MDM2-Mediated Degradation of p14ARF: A Novel Mechanism to Control ARF Levels in Cancer Cells
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{"title"=>"MDM2-mediated degradation of p14ARF: A novel mechanism to control ARF levels in cancer cells", "type"=>"journal", "authors"=>[{"first_name"=>"Maria", "last_name"=>"Vivo", "scopus_author_id"=>"6602824237"}, {"first_name"=>"Maria", "last_name"=>"Matarese", "scopus_author_id"=>"56610115700"}, {"first_name"=>"Maria", "last_name"=>"Sepe", "scopus_author_id"=>"40361631000"}, {"first_name"=>"Rosaria Di", "last_name"=>"Martino", "scopus_author_id"=>"56531517300"}, {"first_name"=>"Luisa", "last_name"=>"Festa", "scopus_author_id"=>"26632837600"}, {"first_name"=>"Viola", "last_name"=>"Calabrò", "scopus_author_id"=>"7003809014"}, {"first_name"=>"Girolama", "last_name"=>"La Mantia", "scopus_author_id"=>"6603724632"}, {"first_name"=>"Alessandra", "last_name"=>"Pollice", "scopus_author_id"=>"7004572048"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "sgr"=>"84923807954", "doi"=>"10.1371/journal.pone.0117252", "scopus"=>"2-s2.0-84923807954", "pui"=>"602566036", "pmid"=>"25723571"}, "id"=>"8858dfce-afd5-39f2-9624-bf4982a034fc", "abstract"=>"We here show a new relationship between the human p14ARF oncosuppressor and the MDM2 oncoprotein. MDM2 overexpression in various cancer cell lines causes p14ARF reduction inducing its degradation through the proteasome. The effect does not require the ubiquitin ligase activity of MDM2 and preferentially occurs in the cytoplasm. Interestingly, treatment with inhibitors of the PKC (Protein Kinase C) pathway and use of p14ARF phosphorylation mutants indicate that ARF phosphorylation could play a role in MDM2 mediated ARF degradation reinforcing our previous observations that ARF phosphorylation influences its stability and biological activity. Our study uncovers a new potentially important mechanism through which ARF and MDM2 can counterbalance each other during the tumorigenic process.", "link"=>"http://www.mendeley.com/research/mdm2mediated-degradation-p14arf-novel-mechanism-control-arf-levels-cancer-cells", "reader_count"=>17, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Student > Doctoral Student"=>2, "Researcher"=>3, "Student > Ph. D. Student"=>5, "Other"=>2, "Student > Master"=>1, "Professor"=>1, "Unspecified"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Student > Doctoral Student"=>2, "Researcher"=>3, "Student > Ph. D. Student"=>5, "Other"=>2, "Student > Master"=>1, "Professor"=>1, "Unspecified"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>2, "Biochemistry, Genetics and Molecular Biology"=>7, "Agricultural and Biological Sciences"=>5, "Chemistry"=>2, "Unspecified"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>2}, "Chemistry"=>{"Chemistry"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>5}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Russia"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1926626"], "description"=>"<p><b>A</b> U2OS cells were transfected with 1,5 μg of either wt or mutants ARF expressing plasmids. 16 hours upon transfection, cells were treated with either DMSO or 10 μM MG132 for 5 hours and subjected to IF with anti-ARF antibody. Representative images showing ARF and ARF mutants subcellular localization in U2OS cells with or without MG132 treatment are shown. Images were taken with a Nikon fluorescence microscope at similar exposure conditions. <b>B</b> The histogram, representing the mean of three independent experiments, reports the percentage of transfected cells showing each localization pattern. Standard deviations are also shown. <b>C</b> Western blot analysis of equal amounts of both cytoplasmic (30 μg) and nuclear (6 μg) protein extracts of transfected U2OS cells treated or not with MG132. Efficiency of cellular fractionation was checked with anti-lamin A/C and anti-tubulin antibodies. Normalized ARF band intensities, shown below each corresponding band, are expressed as fold enrichment respect to untreated samples arbitrarily set to 1 (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117252#sec008\" target=\"_blank\">Materials and Methods</a> for details).