Disease relevance of PRKDC

• 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 [1].
• These facts, together with the demonstration of the ability of doxazosin to bind DNA, allowed us to propose a novel mechanism of action for doxazosin in prostate cancer cells that implies DNA-damage mediated apoptosis by down-regulation of XRCC5 and PRKDC genes [2].
• However, IR-induced phosphorylation of Ser46 was seen even in M059J, a human glioma cell line lacking DNA-PKcs, and it was, at most, only slightly less than in control M059K [3].
• We found that at 15 minutes after irradiation, the levels of NHEJ (as measured by an in vitro DSB end-ligation assay) and DNA-PK catalytic subunit (DNA-PKcs) activity were, respectively, 2-fold and 4-fold higher in radio-resistant than in radio-sensitive B-CLL cells or Epstein-Barr virus (EBV)-transformed B cells [4].
• Immunoreactivity and immunoblot results correlated with the loss of DNA-PKcs allotype in soft tissue sarcoma (Fisher's exact test p = 0.0037) [5].

High impact information on PRKDC

• Thus, DNA-PKcs regulates Artemis by both phosphorylation and complex formation to permit enzymatic activities that are critical for the hairpin-opening step of V(D)J recombination and for the 5' and 3' overhang processing in nonhomologous DNA end joining [6].
• The involvement of inositol phosphate in DNA-PK-dependent NHEJ is of particular interest since the catalytic domain of DNA-PKcs is similar to that found in the phosphatidylinositol 3 (PI 3)-kinase family [7].
• Mutation of the gene for DNA-PK catalytic subunit (Prkdc) cases defective antigen receptor V(D)J recombination and arrests B- and T-lymphocyte development in severe combined immune-deficiency (SCID) mice [8].
• DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-stranded break repair and V(D)J recombination, comprises a DNA-targeting component called Ku and an approximately 460 kDa catalytic subunit, DNA-PKcs [9].
• Strikingly, DNA-PKcs is most similar to PI kinase family members involved in cell cycle control, DNA repair, and DNA damage responses [9].

Chemical compound and disease context of PRKDC

• Curcumin induced melanoma cell apoptosis and cell cycle arrest, which is associated with the downregulation of NFkappaB activation, iNOS and DNA-dependent protein kinase catalytic subunit expression, and upregulation of p53, p21(Cip1), p27(Kip1) and checkpoint kinase 2 [10].

Biological context of PRKDC

• Differentially expressed probe sets within the DNA repair pathway were consistently overexpressed (93%), with strong effects observed for PRKDC, PCNA, and CHEK1 [11].
• The promoters for human DNA-PKcs (PRKDC) and MCM4: divergently transcribed genes located at chromosome 8 band q11 [12].
• M059J is a radiosensitive cell line established from a human glioblastoma tumor that fails to express the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs, now known as PRKDC) [13].
• Thus the radiosensitive phenotype of M059J cells appears to be due to a defect in PRKDC and a truncating ATM mutation [14].
• No other sequence differences in PRKDC between M059J and M059K cells were observed in approximately 15,000 bp of genomic sequence including the sequences of exons 5 through 38 and surrounding intron sequence, suggesting a possible reduction to homozygosity at this locus prior to acquisition of the mutation leading to the M059J cell line [15].

Anatomical context of PRKDC

• Two hybrid cell lines that retain an extra copy of PRKDC display active kinase activity and are radioresistant, demonstrating that the primary defect in M059J cells is in PRKDC [13].
• The DNA-dependent protein kinase catalytic subunit (p460) is cleaved during Fas-mediated apoptosis in Jurkat cells [16].
• We found that tumour-transformed tissue shows positive immunostaining of DNA-PKcs, Ku86 and Ku70, while non-neoplastic squamous epithelium and tumour-free cervix glands show negative immunoreactivity [17].
• Similarly, a Chinese hamster cell line deficient in DNA-PKcs (irs-20) showed little evidence for recovery of DNA replication inhibition up to 6 h after irradiation, whereas the parental CHO cells showed significant recovery and an irs-20 derivative expressing the human DNA-PKcs complete recovery within 4 h [18].
• A large reduction of DNA-PKcs was achieved in normal human fibroblasts after transfection with two DNA-PKcs small interfering RNA sequences [19].

Associations of PRKDC with chemical compounds

• Enhanced phosphorylation of p53 serine 18 following DNA damage in DNA-dependent protein kinase catalytic subunit-deficient cells [20].
• Cytosolic extracts derived from apoptotic cells were able to cleave DNA-PKcs into polypeptides of sizes identical with those seen in vivo, and this cleavage was inhibited by the cysteine protease inhibitors iodoacetamide and N-ethylmaleimide [16].
• Magnesium-ATP and the protein kinase activity of DNA-PKcs are essential for DNA double-strand break repair [21].
• In contrast, 10-fold higher concentrations of caffeine were required to inhibit DNA-PK autophosphorylation in vitro and caffeine failed to inhibit DNA-PKcs dependent double-strand break repair in vivo [22].
• We studied the effect on replicon initiation of another member of the PI-3 family of protein kinases, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) by measuring either total DNA synthesis, or size distribution of nascent DNA using alkaline sucrose gradient centrifugation [18].

