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PubMed1993

Purification and characterization of a template-associated protein kinase that phosphorylates RNA polymerase II.

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.

Amino Acid Sequence↗
PubMed1997

The DNA-dependent protein kinase catalytic subunit (p460) is cleaved during Fas-mediated apoptosis in Jurkat cells.

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase linked to DNA repair and V(D)J recombination. It is composed of a 460-kDa catalytic subunit (DNA-PKcs) and a 70/86-kDa heterodimeric regulatory component that is identical with the human autoantigen Ku. The regulatory subunit targets the catalytic subunit to the free ends of dsDNA breaks. Since apoptosis is associated with internucleosomal chromatin fragmentation and creation of dsDNA breaks, we examined whether the biochemical amounts of either DNA-PKcs or Ku changed during apoptosis mediated by the cell surface receptor Fas. We found that the catalytic subunit was cleaved into several smaller polypeptides early in apoptosis. In contrast to DNA-PKcs, Ku was neither cleaved nor decreased in amount during apoptosis. We then extended our in vivo results to a cellfree system. Cytosolic extracts derived from apoptotic cells were able to cleave DNA-PKcs into polypeptides of sizes identical with those seen in vivo, and this cleavage was inhibited by the cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. Furthermore, DNA-PKcs was cleaved in vitro by purified apopain (CPP32), but not IL-1beta-converting enzyme. Cleavage was also inhibited by the specific tetrapeptide DEVD (amino acids 2709-2712 of the DNA-PKcs sequence), suggesting a candidate position for protease action. Finally, we found that the catalytic activity of DNA-PKcs was decreased in apoptotic cells. We conclude that DNA-PKcs is subject to selective cleavage by proteases during apoptosis. Cleavage of DNA-PKcs may represent a mechanism for regulating the function of DNA-dependent kinase during programmed cell death.

Apoptosis↗
PubMed1997

Functional properties of the separate subunits of human DNA helicase II/Ku autoantigen.

The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II). HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions. To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli. The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A. (1994) EMBO J. 13, 4991-5001). Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195). The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits. The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends. The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity.

Adenosine Triphosphatases↗
PubMed1996

Disruption of DNA-PK in Ku80 mutant xrs-6 and the implications in DNA double-strand break repair.

The Chinese hamster ovary (CHO) mutant cell line xrs-6C is highly sensitive to radiation and is deficient in DNA double-strand break (DSB) repair. The repair defect of xrs-6C is complemented by the human DSB repair gene designated as XRCC5. This gene was recently identified as Ku80, which encodes the human autoantigen protein Ku p80. Ku80 protein forms heterodimer with the Ku70 subunit to form a complex that possesses a DNA end-binding activity. Ku70/Ku80 heterodimer can recruit the catalytic p350 subunit of the DNA-dependent protein kinase. It is demonstrated here that, while the Ku70 mRNA expression is normal in the xrs-6C mutant, Ku70 protein is undetectable. However, introduction of human Ku80 gene into the mutant lead to increased expression of Ku70 protein and restored Ku70 binding to DNA ends, suggesting that mutation of the Ku80 gene affected the formation of Ku70/Ku80 dimers and the stability of the Ku70 protein. We also demonstrated that, although p350 protein expression in the mutants was unaffected, the capacity of p350 to bind to DNA ends was impaired in the mutants. After introduction of the human Ku80 into the mutant, the association of p350 with DNA end was restored, accompanied by recovery in cell survival and DNA double-strand break repair. The results in this report show that mutation of the Ku80 gene disrupts formation of the Ku70/Ku80 dimer and compromises the ability of Ku protein to recruit the DNA-PK p350 subunit to DNA double-strand breaks, causing a dysfunction of DNA DSB repair in the cell.

Animals↗
PubMed1992

Antibodies to the p70/p80 (Ku) antigens in systemic lupus erythematosus.

