Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids.

The Ku protein-DNA-dependent protein kinase system is one of the major pathways by which cells of higher eukaryotes respond to double-strand DNA breaks. The components of the system are evolutionarily conserved and homologs are known from a number of organisms. The Ku protein component binds directly to DNA ends and may help align them for ligation. Binding of Ku protein to DNA also nucleates formation of an active enzyme complex containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The interaction between Ku protein, DNA-PKcs and nucleic acids has been extensively investigated. This review summarizes the results of these biochemical investigations and relates them to recent molecular genetic studies that reveal highly characteristic repair and recombination defects in mutant cells lacking Ku protein or DNA-PKcs.     

Functional analysis of c-Myb protein in T-lymphocytic cell lines shows that it trans-activates the c-myc promoter.

The function of c-Myb protein was revealed by transfecting an expression vector containing the entire c-Myb protein-coding sequence into the murine CTLL-2 T-cell line. Expressions of high levels of c-Myb protein did not alter the expression of several T-cell markers, c-fos mRNA expression, responses to interleukin-2, and growth characteristics of these cells. Interestingly, expression of the c-myc gene was drastically increased in this clone. Further, the c-myb expression plasmid, but not a frameshift mutant of c-myb, enhanced the expression of a hybrid construct of c-myc promoter linked to a reporter gene by 8- to 14-fold. These results demonstrate a role of c-Myb protein in c-myc gene expression.     

Identification of nuclear factor IV/Ku autoantigen in a human 2D-gel protein database. Modification of the large subunit depends on cellular proliferation

Nuclear Factor IV (NFIV) is a heterodimeric DNA-binding protein from HeLa cells, recognizing molecular ends and is identical to the autoantigenic target Ku. We have identified the two NFIV/Ku subunits, by comigration, in the 2D-gel database of transformed human amnion cell (AMA) proteins. We observed that the large subunit of NFIV/Ku consists of at least 3 charge variants that correspond to SSP IEFs 5705 (81.2 kDa, pI 5.74), 6707 (81.2 kDa, pI 5.67) and 6706 (81.9 kDa, pI 5.60) in the AMA catalogue. The relative amounts of the 2 major variants (IEFs 5705 and 6707) was dependent on the state of cell proliferation. Inhibition of DNA-synthesis by hydroxyurea also changed the relative levels of the variants, whereas aphidicolin or a thymidine block had no effect. These results suggest a possible role for NFIV/Ku in DNA replication.     

Hypermethylation of the Keap1 gene in human lung cancer cell lines and lung cancer tissues

Low expression of the oxidative stress sensor Keap1 is thought to be involved in carcinogenesis. However, the mechanisms responsible for inactivation of the Keap1 gene remain unknown. We investigated Keap1 expression using RT-PCR and found that it was downregulated in lung cancer cell lines and tissues when compared with a normal bronchial epithelial cell line. Treatment with 5-Aza-2′-deoxycytidine restored Keap1 expression in lung cancer cell lines, indicating the silencing mechanism to be promoter methylation. Moreover, we evaluated cytosine methylation in the Keap1 promoter and demonstrated that the P1 region, including 12 CpG sites, was highly methylated in lung cancer cells and tissues, but not in normal cells. Importantly, we found evidence that three specific CpG sites (the 3rd, 6th, and 10th CpGs of P1) might be binding sites for proteins that regulate Keap1 expression. Thus, our results suggest for the first time that Keap1 expression is regulated by an epigenetic mechanism in lung cancer.     

The C terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit

Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3' deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity.     

Photoaffinity labeling of a 33 kDa protein subunit of the delta-opioid receptor in neuroblastoma and hybrid cell lines

Tritiated DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) binds with high affinity, specificity and saturability to neuroblastoma N18TG2 and hybrid neuroblastoma x glioma NG108-15 and NG108-5 intact cells. The delta-opioid receptor density in cells cultured in chemically defined medium was increased about 2 times compared to that in cells cultured in 10% fetal calf serum. A major and a minor protein species covalently and specifically bound to [125I]azido-DTLET (Tyr-D-Thr-Gly-pN3Phe-Leu-Thr), photoactivatable ligand, migrated on SDS-gel electrophoresis with Mr values near 33,000 and 58,000, respectively.     

Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein

Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrP(C)-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrP(C) than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrP(Sc), in the agent-inoculated PrP-overexpressing cells, while no PrP(Sc) was detectable in the untransfected cells after 3 passages. Production of PrP(Sc) in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrP(Sc)-A strains Chandler, 139A, and 22L but not to PrP(Sc)-B strains 87V and 22A. We further demonstrate the successful transmission of PrP(Sc) from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrP(C) and the sequence of PrP(Sc). This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.     

Purification of the large ribosomal subunit via its association with the small subunit

We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.     

