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Valosin-containing protein regulates the proteasome-mediated degradation of DNA-PKcs in glioma cells.

Jiang N, Shen Y, Fei X, Sheng K, Sun P, Qiu Y, Larner J, Cao L, Kong X, Mi J - Cell Death Dis (2013)

Bottom Line: However, it is not clear whether VCP is involved in DNA-PKcs (DNA-PK catalytic subunit) degradation or whether it regulates the radiosensitivity of glioblastoma.Our data demonstrated that DNA-PKcs was ubiquitinated and bound to VCP.VCP knockdown resulted in the accumulation of the DNA-PKcs protein in glioblastoma cells, and the proteasome inhibitor MG132 synergised this increase.

View Article: PubMed Central - PubMed

Affiliation: Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
DNA-dependent protein kinase (DNA-PK) has an important role in the repair of DNA damage and regulates the radiation sensitivity of glioblastoma cells. The VCP (valosine-containing protein), a chaperone protein that regulates ubiquitin-dependent protein degradation, is phosphorylated by DNA-PK and recruited to DNA double-strand break sites to regulate DNA damage repair. However, it is not clear whether VCP is involved in DNA-PKcs (DNA-PK catalytic subunit) degradation or whether it regulates the radiosensitivity of glioblastoma. Our data demonstrated that DNA-PKcs was ubiquitinated and bound to VCP. VCP knockdown resulted in the accumulation of the DNA-PKcs protein in glioblastoma cells, and the proteasome inhibitor MG132 synergised this increase. As expected, this increase promoted the efficiency of DNA repair in several glioblastoma cell lines; in turn, this enhanced activity decreased the radiation sensitivity and prolonged the survival fraction of glioblastoma cells in vitro. Moreover, the VCP knockdown in glioblastoma cells reduced the survival time of the xenografted mice with radiation treatment relative to the control xenografted glioblastoma mice. In addition, the VCP protein was also downregulated in ~25% of GBM tissues from patients (WHO, grade IV astrocytoma), and the VCP protein level was correlated with patient survival (R(2)=0.5222, P<0.05). These findings demonstrated that VCP regulates DNA-PKcs degradation and increases the sensitivity of GBM cells to radiation.

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VCP was associated with DNA-PK. (a) The VIM and the protein sequence alignment among humans, monkeys, cows and wolves. (b) The VCP was pulled down by DNA-PKcs IP in different GBM cell lines (WL: whole-cell lysates). DNA-PKcs-deficient M059J cells were considered as negative controls. (c) The schematic drawing of the truncated VCP protein. All truncates were tagged with the Flag epitope (FL: full-length). (d) The plasmids expressing different domains of VCP were separately transfected into 293T cells (con: empty vector), and flag immunmoprecipitation was performed. Both DNA-PKcs and flag were detected in whole-cell lysates (bottom panels, shown as WL) or flag IPs (top panels)
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fig1: VCP was associated with DNA-PK. (a) The VIM and the protein sequence alignment among humans, monkeys, cows and wolves. (b) The VCP was pulled down by DNA-PKcs IP in different GBM cell lines (WL: whole-cell lysates). DNA-PKcs-deficient M059J cells were considered as negative controls. (c) The schematic drawing of the truncated VCP protein. All truncates were tagged with the Flag epitope (FL: full-length). (d) The plasmids expressing different domains of VCP were separately transfected into 293T cells (con: empty vector), and flag immunmoprecipitation was performed. Both DNA-PKcs and flag were detected in whole-cell lysates (bottom panels, shown as WL) or flag IPs (top panels)

Mentions: According to bioinformatic analysis, the DNA-PKcs protein contains a VCP-interacting motif (VIM), which is conserved in mammals such as humans, monkeys, cows and wolves (Figure 1a). To determine whether VCP is associated with DNA-PK, co-immunoprecipitation (co-IP) was performed using the DNA-PKcs antibody in several glioblastoma cell lines; the M059J cell is DNA-PKcs-deficient, and was used as a negative control. Western blots following co-IP showed that VCP is pulled down with DNA-PKcs (Figure 1b); moreover, in agreement with our previous study,24 the more sensitive the cell is, the more VCP associates with DNA-PK, indicating that VCP may be related to radiation resistance. However, radiation itself does not significantly affect the binding of VCP with DNA-PKcs (data not show).


