Cloning and expression analysis of full length mouse cDNA sequences encoding the transformation associated protein p53

Jenkins, Jennifer; Rudge, K.; Redmond, Shelagh; Wade-Evans, A.M.

doi:10.1093/nar/12.14.5609

Cited by 80 publications

(68 citation statements)

References 44 publications

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“…p53 [60], bax [31], and glyceraldehyde-3-P dehydrogenase cDNAs [61] were amplified in bacterial strain DH-5A and purified by two continuous CsCl ultracentrifugations according to Ausubel et al [62]. The inserts of p53, bax, and glyceraldehyde-3-P dehydrogenase cDNAs were recovered by double-gel purification [62] and labeled with A-32 P-dATP (Amersham PB10204) according to Feinberg and Vogelstein [63].…”

Section: Methodsmentioning

confidence: 99%

Okadaic acid‐induced lens epithelial cell apoptosis requires inhibition of phosphatase‐1 and is associated with induction of gene expression including p53 and bax

Fass

Huizar

et al. 1998

European Journal of Biochemistry

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It is well established that phosphorylation and dephosphorylation are key cellular events which regulate important metabolic activities such as gene expression, cell cycle progression, and apoptosis. The polyether fatty acid, okadaic acid has been shown previously to activate apoptosis in a variety of cell lines. Although this marine sponge toxin is known to inhibit protein phosphatase (PP)-2A and PP-1, it is not certain in most cases whether inhibition of PP-1 or PP-2A is necessary to activate apoptosis. Furthermore, it is not clear how inhibition of these phosphatases leads to apoptosis. Here we present evidence that inhibition of PP-2A by okadaic acid does not activate apoptosis in the lens system. However, when PP-1 is inhibited by okadaic acid, rabbit lens epithelial cells undergo rapid apoptosis. Associated with this process is the several-fold up-regulation of the tumor suppressor gene p53 and the pro-apoptotic gene bax at both mRNA and protein levels. Analyses of the temporal pattern of expression of the two genes reveal that the up-regulation is maximized in a few hours after treatment with okadaic acid, when the majority of the treated cells become committed to apoptosis. A brief treatment of the cells with a protein synthesis inhibitor can abolish okadaic acid-induced up-regulation of both P53 and Bax proteins. Concomitant with this inhibition, okadaic acid-induced apoptosis is also temporarily blocked. These results suggest that okadaic acid-induced expression of p53, bax, and other genes are necessary for the activation of the apoptotic programs in lens systems.Keywords : apoptosis; p53 ; bax ; phosphatase-1; lens.Apoptosis is a distinct form of cell death regulated by internal genetic programs [1,2]. The morphological changes associated with apoptosis include blebbing of cytoplasmic membranes, condensation of nucleoplasm and cytoplasm, and fragmentation of the dying cell into apoptotic bodies which are phagocytosed by neighboring cells [3,4]. The biochemical hallmarks of apoptosis are marked by DNA fragmentation into nucleosomal fragments [4,5], activation of the interleukin-1β-converting enzyme family of proteases [6], and cleavage of various substrates of that family of proteases such as poly(ADPribose) polymerase [7,8] and nuclear lamin [9].Apoptosis normally occurs as a physiological process during certain stages of animal development or during tissue homeostasis in an adult organism [10,11]. It can also be triggered by various external signals such as hormone [4], γ-irradiation [12] and withdrawal of growth factors [13] and other factors (reviewed in [2]). Normal physiological apoptosis helps to remove

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Section: Methodsmentioning

confidence: 99%

Okadaic acid‐induced lens epithelial cell apoptosis requires inhibition of phosphatase‐1 and is associated with induction of gene expression including p53 and bax

Fass

Huizar

et al. 1998

European Journal of Biochemistry

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“…Of the 35 tryptic peptides of murine p53, 18 contain sefine residues (14,29,37). We compared these peptides with the observed phosphopeptides by determining the presence of particular amino acids within each phosphopeptide as follows.…”

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confidence: 99%

“…The results from these studies are summarized in the amino acid numbers used are based on the predicted amino acid sequence of murine p53 (14,29,37). The amino acid sequences of the candidate phosphopeptides are given in Table 2.…”

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confidence: 99%

“…1) sequencing. (iii) Even if we could identify the phosphorylation sites by sequencing, we would expect to have difficulty in interpreting whether the release of phosphate occurs at residue 9 or 12 in peptide T3 (amino acids 1 to 27), because it is not known whether the initiation codon of mouse p53 is at codon 1 or 4 of the predicted protein sequence (14,29,37).…”

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confidence: 99%

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Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells.

Meek¹,

Eckhart²

1988

Mol. Cell. Biol.