</p>", "links"=>[], "tags"=>["p 14ARF phosphorylation mutants", "ubiquitin ligase activity", "MDM 2 overexpression", "pkc", "p 14ARF oncosuppressor", "MDM 2 oncoprotein", "Mdm 2", "cancer cell lines causes p 14ARF reduction", "protein kinase C", "Control ARF Levels", "ARF phosphorylation influences"], "article_id"=>1319693, "categories"=>["Biological Sciences"], "users"=>["Maria Vivo", "Maria Matarese", "Maria Sepe", "Rosaria Di Martino", "Luisa Festa", "Viola Calabrò", "Girolama La Mantia", "Alessandra Pollice"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0117252.g007", "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ARF_accumulates_in_the_cytoplasm_upon_proteasome_inhibition_/1319693", "title"=>"ARF accumulates in the cytoplasm upon proteasome inhibition.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-27 02:47:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/1926616"], "description"=>"<p><b>A</b> Half-life analysis in presence or absence of MDM2 overexpression. HeLa cells were mock transfected or transfected with MDM2 expression plasmid (0.6 μg). 24 hours after, cycloheximide was added and cells were harvested at the indicated time points. Lysates were analysed by Western Blot with anti-ARF, anti-actin and anti-MDM2. <b>B</b> The plot represents half-life analysis of ARF in presence or absence of MDM2 overexpression. Band intensities were quantified by Image J software and actin normalized before being plotted in graph. The amount of protein at different time points is expressed as percentage of total protein, i.e. protein amount at t0. Each profile represents the mean of three independent transfections and Western Blot experiments. Standard deviations are also shown. <b>C</b> U2OS cells were transfected with a fixed amount of p14ARF expression plasmid (0.3 μg) and increasing amounts of MDM2 expression plasmid where indicated (0.3–0.6 μg). 24 hours after transfection, cells were treated with 10 μM MG132 (lanes 4–6) or DMSO (lanes 1–3). Levels of p14ARF were analysed by Western Blot on whole protein lysates, probed with anti-ARF and, as control, with anti-MDM2 and anti-actin.</p>", "links"=>[], "tags"=>["p 14ARF phosphorylation mutants", "ubiquitin ligase activity", "MDM 2 overexpression", "pkc", "p 14ARF oncosuppressor", "MDM 2 oncoprotein", "Mdm 2", "cancer cell lines causes p 14ARF reduction", "protein kinase C", "Control ARF Levels", "ARF phosphorylation influences"], "article_id"=>1319687, "categories"=>["Biological Sciences"], "users"=>["Maria Vivo", "Maria Matarese", "Maria Sepe", "Rosaria Di Martino", "Luisa Festa", "Viola Calabrò", "Girolama La Mantia", "Alessandra Pollice"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0117252.g003", "stats"=>{"downloads"=>0, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_p14ARF_turnover_analysis_/1319687", "title"=>"p14ARF turnover analysis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-27 02:47:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/1926617"], "description"=>"<p><b>A</b> U2OS cells were transfected with equal amount (1 μg) of MDM2 and either 3xFlagARF<sub>1–65</sub> or 3xFlagARF<sub>65–132</sub> expressing plasmids, as indicated. Equal amounts of protein extract were immunoprecipitated with anti-MDM2 antibody and revealed by Western Blot with anti-MDM2 and anti-Flag to reveal ARF. <b>B</b> U2OS cells were transfected with 0.3 μg of 3xFlagARF<sub>1–65</sub> (expressing exon 1β of p14ARF) (first lane) alone or with increasing amounts of MDM2 expression plasmid (0.3-0.6-0.9 μg). Levels of p14ARF were analysed by Western Blot on whole protein lysates, probed with anti-ARF and, as control, with anti-MDM2 and anti-actin. <b>C</b> U2OS cells were transfected with 0.3 μg of 3xFlag ARF<sub>65–132</sub> plasmid (expressing exon 2 of p14ARF) (first lane) alone or with increasing amounts of MDM2 expression plasmid (0.3-0.6-0.9 μg). Levels of p14ARF were analysed by Western Blot on whole protein lysates probed with anti-ARF and, as control, with anti-MDM2 and anti-actin. <b>D</b> U2OS cells were transfected with 1 μg of both ARFΔ<sub>2-14/82-101</sub> and MDM2 expression plasmid as indicated. Equal amounts of protein extract were immunoprecipitated with anti-ARF antibody and revealed by Western Blot with anti-MDM2 and anti-ARF. <b>E</b> U2OS cells were transfected with 0.3 μg ARFΔ<sub>2-14/82-101</sub> and increasing amounts of MDM2 expression plasmid where indicated (0.6–0.9 μg). Levels of endogenous p14ARF were analysed by Western Blot on whole protein lysates probed with anti-ARF and, as control, with anti-MDM2 and anti-actin.