Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human vascular smooth muscle cells (HVSMC) cultured from restenotic lesions are resistant to inhibition of proliferation by heparin. We examined if altered expression of p53 protein might be related to this phenomenon. HVSMC were cultured from saphenous vein and p53 protein levels examined using immunocytochemistry, and by immunoprecipitation with antibodies specific for wild-type or mutant conformations followed by SDS PAGE and immunoblotting. Inhibition of proliferation by heparin was measured in 14-day growth assays. Elevated levels of p53 were found in five out of 41 HVSMC strains. The accumulated p53 protein was not precipitated by a mutant conformation-specific anti-p53 antibody in any of the five strains. In one of the five positive strains there was concomitant human cytomegaloviral infection. No significant difference was found between efficacy of heparin in the p53-positive and -negative strains. Furthermore heparin had no detectable effect on p53 protein levels. Although aberrant levels of p53 are detectable in a minority of HVSMC strains, these data do not support a role for altered p53 expression in resistance to the growth inhibitory effects of heparin in these cells.
 ...
PMID:Inhibition of proliferation by heparin and expression of p53 in cultured human vascular smooth muscle cells. 936 85

The contribution of each of the structural domains of p53 to its function has been discussed widely in the literature. Crystallographic studies have revealed much about the structure of the core DNA binding domain, but as it has not been possible to use this approach for the intact protein, the effect of the domains flanking the core must be investigated by more indirect techniques. In this study a series of truncated murine p53 proteins has been investigated for DNA binding activity at 4 degrees C and 37 degrees C, transcriptional activation, and tumour suppression activity. Full-length p53, and truncations lacking the N terminus, purified from a baculovirus expression system all show latency for DNA binding; that is, they must be activated to bind by association with a C-terminal antibody such as PAb421. This demonstrates that latency for DNA binding is independent of the N terminus. Truncations lacking the C-terminal oligomerisation domain, and the isolated core domain, can only be activated to bind DNA and PAb1620 (an antibody recognising the wild-type conformation of the core domain) in the presence of cross-linking antibodies, while murine core only binds to DNA in the presence of PAb1620. An analysis of the thermostability of DNA binding revealed that antibodies that bind the N terminus of p53 could protect the protein against loss of activity at 37 degrees C. C-terminal antibodies, however, were ineffective unless the N-terminal 37 amino acid residues were absent. The N terminus may retain some secondary structure, since it is the main contributor to the anomalous migration in SDS-polyacrylamide gels. Our results suggest that the N terminus has a destabilising effect that influences conformation of p53 at 37 degrees C, so cellular proteins binding to the N terminus in vivo may modulate p53 conformation and stability. The effects on thermostability are also direct evidence showing that antibodies binding to N-terminal deletions create a conformational change in the rest of the molecule. In addition, longer deletions of the C terminus reduce the ability of p53 to transactivate target genes and inactivate tumour suppression activity, while truncations of the N terminus retain partial tumour suppression activity. Our results clearly show participation of both the N and C termini in the regulation of all the functions of p53 at 37 degrees C, indicating that distinct, independent domains interact with each other within, the flexible structure of p53.
 ...
PMID:The N terminus of the murine p53 tumour suppressor is an independent regulatory domain affecting activation and thermostability. 946 32

The tumor suppressor p53 and its target the CDK inhibitor p21 (Cip1/Waf1) are key components of the cellular response to DNA damage. Insight into how p21 is regulated in normal cells, and how it may be deregulated in tumor cells is important for the understanding of tumorigenesis. p21 was induced in normal human diploid fibroblasts after UV irradiation-induced DNA damage, but, at a high dose of UV irradiation, a faster mobility form of p21 on SDS-PAGE (designated p21delta) was expressed. Surprisingly, in a variety of growing transformed cell lines, the level of p21 was low but p21delta was prominent. We found that p21delta appeared to be derived through a loss of around 10 amino acids from the C-terminus of p21, which theoretically would remove the PCNA binding domain, a second cyclin binding domain and the nuclear localization signal sequence. Several characteristics distinguish p21 from p21delta. Both the full length p21 and p21delta could be stabilized by a proteasome inhibitor, but only the full length p21 was associated with Cdk2 and PCNA. Consistent with this, gel filtration chromatography revealed that all the full length p21 in the cell was complexed to other proteins, whereas a significant portion of p21delta was in monomeric form. Moreover, p21 was mainly localized to the nucleus, but p21delta was mainly localized to the cytoplasm. We propose that the decrease in p21 and increase in p21delta could contribute to the deregulation of the cell cycle, and could be a mechanism involved in cellular transformation.
 ...
PMID:Expression of a novel form of p21Cip1/Waf1 in UV-irradiated and transformed cells. 954 35

