Figure 1. DNA-PK signaling pathway
DNA-dependent protein kinase (DNA-PK)
The DNA-dependent protein kinase (DNA–PK) is a nuclear serine/threonine protein kinase which is activated after the interaction with its target DNA. DNA–PK is a complex composed of a large catalytic subunit named DNA–PKcs and a heterodimer of Ku proteins (Ku70/80) that is a regulatory factor. In many studies, mammalian DNA–PK, as a critical component of the response to DNA damage, has been shown to play an important role in the DNA double-strand break (DSB) repair and recombination. It can cooperate with ATM and ATR to activate related proteins via phosphorylation during the DNA damage response and further processes. In addition, DNA-PK also takes part in the modulation of chromatin structure and the maintenance of telomere.
DNA-dependent protein kinase catalytic subunit (DNA-PKcs)
DNA–PKcs is a member of the phosphatidylinositol 3 (PI 3)-kinase family. It is a ∼470-kD polypeptide with several domains. The 500 residues of DNA–PKcs in carboxyl terminus comprise the highly conserved catalytic kinase domain. There are two domains named FAT and FATC that surround the catalytic domain. They significantly enhance the stabilization of conformational changes to the catalytic core and regulate DNA-PK’s activity. The N-terminal domain is large, mostly containing helical elements and DNA repeats called HEAT, including Huntingtin, Elongation Factor 3, PP2A, and TOR1 repeats as well as groups of phosphorylation sites with important regulatory functions, namely the JK, PQR, and ABCDE phosphorylation clusters. DNA-PKcs is indispensable for the non-homologous end joining (NHEJ) as it induces a conformational change that enables related proteins such as enzymes and factors to access the ends of the double-strand break. It is also required for the unique genetic recombination named V(D)J recombination that utilizes NHEJ to promote immune system diversity during the early stages of T and B cell maturation.
Ku70 and Ku80 (also called Ku86) are encoded by the XRCC6 and XRCC5 genes among human genome respectively and they show a high affinity for available ends of double-stranded DNA. The binding is a sequence-independent manner, since Ku70/80 binds to the sugar backbone of DNA rather than the bases. Depending on the length of the DNA substrate, multiple Ku molecules can slide onto the naked DNA in vitro, but typically only one Ku heterodimer is bound to each end of the DSB in vivo. Ku plays a significant role in non-homologous end joining (NHEJ). Ku heterodimer (Ku70/80) senses and binds double-strand breaks, which initiates NHEJ. It recruits the NHEJ machinery to the DNA lesion as a scaffold which interacts with every canonical NHEJ factor directly, such as DNA-PKcs, XRCC4, DNA ligase IV and XLF, and with the majority of the DNA end processing factors. In addition to the primary function of recruitment, the second function of Ku heterodimer is to maintain the stability of the ends of the broken DNA molecule when a double-strand break occurs. The Ku heterodimer forms a ring-shaped protein that slides onto the ends of the broken DNA molecule, maintaining the two ends of the broken DNA molecule together, which stops non-specific processing.
Figure 2. DNA-PK in double-strand break repair
DNA-PK signaling pathway
DNA-PK signaling cascade
DNA-PK is significantly crucial in non-homologous end joining (NHEJ) that can summon specific enzymes to mediate and process the repair of a wide range of DNA-strand breaks. After induction of the DNA-strand break, the broken DNA is quickly recognized and bound by the Ku70/80 heterodimer. The heterodimer forms a ring-like protein complex at the lesion site, which stabilizes the broken DNA double strands. At the same time, the Ku heterodimer recruits related processing proteins including the other part of DNA-PK (DNA-PKcs), XRCC4, DNA ligase IV, XLF and APLF. The Ku heterodimer also serves to maintain the stabilization of this NHEJ complex at the DNA damage site. Then the NHEJ complex bridges the DNA ends and further promotes the end stability. The interaction of DNA-PKcs and Ku heterodimer leads to translocation of the Ku heterodimer inward on the dsDNA strand and ultimately results in activation of the DNA-PKcs kinase activity. The activated DNA-PKcs phosphorylates many factors other than Ku heterodimers, such as Artemis, polynucleotide kinase/phosphatase (PNKP), Werner syndrome ATP-dependent helicase (WRN), DNA polymerase and Aprataxin. After the DNA ligase IV-X-ray cross complementing protein 4 (XRCC4) complex ligates the broken ends with the assistance of the XRCC4-like factor (XLF), NHEJ complex leaves the DNA, which marks the completion of the NHEJ.