</p>", "links"=>[], "tags"=>["p 14ARF phosphorylation mutants", "ubiquitin ligase activity", "MDM 2 overexpression", "pkc", "p 14ARF oncosuppressor", "MDM 2 oncoprotein", "Mdm 2", "cancer cell lines causes p 14ARF reduction", "protein kinase C", "Control ARF Levels", "ARF phosphorylation influences"], "article_id"=>1319688, "categories"=>["Biological Sciences"], "users"=>["Maria Vivo", "Maria Matarese", "Maria Sepe", "Rosaria Di Martino", "Luisa Festa", "Viola Calabrò", "Girolama La Mantia", "Alessandra Pollice"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0117252.g004", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ARF_degradation_by_MDM2_requires_protein_protein_interaction_/1319688", "title"=>"ARF degradation by MDM2 requires protein-protein interaction.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-27 02:47:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/1926624"], "description"=>"<p><b>A</b> U2OS cells were transfected with 0.5 μg of ARF expression plasmid alone (lanes 1, 2) or in combination with 1 μg of MDM2 expression plasmid (lanes 3, 4). 24 hours after transfection cells were treated with Leptomycin B (lanes 1 and 3) or Methanol (lanes 2 and 4). Total cell extracts have been subjected to Western Blot and analyzed with anti-ARF, anti-MDM2 and anti-actin antibodies. <b>B</b> HeLa cells were transfected with increasing amounts of MDM2 expression plasmid (1 and 1.5 μg). 24 hours after transfection, cells were treated with either DMSO, TPA or Bisindolylmalemide and analyzed with anti-ARF, anti-MDM2 and anti-actin antibodies in Western Blot. <b>C</b> HeLa cells were transfected with PKCα siRNA or negative control siRNA (siRNActrl). 48 hours later, cells were mock transfected or transfected with increasing amounts of MDM2 expressing plasmid where indicated (0.6 and 0.8 μg plasmid DNA). Total cell extracts were analyzed by Western blot with anti ARF, anti-MDM2 and anti-actin antibodies. <b>D</b> U2OS cells were transfected with 0.3 μg of either ARFT8A or ARFT8D alone (first lanes) or in combination with increasing amounts of MDM2 expression plasmids (0.3-0.6-0.9 μg). 24 hours after transfection cell extracts were subjected to Western Blot with anti ARF, anti-MDM2 and anti-actin.</p>", "links"=>[], "tags"=>["p 14ARF phosphorylation mutants", "ubiquitin ligase activity", "MDM 2 overexpression", "pkc", "p 14ARF oncosuppressor", "MDM 2 oncoprotein", "Mdm 2", "cancer cell lines causes p 14ARF reduction", "protein kinase C", "Control ARF Levels", "ARF phosphorylation influences"], "article_id"=>1319691, "categories"=>["Biological Sciences"], "users"=>["Maria Vivo", "Maria Matarese", "Maria Sepe", "Rosaria Di Martino", "Luisa Festa", "Viola Calabrò", "Girolama La Mantia", "Alessandra Pollice"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0117252.g006", "stats"=>{"downloads"=>0, "page_views"=>34, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ARF_subcellular_localization_and_phosphorylation_status_influences_its_turnover_/1319691", "title"=>"ARF subcellular localization and phosphorylation status influences its turnover.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-27 02:47:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/1926623"], "description"=>"<p><b>A</b> The scheme indicates MDM2 mutants described in this figure [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117252#pone.0117252.ref025\" target=\"_blank\">25</a>]. <b>B</b> U2OS cells were transfected with a fixed amount of p14ARF expressing plasmid (0.3 μg) and increasing amounts of MDM2<sub>1–441</sub> expressing plasmid (0.3-0.6-0.9 μg). Levels of p14ARF were analysed by Western Blot on whole protein lysates probed with anti-ARF and, as control, with anti-MDM2 and anti-actin. <b>C</b> U2OS cells were transfected with a fixed amount of p14ARF expressing plasmid (0.1 μg) and increasing amounts of MDM2Δ<sub>150–230</sub> expressing plasmid (0.5-1-1.5-2-2,5-3 μg). Levels of p14ARF were analysed by Western Blot on whole protein lysates probed with anti-ARF and, as control, with anti-MDM2 and anti-actin. <b>D</b> U2OS cells were transfected with equal amount (1 μg) of p14ARF expressing plasmid and MDM2<sub>1–441</sub> or MDM2Δ<sub>150–230</sub> expressing plasmid. Equal amounts of protein extract were immunoprecipitated with anti-ARF antibody and revealed by Western Blot with anti-MDM2 and anti-ARF.