After more than a year had elapsed since a single oral exposure to 2 and 4 microgram 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg, there was an apparent dose-related increased incidence of significant endocervical squamous metaplasia in a group of cynomolgus macaques (Scott et al., 1998). In the present experiments we investigated the mechanisms by which chemicals like TCDD could induce epithelial cell transdifferentiation in the primate endocervix. One focus of investigation was epidermal growth factor receptor (EGFR) and the key cytosolic signaling kinases, c-Src and protein tyrosine kinase (PTK), whose responses to TCDD are well characterized. A second focus was the distal kinase Erk2 that transduces the cytosolic signal into a nuclear signal, and which in combination with nuclear casein kinase II (CKII), can lead to activation of p53. Finally, we studied three key target proteins of activated p53 (wafl/p21, Cdc2 p34, and Cdk4), whose modulation could produce cell cycle effects. The studies were carried out using primary cell cultures prepared from endocervical epithelium recovered at necropsy from TCDD-treated (2 and 4 microgram TCDD/kg) and untreated macaques. There was a significant decrease in EGFR binding activity in cells from TCDD-treated animals as compared to controls. A marked increase in the protein amount of H-Ras and a significant increase in the activity of c-Src kinase, PTK, and Erk2 were found in cells from TCDD-treated animals. A significant decrease in the activity of CKII and in the protein amount of p53, wafl/p21, and Cdc2 p34 was found. On the other hand, a substantial increase in the protein amount of Cdk4 and DNA binding activity of AP-1 was found in cells from TCDD-treated animals. In vitro experiments using primary cultures of endocervical cells from untreated macaques revealed that these cells have AhR, and that c-Src protein is functionally attached to the AhR and is specifically activated upon ligand binding as judged by the following criteria. (1) A structure-activity relationship study with TCDD and three dioxin congeners revealed a rank order for their potency in activation of AhR-associated c-Src kinase from cervical cells which was identical to that of previously determined toxicity indices. (2) TCDD-induced, AhR-associated c-Src kinase activity was abolished when an AhR immunoprecipitate from cervical cells was preincubated with alpha-naphthoflavone (AhR blocker) or geldanamycin (Src kinase inhibitor) prior to the addition of TCDD. (3) The analysis of the AhR complex showed three proteins of molecular weights of 100 (AhR), 90, and 60 kDa. (4) The same protein with molecular weight 60 kDa was found when the immunoprecipitate with anti AhR-antibody was analyzed by SDS-PAGE, then transferred into nitrocellulose membrane followed by immunobloting the membrane with anti c-Src-antibody. Our data suggest that TCDD induced pathology in endocervical cells through changes in growth factor receptor signaling, other cytosolic signaling proteins, tumor suppressor proteins, and cell cycle proteins.
 ...
PMID:Alterations in the growth factor signal transduction pathways and modulators of the cell cycle in endocervical cells from macaques exposed to TCDD. 970 5