Valosin-containing protein regulates the proteasome-mediated degradation of DNA-PKcs in glioma cells.

Jiang N, Shen Y, Fei X, Sheng K, Sun P, Qiu Y, Larner J, Cao L, Kong X, Mi J - Cell Death Dis (2013)

VCP was associated with DNA-PK. (a) The VIM and the protein sequence alignment among humans, monkeys, cows and wolves. (b) The VCP was pulled down by DNA-PKcs IP in different GBM cell lines (WL: whole-cell lysates). DNA-PKcs-deficient M059J cells were considered as negative controls. (c) The schematic drawing of the truncated VCP protein. All truncates were tagged with the Flag epitope (FL: full-length). (d) The plasmids expressing different domains of VCP were separately transfected into 293T cells (con: empty vector), and flag immunmoprecipitation was performed. Both DNA-PKcs and flag were detected in whole-cell lysates (bottom panels, shown as WL) or flag IPs (top panels)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3674378&req=5

fig1: VCP was associated with DNA-PK. (a) The VIM and the protein sequence alignment among humans, monkeys, cows and wolves. (b) The VCP was pulled down by DNA-PKcs IP in different GBM cell lines (WL: whole-cell lysates). DNA-PKcs-deficient M059J cells were considered as negative controls. (c) The schematic drawing of the truncated VCP protein. All truncates were tagged with the Flag epitope (FL: full-length). (d) The plasmids expressing different domains of VCP were separately transfected into 293T cells (con: empty vector), and flag immunmoprecipitation was performed. Both DNA-PKcs and flag were detected in whole-cell lysates (bottom panels, shown as WL) or flag IPs (top panels)
Mentions: According to bioinformatic analysis, the DNA-PKcs protein contains a VCP-interacting motif (VIM), which is conserved in mammals such as humans, monkeys, cows and wolves (Figure 1a). To determine whether VCP is associated with DNA-PK, co-immunoprecipitation (co-IP) was performed using the DNA-PKcs antibody in several glioblastoma cell lines; the M059J cell is DNA-PKcs-deficient, and was used as a negative control. Western blots following co-IP showed that VCP is pulled down with DNA-PKcs (Figure 1b); moreover, in agreement with our previous study,24 the more sensitive the cell is, the more VCP associates with DNA-PK, indicating that VCP may be related to radiation resistance. However, radiation itself does not significantly affect the binding of VCP with DNA-PKcs (data not show).

Bottom Line: However, it is not clear whether VCP is involved in DNA-PKcs (DNA-PK catalytic subunit) degradation or whether it regulates the radiosensitivity of glioblastoma.Our data demonstrated that DNA-PKcs was ubiquitinated and bound to VCP.VCP knockdown resulted in the accumulation of the DNA-PKcs protein in glioblastoma cells, and the proteasome inhibitor MG132 synergised this increase.

View Article: PubMed Central - PubMed

Affiliation: Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
DNA-dependent protein kinase (DNA-PK) has an important role in the repair of DNA damage and regulates the radiation sensitivity of glioblastoma cells. The VCP (valosine-containing protein), a chaperone protein that regulates ubiquitin-dependent protein degradation, is phosphorylated by DNA-PK and recruited to DNA double-strand break sites to regulate DNA damage repair. However, it is not clear whether VCP is involved in DNA-PKcs (DNA-PK catalytic subunit) degradation or whether it regulates the radiosensitivity of glioblastoma. Our data demonstrated that DNA-PKcs was ubiquitinated and bound to VCP. VCP knockdown resulted in the accumulation of the DNA-PKcs protein in glioblastoma cells, and the proteasome inhibitor MG132 synergised this increase. As expected, this increase promoted the efficiency of DNA repair in several glioblastoma cell lines; in turn, this enhanced activity decreased the radiation sensitivity and prolonged the survival fraction of glioblastoma cells in vitro. Moreover, the VCP knockdown in glioblastoma cells reduced the survival time of the xenografted mice with radiation treatment relative to the control xenografted glioblastoma mice. In addition, the VCP protein was also downregulated in ~25% of GBM tissues from patients (WHO, grade IV astrocytoma), and the VCP protein level was correlated with patient survival (R(2)=0.5222, P<0.05). These findings demonstrated that VCP regulates DNA-PKcs degradation and increases the sensitivity of GBM cells to radiation.

Show MeSH
Related in: MedlinePlus