Protein phosphatases regulate DNA-dependent protein kinase activity

DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA end-binding Ku70/Ku80 heterodimer. DNA-PK is required for DNA double strand break repair by the process of nonhomologous end joining. Nonhomologous end joining is a major mechanism for the repair of DNA double strand breaks in mammalian cells. As such, DNA-PK plays essential roles in the cellular response to ionizing radiation and in V(D)J recombination. In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku70 and Ku80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK. Here we show that phosphorylation-induced loss of the protein kinase activity of DNA-PK is restored by the addition of the purified catalytic subunit of either protein phosphatase 1 or protein phosphatase 2A (PP2A) and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin. We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, causes a 50-60% decrease in DNA-PK protein kinase activity, although the protein phosphatase 1 activity in these cells was unaffected. In vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was observed when cells were labeled with [(32)P]inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid. Together, our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity and that the protein phosphatase responsible for reactivation in vivo is a PP2A-like enzyme.     

Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies.

DNA-dependent protein kinase (DNA-PK or the scid factor) and Ku are critical for DNA end-joining in V(D)J recombination and in general non-homologous double-strand break repair. One model for the function of DNA-PK is that it forms a complex with Ku70/86, and this complex then binds to DNA ends, with Ku serving as the DNA-binding subunit. We find that DNA-PK can itself bind to linear DNA fragments ranging in size from 18 to 841 bp double-stranded (ds) DNA, as indicated by: (i) mobility shifts; (ii) crosslinking between the DNA and DNA-PK; and (iii) atomic-force microscopy. Binding of the 18 bp ds DNA to DNA-PK activates it for phosphorylation of protein targets, and this level of activation is not increased by addition of purified Ku70/86. Ku can stimulate DNA-PK activity beyond this level only when the DNA fragments are long enough for the independent binding to the DNA of both DNA-PK and Ku. Atomic-force microscopy indicates that under such conditions, the DNA-PK binds at the DNA termini, and Ku70/86 assumes a position along the ds DNA that is adjacent to the DNA-PK.     

Ku protein in human T and B lymphocytes: full length functional form and signs of degradation

DNA-dependent protein kinase (DNA-PK) has been shown to take part in cell cycle regulatory signal transduction and in the repair of X-ray-induced DNA double-strand breaks. Functional DNA-PK is furthermore needed for the generation of antigen specificity during lymphocyte maturation. The Ku86 subunit of DNA-PK has been reported to exist in human B lymphocytes in a truncated form capable of binding to broken DNA but lacking the ability to activate the kinase function of DNA-PK. In the present work the Ku70 and Ku86 dimer proteins in T and B lymphocytes from human blood donors were analysed by immunoblotting and were observed apparently to be of full length. Also, nuclear protein extracted from B and non-B lymphocytes displayed DNA-dependent kinase activity. However, a minor fraction of Ku86 in lymphocytes was observed to be truncated with a molecular mass of approx. 70 kDa.     

Involvement of DNA-dependent protein kinase in regulation of stress-induced JNK activation.

DNA-dependent protein kinase (DNA-PK) is composed of a 460-kDa catalytic subunit and the regulatory subunits Ku70 and Ku80. The complex is activated on DNA damage and plays an essential role in double-strand-break repair and V(D)J recombination. In addition, DNA-PK is involved in S-phase checkpoint arrest following irradiation, although its role in damage-induced checkpoint arrest is not clear. In an effort to understand the role of DNA-PK in damage signaling, human and mouse cells containing the DNA-PK catalytic subunit (DNA-PKcs proficient) were compared with those lacking DNA-PKcs for c-Jun N-terminal kinase (JNK) activity that mediates physiologic responses to DNA damage. The DNA-PKcs-proficient cells showed much tighter regulation of JNK activity after DNA damage, while the level of JNK protein in both cell lines remained unchanged. The JNK proteins physically associated with DNA-PKcs and Ku70/Ku80 heterodimer, and the interaction was significantly stimulated after DNA damage. Various JNK isoforms not only contained a DNA-PK phosphorylation consensus site (serine followed by glutamine) but also were phosphorylated by DNA-PK in vitro. Together, our results suggest that DNA damage induces physical interaction between DNA-PK and JNK, which may in turn negatively affect JNK activity through JNK phosphorylation by DNA-PK.     

The DNA-dependent protein kinase catalytic activity regulates DNA end processing by means of Ku entry into DNA

The DNA-dependent protein kinase (DNA-PK) is required for double-strand break repair in mammalian cells. DNA-PK contains the heterodimer Ku and a 460-kDa serine/threonine kinase catalytic subunit (p460). Ku binds in vitro to DNA termini or other discontinuities in the DNA helix and is able to enter the DNA molecule by an ATP-independent process. It is clear from in vitro experiments that Ku stimulates the recruitment to DNA of p460 and activates the kinase activity toward DNA-binding protein substrates in the vicinity. Here, we have examined in human nuclear cell extracts the influence of the kinase catalytic activity on Ku binding to DNA. We demonstrate that, although Ku can enter DNA from free ends in the absence of p460 subunit, the kinase activity is required for Ku translocation along the DNA helix when the whole Ku/p460 assembles on DNA termini. When the kinase activity is impaired, DNA-PK including Ku and p460 is blocked at DNA ends and prevents their processing by either DNA polymerization, degradation, or ligation. The control of Ku entry into DNA by DNA-PK catalytic activity potentially represents an important regulation of DNA transactions at DNA termini.     

