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)

The carboxyl-terminal domain of RNA polymerase II contains a tandemly repeated heptapeptide sequence. Previous work has shown that this sequence is phosphorylated at multiple sites by a template-associated protein kinase, in a reaction that is closely associated with the initiation of RNA synthesis. We have purified this kinase to apparent homogeneity from human (HeLa) cells. The purified kinase phosphorylates native RNA polymerase II only in the presence of DNA and the general transcription factors TFIID (TBP), TFIIB, and TFIIF. Two kinase components are required for full activity: a catalytic component and a DNA-binding regulatory component. The regulatory component has been identified as Ku autoantigen, based on the molecular weights of its component polypeptides, its DNA-binding properties, and its reactivity with anti-Ku monoclonal antibodies. The Ku autoantigen recruits the catalytic component of the kinase to the template. Ku autoantigen has been previously proposed to interact with DNA by a characteristic bind-and-slide mechanism. This mode of interaction may provide a mechanism for targeting the kinase to the transcription complex and other DNA-bound substrates.
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PMID:Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II. 146 19

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

DNA-PK is a DNA-activated serine/threonine protein kinase capable of phosphorylating a number of nuclear DNA-binding proteins. Purified human DNA-PK has two subunits, a 350-kDa polypeptide, Prkdc, which binds ATP and is presumed to contain the catalytic site, and the Ku autoantigen which mediates DNA binding and activation. Previous studies have shown that DNA-PK is activated in vitro by linear double-stranded DNA fragments; however, the Ku subunit binds a broader range of DNA structures. Here we show that EBP-80, a protein originally isolated as a transcription factor for a retroviral long terminal repeat element and subsequently found to be similar to if not identical with Ku, also mediates kinase activation. The EBP-80-Prkdc complex is activated by nanomolar concentrations of DNA constructs containing single-to-double strand transitions, including a closed stem-loop structure and single strand gaps of 0 (nick), 6, and 30 nucleotides. Kinase activation parallels the ability of EBP-80 to bind these and other constructs. Our results extend the range of DNA configurations known to activate DNA-PK and are consistent with the participation of this enzyme complex in several nuclear functions.
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PMID:DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps. 820 88

We have recently shown that a template-associated protein kinase, which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II, is a two-component system. We describe here the purification of these two components to apparent homogeneity from human (HeLa) cell nuclear extract. Kinase component A has a 340-kDa native molecular mass, consists of a single large polypeptide, and contains the kinase active site. Kinase component B, which is identical to the Ku autoantigen, has a 180-kDa native molecular mass, and consists of apparently equimolar 67- and 83-kDa polypeptides. Component B stimulates the activity of component A, and under some conditions, confers DNA dependence on the reaction. The purified kinase converts the CTD to the multiply phosphorylated CTD0 form. Conversion occurs processively, and this processivity is an inherent property of component A. The in vitro phosphorylated CTD0 form contains approximately equimolar phosphoserine and phosphothreonine, but no detectable phosphotyrosine.
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PMID:Purification and characterization of a template-associated protein kinase that phosphorylates RNA polymerase II. 848 98

We have identified a series of proteins based on an affinity for cisplatin-damaged DNA. One protein termed DRP-1 has been purified to homogeneity and was isolated as two distinct complexes. The first complex is a heterodimer of 83- and 68-kDa subunits, while the second complex is a heterotrimer of 350-, 83-, and 68-kDa subunits in a 1:1:1 ratio. The 83- and 68-kDa subunits in each complex are identical. The 83-kDa subunit of DRP-1 was identified as the p80 subunit of Ku autoantigen by N-terminal protein sequence analysis and reactivity with a monoclonal antibody directed against human Ku p80 subunit. The 68-kDa subunit of DRP-1 cross-reacted with monoclonal antisera raised against the Ku autoantigen p70 subunit. The 350-kDa subunit was identified as DNA-PKcs, the catalytic subunit of the human DNA-activated protein kinase, DNA-PK. DRP-1/Ku DNA binding was assessed in mobility shift assays and competition binding assays using cisplatin-damaged DNA. Results indicate that DNA binding was essentially unaffected by cisplatin-DNA adducts in the presence or absence of DNA-PKcs. DNA-PK activity was only stimulated with undamaged DNA, despite the ability of Ku to bind to cisplatin-damaged DNA. The lack of DNA-PK stimulation by cisplatin-damaged DNA correlated with the extent of cisplatin-DNA adduct formation. These results demonstrate that Ku can bind cisplatin-damaged DNA but fails to activate DNA-PK. These results are discussed with respect to the repair of cisplatin-DNA adducts and the role of DNA-PK in coordinating DNA repair processes.
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PMID:Human Ku autoantigen binds cisplatin-damaged DNA but fails to stimulate human DNA-activated protein kinase. 866 30