Physical interactions of PRKDC

• DNA-PKcs interacted directly with RPA1 in vitro [23].
• The atomic structure of a truncated Ku70-Ku80 was determined; however, the subunit-specific carboxy-terminal domain of Ku80-essential for binding to DNA-PKcs-was determined only in isolation, and the C-terminal domain of Ku70 was not resolved in its DNA-bound conformation [24].
• Here, we report that Artemis forms a complex with the 469 kDa DNA-dependent protein kinase (DNA-PKcs) in the absence of DNA [6].
• Here, we show that MDC1 directly interacts with the Ku/DNA-PKcs complex [25].
• First, we showed that WRN forms a complex with DNA-PKcs and Ku in solution [26].

Enzymatic interactions of PRKDC

• Furthermore, DNA-PKcs was cleaved in vitro by purified apopain (CPP32), but not IL-1beta-converting enzyme [16].
• WRN is phosphorylated in vivo after treatment of cells with DNA-damaging agents in a pathway that requires DNA-PKcs [27].
• Upon complex formation, DNA-PKcs phosphorylates Artemis, and Artemis acquires endonucleolytic activity on 5' and 3' overhangs, as well as hairpins [6].
• DNA-PKcs that was bound to EBNA-LP phosphorylated p53 or EBNA-LP in vitro, and the phosphorylation of EBNA-LP was inhibited by Wortmannin, a specific in vitro inhibitor of DNA-PKcs [28].
• Here we show that replication stress leads to focus formation of Rad51 and phosphorylated DNA-PKcs, key components of the homologous recombination (HR) and nonhomologous end-joining (NHEJ), double-strand break (DSB) repair pathways, respectively [29].

Co-localisations of PRKDC

• Phosphorylated DNA-PKcs colocalizes with both gamma-H2AX and 53BP1 after DNA damage [30].

Regulatory relationships of PRKDC

• Likewise, radiation-induced autophosphorylation of DNA-PKcs at Thr2609 was only blocked in A549 cells by these two inhibitors and AKT1 small interfering RNA transfection [31].
• Ku70 and Ku80 have been shown to form a heterodimeric complex that can bind tightly to free DNA ends and activate the protein kinase DNA-PKcs [32].
• The kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is necessary in activating Artemis as an endonuclease [33].
• Interestingly, DNA-PKcs stimulated intermolecular ligation by DNA ligase IV-XRCC4 [34].
• Survival of patients with tumors expressing high levels of DNA-PKcs was significantly better than survival of patients with tumors expressing low levels of DNA-PKcs (p =.0024). p53 and MDM2 status alone did not correlate with survival of patients [35].

Other interactions of PRKDC

• Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit modulate RAG-mediated cleavage: implications for the enforcement of the 12/23 rule [36].
• Modulation of cleavage by Ku70/80 and DNA-PKcs results in preferential inhibition of 12/12 and 23/23 DNA cleavage, thus increasing 12/23 rule specificity [36].
• In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form [37].
• CONCLUSION: Targeting of the EGFR-dependent PI3K-AKT pathway in K-RAS-mutated A549 cells significantly affects postradiation survival by affecting the activation of DNA-PKcs, resulting in a decreased DSB repair capacity [31].
• The MCM4 and DNA-PKcs promoters are in CpG islands separated by approximately 700 bp, and transcription from each initiates at multiple, closely spaced sites [12].
• Although DNA-PKcs autophosphorylates itself at Ser-2056 after IR, we have reported here that ATM mediates DNA-PKcs phosphorylation at Thr-2609 as well as at the adjacent (S/T)Q motifs within the Thr-2609 cluster [38].

Analytical, diagnostic and therapeutic context of PRKDC

• Sequence analysis of PCR-amplified exons revealed the loss in M059J cells of a single "A" nucleotide in exon 32, corresponding to the first nucleotide of codon 1351 (ACC, Thr) of PRKDC [15].
• 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 [37].
• 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 [37].
• Here we show, using electron spectroscopic imaging, that DNA-PKcs and Ku interact with multiple DNA molecules to form large protein-DNA complexes that converge at the base of multiple DNA loops [21].
• The primary aim of this study was to investigate if the expression of the DNA damage identifying protein DNA-PKcs known to be involved in DNA repair after treatment with ionising radiation can be used as a predictive marker for radiotherapy (RT) response in cervical cancer [17].

References