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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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Subcellular localization of DRAM.(A–E) Colocalization of DRAM in different Golgi-endosome-lysosome compartments. (F) TSG101 and DRAM do not colocalize in endoplasmic vesicles. (G) TSG101 and VRK1 colocalize in different locations in the absence of DRAM. In these experiments, H1299 cells were transfected with plasmid pCDNA3-DRAM-Myc-His and plated on 10-cm2 dishes (5×105) containing 1-cm-diameter sterile glass coverslips. The coverslips were stained twenty-four hours after DRAM transfection with specific antibodies for the endogenous proteins: VRK1 (1F6), giantin, GM130, EEA1 and LAMP2. DRAM was detected with an anti-myc epitope antibody. TSG101 was detected with a polyclonal antibody.
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pone-0017320-g003: Subcellular localization of DRAM.(A–E) Colocalization of DRAM in different Golgi-endosome-lysosome compartments. (F) TSG101 and DRAM do not colocalize in endoplasmic vesicles. (G) TSG101 and VRK1 colocalize in different locations in the absence of DRAM. In these experiments, H1299 cells were transfected with plasmid pCDNA3-DRAM-Myc-His and plated on 10-cm2 dishes (5×105) containing 1-cm-diameter sterile glass coverslips. The coverslips were stained twenty-four hours after DRAM transfection with specific antibodies for the endogenous proteins: VRK1 (1F6), giantin, GM130, EEA1 and LAMP2. DRAM was detected with an anti-myc epitope antibody. TSG101 was detected with a polyclonal antibody.

Mentions: VRK1 is mostly nuclear, but there is always a subpopulation that is cycling in the cytosol [38] and can be detected in cytosolic vesicles, mainly in the Golgi apparatus [39], and can be detected with a specific antibody [38]. This VRK1 subpopulation enters a degradation pathway that ends in the lysosome [23]. Overexpressed DRAM was detected in lysosomes colocalizing with cathepsin D [40]. To ascertain if cytosolic VRK1 could also be detected in a common intracellular compartment with DRAM protein, the subcelular location of DRAM was determined in combination with several components of the Golgi-endosome-lysosome vesicular traffic that were used as markers. VRK1 is already known to be present in Golgi, colocalizing with giantin [38], [39]. DRAM protein was partially detected colocalizing with both VRK1 and giantin in cytosolic vesicles (Fig. 3A, B). DRAM also colocalized with GM130 (Fig. 3C), a marker of cis-Golgi; with EEA1, a marker for early endosome vesicles (Fig. 3D) and with LAMP2, a lysosomal marker (Fig. 3E). DRAM was detected in all these compartments suggesting that DRAM localization is more widely expressed than previously reported [30], and is not limited to lysosomes, but also colocalize with the Golgi as part of the degradation pathway of endosomal-lysosomal intracellular traffic.


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)

Subcellular localization of DRAM.(A–E) Colocalization of DRAM in different Golgi-endosome-lysosome compartments. (F) TSG101 and DRAM do not colocalize in endoplasmic vesicles. (G) TSG101 and VRK1 colocalize in different locations in the absence of DRAM. In these experiments, H1299 cells were transfected with plasmid pCDNA3-DRAM-Myc-His and plated on 10-cm2 dishes (5×105) containing 1-cm-diameter sterile glass coverslips. The coverslips were stained twenty-four hours after DRAM transfection with specific antibodies for the endogenous proteins: VRK1 (1F6), giantin, GM130, EEA1 and LAMP2. DRAM was detected with an anti-myc epitope antibody. TSG101 was detected with a polyclonal antibody.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046209&req=5

pone-0017320-g003: Subcellular localization of DRAM.(A–E) Colocalization of DRAM in different Golgi-endosome-lysosome compartments. (F) TSG101 and DRAM do not colocalize in endoplasmic vesicles. (G) TSG101 and VRK1 colocalize in different locations in the absence of DRAM. In these experiments, H1299 cells were transfected with plasmid pCDNA3-DRAM-Myc-His and plated on 10-cm2 dishes (5×105) containing 1-cm-diameter sterile glass coverslips. The coverslips were stained twenty-four hours after DRAM transfection with specific antibodies for the endogenous proteins: VRK1 (1F6), giantin, GM130, EEA1 and LAMP2. DRAM was detected with an anti-myc epitope antibody. TSG101 was detected with a polyclonal antibody.
Mentions: VRK1 is mostly nuclear, but there is always a subpopulation that is cycling in the cytosol [38] and can be detected in cytosolic vesicles, mainly in the Golgi apparatus [39], and can be detected with a specific antibody [38]. This VRK1 subpopulation enters a degradation pathway that ends in the lysosome [23]. Overexpressed DRAM was detected in lysosomes colocalizing with cathepsin D [40]. To ascertain if cytosolic VRK1 could also be detected in a common intracellular compartment with DRAM protein, the subcelular location of DRAM was determined in combination with several components of the Golgi-endosome-lysosome vesicular traffic that were used as markers. VRK1 is already known to be present in Golgi, colocalizing with giantin [38], [39]. DRAM protein was partially detected colocalizing with both VRK1 and giantin in cytosolic vesicles (Fig. 3A, B). DRAM also colocalized with GM130 (Fig. 3C), a marker of cis-Golgi; with EEA1, a marker for early endosome vesicles (Fig. 3D) and with LAMP2, a lysosomal marker (Fig. 3E). DRAM was detected in all these compartments suggesting that DRAM localization is more widely expressed than previously reported [30], and is not limited to lysosomes, but also colocalize with the Golgi as part of the degradation pathway of endosomal-lysosomal intracellular traffic.

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