Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Members of the phosphatidylinositol (PI) 3-kinase gene family, including the ataxia telangiectasia gene and the DNA-dependent protein kinase (DNA-PK), are involved in regulating cellular radiosensitivity. We have investigated two structurally unrelated PI 3-kinase inhibitors, wortmannin and LY294002, to determine whether they inhibit DNA-PK and increase cellular radiosensitivity. The PI 3-kinase inhibitors wortmannin and LY294002 were effective radiosensitizers of human tumor cells, with sensitizer enhancement ratios (at 10% survival) of 2.8 and 1.9, respectively, in SW480 cells. Wortmannin and LY294002 inhibited the kinase activity of purified DNA-PK and inactivated cellular DNA-PK kinase activity. Inhibition of cellular DNA-PK activity occurred at the same concentrations of wortmannin that caused radiosensitization, and this correlation was found in a range of tumor cell lines. However, cells deficient in either DNA-PK (scid cells) or the ataxia telangiectasia protein were also partly sensitized to radiation by wortmannin, indicating the involvement of more than one protein kinase in the mechanism of action of wortmannin. Wortmannin also affected the G2-M checkpoint. SW480 cells had a reversible G2-M delay of 20 h following irradiation. However, wortmannin-treated SW480 cells had a prolonged G2-M delay; more than 75% of cells were arrested in G2 at 50 h postirradiation. This suggests the accumulation of significant unrepaired DNA damage following inhibition of PI 3-kinase family members. Therefore, PI 3-kinase inhibitors may represent a new class of radiosensitizers that inhibit the repair of DNA damage.
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PMID:Radiosensitization of human tumor cells by the phosphatidylinositol3-kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G2-M delay. 981 94

Mammalian cells defective in DNA end-joining are highly sensitive to ionizing radiation and are immunodeficient because of a failure to complete V(D)J recombination. By using cell-free extracts prepared from human lymphoblastoid cell lines, an in vitro system for end-joining has been developed. Intermolecular ligation was found to be accurate and to depend on DNA ligase IV/Xrcc4 and requires Ku70, Ku86, and DNA-PKcs, the three subunits of the DNA-activated protein kinase DNA-PK. Because these activities are involved in the cellular resistance to x-irradiation and V(D)J recombination, the development of this in vitro system provides an important advance in the study of the mechanism of DNA end-joining in human cells.
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PMID:DNA end-joining catalyzed by human cell-free extracts. 982 54

The DNA-dependent protein kinase (DNA-PK) plays an important role in mammalian DNA double-strand break repair and immunoglobulin gene rearrangement. The DNA-PK holoenzyme is activated by assembly at DNA ends and is comprised of DNA-PKcs, a 460 kDa protein kinase catalytic subunit, and Ku, a 70 kDa/80 kDa heterodimeric DNA-targeting component. We have solved the three-dimensional structure of DNA-PKcs to approximately 21 A resolution by analytically combining images of nearly 9500 individual particles extracted from cryo-electron micrographs. The DNA-PKcs protein has an open, pseudo 2-fold symmetric structure with a gap separating a crown-shaped top from a rounded base. Columns of density are observed to protrude into the gap from both the crown and the base. Measurements of the enclosed volume indicate that the interior of the protein is largely hollow. The structure of DNA-PKcs suggests that its association with DNA may involve the internalization of double-stranded ends.
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PMID:Cryo-EM imaging of the catalytic subunit of the DNA-dependent protein kinase. 983 27

Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and p11 respectively) and functions in all three major DNA metabolic processes: replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase alpha-stimulation domain and a single-stranded DNA-binding domain. It also contains a putative metal-binding domain of the 4-cysteine type (Cys-Xaa4-Cys-Xaa13-Cys-Xaa2-Cys) that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, we created various p70 mutants that lack the zinc-finger motif (by Cys-->Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPA's function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of zinc-finger mutant RPA in NER may be explained by the observation that wild-type RPA significantly stimulated DNA polymerase delta activity, whereas only marginal stimulation was observed with zinc-finger mutant RPA. We also observed that ZFM reduced RPA's single-stranded DNA-binding activity by 2-3-fold in the presence of low amounts of RPA. Interestingly, the ZFM abolished phosphorylation of the p34 subunit by DNA-dependent protein kinase, but not that by cyclin-dependent kinase. Taker together, our results strongly suggest a positive role for RPA's zinc finger domain in its function.
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PMID:In vitro analysis of the zinc-finger motif in human replication protein A. 988 30

PCAF histone acetylase is found in a complex with more than 20 associated polypeptides. Here we report cloning and characterization of the 400 kDa PCAF-associated factor referred to as PAF400. PAF400 is almost identical to TRRAP, which binds to c-Myc and E2F, and has significant sequence similarities to the ATM superfamily including FRAP, ATM, ATR, and the catalytic subunit of DNA-PK. Remarkably, PAF400 and FRAP share sequence similarity in broad regions that cover 80% of the entire PAF400 sequence. However, unlike the other members of the ATM superfamily, PAF400 is not a protein kinase as judged from the lack of kinase motif and autophosphorylation activity. We discuss the possibility that PAF400 may play a role in signaling of DNA damage to p53 by stimulation of p53 acetylation.
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PMID:The 400 kDa subunit of the PCAF histone acetylase complex belongs to the ATM superfamily. 988 74