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We observed six major tryptic phosphopeptides in p53 from simian virus 40-transformed and normal NIH 3T3 cells. Analyses of the phosphopeptides indicated that serines 37, 310 and/or 312, 389 and one or more of serines 7, 9, 12, 18, and 23 were phosphorylated. Phosphorylation of serines 310 and/or 312 was twofold higher in the simian virus 40-transformed cells as compared with that in normal NIH 3T3 cells.p53 is a cellular protein of apparent molecular weight 53,000, which is present at low levels in normal cells and at high levels in a wide range of primary tumors and transformed cell lines (for reviews, see references 5, 26, and 31). The molecular function of p53 is unknown. However, it is a cell cycle-related nuclear protein which appears to be essential for the progression of cells from a quiescent to an actively growing state and therefore may be an important regulatory element in growth control (15, 20-23, 25, 30). p53 has also been classed as an oncogene based on its ability to rescue primary cells from senescence (12,13); to cooperate, in place of myc, with an activated ras oncogene to transform primary rat embryo fibroblasts (7,13,28); and to transform previously immortalized cells to a tumorigenic phenotype without a significant change in cell morphology (6,16,35). The protein also forms stable complexes with the Elb tumor antigen of adenovirus (33) and the large tumor antigen (large T) of simian virus 40 (SV40; 17-19) and may have a direct involvement in SV40-mediated transformation (24).p53 is a phosphoprotein (5,26,31) which lacks intrinsic protein kinase activity (34). Two phosphorylated amino acids, serines 312 and 389, have been identified in mouse p53 by using Edman sequencing (32); additional phosphorylation sites have been tentatively mapped to the amino terminus between amino acid residues 28 and 98. The functional significance of these phosphorylations is unclear. However, serine 389 may be important for transformation by SV40, since a reduction in phosphorylation of this residue at the nonpermissive temperature in SV40-tsA58-transformed cells correlates with a failure to form the large T/p53 complex (2, 8, 32).To further characterize the phosphorylation sites of p53, we labeled SV40-transformed NIH 3T3 (SV3T3) cells for 16 h in phodphate-free Dulbecco-Vogt modified Eagle mediumn supplemented with 10% dialyzed calf serum and 2.5 mCi of 32p; per ml. Proteins were immunoprecipitated with anti-p53 monoclonal antibody PAb122 or PAb246 (36; kindly proAided by E. Gurney, University of Utah, and J. Yewdell, Wistar Institute, respectively), resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected by autoradiography. p53 was eluted from the gel and oxidized as described previously (1) by using 20 ,ug of RNase A as a carrier. Digestion with tolylsulfonyl phenylalanyl chloromethyl ketone-treated trypsin was carried out by using a trypsin/carrier ratio of 1:50 (wt/wt) to minimize traces of * Corresponding author. chymotryptic activity. Phosphopeptides were separated in two dime...

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“…Transcription and translation ofp53 cDNA An expression plasmid containing full length murine p53 cDNA, designated pSP65m53 (Jenkins et al, 1984) was transfected into E. coli, amplified and purified as detailed in Gamble and Milner (1988). The plasmid was linearised with HindIll and aliquots containing 1.0fig cDNA il-' in double distilled water were stored at -20'C.…”

Section: Methodsmentioning

confidence: 99%

Evidence for allosteric variants of wild-type p53, a tumour suppressor protein

Cook

Milner²

1990

Br J Cancer

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A tumour suppressor function for p53 is indicated in human lung cancer and in carcinoma of the colorectum. Loss of suppressor function, by mutation of the p53 gene, is associated with activation of p53 as an oncogene. The suppressor (wild type) and oncogenic (mutant) forms of the murine p53 protein are distinguishable at the molecular level by reactivity with anti-p53 monoclonal antibodies. For example, activated mutant p53 fails to react with PAb246 (p53-246 degrees). We now demonstrate that wild type p53 mRNA can be expressed either as p53-246+ or p53-246 degrees. We propose that p53-246 degrees may represent an allosteric variant of wild type p53 compatible with positive growth control. Thus, for wild type p53 the variants p53-246+ and p53-246 degrees may reflect suppressor and activator functions of p53 in the normal control of cell proliferation. For human p53 we present evidence that the epitope recognised by PAb1620 is analogous to that for PAb246 on murine p53. Thus the epitope for PAb1620 may prove to be of use as a marker for wild type human p53 with anti-oncogenic function. Images Figure 1 Figure 2 Figure 5

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Cloning and expression analysis of full length mouse cDNA sequences encoding the transformation associated protein p53

Cited by 80 publications

References 44 publications

Okadaic acid‐induced lens epithelial cell apoptosis requires inhibition of phosphatase‐1 and is associated with induction of gene expression including p53 and bax

Okadaic acid‐induced lens epithelial cell apoptosis requires inhibition of phosphatase‐1 and is associated with induction of gene expression including p53 and bax

Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells.

Evidence for allosteric variants of wild-type p53, a tumour suppressor protein

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