Resistance against cycloheximide in cell lines from Chinese hamster and human cells is conferred by the large subunit of cytoplasmic ribosomes

Summary Cell lines from Chinese hamster ovary [CHO-K1-D3] and human fibroblast cells [46, XX, 18p-] were mutagenized with N-nitrosomethylurea followed by a selection for cycloheximide resistance. Two mutants resistant against the durg were selected from either wildtype. 80S ribosomes and their ribosomal subunits were isolated from all mutant and wildtype cells. 80S ribosomes reassociated from the isolated subunits were as active as isolated 80S couples in the poly (U) dependent poly (Phe) synthesis. Hybrid 80S ribosomes constructed from subunits of the various cell lines of the same species were fully active, whereas the interspecies 80S hybrids were not active at all in poly (Phe) synthesis.Hybrid 80S ribosomes from subunits of mutant and the ocrresponding wildtype cells were tested in the poly (U) assay in the presence and absence of cycloheximide. The results strikingly indicate that in all four mutant cell lines the resistance against cycloheximide is conferred by the large subunit of cytoplasmic ribosomes.Abbreviations CHM Cycloheximide - CHO Chinese hamster ovarien - FBS foetal bovine serum - Eagle MEM Eagle minimal essential medium - EMS Ethyl-metansulfonate - NMU N-nitrosomethylurea     

N-terminal constraint activates the catalytic subunit of the DNA-dependent protein kinase in the absence of DNA or Ku

The DNA-dependent protein kinase (DNA-PK) was identified as an activity and as its three component polypeptides 25 and 15 years ago, respectively. It has been exhaustively characterized as being absolutely dependent on free double stranded DNA ends (to which it is directed by its regulatory subunit, Ku) for its activation as a robust nuclear serine/threonine protein kinase. Here, we report the unexpected finding of robust DNA-PKcs activation by N-terminal constraint, independent of either DNA or its regulatory subunit Ku. These data suggest that an N-terminal conformational change (likely induced by DNA binding) induces enzymatic activation.     

Interplay between Ku, Artemis, and the DNA-dependent protein kinase catalytic subunit at DNA ends

Repair of DNA double strand breaks (DSB) by the nonhomologous end-joining pathway in mammals requires at least seven proteins involved in a simplified two-step process: (i) recognition and synapsis of the DNA ends dependent on the DNA-dependent protein kinase (DNA-PK) formed by the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs in association with Artemis; (ii) ligation dependent on the DNA ligase IV.XRCC4.Cernunnos-XLF complex. The Artemis protein exhibits exonuclease and endonuclease activities that are believed to be involved in the processing of a subclass of DSB. Here, we have analyzed the interactions of Artemis and nonhomologous end-joining pathway proteins both in a context of human nuclear cell extracts and in cells. DSB-inducing agents specifically elicit the mobilization of Artemis to damaged chromatin together with DNA-PK and XRCC4/ligase IV proteins. DNA-PKcs is necessary for the loading of Artemis on damaged DNA and is the main kinase that phosphorylates Artemis in cells damaged with highly efficient DSB producers. Under kinase-preventive conditions, both in vitro and in cells, Ku-mediated assembly of DNA-PK on DNA ends is responsible for a dissociation of the DNA-PKcs.Artemis complex. Conversely, DNA-PKcs kinase activity prevents Artemis dissociation from the DNA-PK.DNA complex. Altogether, our data allow us to propose a model in which a DNA-PKcs-mediated phosphorylation is necessary both to activate Artemis endonuclease activity and to maintain its association with the DNA end site. This tight functional coupling between the activation of both DNA-PKcs and Artemis may avoid improper processing of DNA.     

Regulation by ceramide of epidermal growth factor signal transduction and mitogenesis in cell lines overexpressing the growth factor receptor.

Ceramide has emerged as a pleiotropic signal mediator of cellular responses including differentiation, proliferation, cell cycle arrest and apoptosis. In the present study we evaluated the effect of cell permeant ceramide analogues on ligand-induced tyrosine phosphorylation of the EGF receptor (EGFR), phospholipase Cy (PLCgamma) activity and cell proliferation. Treatment with N-acetylsphingosine (C2-cer) and N-hexanoylceramide (C6-cer) prevented EGF-induced tyrosine trans-phosphorylation of the receptor in two different cell lines overexpressing the human EGFR (A431 and EGF-T17 cells). In contrast, treatment of A431 and EGFR-T17 cells with C2-cer or C6-cer did not affect the ligand binding capacity of the receptor, an effect that was however observed after TPA-induced activation of PKC. In addition EGF-stimulated PLCgamma activity was transiently decreased in A431 cells treated with C6-cer and only a modest, albeit significant reduction on ligand-induced 3H-InsP3 generation was observed in EGFR-T17 cells pretreated with ceramide. We also examined the effect of C2-cer on serum (A431)- or EGF (EGFR-T 17)-induced cell proliferation. Treatment of EGFR-TI7 cells with C2-cer (0.1-10 microM) did not affect cell viability, but prevented EGF-induced 3H-thymidine incorporation in a dose-dependent manner. In contrast, 3H-thymidine incorporation in serum-stimulated A431 cells decreased only at the higher doses of C2-cer used (1-10 microM), being this effect accompanied by a slight, albeit significant (20-25%), reduction in cell viability.