developing murine tissues.

The induction of the p53 response to ionising radiation has been studied during murine development and in the adult animal. The response has been assessed by precise quantitative assay of p53 protein levels in tissues and by immunohistochemistry. Newly developed transgenic mice in which a lacZ transgene is driven by a p53 response element have also been used to directly assess the transcriptional activity of the induced protein. There is striking developmental control of the p53 response so that in early development all tissues accumulate high levels of p53 following radiation and indeed p53 is present at elevated levels in some unirradiated tissues. Later in development clear heterogeneity of the p53 response becomes apparent, both in terms of the responses of individual tissues and of cell populations, within those tissues. The study of lacZ transgene expression and the occurrence of apoptosis in different tissues that accumulate p53 protein point to a further level of control regulating the nature and degree of the downstream response to elevated levels of p53 in cells. These findings have important implications for the susceptibility of different tissue types to carcinogenic and other insults. The early expression of the p53 response is consistent with novel models of p53 function that suggest it may have evolved principally as a defense against teratogenic insult that permits plasticity of development.

Age Factors↗
PubMed1996

Modification of two distinct COOH-terminal domains is required for murine p53 activation by bacterial Hsp70.

Activation of the latent DNA binding function of human p53 protein by the bacterial Hsp70, DnaK, represents a unique reaction in which a heat shock protein can interact with a native protein to affect its function. We have localized a likely DnaK interaction site on native human p53 tetramers to a motif flanking the COOH-terminal casein kinase II and protein kinase C phosphorylation sites. Murine p53 is less efficiently activated by DnaK, which has permitted a search for factors that might cooperate in p53 activation by DnaK. We show that optimal activation by DnaK may be dependent upon the phosphorylation state of murine p53, in particular, modification of p53 at the cdc2 phosphorylation site by point mutation decreases the extent of activation by DnaK. Additionally, the monoclonal antibody PAb241, binding in the vicinity of the cdc2 phosphorylation site, is able to activate the specific DNA binding function of p53. This has led us to propose a second regulatory motif flanking the tetramerization domain of p53 that cooperates with factors binding at the negative regulatory domain in the extreme COOH terminus.

Amino Acid Sequence↗
PubMed1996

Domains of DnaA protein involved in interaction with DnaB protein, and in unwinding the Escherichia coli chromosomal origin.

DnaA protein of Escherichia coli is a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriC plasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA protein-binding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.

Adenosine Triphosphate↗
PubMed1996

Epitope analysis of the murine p53 tumour suppressor protein.

The identification and characterisation of the p53 tumour suppressor has relied extensively on the use of immunological reagents. To facilitate further characterisation of the murine p53 protein (Mp53), and its interaction with other proteins, we have characterised the antigenic sites of Mp53 in fine detail. Using an overlapping Mp53 peptide library we report the identification by Pepscan ELISA of the epitopes of nine antibodies. We have also used this technique to determine whether corresponding epitopes were present in a human p53 (Hp53) peptide library. This comparison was extended to include polyclonal sera of mice immunized with either Mp53 or Hp53, to compare the overall range of antigenic sites. The range of antigenic sites identified by polyclonal sera is very similar, although the N-terminus of Mp53 is clearly not an immunodominant region, in contrast to the N-terminus of Hp53. However, the N-terminus of Mp53 is immunogenic in rabbits as demonstrated by the Pepscan ELISA of CM5 serum (a rabbit anti-Mp53 serum used in analysing p53 expression in mice). Since, very few new antigenic sites were identified in either Mp53 or Hp53, new approaches will have to be employed to identify novel immunological reagents against human and murine p53.

Animals↗
PubMed1996

Modification of an N-terminal regulatory domain of T antigen restores p53-T antigen complex formation in the absence of an essential metal ion cofactor.

