Protein Kinase Catalytic Subunit Gene Research Articles

Transcriptional activation of DNA‐dependent protein kinase catalytic subunit gene expression by oestrogen receptor‐α

The cellular response to DNA double-strand break (DSB) occurs through an integrated sensing and signalling network that maintains genomic stability. Oestrogen (E2), among its many functions, is known to have a positive effect on global genomic DNA repair; however, the mechanism by which it functions is unclear. A central enzyme involved in DNA DSB repair in mammalian cells is the DNA-dependent protein kinase (DNA-PK). Here, we show that E2 enhances DNA-PK catalytic subunit (DNA-PKcs) promoter activity with subsequent transcriptional and translational upregulation of DNA-PKcs in a breast cancer cell line. We identify two potential E2 receptor-alpha (ERalpha)-binding sites in a region upstream from the DNA-PKcs initiation site. By using small interfering RNA and the specific E2 receptor antagonist ICI 182,780, we demonstrate that ERalpha knockdown reduces E2-induced upregulation of DNA-PKcs expression and activity in breast carcinoma cells. E2-induced DNA-PK transactivation results in an increased ability of the cells to repair DNA DSB. This previously unknown mechanism of DNA-PK regulation sheds new light on tumour biology and reveals new possibilities for the prevention and therapy of E2-sensitive proliferative diseases.

Existence of a threshold-like dose for γ-ray induction of thymic lymphomas and no susceptibility to radiation-induced solid tumors in SCID mice

Severe combined immune deficiency (SCID) mice exhibit limited repair of DNA double-strand breaks and are sensitive to ionizing radiation due to a mutation of the DNA-dependent protein kinase catalytic subunit gene. To elucidate the effects of deficient DNA double-strand break repair on radiation-induced carcinogenesis, the dose–response relationship for the induction of all tumor types was examined in wild-type and SCID mice. In wild-type mice, the incidence of thymic lymphomas at γ-ray doses up to 1 Gy was almost equal to the background level, increased gradually above 1 Gy, and reached a maximum of 12.5% at 5 Gy, which is indicative of a threshold dose of less than 1 Gy. SCID mice were extremely susceptible to the induction of spontaneous and radiation-induced thymic lymphomas. The incidence of thymic lymphomas in SCID mice irradiated with 0.1 Gy or less was similar to the background level; that is, it increased markedly from 31.7% at 0.1 Gy to 51.4% at 0.25 Gy, and reached a maximum of 80.6% at 2 Gy, suggesting the presence of a threshold-like dose at low γ-ray doses, even in radiosensitive SCID mice. As the average latency for the induction of thymic lymphomas at 0.1 Gy was significantly shortened, the effect of 0.1 Gy γ-rays on thymic lymphoma induction was marginal. The high susceptibility of SCID mice to develop thymic lymphomas indicates that thymic lymphomas are induced by a defect in DNA double-strand break repair or V(D)J recombination. Excessive development of tumors other than thymic and nonthymic lymphomas was not observed in SCID mice. Furthermore, our data suggest that the defective double-strand break repair in SCID mice is not a major determinant for the induction of nonlymphoid tumors.

Isolation and characterisation of a cAMP-dependent protein kinase catalytic subunit gene from Plasmodium falciparum

Isolation and characterisation of a cAMP-dependent protein kinase catalytic subunit gene from Plasmodium falciparum

Characterization of the promoter region, first ten exons and nine intron-exon boundaries of the DNA-dependent protein kinase catalytic subunit gene, DNA-PKcs (XRCC7).

The gene, DNAPKcs (XRCC7), for the human DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a strong candidate that complements a severe combined immunodeficiency (scid) and hypersensitivity to ionizing radiation in mice. We constructed a cosmid library from a previously identified, X RCC7-covering YAC clone (943G4). From the library, we isolated three cosmid clones containing the 5'-region of XRCC7. Sequence analysis with primer walking on a 6.3-kb segment of these cosmids identified the promoter region, the first ten exons and nine intron-exon boundaries of XRCC7. The promoter region contains several potential Sp1 protein-binding sites and a high G+C content but no TATA or CCAAT boxes. These findings are consistent with the TATA-less housekeeping gene promoter and provides the basis for transcriptional regulatory studies. Since nine other exons spanning an 8-kb segment are already known, a total of 19 exons in the gene have been identified. The cosmids isolated and the primer sets designed in the present study are useful for mutation analysis in patients with a SCID phenotype.