p21waf1/cip1 mRNA and protein accumulate in intact cells exposed to oxidizing agents through a p53-independent, MAPK-dependent mechanism. Treatment with oxidizing agents also yields a second form of this protein (FM p21), characterized by a faster migration on SDS-PAGE. This phenomenon depends on the modification of intracellular redox conditions induced by diethylmaleate, a glutathione-depleting agent, being prevented by the pretreatment with the glutathione precursor N-acetylcysteine. The appearance of this FM p21 form is very early, being observed 5 min after exposure to diethylmaleate, long before the already observed accumulation of p21 induced by oxidative stress. Furthermore, experiments with dominant negative mutants of MEK demonstrate that, in contrast with that observed for the oxidative stress-induced accumulation of p21 mRNA and protein, the appearance of FM p21 form is not dependent from the activation of the MAPK pathway. It was previously observed (Tchou et al, 1996) that in some lung carcinoma cells long exposure to high doses of phorbol esters also induces the appearance of a faster-migrating p21 electrophoretic band and it was suggested that this could result from a different phosphorylation or from a proteolytic processing at the C-terminus of the protein. The latter is not the case for the diethylmaleate-induced FM p21 whose C-terminus is intact, as demonstrated by the expression of a C-terminus tagged p21 cDNA. On the contrary, the observed migration shift seems to be dependent on the hypophosphorylation of the protein; in fact, a pretreatment of cells with okadaic acid, an inhibitor of (serine/threonine) phosphatases, inhibits the oxidation-dependent appearance of the FM p21 and the block of protein synthesis, caused by cycloeximide, does not affect the appearance of FM p21, that thus could derive from the dephosphorylation of preexisting protein.
 ...
PMID:A new p21waf1/cip1 isoform is an early event of cell response to oxidative stress. 984 80

The conformation and activity of pRb, the product of the retinoblastoma susceptibility gene, is dependent on the phosphorylation status of one or more of its 16 potential cyclin-dependent kinase (cdk) sites. However, it is not clear whether the phosphorylation status of one or more of these sites contributes to the determination of the various conformations and activity of pRb. Moreover, whether and how the conformation of pRb may regulate the phosphorylation of the cdk sites is also unclear. In the process of analyzing the function and regulation of pRb, we uncovered the existence of an unusual structural motif, m89 (amino acids 880-900), the mutation of which confers upon pRb a hypophosphorylated conformation. Mutation of this structural domain activates, rather than inactivates, the growth suppressor function of pRb. In order to understand the effect of the mutation of m89 on the phosphorylation of cdk sites, we identified all the cdk sites (Thr-356, Ser-807/Ser-811, and Thr821) the phosphorylation of which drastically modify the conformation of pRb. Mutation of each of these four sites alone or in combinations results in the different conformations of pRb, the migration pattern of which, on SDS-polyacrylamide gel electrophoresis, resembles various in vivo hypophosphorylated forms. Each of these hypophosphorylated forms of pRb has enhanced growth suppressing activity relative to the wild type. Our data revealed that the m89 structural motif controls the exposure of the cdk sites Ser-807/Ser-811 in vitro and in vivo. Moreover, the m89 mutant has enhanced growth suppressing activity, similar to a mutant with alanine substitutions at Ser-807/Ser-811. Our recent finding, that the m89 region is part of a structural domain, p5, conserved antigenically and functionally between pRb and p53, suggests that the evolutionarily conserved p5 domain may play a role in the coordinated regulation of the activity of these two tumor suppressors, under certain growth conditions.
 ...
PMID:Discovery of a regulatory motif that controls the exposure of specific upstream cyclin-dependent kinase sites that determine both conformation and growth suppressing activity of pRb. 1009 28

The unique behavior of green fluorescent protein (GFP) on SDS-PAGE was applied to the detection of a single amino acid substitution in GFP-tagged polypeptides. This simple detection method using SDS/urea gels was designated GFP-display. The N-terminal 18 or 37 amino acids of K-Ras was used as a model GFP-tagged polypeptide. K-ras exon 1 was fused to a gfp cDNA at each end and expressed in Escherichia coli. Amino acid number 12 of K-Ras (wild type; Gly) was changed to Ser, Arg, Cys, Asp, Ala, or Val, and the mobility shift of the greenish fluorescent bands in the SDS/urea gel was analyzed. These mutants were easily detected by GFP-display; however, detection depended strongly on the urea concentration and electrophoresis temperature. Subsequently, GFP-display was applied to the 36 amino acids encoding human p53 exon 7. Amino acid number 248 (wild type; Arg) was changed to Gly, Trp, Gln, Pro, or Leu, and similar mobility shifts were observed. GFP-display could be coupled with an in vitro translation system. Fluorescent active GFP and GFP-Ras fusion proteins were synthesized within a few hours. GFP-display shows potential as a modern approach to gene mutation analysis at the protein level, and is a useful method for protein engineering studies.
 ...
PMID:A new approach to gene mutation analysis using "GFP-Display". 1073 55

