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

Human DNA-PK is a nuclear, serine/threonine protein kinase that, when activated by DNA, phosphorylates several DNA-binding substrates, including the tumor suppressor protein p53. To identify which p53 residues are phosphorylated, we examined DNA-PK's ability to phosphorylate synthetic peptides corresponding to human p53 sequences. Serines 15 and 37 in the amino-terminal transactivation domain of human p53, and serines 7 and 18 of mouse p53, were phosphorylated by DNA-PK in the context of synthetic peptides. Other serines in these p53 peptides, and serines in other p53 peptides, including peptides containing the serine 315 p34cdc2 site and the serine 392 casein kinase II site, were not recognized by DNA-PK or were phosphorylated less efficiently. Phosphorylation of the conserved serine 15 in human p53 peptides depended on the presence of an adjacent glutamine, and phosphorylation was inhibited by the presence of a nearby lysine. Phosphorylation of recombinant wild-type mouse p53 was inhibited at high DNA concentrations, suggesting that DNA-PK may phosphorylate p53 only when both are bound to DNA at nearby sites. Our study suggests that DNA-PK may have a role in regulating cell growth and indicates how phosphorylation of serine 15 in DNA-bound p53 could alter p53 function.
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PMID:Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. 140 79

HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA). Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators. The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation. During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner. We conclude that this 350-kDa polypeptide is likely to be DNA-PK. Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S. P. Lees-Miller and C. W. Anderson, J. Biol. Chem. 264:17275-17280, 1989). Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa. Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth.
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PMID:Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. 224 67

Screening for inhibitors of DNA-dependent protein kinase (DNA-PK) revealed 3-cyano-5-(4-pyridyl)-6-hydrazonomethyl-2-pyridone, designated OK-1035, to be a potent and selective inhibitor. When a synthetic peptide was used as a substrate, OK-1035 caused 50% inhibition of DNA-PK activity at 8 microM, a concentration more than 50 times lower than those required against seven other protein kinases tested. OK-1035 inhibited the phosphorylation by DNA-PK of consensus peptide as well as that of recombinant human wild type-p53. Kinetic studies indicated that OK-1035 inhibited DNA-PK activity in an ATP-competitive manner.
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PMID:OK-1035, a selective inhibitor of DNA-dependent protein kinase. 757 20

The Ku autoantigen is a heterodimer of 70 kDa (p70) and -80 kDa (p80) subunits that is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK). The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates Sp-1, Oct-1, p53 and RNA polymerase II in vitro, but the precise cellular role of DNA-PK remains unclear. In the present studies, the assembly of p70/p80 heterodimers and the interaction of Ku with DNA was investigated using recombinant vaccinia viruses directing the synthesis of human p70 (p70-vacc) and p80 (p80-vacc), and monoclonal antibodies (mAbs). Expression of human Ku antigens in rabbit kidney (RK13) cells could be demonstrated by immunofluorescent staining because this cell line contains little endogenous Ku. A novel mAb designated 162 stained the nuclei of RK13 cells coinfected with p70-vacc and p80-vacc, but not cells that were infected with either virus alone, suggesting that it recognized the p70/p80 heterodimer but not monomeric p70 or p80. In agreement with the immunofluorescence data, 162 immunoprecipitated both p70 and p80 from extracts of coinfected cells, but did not immunoprecipitate either subunit by itself from extracts of cells infected with p70-vacc or p80-vacc, respectively. Conversely, the binding of 162 to Ku isolated from human K562 cells stabilized the p70/p80 heterodimer under conditions that normally dissociate p70 from p80. The nuclei of cells infected with p70-vacc alone could be stained with mAb N3H10 (anti-p70) and cells infected with p80-vacc alone could be stained with mAb 111 (anti-p80), indicating that the formation of p70/p80 heterodimers was not required for nuclear transport. Finally, free recombinant and cellular p70 both bound to DNA efficiently in vitro, suggesting that free p70, like the p70/p80 heterodimer, serves as a DNA-binding factor. Moreover, free human p70 could be released from the nuclei of p70-vacc-infected RK13 cells by deoxyribonuclease I treatment, suggesting that it was associated with chromatin in vivo. The nuclear transport of free p70 and the association of free p70 with chromatin in vivo raise the possibility that newly synthesized cellular p70 might undergo nuclear transport and DNA-binding prior to dimerization with p80 or assembly with p350.
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PMID:Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers. 769 19