The Ku (p70/p80) autoantigen, a heterodimer consisting of 70 kDa (p70) and 80 kDa (p80) protein subunits, is one of a group of DNA-associated autoantigens identified as targets of autoantibodies produced by patients with SLE and related disorders. Many of these DNA-protein antigens are involved in organizing the genome into transcriptionally active (euchromatin) and inactive (heterochromatin) domains. The bulk of available evidence indicates that the Ku antigen is also involved in organizing the genome, although its precise role remains unclear. Molecular cloning of the protein subunits of Ku has revealed that the structure of p70 resembles that of certain transcriptional activator proteins, and there is some evidence in vitro that Ku may increase transcriptional activity from at least two promoters. Moreover, examination of the distribution of Ku in the polytene chromosomes of insects suggests an association with transcriptionally active chromatin. The DNA-binding domain of Ku has been localized to the C-terminus of p70, whereas p80 does not appear to bind DNA, and may be involved in interactions with other proteins. Epitope mapping and mutagenesis experiments have shown that the immunodominant epitope of p70 lies within the DNA-binding domain. Surprisingly, this autoepitope is not conserved between humans and mice, raising the possibility that the interaction of Ku with DNA might exhibit species specific functional differences. At least seven additional autoepitopes have been identified on the Ku particle, located on p70, p80, or both subunits. Autoantibodies to p70, p80, and DNA are produced tandemly by patients with SLE, providing evidence for an antigen-driven immune response targeting the entire Ku particle. The multiple specificities of anti-Ku autoantibodies and the tandem production of antibodies to the various constituents of the Ku particle are consistent with a role of either "molecular mimicry" or "intermolecular help" in the generation of autoimmunity to this antigen.

Animals↗
PubMed2005

Retention and repression: fates of hyperedited RNAs in the nucleus.

Double-stranded RNA (dsRNA) is often formed in the nuclei of mammalian cells, but in this compartment it does not induce the effects characteristic of cytoplasmic dsRNA. Rather, recent work has suggested that nuclear dsRNA is a target for the ADAR class of enzymes, which deaminate adenosines to inosines. Further, there are a number of distinct fates of such edited RNA, including nuclear retention and perhaps also gene silencing.

Active Transport, Cell Nucleus↗
PubMed1996

Structures targeted by the immune system in myositis.

Polymyositis and dermatomyositis are characterized by the production of a series of autoantibodies to various cellular constituents. Some of these autoantibodies are found specifically in patients with polymyositis or dermatomyositis or myositis overlap syndrome and predict clinical subgroups and prognosis. If we are to understand the etiology and pathogenic mechanisms of polymyositis and dermatomyositis, it will be particularly important to elucidate the structure and function of target autoantigens recognized by these myositis-specific autoantibodies. In recent years, many autoantigens and some epitopes have been identified using molecular biology approaches. During the 1-year period reviewed here, the nature and function of the Mi-2 and the Ku(p70/p80) antigens which are recognized by autoantibodies in patients with dermatomyositis and with the myositis overlap syndrome, respectively, have been elucidated. Several new autoantibodies that are not specific for but that are associated with myositis have also been described.

Amino Acyl-tRNA Synthetases↗
PubMed1998

Physical interaction between epidermal growth factor receptor and DNA-dependent protein kinase in mammalian cells.

Binding of extracellular ligands to epidermal growth factor receptors (EGFR) activate signal transduction pathways associated with cell proliferation, and these events are inhibited by monoclonal antibodies against EGFR. Since efficient DNA repair in actively growing cells may require growth factor signaling, it was of interest to explore any linkage between EGFR-mediated signaling and DNA-dependent protein kinase (DNA-PK), an enzyme believed to be involved in repairing double strand breaks and V(D)J recombination. We report that anti-EGFR monoclonal antibodies (mAbs), and not EGFR ligands, trigger a specific early physical interaction between EGFR and a 350-kDa catalytic subunit of DNA or its regulatory heterodimeric complex Ku70/80, in a variety of cell types, both in vivo and in vitro. Inhibition of EGFR signaling by anti-EGFR mAb was accompanied by a reduction in the levels of the DNA-PK and its activity in the nuclear fraction. Confocal imaging revealed that a substantial amount of DNA-PK was co-localized with EGFR in anti-EGFR mAb-treated cells. Anti-EGFR mAb-induced physical interaction between EGFR and DNA-PK or Ku70/80 was dependent on the presence of EGFR, but not on the levels of EGFR. The EGFR associated with DNA-PK or Ku70/80 retains its intrinsic kinase activity. Our findings demonstrate the existence of a novel cellular pathway in mammalian cells that involves physical interactions between EGFR and DNA-PK or Ku70/80 in response to inhibition of EGFR signaling. Our present observations suggest a possible role of EGFR signaling in maintenance of the nuclear levels of DNA-PK, and interference in EGFR signaling may possibly result in the impairment of DNA repair activity in the nuclei in anti-EGFR mAb-treated cells.

