DNA-dependent protein kinase catalytic subunit modulates the stability of c-Myc oncoprotein

Abstract

BackgroundC-Myc is a short-lived oncoprotein that is destroyed by ubiquitin-mediated proteolysis. Dysregulated accumulation of c-Myc commonly occurs in human cancers. Some of those cases with the dysregulated c-Myc protein accumulation are attributed to gene amplification or increased mRNA expression. However, the abnormal accumulation of c-Myc protein is also a common finding in human cancers with normal copy number and transcription level of c-Myc gene. It seems that the mechanistic dysregulation in the control of c-Myc protein stabilization is another important hallmark associated with c-Myc accumulation in cancer cells. Here we report a novel mechanistic pathway through which DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulates the stability of c-Myc protein.ResultsFirstly, siRNA-mediated silencing of DNA-PKcs strikingly downregulated c-Myc protein levels in HeLa and HepG2 cells, and simultaneously decreased cell proliferation. The c-Myc protein level in DNA-PKcs deficient human glioma M059J cells was also found much lower than that in DNA-PKcs efficient M059K cells. ATM deficiency does not affect c-Myc expression level. Silencing of DNA-PKcs in HeLa cells resulted in a decreased stability of c-Myc protein, which was associated the increasing of c-Myc phosphorylation on Thr58/Ser62 and ubiquitination level. Phosphorylation of Akt on Ser473, a substrate of DNA-PKcs was found decreased in DNA-PKcs deficient cells. As the consequence, the phosphorylation of GSK3 β on Ser9, a negatively regulated target of Akt, was also decreased, and which led to activation of GSK 3β and in turn phosphorylation of c-Myc on Thr58. Moreover, inhibition of GSK3 activity by LiCl or specific siRNA molecules rescued the downregulation of c-Myc mediated by silencing DNA-PKcs. Consistent with this depressed DNA-PKcs cell model, overexpressing DNA-PKcs in normal human liver L02 cells, by sub-chronically exposing to very low dose of carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), increased c-Myc protein level, the phosphorylation of Akt and GSK3 β, as well as cell proliferation. siRNA-mediated silencing of DNA-PKcs in this cell model reversed above alterations to the original levels of L02 cells.ConclusionA suitable DNA-PKcs level in cells is necessary for maintaining genomic stability, while abnormal overexpression of DNA-PKcs may contribute to cell proliferation and even oncogenic transformation by stabilizing the c-Myc oncoprotein via at least the Akt/GSK3 pathway. Our results suggest DNA-PKcs a novel biological role beyond its DNA repair function.

Inactivation of DNA-Dependent Protein Kinase Leads to Spindle Disruption and Mitotic Catastrophe with Attenuated Checkpoint Protein 2 Phosphorylation in Response to DNA Damage

“…All of the antibodies are commercially available: anti-DNA-PKcs total (sc-9051, H163, Santa Cruz), anti-phosphor- siRNAs and transfection. For the knockdown of Ku70 and Ku80 expression, the siRNA molecules used are adopted from previous reports: annealed Ku70 siRNAs sense strand 5′-GGAAGAGAUAGUUUGAUUUdTdT-3′, antisense strand 5′-AAAUCAAACUAUCUCUUCCTG-3′ (33); Ku80 siRNAs sense strand 5′-ACAAAAUCCAGCCAAGUUCdTdT-3′, antisense strand 5′-GAACUUGGCUGGAUUUUGUTG-3′ (34); and the silencer negative control siRNA sense strand 5′-UUCUCC-GAACGUGUCACGUTT-3′ (32). The siRNA molecules were synthesized and purified by Shanghai GenePharma Co.; siRNA transfection was performed using Lipofectamine 2000 reagent according to the procedures reported (32).…”

“…HeLa, HeLa-NC, HeLa-H1, and ATM-deficient AT5BIVA cells were maintained in DMEM containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C in 5% CO 2 . HeLa-H1 and HeLa-NC were generated from HeLa cells by stably transfecting with a DNA-PKcs-specific siRNA construct targeting the catalytic motif (nucleotides 11637-11655, H1) and a control siRNA construct (NC), respectively (32). A cobalt-60 γ-ray source was used to irradiate the cells at a dose rate of 1.74 Gy/min at room temperature.…”

The involvement of c-Myc in the DNA double-strand break repair via regulating radiation-induced phosphorylation of ATM and DNA-PKcs activity

“…These results suggest that the interaction between DNA-PKcs and c-Myc provides a DNA damage-responsive complex through which c-Myc may be directly involved in DSB repair. At the same time, DNAPKcs regulates the stability of c-Myc [21], but c-Myc may also act as a weak inhibitor to regulate the autophosphorylation of DNA-PKcs at S2056. It has been reported that the autophosphorylation of DNA-PKcs causes DNA-PKcs to separate from Ku, which inhibits the catalytic activity of DNA-PK [34].…”

“…In a previous work, we found that the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and c-Myc proteins interact with each other, and we proposed a new signalling pathway for maintaining the stability of c-Myc, the DNA-PKcs/Akt/GSK3/c-Myc pathway [20,21]. DNAPKcs is a protein kinase requiring DNA ends as a cofactor, and it is thus also known as the DNA break-sensing molecule, which is crucial in the non-homologous end joining (NHEJ) pathway of DNA DSB repair [22,23].…”

Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities

“…Interestingly, previous studies demonstrated that c-Myc interacts with DNA-PKcs (28) and that the inactivation of DNA-PKcs results in a marked reduction of c-Myc protein expression via the ubiquitin/ proteasome pathway (29,30). We examined whether MeV infection and the consequent DNA-PK inactivation also induce c-Myc degradation.…”