Targeted Ubiquitination and Degradation of G-Protein-Coupled Receptor Kinase 5 by the DDB1-CUL4 Ubiquitin Ligase Complex
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{"title"=>"Targeted ubiquitination and degradation of G-protein-coupled receptor kinase 5 by the DDB1-CUL4 Ubiquitin Ligase complex", "type"=>"journal", "authors"=>[{"first_name"=>"Ziyan", "last_name"=>"Wu", "scopus_author_id"=>"35785535800"}, {"first_name"=>"Yuejun", "last_name"=>"Chen", "scopus_author_id"=>"7601439809"}, {"first_name"=>"Tong", "last_name"=>"Yang", "scopus_author_id"=>"57197380374"}, {"first_name"=>"Qinqin", "last_name"=>"Gao", "scopus_author_id"=>"57199950199"}, {"first_name"=>"Man", "last_name"=>"Yuan", "scopus_author_id"=>"36464612800"}, {"first_name"=>"Lan", "last_name"=>"Ma", "scopus_author_id"=>"7403574019"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"22952844", "sgr"=>"84865407821", "doi"=>"10.1371/journal.pone.0043997", "scopus"=>"2-s2.0-84865407821", "pui"=>"365513921", "issn"=>"19326203"}, "id"=>"5c27b1f2-fa51-3738-925a-47ffca29a0fd", "abstract"=>"The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors (GPCRs) and desensitize GPCR-mediated signaling. Recent studies indicate they also function non-catalytically via interaction with other proteins. In this study, a proteomic approach was used to screen interacting proteins of GRK5 in MDA-MB-231 cells and HUVEC cells. Mass spectrometry analysis reveals several proteins in the GRK5 immunocomplex including damaged DNA-binding protein 1 (DDB1), an adaptor subunit of the CUL4-ROC1 E3 ubiquitin ligase complex. Co-immunoprecipitation experiments confirmed the association of GRK5 with DDB1-CUL4 complex, and reveal that DDB1 acts as an adapter to link GRK5 to CUL4 to form the complex. Overexpression of DDB1 promoted, whereas knockdown of DDB1 inhibited the ubiquitination of GRK5, and the degradation of GRK5 was reduced in cells deficient of DDB1. Furthermore, the depletion of DDB1 decreased Hsp90 inhibitor-induced GRK5 destabilization and UV irradiation-induced GRK5 degradation. Thus, our study identified potential GRK5 interacting proteins, and reveals the association of GRK5 with DDB1 in cell and the regulation of GRK5 level by DDB1-CUL4 ubiquitin ligase complex-dependent proteolysis pathway.", "link"=>"http://www.mendeley.com/research/targeted-ubiquitination-degradation-gproteincoupled-receptor-kinase-5-ddb1cul4-ubiquitin-ligase-comp", "reader_count"=>17, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Researcher"=>2, "Student > Ph. D. Student"=>6, "Student > Master"=>7, "Student > Bachelor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Researcher"=>2, "Student > Ph. D. Student"=>6, "Student > Master"=>7, "Student > Bachelor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>12, "Medicine and Dentistry"=>2, "Social Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Social Sciences"=>{"Social Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>12}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"United States"=>1, "Chile"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/585915"], "description"=>"<p>(A,B) MDA-MB-231 cells stably expressing GFP or GRK5-Flag (A), or 293 T cells transiently transfected with GFP or GRK5-Flag (B) were lysed, immunoprecipitated with M2 affinity gel, and analyzed by Western blotting with the indicated antibodies. (C) Endogenous DDB1 complexes were immunoprecipitated from untransfected 293 T cell lysate using the anti-DDB1 antibody and analyzed by Western blotting with GRK5 antibody. Mouse IgG was included as a control. * indicates mouse IgG.</p>", "links"=>[], "tags"=>["associates", "ddb1-cul4", "ubiquitin", "ligase"], "article_id"=>256414, "categories"=>["Biochemistry"], "users"=>["Ziyan Wu", "Yuejun Chen", "Tong Yang", "Qinqin Gao", "Man Yuan", "Lan Ma"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043997.g002", "stats"=>{"downloads"=>2, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_GRK5_associates_with_DDB1_CUL4_ubiquitin_ligase_complex_/256414", "title"=>"GRK5 associates with DDB1-CUL4 ubiquitin ligase complex.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-27 01:46:54"}