Animals↗
PubMed1999

Morphological and biochemical analysis of anti-nuclear matrix protein antibodies in human sera.

Autoimmune sera have been used in the diagnosis of autoimmune diseases as well as the analysis of nuclear substructures. In an attempt to study the biological characteristics of the nuclear matrix, we screened human sera using immunofluorescent staining and immunoblot. We detected antibodies against nuclear matrix (NM), a remnant nonchromatin protein compartment after the treatment of detergent, salt and nuclease, in 212 out of 284 tested sera (74.6%) by immunoblot. Peptides with molecular weights of 70 kDa, 50 kDa and 25 kDa were detected in the order of frequency. Clinical informations of 198 out of 212 cases were available and went as follows: 38 cases were autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; 132 non-autoimmune and non-neoplastic diseases; 16 neoplastic diseases and 12 cases unclassified. The immunofluorescent staining intensity by anti-nuclear matrix protein (NMP) antibodies decreased variably, but fibrillogranular, speckled and nucleolar immunolocalization patterns were retained after in situ fractionation. Ku70 and La protein were detected by anti-NMP antibodies. Immunolocalization by anti-NMP antibodies indicates that the NMPs constitute a variety of characteristic nuclear substructures and may serve as autoantigens in diverse human diseases. In addition, the presence of Ku70 and La protein as NMPs suggests that the NM can be functionally active in association with DNA or RNA.

Antigens, Nuclear↗
PubMed2001

Hypoxia-activated ligand HAL-1/13 is lupus autoantigen Ku80 and mediates lymphoid cell adhesion in vitro.

Hypoxia is known to induce extravasation of lymphocytes and leukocytes during ischemic injury and increase the metastatic potential of malignant lymphoid cells. We have recently identified a new adhesion molecule, hypoxia-activated ligand-1/13 (HAL-1/13), that mediates the hypoxia-induced increases in lymphocyte and neutrophil adhesion to endothelium and hypoxia-mediated invasion of endothelial cell monolayers by tumor cells. In this report, we used expression cloning to identify this molecule as the lupus antigen and DNA-dependent protein kinase-associated nuclear protein, Ku80. The HAL-1/13-Ku80 antigen is present on the surface of leukemic and solid tumor cell lines, including T and B lymphomas, myeloid leukemias, neuroblastoma, rhabdomyosarcoma, and breast carcinoma cells. Transfection and ectopic expression of HAL-1/13-Ku80 on (murine) NIH/3T3 fibroblasts confers the ability of these normally nonadhesive cells to bind to a variety of human lymphoid cell lines. This adhesion can be specifically blocked by HAL-1/13 or Ku80-neutralizing antibodies. Loss of expression variants of these transfectants simultaneously lost their adhesive properties toward human lymphoid cells. Hypoxic exposure of tumor cell lines resulted in upregulation of HAL-1/13-Ku80 expression at the cell surface, mediated by redistribution of the antigen from the nucleus. These studies indicate that the HAL-1/13-Ku80 molecule may mediate, in part, the hypoxia-induced adhesion of lymphocytes, leukocytes, and tumor cells.

3T3 Cells↗
PubMed1996

Characterization of a Ku86 variant protein that results in altered DNA binding and diminished DNA-dependent protein kinase activity.

Three proteins known to play a critical role in mammalian DNA double-strand break repair and lymphoid V(D)J recombination are the autoantigens Ku86 and Ku70 and a 465-kDa serine/threonine protein kinase catalytic subunit (DNA-PKcs). These proteins physically associate to form a complex (DNA.PK) with DNA-dependent protein kinase activity. In this study, we demonstrate using electrophoretic mobility shift assays (EMSAs) that the nuclear DNA end-binding activity of Ku is altered in the human promyelocytic leukemic HL-60 cell line. Western blot and EMSA supershift analyses revealed that HL-60 cells expressed both full-length and variant Ku86 proteins. However, a combined EMSA and immunoanalysis revealed that the Ku heterodimers complexed with DNA in HL-60 cells contained only the variant Ku86 proteins. Finally, UV cross-linking experiments and DNA.PK assays demonstrated that the Ku complexes containing variant Ku86 had a greatly reduced ability to interact with DNA-PKcs and that consequently HL-60 cells had severely diminished DNA.K activity. These data provide important insights into the interaction between Ku and DNA-PKcs and into the role of DNA.PK in DNA double-strand break repair.

