Cell cycle‐dependent regulation of the DNA‐dependent protein kinase

Nilsson, Anders; Sirzén, Florin; Lewensohn, Rolf; Wang, N.; Skog, Sven

doi:10.1046/j.1365-2184.1999.3240239.x

Cited by 18 publications

(11 citation statements)

References 21 publications

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“…We note, however, that single-stranded DNA structures likely occur at their highest concentration during S phase, which is coincident with a reduction in DNA-PK activity that occurs in the absence of a changes in the levels of Ku or DNA-PK cs (19,33). Although DNA-PK cs is a phosphoprotein in cellular extracts, it is not yet clear to what extent the kinase is maintained in a catalytically inactive state.…”

Section: Discussionmentioning

confidence: 93%

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: 93%

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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“…Several reports suggest that DNA-PKc exists in the cytoplasm in mitotic phase. 42,43 Although p53 is predominantly a nuclear protein, it also travels from the nucleus to the mitochondria in response to death stress. 44 Moreover, it seems that, in dividing Cbl cos cells, DNA-PKc is more in the nucleus than in the cytoplasm, whereas in dying cells (in which the nucleus appears fragmented, a sign of late-stage apoptosis), it is present more in the cytoplasm (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Gene network analysis of oxidative stress-mediated drug sensitivity in resistant ovarian carcinoma cells

Maiti

2009

Pharmacogenomics J

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Drug resistance in cancer cells involves complex molecular mechanisms and ovarian carcinoma cells become resistant to chlorambucil (Cbl) after continuous treatment. This drug-and ionizing radiation-resistant cells have lower level of endogenous ROS (reactive oxygen species) compared with sensitive cells. Elevation of the cellular ROS level by exogenous ROS generation increases the sensitivity of Cbl to resistant cells. In contrast, antioxidants prevent the sensitization of resistant cells to Cbl by H 2 O 2 , COS (chronic oxidative stress) or NOO À . The molecular mechanism of drug sensitivity with COS has been investigated by microarray gene expressions followed by gene network analysis and it reveals that a cdc42/rac1 guanine exchange factor, ARHGEF6, with p53 and DNA-Pkc (PRKDC) is central to induce apoptosis in Cbl cos (Cbl with COS) cells. mRNA and protein levels of major gene network pathway differ significantly in Cbl cos cells than in Cbltreated cells. Moreover, DNA-PKc physically interacts with ARHGEF6 and p53 mostly in the nucleus of Cbl-treated cells, whereas in Cbl cos -treated cells, its interactions are mostly in the cytoplasm. These results suggest that low doses of Cbl and very low doses of COS together kill Cbl-resistant ovarian carcinoma cells and ARHGEF6 signaling may have an instrumental role in induction of apoptosis in Cbl cos cells.

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“…quires growth factor signaling (32) and that this effect may be due to the physical association of DNA-PK with epidermal growth factor receptor (33). DNA-PK can be detected in both the nucleus and cytoplasm (34,35). Significantly inositol hexakisphosphate (IP 6 ) was reported to bind to DNA-PK (36 -38), suggesting that IP 6 might play a pivotal role in modulating the localization and/or biological properties of DNA-PK.…”

Section: Impairment Of Ser-473 Phosphorylation In Dna-pkcs Sirna-treamentioning

confidence: 99%

Identification of a PKB/Akt Hydrophobic Motif Ser-473 Kinase as DNA-dependent Protein Kinase

Feng

Park

Cron

et al. 2004

Journal of Biological Chemistry

454

339

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Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr-308 and Ser-473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 kinase (S473K), respectively. Although PDK1 has been well characterized, the identification of the S473K remains controversial. A major PKB Ser-473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK co-localized and associated with PKB at the plasma membrane. In vitro, DNA-PK phosphorylated PKB on Ser-473, resulting in a ϳ10-fold enhancement of PKB activity. Knockdown of DNA-PK by small interfering RNA inhibited Ser-473 phosphorylation induced by insulin and pervanadate. DNA-PK-deficient glioblastoma cells did not respond to insulin at the level of Ser-473 phosphorylation; this effect was restored by complementation with the human PRKDC gene. We conclude that DNA-PK is a long sought after kinase responsible for the Ser-473 phosphorylation step in the activation of PKB.The signaling pathway centered on protein kinase B (PKB, 1 also called Akt) has emerged as a critical mediator of diverse cellular processes including metabolism, gene expression, migration, angiogenesis, proliferation, and cell survival (1, 2). PKB is tightly controlled and the consequences of its deregulation have been implicated in the development of cancers and diabetes (1, 2). The activity of PKB is markedly stimulated in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Upon stimulation, PKB is recruited to the plasma membrane through the binding of its N-terminal pleckstrin homology (PH) domain to phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), a lipid product of PI3K. PKB␣ is then activated by phosphorylation on two residues: Thr-308 in the activation loop and Ser-473 in the hydrophobic motif of the C-terminal tail (3). There is convincing evidence that Thr-308 is phosphorylated by 3-phosphoinositol-dependent kinase 1 (PDK1) (4, 5). In embryonic stem cells in which the PDK1 gene has been genetically disrupted, PKB is resistant to growth factor stimulation as consequence of loss of Thr-308 phosphorylation (5), but phosphorylation on Ser-473 still occurs. Like PKB, PDK1 also contains a PH domain that binds to PIP 3 (4, 6). Phosphorylation of Thr-308 in vivo is dependent on PI3K activity, but it is unclear if this requirement is necessary for the unfolding of PKB to allow access of PDK1 to Thr-308 site or direct activation of PDK1 through its PH domain (6, 7). Other results indicate that PI3K is important for PKB on Ser-473 because analysis of knock-in embryonic stem cells expressing PDK1 with a mutation in its PH domain revealed that PKB is not activated by insulin-like growth factor-1 (IGF-1), whereas ribosomal S6 kinase (RSK) is activated normally, indicating the importance of colocalization of PKB with PDK1 at the plasma membrane (8).Identification of the kinase responsible for phosphorylating Ser-473 has been a major challenge for a number of years but remains elusive. Several kinases have been r...

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Cell cycle‐dependent regulation of the DNA‐dependent protein kinase

Cited by 18 publications

References 21 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

Gene network analysis of oxidative stress-mediated drug sensitivity in resistant ovarian carcinoma cells

Identification of a PKB/Akt Hydrophobic Motif Ser-473 Kinase as DNA-dependent Protein Kinase

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