The double-stranded DNA-activated protein kinase (DNA-PK) is a serine-threonine protein kinase that is composed of a large catalytic subunit (p350) and a DNA-binding protein of 70 and 80 kDa subunits known as the Ku autoantigen. When targeted to DNA by free DNA ends, DNA-PK phosphorylates many DNA-binding proteins and transcription factors. Previously, DNA-PK had only been purified and characterized from transformed human tissue culture cells. Here we report that DNA-PK is an abundant protein in human placenta and lymphocytes. We have purified the placental DNA-PK to homogeneity and show that its biochemical properties are similar to those of the HeLa cell enzyme.
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PMID:Purification and characterization of the double-stranded DNA-activated protein kinase, DNA-PK, from human placenta. 903 91

The DNA-dependent protein kinase (DNA-PK) is a trimeric enzyme consisting of a 460-kDa catalytic subunit (DNA-PKcs) and a heterodimeric regulatory complex called Ku, which is comprised of 70 (Ku70) and 86 (Ku80) kDa subunits. Mutations that affect the expression of the catalytic or Ku80 subunits of DNA-PK disrupt both V(D)J recombination and DNA double-stranded break repair pathways. In this report, we show that two previously uncharacterized rodent cell lines that are defective in DNA double-stranded break repair express catalytically inactive DNA-PK. The DNA-PKcs from the DNA double-stranded break repair mutant cell lines IRS-20 and SX-9 assembles on double-stranded DNA but fails to function as a protein kinase. In addition to the kinase defect, the abundance of the DNA-PKcs from both of these cell lines is reduced relative to wild-type controls. These results suggest that the DNA-PKcs gene from each of these cell lines contains mutations that inactivate the enzymatic activity and the expression or stability of the gene product. These data further strengthen the hypothesis that DNA-PK-mediated protein phosphorylation is a necessary component of the DNA double-stranded break repair pathway.
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PMID:Characterization of two DNA double-stranded break repair-deficient cell lines that express inactive DNA-dependent protein kinase catalytic subunits. 909 71

The DNA-PKcs gene encodes the 465-kDa catalytic subunit of DNA-dependent protein kinase (DNA-PK), which associates with heterodimeric autoantigens Ku70 and Ku80 and exhibits protein kinase activity depending on DNA double-strand breaks. The gene is also responsible for the aberration in severe combined immune deficiency (SCID) mice, which exhibit a high sensitivity to ionizing radiation and abnormal DNA rearrangement of immunoglobulin and T cell receptor genes. There is further evidence that DNA-PKcs phosphorylates various proteins involved in DNA replication, transcription, repair, and recombination. Nevertheless the structure/function relationship in this huge molecule is virtually unknown. We determined the exons and introns of the murine DNA-PKcs gene by the long-distance polymerase chain reaction method. The murine DNA-PKcs gene consists of 86 exons distributed in a region of more than 250 kb. The average size of the exons is 140 bp. All the splicing sites conform to the GT/AG rule. The SCID mutation site (Tyr4046) has been identified in exon 85. The genomic structure of the DNA-PKcs gene provides clues for the study of various functional domains in this macromolecule.
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PMID:The murine DNA-PKcs gene consists of 86 exons dispersed in more than 250 kb. 933 76

Heterodimers of the 70 and 80 kDa Ku autoantigens (Ku70 and Ku80) activate the DNA-dependent protein kinase (DNA-PK). Mutations in any of the three subunits of this protein kinase (Ku70, Ku80 and DNA-PKcs) lead to sensitivity to ionizing radiation (IR) and to DNA double-strand breaks, and V(D)J recombination product formation defects. Here we show that the IR repair, DNA end binding and DNA-PK defects in Ku70-/- embryonic stem cells can be counteracted by introducing epitope-tagged wild-type Ku70 cDNA. Truncations and chimeras of Ku70 were used to identify the regions necessary for DNA end binding and IR repair. Site-specific mutational analysis revealed a core region of Ku70 responsible for DNA end binding and heterodimerization. The propensity for Ku70 to associate with Ku80 and to bind DNA correlates with the ability to activate DNA-PK, although two mutants showed that the roles of Ku70 in DNA-PK activation and IR repair are separate. Mutation of DNA-PK autophosphorylation sites and other structural motifs in Ku70 showed that these sites are not necessary for IR repair in vivo. These studies reveal Ku70 features required for double-strand break repair.
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PMID:Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions. 936

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


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