We have discovered that the ability of the tumor suppressor protein p53 to bind to the viral large T antigen (TAg) oncogene product is regulated by divalent cations. Both proteins were purified from an insect cell line infected with the appropriate baculovirus expression vector. In a two-site capture enzyme-linked immunosorbent assay, complex formation between the purified proteins is strictly dependent on the addition of specific concentrations of divalent metal ions, notably zinc, copper, cadmium, cobalt, manganese, and nickel. In the presence of zinc the pattern of proteolytic fragments obtained when TAg was subjected to proteolysis by endoproteinase Glu-C (V8) was strikingly different, supporting the idea that a conformational change in TAg associated with ion binding is required for it to complex with p53. Monoclonal antibody analysis provides supporting evidence for a conformational change. When TAg was captured onto an enzyme-linked immunosorbent assay plate coated with PAb 419 as opposed to many other anti-TAg antibodies, complex formation was completely independent of the presence of additional divalent cations. Our results suggest that the ability of p53 and TAg to form a stable complex in vitro is dependent upon a regulatory domain residing in the N terminus of TAg, zinc ions or the binding of a specific monoclonal antibody (PAb 419) provoking a conformational change in TAg that facilitates and supports complex formation.

Animals↗
PubMed1996

Allosteric regulation of the thermostability and DNA binding activity of human p53 by specific interacting proteins. CRC Cell Transformation Group.

Conformational stability is a prerequisite for the physiological activity of the tumor suppressor protein p53. p53 protein can be allosterically activated for DNA binding by phosphorylation or through noncovalent interaction with proteins such as DnaK, the Escherichia coli homologue of the heat shock protein Hsp70. We present in vitro evidence for a rapid temperature-dependent change in the conformation and tetrameric nature of wild-type p53 upon incubation at 37 degrees C, which correlates with a permanent loss in DNA binding activity. We show that p53 is allosterically regulated for stabilization of the wild-type conformation and DNA binding activity at 37 degrees C by binding of two classes of ligands to regulatory sites on the N and C terminus of the molecule through which an intrinsic instability of p53 is neutralized. Deletion of the domain conferring instability at the C terminus is sufficient to confer enhanced stability to the total protein. DnaK binding to the C terminus can profoundly protect p53 at 37 degrees C from a temperature-dependent loss of the DNA binding activity but does not renature or activate denatured p53. In contrast, another activator of the DNA binding activity of latent p53, the monoclonal antibody PAb421, which also interacts with the C terminus of the protein, is not able to protect p53 from thermal denaturation. Two monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, do not activate the latent DNA binding function of p53 but can protect the p53 wild-type conformation at 37 degrees C. Thus, activation of the DNA binding function of p53 is not synonymous with protection from thermal denaturation, and therefore, both of these pathways may be used in cells to control the physiological activity of p53. The protection of p53 conformation from heat denaturation by interacting proteins suggests a novel mechanism by which p53 function could be regulated in vivo.

Allosteric Regulation↗
PubMed1995

Small peptides activate the latent sequence-specific DNA binding function of p53.

Normal cells contain p53 protein in a latent state that can be activated for sequence-specific transcription by low levels of UV radiation without an increase in protein levels. Microinjection of cells with an antibody specific to the C-terminal negative regulatory domain can activate the function of p53 as a specific transcription factor in the absence of irradiation damage, suggesting that posttranslational modification of a negative regulatory domain in vivo is a rate-limiting step for p53 activation. Small peptides derived from the negative regulatory domain of p53 have been used as biochemical tools to distinguish between allosteric and steric mechanisms of negative regulation of p53 tetramer activity. Presented is the development of a highly specific peptide activation system that is consistent with an allosteric mechanism of negative regulation and that forms a precedent for the synthesis of novel low molecular mass modifiers of the p53 response.

Allosteric Regulation↗
PubMed1995

Two distinct signaling pathways activate the latent DNA binding function of p53 in a casein kinase II-independent manner.

Post-translational modification of a carboxyl-terminal negative regulatory domain in vitro by either casein kinase II or protein kinase C allosterically activates the latent sequence-specific DNA binding function of p53. Reported here is a biochemical approach to determine the types of signaling pathways and enzymes that are involved in p53 activation in cells. Using a novel chromatographic method, we have been able to separate three distinct biochemical forms of p53 that have been synthesized in vivo; two are in an activated state, and one is in a latent state for sequence-specific DNA binding. The two activated forms of p53 appear to be controlled individually by either a constitutive or a UV-inducible signaling pathway. p53 lacking the COOH-terminal casein kinase II site (p53 delta 4) was characterized biochemically and used to determine the affects of deletion of the casein kinase II motif on the production of the two activated forms of p53 in vivo. As observed with full-length p53, the production of two distinct chromatographic forms of activated p53 delta 4 occurs in vivo, indicating that p53 activation can occur through a casein kinase II-independent pathway and suggesting that two other factors are involved in activation of p53 in vivo.

Amino Acid Sequence↗