To investigate the effect of phosphorylation on the transcription activity of p53, ten phosphorylation mutants were constructed covering all the identified phosphorylation sites of rat p53. These included mutants of two casein kinase I sites (Ser6 and Ser9), two DNA-PK sites (Ser15 and Ser39), a p34cdc2 site (Ser313), the adjacent Ser312 and a casein kinase II site (Ser390). Two double phosphorylation mutants (Ser4, 6 and Ser15, 390) and one triple phosphorylation mutant (Ser4, 6 and 15) were also constructed. The transcription activity of all the p53 phosphorylation mutants was tested by transfection into two different types of cells, Saos-2 cells and p53(-/-) fibroblasts derived from p53 knock out mice, which both lack endogenouse p53. Surprisingly, all the p53 phosphorylation mutants retain transcription activity and the seven mutants tested can also suppress cell growth.
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PMID:p53 phosphorylation mutants retain transcription activity. 786 59

Overexpression of wild-type p53 prevents cells from entering the S phase of the cell cycle. The amino-terminal transactivation region of p53 is phosphorylated by several protein kinases, including DNA-PK, a nuclear serine/threonine protein kinase that in vitro requires DNA for activity. DNA-PK was recently shown to phosphorylate serines 15 and 37 of human p53 (Lees-Miller et al., 1992. Mol. Cell. Biol., 12, 5041-5049). To prevent phosphorylation at these sites, mutants were constructed that changed the codons for serine 15 or serine 37 to alanine codons. Expression of p53-Ala-37 in stably transformed T98G cells blocked progression of the cells into S phase as well as did the expression of wild-type p53. In contrast, p53-Ala-15 was partially defective in blocking cell cycle progression. Several cell clones transformed with the mutant p53-Ala-15 gene expressed normal levels of p53 mRNA but accumulated little or no detectable p53 protein. However, by using a transient expression system driven by a strong cytomegalovirus promoter, we showed that the inability of p53-Ala-15 to fully block cell cycle progression was not due to inadequate levels of expression or to a failure of the mutant protein to accumulate in the nucleus. These results suggest that phosphorylation of Ser-15 may affect p53 function.
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PMID:Mutation of the serine 15 phosphorylation site of human p53 reduces the ability of p53 to inhibit cell cycle progression. 850 77

The murine scid mutation affects both V(D)J recombination and DNA repair. This mutation has been mapped to the gene encoding the catalytic subunit of the DNA-dependent protein kinase (DNA-PK), which is activated by DNA damage in normal cells. In scid mice, antigen receptor gene rearrangements are initiated normally, but impaired joining of coding ends prevents assembly of functional receptor genes, resulting in arrest of B- and T-cell development. Others have shown that exposure of scid mice to genotoxic agents such as gamma-irradiation rescues rearrangement at the T-cell receptor (TCR) beta locus and promotes thymocyte development. Here we demonstrate that irradiation rescues rearrangements at multiple TCR loci, suggesting a general effect on the recombination mechanism. Furthermore, our data show that p53 is required for irradiation-mediated rescue of both thymocyte development and V(D)J recombination. We also find that thymocyte proliferation and differentiation in the absence of DNA damage do not require p53 and are not sufficient to rescue V(D)J recombination. These results suggest that exposure to ionizing radiation facilitates a partial bypass of the scid defect, perhaps by inducing p53-dependent DNA damage response pathways.
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PMID:p53 is required for both radiation-induced differentiation and rescue of V(D)J rearrangement in scid mouse thymocytes. 859 86