Stimulation of transfected HepG2 cells (TFG2) with the alpha(1)-adrenergic agonist phenylephrine (PE) significantly activated p21(waf1/cip1) gene expression without affecting p53 gene expression. Northern blotting and reporter assay demonstrated that this induction was due to PE stimulation of p21(waf1/cip1) mRNA stability. To further define the underlying mechanism, we prepared a chloramphenicol acetyltransferase (CAT)-p21(waf1/cip1) 3'-untranslated region (3'-UTR) hybrid construct by inserting the 3'-UTR of p21(waf1/cip1) mRNA just downstream from the CAT coding sequence and transfected it into TFG2 cells. PE treatment enhanced the activity of this construct by 6-fold. Deletion analyses indicated that an AU-rich element (AURE) located between 553 to 625 within the p21(waf1/cip1) 3'-UTR was required for this induction. RNA gel shift assays demonstrated that this AURE bound an RNA-binding protein. This protein has been purified 5000-fold from PE-treated TFG2 cells by heparin-Sepharose and RNA affinity chromatography. SDS-polyacrylamide gel electrophoresis, UV cross-linking, and Northwestern analyses indicated the molecular mass of this protein as 24 and 52 kDa. Finally, PE treatment markedly enhanced this RNA-protein binding by a p42/44 mitogen-activated protein kinase-dependent mechanism. These data suggest that the AURE located between 553 and 625 within the p21(waf1/cip1) mRNA 3'-UTR, which binds an RNA-binding protein, is responsible for PE-induced p21(waf1/cip1) mRNA stability.
 ...
PMID:Alpha(1) adrenergic agonist induction of p21(waf1/cip1) mRNA stability in transfected HepG2 cells correlates with the increased binding of an AU-rich element binding factor. 1076 10

Mouse mortalin proteins, mot-1 and mot-2, differ by only two amino acid residues in their C-terminus. In previous studies we showed that they differ in their subcellular distributions and interactions with the tumor suppressor protein, p53. By using mot-1 deletion mutants and amino acid substitution constructs, we report here that inability of mot-1 to affect p53 activity in vivo is dependent on the presence of both of the unique mot-1 amino acids and all three of the predicted hsp70, EF hand, and leucine zipper motif regions. The two proteins and their single amino acid mutants showed different mobilities on SDS-polyacrylamide gel presenting an evidence for their different secondary structures. Taken together, the data suggest that each of the two differing amino acids between mot-1 and mot-2 is an important determinant of their secondary structures and in vivo activities.
 ...
PMID:Transcriptional inactivation of p53 by deletions and single amino acid changes in mouse mot-1 protein. 1111 32

We previously identified a non-p53, p53-responsive DNA element (p53RE)-binding protein named NBP, functionally analogous to p53, from human cervical carcinoma Hela cells. Here we report a biochemical study demonstrating that this activity is the recently cloned p53 analog p63. NBP was purified through conventional and DNA affinity chromatography to apparent homogeneity with a prominent polypeptide migrating in between the 43 and 68 kDa positions on a SDS gel. This polypeptide immunoreacted with monoclonal anti-p63 but not anti-p53 or anti-p73 antibodies. Also, NBP co-purified with p63 through each step of fractionation, as detected with anti-p63 antibodies. DNA-protein complexes formed with purified NBP and p53RE-containing oligomers derived from the p21(waf1) promoter were supershifted by anti-p63 but not anti-p53 antibodies. Thus, these results demonstrate that NBP is encoded by the p53 homolog p63 gene.
 ...
PMID:NBP is the p53 homolog p63. 1118 41


<< Previous 1 2 3 4 5 6 7 8 Next >>