Figure 3. DNA-PK signaling cascade in NHEJ
DNA-PK not only senses DNA damage and mediates non-homologous end joining but also participates in the detection of the invasion of foreign DNA in the cytoplasm and results in the further activation of immune signaling.
DNA-PK can also act as pathogen recognition receptors (PRRs) for cytosolic DNA from pathogens. In response to foreign DNA, Ku70 increases the level of IFNl1 through the activation of IFN regulatory factor (IRF)-1 and IRF-7. At the same time, DNA-PK promotes the expression of cytokine, IFN-b, and chemokine genes through the activation of TBK1, IRF-3, and STING.
In addition, single-stranded endosomal DNA initiates TLR9 signaling in which TLR9 recruits the myeloid differentiation marker 88 (MyD88). By engaging with TANK-binding kinase 1 (TBK1), it induces the transcription of nuclear factor kappa B (NF-kB) and IFN-regulatory factor (IRF).
The full activation of DNA-PK catalytic subunit is through the translocation of the Ku heterodimer inward on the dsDNA which allows DNA-PKcs to interact directly with DSB end. Without Ku70/80 and the induction of double-strand break, DNA-PKcs cannot be activated and thus the further signaling pathways are inhibited as well. Although the precise mechanism that how the association with the Ku–DNA complex stimulates the catalytic activity of DNA-PKcs is not well described, it is likely that many regions of the protein take part in this process. The direct interaction with the Ku–DNA complex induces a conformational change in the FAT and FATC domains that surround the catalytic domain. According to the structure results, it is revealed that this conformation change leads to the alteration of the catalytic groups and the ATP binding pocket of DNA-PKcs, which results in the ultimately full activation of its kinase activity. In addition to C-terminus, the N-terminus also exerts impacts on modulating the enzymatic activity of DNA-PKcs. It has been shown that the N terminal region keeps DNA-PKcs at a low basal activity. A conformational change occurs after the perturbation of the N-terminus, which leads to an increase in basal kinase activity. EGFR and the downstream PI3K-Akt signaling pathway can also affect DNA-PKcs activation and radio-sensitivity and then regulate related pathways. It is shown that the activation of nuclear Akt promotes its direct interaction with DNA-PKcs through the C terminal domain of Akt, which activates DNA-PKcs. Chromatin status and modulators such as isoform of heterochromatin protein 1 (HP1) also participate in the regulation of DNA-PK activity. Another important factor is CK2 kinase that is a versatile kinase involved in many cellular regulations including chromatin modulation in DNA damage sensing and repair.
Relationship with diseases
Given the paramount importance of DNA repair to our health and the vital role that DNA-PK plays in it, the mutations and changes of DNA-PK contribute to many diseases. It is universally acknowledged that aging increases DNA breaks and it actually activates DNA-PK as well. The activation of DNA-PK in skeletal muscle greatly suppresses mitochondrial function, energy metabolism, and physical fitness. DNA-PK also phosphorylates HSP90a, which inhibit its chaperone function for AMP-activated protein kinase (AMPK). This leads to the mitochondrial and matabolic decline as AMPK is critical for mitochondrial biogenesis and energy metabolism. DNA-PK is also highly associated with Alzheimer’s disease (AD). In AD brains, the level of DNA-PK is significantly lower than that in normal brains. The deficiency of Ku80 disrupts somatostatin signaling since Ku80 is an important somatostatin receptor, which induces the generation of amyloid beta (Aβ).