Sequence-specific DNA Binding and Transcription Factor Phosphorylation by Ku Autoantigen/DNA-dependent Protein Kinase

Giffin, Ward; Kwast‐Welfeld, Joanna; Rodda, David J.; Préfontaine, Gratien G.; Traykova-Andonova, Maya; Zhang, Yixian; Weigel, Nancy L.; Lefebvre, Yvonne A.; Haché, Robert J.G.

doi:10.1074/jbc.272.9.5647

Cited by 75 publications

(94 citation statements)

References 78 publications

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“…Our results with p53 and RPA indicate that the phosphorylation of heterologous substrates by DNA-PK from structured single-stranded DNA is likely to be significantly enhanced through mechanisms that assist in the recruitment of the substrate to the kinase. We have previously shown a similar result for the phosphorylation of glucocorticoid receptor by DNA-PK in cis from specific DNA sequences (21). Such interactions may prove to be significant for determining specific substrate phosphorylation by DNA-PK in vivo and could compensate for the high k m of DNA-PK for substrates (25) and its very relaxed specificity (S͞TQ) (35).…”

Section: Discussionmentioning

confidence: 55%

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Activation and autoregulation of DNA-PK from structured single-stranded DNA and coding end hairpins

Soubeyrand

Torrance

Giffin

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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DNA-dependent protein kinase (DNA-PK) acts through an essential relationship with DNA to participate in the regulation of multiple cellular processes. Yet the role of DNA as a cofactor in kinase activity remains to be completely elucidated. For example, although DNA-PK activity appears to be required for the resolution of hairpin coding ends in variable diversity joining recombination, kinase activity remains to be demonstrated from hairpin ends or other DNA structures. In the present study we report that DNA-PK is strongly activated from hairpin ends and structured singlestranded DNA, but that the phosphorylation of many heterologous substrates is blocked efficiently by inactivation of the kinase through autophosphorylation. However, substrates that bound efficiently to single-stranded DNA such as p53 and replication protein A were efficiently phosphorylated by DNA-PK from structured DNA. DNA-PK also was found to be active toward heterologous substrates from hairpin ends on double-stranded DNA under conditions where autophosphorylation was minimized. These results suggest that the role of DNA-PK in resolving coding end hairpins is likely to be enzymatic rather than structural, expand understanding of how DNA-PK binding to structured DNA relates to enzyme activity, and suggest a mechanism for autoregulatory control of its kinase activity in the cell. D NA-dependent protein kinase (DNA-PK) is a Ser͞Thr kinase required for the resolution of the hairpin coding ends in variable diversity joining [V(D)J] recombination and for correct DNA end joining in nonhomologous DNA (1). Roles for DNA-PK also have been proposed in DNA replication and the regulation of specific gene transcription by RNA polymerases I and II (2-4). Although physiological substrates for DNA-PK remain to be demonstrated, kinase activity appears to be essential to DNA-PK function in recombination, DNA repair, and transcriptional regulation (5).DNA-PK is comprised of two components: a large catalytic subunit (DNA-PK cs ), which binds DNA with low affinity (6), and the Ku antigen (Ku70͞Ku80), which binds specifically to DNA ends, sequences, and structural transitions in B-form DNA with high affinity (3, 7). DNA-PK cs is a member of the large phosphatidylinositol 3-kinase-related kinase family with several other kinases, including the ataxia telangiectasia gene product and ataxia telangectasia and RAD-3-related kinase (8). Ku appears to be essential for DNA-PK cs function in vivo and likely acts by promoting the recruitment of DNA-PK cs to DNA ends and sequences from which the kinase is activated (3, 9, 10). Ku also contains limited DNA helicase activity and can induce structural transitions in DNA flanking sequence-specific DNA-PK binding sites (11,12). Whether Ku helicase activity contributes to the activation of DNA-PK cs from DNA ends is not known.DNA-PK cs is activated at DNA ends in the presence and absence of Ku and from specific Ku DNA binding sites when recruited by Ku (6, 13). Activation of DNA-PK cs from DNA ends is further stimulated by the p...

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Section: Discussionmentioning

confidence: 55%

“…Previously, we and others have shown that colocalization to DNA and protein-protein interactions can dramatically enhance substrate phosphorylation by DNA-PK (9,21,26). Full-length recombinant p53 binds strongly to single-stranded DNA, but high affinity binding to doublestranded DNA depends on additional postranslational modification (27).…”

Section: Resultsmentioning

confidence: 97%

Activation and autoregulation of DNA-PK from structured single-stranded DNA and coding end hairpins

Soubeyrand

Torrance

Giffin

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…lacking double strand breaks (34). These findings suggest that the mechanism of Ku-mediated repression of heat shock gene expression may be different than originally proposed.…”

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confidence: 73%

Heat Shock Transcription Factor 1 Binds Selectively in Vitro to Ku Protein and the Catalytic Subunit of the DNA-dependent Protein Kinase

Huang

Nueda

Yoo

et al. 1997

Journal of Biological Chemistry

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Heat shock transcription factor 1 (HSF1) functions as the master regulator of the heat shock response in eukaryotes. We have previously shown that, in addition to its role as a transcription factor, HSF1 stimulates the activity of the DNA-dependent protein kinase (DNA-PK). DNA-PK is composed of two components: a 460-kDa catalytic subunit and a 70-and 86-kDa heterodimeric regulatory component, also known as the Ku protein. We report here that HSF1 binds specifically to each of the two components of DNA-PK. Binding occurs in the absence of DNA. The complex with the Ku protein is stable and forms at a stoichiometry close to unity between the Ku protein heterodimer and the active HSF1 trimer. The binding is blocked by antibodies against HSF1. Our results show that HSF1 also binds directly, but more weakly, to the catalytic subunit of DNA-PK. Both interactions are dependent on a specific region within the HSF1 regulatory domain. This sequence is necessary but not sufficient for HSF1 stimulation of DNA-PK activity. The ability of HSF1 to interact with both components of DNA-PK provides a potential mechanism for the activation of DNA-PK in response to heat and other forms of stress.