Replication protein A (RPA) is a trimeric single-stranded DNA (ssDNA)-binding complex of eukaryotic cells that plays an important role in DNA metabolism by stabilising single-stranded regions of DNA. The functionally important binding activity towards ssDNA is mainly localised on the large subunit, RPA70, whereas the middle subunit, RPA32, appears to have a regulatory function. It has been shown previously that RPA32 is phosphorylated both during the S-phase of a normal cell cycle and in response to DNA damage. In this study we demonstrate that phosphorylation of RPA32 is rapidly induced during apoptotic cell death of Jurkat T-lymphocytes, resulting in a hyperphosphorylated form with reduced electrophoretic mobility. In contrast, the large subunit of RPA is neither modified nor cleaved during apoptosis. Phosphorylation of RPA32 begins in parallel to the degradation of DNA to high molecular weight fragments, and slowly continues until late apoptosis. Experiments with specific kinase inhibitors indicate that RPA32 hyperphosphorylation requires the activities of DNA-dependent protein kinase and of a cyclin-dependent protein kinase. Interestingly, the hyperphosphorylated, but not the less phosphorylated forms of RPA32, sediments independently from the trimeric complex in sucrose gradients under high ionic strength, and is not bound to the complex in immunoprecipitation assays.
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PMID:Hyperphosphorylation of replication protein A middle subunit (RPA32) in apoptosis. 1003 12

The tumour suppressor gene product, p53, is involved in mediating cellular responses to DNA damage including growth arrest and/or apoptosis. The mechanism by which p53 protein senses the presence of damaged DNA is not understood. The possibility that p53 may be post-translationally modified by enzymes that are activated in response to DNA damage including DNA-dependent protein kinase (DNA-PK), poly(ADP-ribose) polymerase and stress activated protein kinase has received considerable attention. Recent studies have indicated that DNA-PK is not required for the transactivation or apoptosis-promoting activities of p53 protein. However, the possibility that other functions of p53 may be dependent on phosphorylation by DNA-PK has not been explored. Here we describe a series of experiments that compares the expression, function and phosphorylation status of p53 protein in normal and DNA-PK-deficient scid cells. While several novel p53 phosphoforms are generated in response to DNA damage in normal cells, the same phosphoforms are observed in scid cells.
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PMID:Phosphorylation of p53 protein in response to ionizing radiation occurs at multiple sites in both normal and DNA-PK deficient cells. 1010 21

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.
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PMID:Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining. 1020 11

In all organisms multiple pathways to repair DNA double-strand breaks (DSB) have been identified. In mammalian cells DSB are repaired by two distinct pathways, homologous and non-homologous (illegitimate) recombination. X-ray-sensitive mutants have provided a tool for the identification and understanding of the illegitimate recombination pathway in mammalian cells. Two (sub-)pathways can be distinguished, the first mediated by DNA-PK-dependent protein kinase (DNA-PK), and the second directed by the hMre11/hRad50 complex. A variety of mutants impaired in DSB repair by illegitimate recombination, with mutations in Ku, DNA-PKcs, XRCC4 or nibrin, have been described. Herein, the characterization of these mutants with respect to the impaired cellular function and the molecular defect is provided. Further studies on these mutants, as well as on new mutants impaired in as-of-yet unidentified pathways, should be helpful to a better understanding of DSB repair and of the processes leading to genome instability and cancer.
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PMID:Mammalian X-ray-sensitive mutants which are defective in non-homologous (illegitimate) DNA double-strand break repair. 1021 15

DNA-PK is a nuclear, serine/threonine protein kinase required for repairing DNA double-strand breaks and for V(D)J recombination. To determine the distribution of DNA-PK in human tissues, we assayed paraffin-embedded sections of normal and cancerous tissues for DNA-PKcs and Ku80 by immunohistochemistry. We also assayed for Brca2, a human tumor suppressor gene that is implicated in the repair of DNA strand-breaks. Brca2 was strongly expressed in epithelial cells of the breast, endometrium, and thymus, in tingible body macrophages of follicular germinal centers of lymphoid tissue, and in reticuloendothelial cells in the spleen. DNA-PKcs and Ku80 expression was usually parallel, but both were expressed in a highly cell- and tissue-specific manner. The highest levels were observed in spermatogenic cells (but not in spermatozoa), and in neurons and glial cells of the central and autonomic nervous system. Neither protein was consistently expressed in liver nor in resting mammary epithelium, but lactating breast epithelium was strongly positive for DNA-PKcs and Ku80. In contrast to established human cell cultures, expression between cells in the same tissue was highly selective in the epidermis, exocrine pancreas, renal glomeruli, the red pulp of the spleen, and within cellular compartments of tonsils, lymph nodes, and thymus. Most cancerous tissues were consistently positive for DNA-PKcs and Ku80, except invasive carcinoma of the breast. DNA-PKcs, Ku80, and Ku70 mRNAs were expressed in all normal tissues with relatively little variation in levels. Our results suggest that the apparent absence of DNA-PKcs and Ku80 from some cells or tissues is a consequence of post-transcriptional mechanisms that regulate protein levels.
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PMID:DNA-PK, the DNA-activated protein kinase, is differentially expressed in normal and malignant human tissues. 1034 Mar 83


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