Differentiation between the Human Hookworms Ancylostoma duodenale and Necator americanus Using PCR-RFLP

There are 2 major species of hookworms that infect humans. Ancylostoma duodenale and Necator americanus. Although traditionally considered to be identical for treatment purposes, there are significant life history differences between the species that must be considered for the rational design of chemotherapeutic and immunoprophylactic control strategies. However, identification of the species infecting a particular population has been problematic, as the eggs of the 2 species cannot be reliably differentiated by classical parasitological methods. A PCR-based technique for the differentiation of hookworm species that infect humans is reported. A fragment of the 3' untranslated region of the cAMP-dependent protein kinase catalytic subunit gene was amplified from A. duodenale and N. americanus genomic DNA using primers derived from the corresponding A. caninum cDNA. Digestion of the amplified DNA with the restriction enzymes HpaII, MboI, TaqI, and ThaI generated specific restriction fragment patterns unique to each species. The technique can distinguish between pure and mixtures of hookworm DNA and can amplify DNA from a single egg. The primers also amplify the fragment from the DNA of several other species of hookworms that infect humans and other animals. The technique is fast, simple, and hookworm specific and represents a considerable savings in time over current methods used for distinguishing between human hookworm infections.

Genetic investigation of cAMP-dependent protein kinase function in Drosophila development.

The cAMP-dependent protein kinase (PKA) has been shown to mediate the vast majority of cellular responses to the intracellular second messenger, cAMP, in eukaryotes. To study the role of cAMP signal transduction in Drosophila development, we have isolated and molecularly characterized mutations of varying severity in the Drosophila PKA gene, DC0. Biochemical measurements indicate that DC0 is either the sole or the major PKA catalytic subunit gene in Drosophila. Adult females heterozygous for a strong and a weak DC0 allele fail to lay eggs and show a striking and novel defect in oogenesis that includes the formation of egg chambers containing multinucleate nurse cells. Females heterozygous for two weak DC0 alleles are fertile but produce offspring showing a variety of defects in embryogenesis, including preblastoderm arrest and alterations in cuticular patterning. Animals zygotically null for DC0 die as morphologically normal first-instar larvae, implying that maternally encoded protein, which perdures for at least 12 hr, suffices for embryogenesis. Animals hemizygous for weak DC0 alleles survive for several days as larvae but grow slowly. Mitotic recombination experiments in the adult eye indicate that the DC0 gene is not required autonomously either for cell viability or normal growth rates. These results argue that cAMP-mediated signal transduction is essential at a variety of stages during the development of a metazoan.

CGMP-dependent Protein Kinase Genes in Drosophila

Two Drosophila genes encoding products related to cGMP-dependent protein kinase have been isolated by cross-hybridization to a Drosophila cAMP-dependent protein kinase catalytic subunit gene. Both genes encode products with putative cGMP binding and kinase domains on the same polypeptide chain, as found for the prototypical bovine lung cGMP-dependent protein kinase. The deduced product of one gene (DG1; cytological position, 21D) is 14% larger than the bovine enzyme and differs substantially in sequence at the amino terminus, the region responsible in the bovine enzyme for dimerization. The second gene (DG2; cytological position, 24A) is transcribed into three major RNA species of different size. The largest (DG2; T1) and smallest (DG2;T3) RNAs encode overlapping polypeptides of similar sequence to the whole length of bovine lung cGMP-dependent protein kinase. The translation product of the third major RNA (DG2;T2) lacks sequences similar to those that constitute the dimerization and kinase inhibitory domains of the bovine enzyme. The percentage amino acid identity between DG1 or DG2 and bovine lung cGMP-dependent protein kinase is 55 and 64%, respectively. A common progenitor of the two cGMP-dependent protein kinase genes, DG1 and DG2, is strongly suggested by the conserved positions of introns in these genes.

Cloning, sequence, and expression of the Drosophila cAMP-dependent protein kinase catalytic subunit gene.

Genomic DNA containing the protein coding region for Drosophila cAMP-dependent protein kinase catalytic subunit has been cloned and sequenced. The probe used to detect and isolate the gene fragment was constructed from two partially complementary synthetic oligonucleotides and contains 60 base pairs that encode (using Drosophila codon preferences) amino acids 195-214 of the beef heart catalytic subunit. In reduced stringency hybridization conditions, the probe recognizes two target sites in fly genomic DNA with 85% homology. One of these sites is in the cAMP-dependent protein kinase catalytic subunit gene, which was isolated as a 3959-base pair HindIII fragment. This fragment contains all of the protein coding portion, 900 base pairs upstream of the initiator ATG, and 2000 base pairs downstream of the termination codon (TAG). The coding portion of the gene contains no introns and yields a protein of 352 amino acids. There is a 2-amino acid insertion near the N terminus of the fly protein relative to the beef and mouse enzymes. Of the remaining 350 amino acids, 273 are invariant in the three species. A probe derived from the coding sequence of the HindIII clone hybridizes strongly to a 5100-base poly(A)+ RNA and weakly to 4100- and 3400-base poly(A)+ RNAs expressed in adult flies. A 2100-base pair EcoRI genomic fragment containing the second site recognized by the 60-base pair probe has also been cloned. DNA sequence analysis demonstrates that this fragment is part of the cGMP-dependent protein kinase gene or a close homolog. The catalytic subunit gene and the cGMP-dependent protein kinase gene have been located in regions 30C and 21D, respectively, of chromosome 2.