DNA-PK-dependent phosphorylation of Ku70/80 is not required for non-homologous end joining

The Ku70/80 heterodimer is a major player in non-homologous end joining and the repair of DNA double-strand breaks. Studies suggest that once bound to a DNA double-strand break, Ku recruits the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) to form the DNA-dependent protein kinase holoenzyme complex (DNA-PK). We previously identified four DNA-PK phosphorylation sites on the Ku70/80 heterodimer: serine 6 of Ku70, serine 577 and 580 and threonine 715 of Ku80. This raised the interesting possibility that DNA-PK-dependent phosphorylation of Ku could provide a mechanism for the regulation of non-homologous end joining. Here, using mass spectrometry and phosphospecific antibodies we confirm that these sites are phosphorylated in vitro by purified DNA-PK. However, we show that neither DNA-PK nor the related protein kinase ataxia-telangiectasia mutated (ATM) is required for phosphorylation of Ku at these sites in vivo. Furthermore, Ku containing serine/threonine to alanine mutations at these sites was fully able to complement the radiation sensitivity of Ku negative mammalian cells indicating that phosphorylation at these sites is not required for non-homologous end joining. Interestingly, both Ku70 and Ku80 were phosphorylated in cells treated with the protein phosphatase inhibitor okadaic acid under conditions known to inactivate protein phosphatase 2A-like protein phosphatases. Moreover, okadaic acid-induced phosphorylation of Ku80 was inhibited by nanomolar concentrations of the protein kinase inhibitor staurosporine. These results suggest that the phosphorylation of Ku70 and Ku80 is regulated by a protein phosphatase 2A-like protein phosphatase and a staurosporine sensitive protein kinase in vivo, but that DNA-PK-mediated phosphorylation of Ku is not required for DNA double-strand break repair.     

The N-terminal Region of the DNA-dependent Protein Kinase Catalytic Subunit Is Required for Its DNA Double-stranded Break-mediated Activation

DNA-dependent protein kinase (DNA-PK) plays an essential role in the repair of DNA double-stranded breaks (DSBs) mediated by the nonhomologous end-joining pathway. DNA-PK is a holoenzyme consisting of a DNA-binding (Ku70/Ku80) and catalytic (DNA-PKcs) subunit. DNA-PKcs is a serine/threonine protein kinase that is recruited to DSBs via Ku70/80 and is activated once the kinase is bound to the DSB ends. In this study, two large, distinct fragments of DNA-PKcs, consisting of the N terminus (amino acids 1–2713), termed N-PKcs, and the C terminus (amino acids 2714–4128), termed C-PKcs, were produced to determine the role of each terminal region in regulating the activity of DNA-PKcs. N-PKcs but not C-PKcs interacts with the Ku-DNA complex and is required for the ability of DNA-PKcs to localize to DSBs. C-PKcs has increased basal kinase activity compared with DNA-PKcs, suggesting that the N-terminal region of DNA-PKcs keeps basal activity low. The kinase activity of C-PKcs is not stimulated by Ku70/80 and DNA, further supporting that the N-terminal region is required for binding to the Ku-DNA complex and full activation of kinase activity. Collectively, the results show the N-terminal region mediates the interaction between DNA-PKcs and the Ku-DNA complex and is required for its DSB-induced enzymatic activity.     

DNA-dependent protein kinase (DNA-PK) phosphorylates nuclear DNA helicase II/RNA helicase A and hnRNP proteins in an RNA-dependent manner

An RNA-dependent association of Ku antigen with nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), was found in nuclear extracts of HeLa cells by immunoprecipitation and by gel filtration chromatography. Both Ku antigen and NDH II were associated with hnRNP complexes. Two-dimensional gel electrophoresis showed that Ku antigen was most abundantly associated with hnRNP C, K, J, H and F, but apparently not with others, such as hnRNP A1. Unexpectedly, DNA-dependent protein kinase (DNA-PK), which comprises Ku antigen as the DNA binding subunit, phosphorylated hnRNP proteins in an RNA-dependent manner. DNA-PK also phosphorylated recombinant NDH II in the presence of RNA. RNA binding assays displayed a preference of DNA-PK for poly(rG), but not for poly(rA), poly(rC) or poly(rU). This RNA binding affinity of DNA-PK can be ascribed to its Ku86 subunit. Consistently, poly(rG) most strongly stimulated the DNA-PK-catalyzed phosphorylation of NDH II. RNA interference studies revealed that a suppressed expression of NDH II altered the nuclear distribution of hnRNP C, while silencing DNA-PK changed the subnuclear distribution of NDH II and hnRNP C. These results support the view that DNA-PK can also function as an RNA-dependent protein kinase to regulate some aspects of RNA metabolism, such as RNA processing and transport.     

Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment

Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA-ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4-ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4-ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4-ligase IV loading to DNA ends since stable DNA-PK/XRCC4-ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process.     

Werner protein is a target of DNA-dependent protein kinase in vivo and in vitro, and its catalytic activities are regulated by phosphorylation

Human Werner Syndrome is characterized by early onset of aging, elevated chromosomal instability, and a high incidence of cancer. Werner protein (WRN) is a member of the recQ gene family, but unlike other members of the recQ family, it contains a unique 3'-->5' exonuclease activity. We have reported previously that human Ku heterodimer interacts physically with WRN and functionally stimulates WRN exonuclease activity. Because Ku and DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), form a complex at DNA ends, we have now explored the possibility of functional modulation of WRN exonuclease activity by DNA-PK. We find that although DNA-PKcs alone does not affect the WRN exonuclease activity, the additional presence of Ku mediates a marked inhibition of it. The inhibition of WRN exonuclease by DNA-PKcs requires the kinase activity of DNA-PKcs. WRN is a target for DNA-PKcs phosphorylation, and this phosphorylation requires the presence of Ku. We also find that treatment of recombinant WRN with a Ser/Thr phosphatase enhances WRN exonuclease and helicase activities and that WRN catalytic activity can be inhibited by rephosphorylation of WRN with DNA-PK. Thus, the level of phosphorylation of WRN appears to regulate its catalytic activities. WRN forms a complex, both in vitro and in vivo, with DNA-PKC. WRN is phosphorylated in vivo after treatment of cells with DNA-damaging agents in a pathway that requires DNA-PKcs. Thus, WRN protein is a target for DNA-PK phosphorylation in vitro and in vivo, and this phosphorylation may be a way of regulating its different catalytic activities, possibly in the repair of DNA dsb.     

Regulation of CHK2 by DNA-dependent protein kinase

Chk2 is a critical mediator of diverse cellular responses to DNA damage. Activation of Chk2 by DNA damage requires phosphorylation at sites including Thr68. In earlier work, we found that an activity present in rabbit reticulocyte lysates phosphorylates and activates Chk2. We now find that hypophosphorylated Chk2 can be phosphorylated at Thr68 by various subcellular fractions of HEK293 cells. This activity is sensitive to the phosphatidylinositol 3'-kinase-like kinase inhibitor wortmannin, but not to caffeine. DNA enhances the Chk2 phosphorylation by cellular fractions in vitro. The wortmannin-sensitive Chk2 kinase activity is present in fractions from ATM-deficient cells. In contrast, Chk2 was not efficiently phosphorylated at Thr68 in vitro by fractions from cells with a defective DNA-dependent protein kinase (DNA-PK) catalytic subunit. Chk2 is phosphorylated by purified DNA-PK in vitro. Endogenous Chk2 coimmunoprecipitates Ku70 and Ku80. In a series of matched cell lines having and lacking functional DNA-PK, Chk2 activation by exposure of cells to ionizing radiation, or to camptothecin was consistently diminished in the absence of DNA-PK. Down-regulation of DNA-PK(cs) by either siRNA or a chemical inhibitor attenuated radiation-induced Chk2 phosphorylation. Ionizing radiation-induced Chk2 phosphorylation was wortmannin-sensitive in ATM-defective cells with depleted ATR. These results suggest that DNA-PK augments ATM and ATR in activation of Chk2 by DNA damage.     

Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells

DNA-dependent protein kinase (DNA-PK) is activated in a two-step process whereby the Ku heterodimer first binds to the DNA double-strand breaks (dsbs) and then the DNA-PK catalytic subunit (cs) is recruited to form a repair complex. Oxidative stress is simultaneously generated along with DNA damage by ionizing radiation or chemotherapeutic agents whose impact on the DNA-PK activity has not previously been investigated. Here we show that the DNA damage-induced kinase activity of DNA-PK was modulated by oxidative stress, which was induced along with DNA dsbs in chlorambucil (Cbl)-exposed cells. Pretreatment with the antioxidants, 2(3)-t-butyl-4-hydroxyanisole or N-acetyl-l-cysteine enhanced the amount of DNA-PKcs phosphorylated at threonine 2609 (DNA-PK^pThr2609) at the DNA dsbs and DNA-PK activity. Conversely, oxidative stress induced by l-buthionine (SR)-sulfoximine or glucose oxidase decreased the DNA-PK activity in Cbl-exposed cells. In addition, DNA-PK^pThr2609 was poorly detectable at the site of DNA dsbs, as shown by colocalization to DNA-end-binding pH2AX or p53BP1. There was no change in the protein levels of DNA-PKcs, Ku70, or Ku86. Data from these studies provide the first evidence that oxidative stress effects posttranslational modification and assembly of DNA-PK complex at DNA dsbs, and thereby repair of DNA dsbs.