</p>", "links"=>[], "tags"=>["p 14ARF phosphorylation mutants", "ubiquitin ligase activity", "MDM 2 overexpression", "pkc", "p 14ARF oncosuppressor", "MDM 2 oncoprotein", "Mdm 2", "cancer cell lines causes p 14ARF reduction", "protein kinase C", "Control ARF Levels", "ARF phosphorylation influences"], "article_id"=>1319690, "categories"=>["Biological Sciences"], "users"=>["Maria Vivo", "Maria Matarese", "Maria Sepe", "Rosaria Di Martino", "Luisa Festa", "Viola Calabrò", "Girolama La Mantia", "Alessandra Pollice"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0117252.g005", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ARF_degradation_by_MDM2_does_not_require_the_ubiquitin_ligase_domain_of_MDM2_nor_its_nuclear_nucleolar_localization_/1319690", "title"=>"ARF degradation by MDM2 does not require the ubiquitin ligase domain of MDM2 nor its nuclear/nucleolar localization.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-27 02:47:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/1926613"], "description"=>"<p>H1299 and HeLa cells were treated with either MDM2 or Negative Control directed siRNA (10 nM final concentration) and, after 72 hours of incubation, collected and analysed by Western blot and and qRT-PCR as described in Materials and Methods.</p>", "links"=>[], "tags"=>["p 14ARF phosphorylation mutants", "ubiquitin ligase activity", "MDM 2 overexpression", "pkc", "p 14ARF oncosuppressor", "MDM 2 oncoprotein", "Mdm 2", "cancer cell lines causes p 14ARF reduction", "protein kinase C", "Control ARF Levels", "ARF phosphorylation influences"], "article_id"=>1319684, "categories"=>["Biological Sciences"], "users"=>["Maria Vivo", "Maria Matarese", "Maria Sepe", "Rosaria Di Martino", "Luisa Festa", "Viola Calabrò", "Girolama La Mantia", "Alessandra Pollice"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0117252.g002", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_MDM2_silencing_causes_increase_of_endogenous_ARF_levels_/1319684", "title"=>"MDM2 silencing causes increase of endogenous ARF levels.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-27 02:47:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/1926610"], "description"=>"<p><b>A</b> H1299 cells were mock transfected (first lane) or transfected with increasing amounts of MDM2 expression plasmid (0.3-0.6-0.9 μg). Effect on p14ARF intracellular levels was evaluated by Western Blot on whole protein lysates probed with anti-ARF and, as control, with anti-MDM2 and anti-actin. <b>B</b> HeLa cells were mock transfected or transfected with MDM2 expression plasmid (0.3–0.6 μg) and analysed as in <i>A</i>. <b>C</b> Real Time relative quantification of ARF expression in H1299 and HeLa cells. Cells were transfected by electroporation with MDM2 plasmid (4x10<sup>6</sup> cells/2.5 μg of MDM2 expression plasmid) and subjected to RNA extraction and qRT-PCR analysis. ARF RNA levels were normalized respect to 18s RNA. Values in the graph represents the average of three independent experiments. Standard deviation are shown. <b>D</b> U2OS, <b>E</b> H1299 and <b>F</b> MEF p53<sup>-/-</sup> MDM2<sup>-/-</sup> cells were transfected with a fixed amount of p14ARF expressing plasmid (0.3 μg first lane) alone or with increasing amounts of MDM2 expressing plasmid (0.3-0.6-0.9 μg). Effect on p14ARF levels was evaluated by Western Blot on whole protein lysates, probed with anti-ARF or anti-Xpress (to detect exogenously expressed ARF) and, as control, with anti-MDM2 and anti-actin. Western Blot shown are representative of at least three independent experiments. ARF band intensities were quantified by Image J software, actin normalized and expressed as fold enrichment respect to untreated samples arbitrarily set to 1 (See <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117252#sec008\" target=\"_blank\">Materials and Methods</a> for details).</p>", "links"=>[], "tags"=>["p 14ARF phosphorylation mutants", "ubiquitin ligase activity", "MDM 2 overexpression", "pkc", "p 14ARF oncosuppressor", "MDM 2 oncoprotein", "Mdm 2", "cancer cell lines causes p 14ARF reduction", "protein kinase C", "Control ARF Levels", "ARF phosphorylation influences"], "article_id"=>1319681, "categories"=>["Biological Sciences"], "users"=>["Maria Vivo", "Maria Matarese", "Maria Sepe", "Rosaria Di Martino", "Luisa Festa", "Viola Calabrò", "Girolama La Mantia", "Alessandra Pollice"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0117252.g001", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_MDM2_overexpression_causes_reduction_of_p14ARF_levels_/1319681", "title"=>"MDM2 overexpression causes reduction of p14ARF levels.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-27 02:47:00"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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