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Downregulation of VRK1 by p53 in response to DNA damage is mediated by the autophagic pathway.

Valbuena A, Castro-Obregón S, Lazo PA - PLoS ONE (2011)

Bottom Line: DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H.Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown.The implication of the autophagic pathway was confirmed by its requirement for Beclin1.

View Article: PubMed Central - PubMed

Affiliation: Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Salamanca, Spain.

ABSTRACT
Human VRK1 induces a stabilization and accumulation of p53 by specific phosphorylation in Thr18. This p53 accumulation is reversed by its downregulation mediated by Hdm2, requiring a dephosphorylated p53 and therefore also needs the removal of VRK1 as stabilizer. This process requires export of VRK1 to the cytosol and is inhibited by leptomycin B. We have identified that downregulation of VRK1 protein levels requires DRAM expression, a p53-induced gene. DRAM is located in the endosomal-lysosomal compartment. Induction of DNA damage by UV, IR, etoposide and doxorubicin stabilizes p53 and induces DRAM expression, followed by VRK1 downregulation and a reduction in p53 Thr18 phosphorylation. DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H. Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown. LC3 and p62 were also downregulated, like VRK1, in response to UV-induced DNA damage. The implication of the autophagic pathway was confirmed by its requirement for Beclin1. We propose a model with a double regulatory loop in response to DNA damage, the accumulated p53 is removed by induction of Hdm2 and degradation in the proteasome, and the p53-stabilizer VRK1 is eliminated by the induction of DRAM that leads to its lysosomal degradation in the autophagic pathway, and thus permitting p53 degradation by Hdm2. This VRK1 downregulation is necessary to modulate the block in cell cycle progression induced by p53 as part of its DNA damage response.

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Knock-down of DRAM and Beclin-1 (BECN1), and addition of leptomycin B prevented downregulation of VRK1 induced by UV light.(A) Human fibroblast WS1 cells were transfected with siControl, siDRAM-01, siBECN1-smart pool, or treated with leptomycin B. After that, these cells were irradiated with UV-C light and the protein levels determined at different time points. The changes in levels of VRK1 protein were detected with the 1B5 mAb. The quantification of the blots is shown in the graph at the right. (B). The effectiveness of the DRAM knock-down was determined by qRT-PCR and the result shown in the bar graph at the bottom, and siBECN1 by western blot (Fig. S2). Knock-down siRNA transfections were performed 48 hours, and addition of leptomycin B was 12 hours, before the start of UV treatment. (C) P62/SQSTM1 and LC3B are proteins degraded by autophagy. P62/SQSTM1 and LC3B proteins are also degraded in response to UV light, following a transient accumulation in autophagosomes after induction of damage [60], [61].
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pone-0017320-g005: Knock-down of DRAM and Beclin-1 (BECN1), and addition of leptomycin B prevented downregulation of VRK1 induced by UV light.(A) Human fibroblast WS1 cells were transfected with siControl, siDRAM-01, siBECN1-smart pool, or treated with leptomycin B. After that, these cells were irradiated with UV-C light and the protein levels determined at different time points. The changes in levels of VRK1 protein were detected with the 1B5 mAb. The quantification of the blots is shown in the graph at the right. (B). The effectiveness of the DRAM knock-down was determined by qRT-PCR and the result shown in the bar graph at the bottom, and siBECN1 by western blot (Fig. S2). Knock-down siRNA transfections were performed 48 hours, and addition of leptomycin B was 12 hours, before the start of UV treatment. (C) P62/SQSTM1 and LC3B are proteins degraded by autophagy. P62/SQSTM1 and LC3B proteins are also degraded in response to UV light, following a transient accumulation in autophagosomes after induction of damage [60], [61].

Mentions: To confirm the implication of DRAM in VRK1 downregulation induced by UV light, the level of endogenous DRAM was knocked-down by specific siRNA in WS1 fibroblasts. VRK1 protein level was higher in response to UV when DRAM was knocked-down (Fig. 5A). SiDRAM-01 was very effective in downregulating DRAM mRNA levels, while DRAM expression was induced in non-transfected or in siControl cells (Fig. 5B). These results confirmed the role of DRAM in UV-light induced downregulation of VRK1.