Amino Acid Sequence↗
PubMed1999

The Ku70 autoantigen interacts with p40phox in B lymphocytes.

Ku70, a regulatory component of the DNA-dependent protein kinase, was identified by a yeast two-hybrid screen of a B lymphocyte cDNA library as a partner of p40phox, a regulatory component of the O2--producing NADPH oxidase. Truncated constructs of p40phox and Ku70 were used to map the interacting sites. The 186 C-terminal amino acids (aa) of Ku70 were found to interact with two distinct regions of p40phox, the central core region (aa 50-260) and the C-terminal extremity (aa 260-339). In complementary experiments, it was observed that Ku70 binds to immobilized recombinant p40phox fusion protein and that p40phox and Ku70 from a B lymphocyte cell extract comigrate in successive chromatographies on Q Separose, Superose 12 and hydroxylapatite columns. Moreover, we report that Ku70 and p40phox colocalize in B lymphocytes and in transfected Cos-7 cells. We also show that the two NADPH oxidase activating factors, p47phox and p67phox are substrates for DNA-PK in vitro and that they are present together with p40phox in the nucleus of B cells. These results may help solve the paradox that the phox protein triad, p40phox, p47phox and p67phox, is expressed equally in B lymphocytes and neutrophils, whereas the redox component of the NADPH oxidase, a flavocytochrome b, which is well expressed in neutrophils, is barely detectable in B lymphocytes.

Animals↗
PubMed1999

Molecular cloning and sequencing of cDNAs encoding homologues of human Ku70 and Ku80 autoantigen from Xenopus and their expression in various Xenopus tissues.

We isolated cDNA clones encoding Ku70 and Ku80 homologues of Xenopus laevis from a cDNA library prepared from Xenopus oocytes. The nucleotide sequences of these Ku70 and Ku80 homologues have coding sequences of 1833 bp and a 611 aa protein, and 2178 bp and a 726 aa protein, respectively. The amino acid sequences deduced from the open reading frame of the Ku70 and Ku80 cDNA clones were highly homologous to those from Ku genes previously isolated, such as human (ca. 65% and ca. 62% identity, respectively) and mouse (ca. 65% and ca. 60%), and show a certain degree of homology to Drosophila (ca. 27% with Ku70), Caenorhabditis elegans (ca. 20% with Ku80) and Saccharomyces cerevisiae (ca. 23% and ca. 19%). Our detailed comparison of the predicted amino acid sequences among these species revealed the highly conserved octa-peptide LPFXXDIR common to both Xenopus Ku70 and Ku80 homologues in the region showing the high homology throughout the species tested. A Northern analysis using specific cDNA probes showed that Ku poly(A)+ mRNAs are expressed at high levels in Xenopus adult oocyte and testis.

Amino Acid Sequence↗
PubMed1995

Molecular structure and function of autoantigens in systemic sclerosis.

Autoantibodies in systemic sclerosis target a limited set of nuclear proteins, principally those of the nucleolus and RNA transcription complexes. These antibodies have proved helpful in diagnosis of this disease, and have been used extensively as probes of nuclear structure and function. Despite these advances, the events that initially trigger autoantibody production in systemic sclerosis are not yet known. While these ANA are not known to disrupt cellular processes by entering living cells, or to cause tissue injury (in contrast to SLE, where autoantibodies may mediate tissue damage), it seems likely that they do not merely represent epiphenomena of the disease. Rather, it is logical to assume that their origin is in some manner tied to etiology of systemic sclerosis, since they segregate by syndrome within the spectrum of this disease (for example, anti-kinetochore antibodies occur in limited cutaneous disease, and anti-topoisomerase I and anti-RNA polymerase antibodies occur in diffuse disease), and since they are distinct from the ANA found in other connective tissue diseases in their selectivity for the nucleolus and RNA polymerases.

Antigens, Nuclear↗