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“…It was subsequently shown that the catalytic subunit of Ku antigen (DNA-dependent protein kinase), along with both regulatory subunits of Ku antigen (p70 and p86), phosphorylates glucocorticoid receptor after binding to its specific binding DNA element called negative response element (36). Although the authors did not refer to the direct role of this type of phosphorylation on gene repression (36), their report prompted us to speculate that presumable phosphorylation of VDR by the Ku antigen, which would have been triggered by the treatment with 1,25-dihydroxyvitamin D 3 , might weaken its activity to bind to nVDRE hPTHrP , leading to vitamin D-mediated gene repression. To examine this possibility, we treated nuclear proteins obtained from vehicle-or 1,25-dihydroxyvitamin D 3 -administered MT2 cells with potato acid phosphatase for different times and examined their activity to bind nVDRE hPTHrP by the Southwestern assay as shown in Fig.…”

Section: Phosphatase Treatment Of Vdr Restores Its Binding To Nvdre Hmentioning

confidence: 99%

A Negative Vitamin D Response DNA Element in the Human Parathyroid Hormone-related Peptide Gene Binds to Vitamin D Receptor Along with Ku Antigen to Mediate Negative Gene Regulation by Vitamin D

Nishishita¹,

Okazaki²,

Ishikawa³

et al. 1998

Journal of Biological Chemistry

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The mechanism by which steroid/thyroid nuclear hormone receptors activate gene transcription has been extensively studied (1-8). Among them there have been so many lines of solid evidence showing that vitamin D receptor (VDR), 1 thyroid hormone receptor, and retinoic acid receptor employ a common machinery to exert their ligand-dependent specific effects; all of them utilize a retinoic acid X receptor (RXR) in common as a partner of a heterodimer (2, 4). However, this mechanism seems to be confined only to gene stimulation but not to gene repression. This situation led us to address one of the key points in hormonal biology, negative feedback mechanism. The levels of almost all the hormones synthesized at a specific organ are under rigid control to keep their levels within a very narrow range. One of the representative mechanisms is the so-called end product inhibition. In the cases of the nuclear hormone receptors, negative transcriptional regulation of several pituitary trophic peptide hormone genes by glucocorticoid or sex steroid hormones is a good example. Although individual nuclear hormone receptors might well be involved in such regulation, detailed unified molecular mechanisms such as the manner of dimerization are largely unknown, with a few exceptions (9 -14). Likewise, the mechanism of negative gene regulation by VDR and vitamin D is only partially understood (15-21). In this case, an active form of vitamin D, 1,25-dihydroxyvitamin D 3 , can be considered an end product of parathyroid hormone (PTH) action, and this metabolite, in turn, inhibits the synthesis of PTH mRNA to keep the blood calcium level constant. Demay et al. (16) first reported that a negative vitamin D response element (nVDRE) exists in the upstream region of the human PTH gene to mediate such gene repression. This element contains a homologous sequence to only one of the two hexameric DNA sequences that form the core sequence of the consensus DNA sequence (VDRE) for positive gene regulation by vitamin D (2,4,(22)(23)(24)(25). In this process, it was shown that VDR, but not RXR, was involved, and the presence of another unknown partner protein(s) of VDR was proposed (17). However, there have been no reports confirming that the nVDRE is conserved among the genes whose expression is negatively regulated by vitamin D. We have found that the human PTH-related peptide (PTHrP) gene, which is assumed to be derived from an ancestoral gene in common with the PTH gene (26), contains a DNA sequence very homologous to the nVDRE of hPTH. Inoue et al. (27) reported that expression of the hPTHrP gene was inhibited by 1,25-dihydroxyvitamin D 3 at the transcriptional level in human adult T cell lymphoma/ leukemia virus-infected T cells, MT2 cells. With these cells, the therapeutic potential of vitamin D to decrease the blood calcium level due to the inhibition of PTHrP synthesis was discussed. Unlike parathyroid cells, from which it is hard to establish cultured cell lines, MT2 cells are transfectable cultured cell lines. Here, we examined the mole...

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Sequence-specific DNA Binding and Transcription Factor Phosphorylation by Ku Autoantigen/DNA-dependent Protein Kinase

Cited by 75 publications

References 78 publications

Activation and autoregulation of DNA-PK from structured single-stranded DNA and coding end hairpins

Activation and autoregulation of DNA-PK from structured single-stranded DNA and coding end hairpins

Heat Shock Transcription Factor 1 Binds Selectively in Vitro to Ku Protein and the Catalytic Subunit of the DNA-dependent Protein Kinase

A Negative Vitamin D Response DNA Element in the Human Parathyroid Hormone-related Peptide Gene Binds to Vitamin D Receptor Along with Ku Antigen to Mediate Negative Gene Regulation by Vitamin D

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