Downregulation of VRK1 by p53 in response to DNA damage is mediated by the autophagic pathway.

Valbuena A, Castro-Obregón S, Lazo PA - PLoS ONE (2011)

Knock-down of DRAM and Beclin-1 (BECN1), and addition of leptomycin B prevented downregulation of VRK1 induced by UV light.(A) Human fibroblast WS1 cells were transfected with siControl, siDRAM-01, siBECN1-smart pool, or treated with leptomycin B. After that, these cells were irradiated with UV-C light and the protein levels determined at different time points. The changes in levels of VRK1 protein were detected with the 1B5 mAb. The quantification of the blots is shown in the graph at the right. (B). The effectiveness of the DRAM knock-down was determined by qRT-PCR and the result shown in the bar graph at the bottom, and siBECN1 by western blot (Fig. S2). Knock-down siRNA transfections were performed 48 hours, and addition of leptomycin B was 12 hours, before the start of UV treatment. (C) P62/SQSTM1 and LC3B are proteins degraded by autophagy. P62/SQSTM1 and LC3B proteins are also degraded in response to UV light, following a transient accumulation in autophagosomes after induction of damage [60], [61].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046209&req=5

pone-0017320-g005: Knock-down of DRAM and Beclin-1 (BECN1), and addition of leptomycin B prevented downregulation of VRK1 induced by UV light.(A) Human fibroblast WS1 cells were transfected with siControl, siDRAM-01, siBECN1-smart pool, or treated with leptomycin B. After that, these cells were irradiated with UV-C light and the protein levels determined at different time points. The changes in levels of VRK1 protein were detected with the 1B5 mAb. The quantification of the blots is shown in the graph at the right. (B). The effectiveness of the DRAM knock-down was determined by qRT-PCR and the result shown in the bar graph at the bottom, and siBECN1 by western blot (Fig. S2). Knock-down siRNA transfections were performed 48 hours, and addition of leptomycin B was 12 hours, before the start of UV treatment. (C) P62/SQSTM1 and LC3B are proteins degraded by autophagy. P62/SQSTM1 and LC3B proteins are also degraded in response to UV light, following a transient accumulation in autophagosomes after induction of damage [60], [61].
Mentions: To confirm the implication of DRAM in VRK1 downregulation induced by UV light, the level of endogenous DRAM was knocked-down by specific siRNA in WS1 fibroblasts. VRK1 protein level was higher in response to UV when DRAM was knocked-down (Fig. 5A). SiDRAM-01 was very effective in downregulating DRAM mRNA levels, while DRAM expression was induced in non-transfected or in siControl cells (Fig. 5B). These results confirmed the role of DRAM in UV-light induced downregulation of VRK1.

Bottom Line: DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H.Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown.The implication of the autophagic pathway was confirmed by its requirement for Beclin1.

View Article: PubMed Central - PubMed

Affiliation: Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Salamanca, Spain.

ABSTRACT
Human VRK1 induces a stabilization and accumulation of p53 by specific phosphorylation in Thr18. This p53 accumulation is reversed by its downregulation mediated by Hdm2, requiring a dephosphorylated p53 and therefore also needs the removal of VRK1 as stabilizer. This process requires export of VRK1 to the cytosol and is inhibited by leptomycin B. We have identified that downregulation of VRK1 protein levels requires DRAM expression, a p53-induced gene. DRAM is located in the endosomal-lysosomal compartment. Induction of DNA damage by UV, IR, etoposide and doxorubicin stabilizes p53 and induces DRAM expression, followed by VRK1 downregulation and a reduction in p53 Thr18 phosphorylation. DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H. Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown. LC3 and p62 were also downregulated, like VRK1, in response to UV-induced DNA damage. The implication of the autophagic pathway was confirmed by its requirement for Beclin1. We propose a model with a double regulatory loop in response to DNA damage, the accumulated p53 is removed by induction of Hdm2 and degradation in the proteasome, and the p53-stabilizer VRK1 is eliminated by the induction of DRAM that leads to its lysosomal degradation in the autophagic pathway, and thus permitting p53 degradation by Hdm2. This VRK1 downregulation is necessary to modulate the block in cell cycle progression induced by p53 as part of its DNA damage response.

Show MeSH
Related in: MedlinePlus