The tumor growth suppressor p21 has been shown to be induced by wild-type p53 (wt-p53) and to be a potent inhibitor of cyclin-dependent kinases and PCNA/DNA polymerase delta. Although wt-p53 is reported to be phosphorylated by several protein kinases, the function and significance of the phosphorylation of wt-p53 are not yet fully understood. Using OK-1035, a selective inhibitor of DNA-dependent protein kinase (DNA-PK), we demonstrated the importance of the phosphorylation of wt-p53 by DNA-PK in the DNA damage-mediated expression of the p21 gene. Treatment of HCT116, a human colon carcinoma cell line, with adriamycin induced the expression of wt-p53 and p21. By addition of OK-1035 to this culture, the induction of p21 protein was significantly decreased in a dose-dependent manner, whereas wt-p53 induction was not affected. Northern blot analysis revealed that suppression of p21 protein expression by OK-1035 resulted from reduction in the level of p21 mRNA. OK-1035 did not directly affect the binding ability of wt-p53 to its consensus DNA sequence. Our observations support the idea that wt-p53 induces the transcriptional activation of the p21 gene only after it is phosphorylated by DNA-PK.
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PMID:DNA-dependent protein kinase inhibitor (OK-1035) suppresses p21 expression in HCT116 cells containing wild-type p53 induced by adriamycin. 861 35

Base excision-repair, which is required for correction of spontaneous hydrolytic and oxidative damage to DNA as well as lesions inflicted by alkylating agents, is a relatively well understood repair pathway. Mammalian factors involved in this pathway are reviewed, with emphasis on current uncertainties. Most DNA replication and repair enzymes in mammalian cell nuclei, e.g. DNA polymerases alpha, beta, delta, and epsilon, have direct counterparts in yeast. In contrast, the abundant enzymes in mammalian cell nuclei that bind and are activated specifically by DNA strand interruptions, poly(ADP-ribose) polymerase and DNA-dependent protein kinase, have not been detected in yeast; nor has p53, which is elevated in response to DNA strand breaks. We have found a family of four distinct DNA ligases in human cell nuclei, whereas only a single DNA ligase has been detected in yeast. It would appear that the cellular responses to DNA strand breaks may differ markedly between higher and lower eukaryotes.
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PMID:Recognition and processing of damaged DNA. 865 50

Double-stranded DNA breaks (DSBs) trigger p53-mediated cell cycle arrest or apoptosis pathways that limit the oncogenic consequences of exposure to genotoxic agents, but p53-mediated responses to DSB generated by normal physiologic events have not been documented. "Broken" V(D)J coding ends accumulate in scid lymphocyte precursors as a consequence of a mutation in DNA-dependent protein kinase (DNA-PK). The ensuing failure to rearrange efficiently antigen receptors arrests lymphoid development. Here we show that scid thymocytes express high levels of p53 protein, attributable to recombinase activating gene (RAG)-dependent generation of DSB adjacent to V, D, and J gene segments. To examine the functional importance of p53 expression in vivo, we bred p53-/- scid mice. The absence of p53 facilitated production of in-frame V(D)Jbeta coding joints and developmental progression of scid thymocytes, in addition to a dramatic accumulation of pro-B cells. All mice developed disseminated pro-B or immature T cell lymphoma/leukemia by 7-12 weeks of age. We present evidence that p53 deficiency prolongs the survival of scid lymphocyte precursors harboring broken V(D)J coding ends, allowing the accumulation of aneuploid cells. These results demonstrate that a p53-mediated DNA damage checkpoint contributes to the immune deficiency characteristic of the scid mutation and limits the oncogenic potential of DSBs generated during V(D)J recombination.
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PMID:V(D)J recombination activates a p53-dependent DNA damage checkpoint in scid lymphocyte precursors. 876 47


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