  • {"files"=>["https://ndownloader.figshare.com/files/586012"], "description"=>"<p>(A–C) 293 T cells were transiently co-transfected with indicated plasmids alone or co-expressing both or all of them. The cell lysates were immunoprecipitated with M2 affinity gel (A) or anti-HA agarose (B and C) and then the immunoprecipitates and cell lysate input were analyzed by Western blotting with the indicated antibodies. (D) 293 T cells were co-transfected with GRK5-Flag and indicated shRNA plasmids. Cells were immunoprecipitated with M2 affinity gel. The immunoprecipitates and cell lysate input were analyzed by Western blotting with the indicated antibodies. (E) 293 T cells expressing Flag-GRK2, Flag-GRK5, Flag-GRK6, or control plasmid were lysed and immunoprecipitated with M2 affinity gel. The immunoprecipitates and the cell lysate input were analyzed by Western blotting with the indicated antibodies.</p>", "links"=>[], "tags"=>["associates", "grk5", "targets"], "article_id"=>256501, "categories"=>["Biochemistry"], "users"=>["Ziyan Wu", "Yuejun Chen", "Tong Yang", "Qinqin Gao", "Man Yuan", "Lan Ma"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043997.g003", "stats"=>{"downloads"=>1, "page_views"=>24, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_DDB1_associates_with_GRK5_and_targets_GRK5_to_CUL4_/256501", "title"=>"DDB1 associates with GRK5 and targets GRK5 to CUL4.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-27 01:48:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/586212"], "description"=>"<p>(A) 293 T cells were infected with lentivirus expressing control RNAi or DDB1 RNAi. Cells were treated with 100 µg/ml CHX for the indicated time. Cell lysates were analyzed by Western blotting with antibodies specific for the indicated proteins. Quantitative graphs are shown. The amount of GRK5 at 0 h was defined as 100%. Data are the mean±SE from 4 independent experiments. *p<0.01. (two-way ANOVA) compared with control RNAi. (B) 293 T cells were treated with geldanamycin at indicated concentration for 24 h at 37°C. Cell lysates were analyzed by Western blotting with antibodies specific for the indicated proteins. (C) 293 T cells were pretreated with or without 10 µM MG132 for 1 h followed by incubation with geldanamycin for 12 h. Cell lysates were analyzed as in B. (D) 293 T cells were infected by lentivirus encoding control or DDB1 RNAi. Cells were treated with 1 µM geldanamycin for the indicated time. Cell lysates were analyzed as in B.</p>", "links"=>[], "tags"=>["regulates", "grk5"], "article_id"=>256701, "categories"=>["Biochemistry"], "users"=>["Ziyan Wu", "Yuejun Chen", "Tong Yang", "Qinqin Gao", "Man Yuan", "Lan Ma"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043997.g005", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_DDB1_regulates_GRK5_degradation_/256701", "title"=>"DDB1 regulates GRK5 degradation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-27 01:51:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/586124"], "description"=>"<p>(A) 293 T cells were co-transfected with GRK5-Flag, HA-Ub, DDB1, and Myc-CUL4A in various combinations as indicated. Cell lysate was immunoprecipitated with M2 affinity gel. The GRK5-ubiquitin complex was detected with anti-HA antibody after immunoblotting. (B) 293 T cells were co-transfected with GRK5-Flag, HA-Ub, control RNAi, DDB1RNAi, CUL4A RNAi, and CUL4B RNAi in various combinations as indicated. Cells were pretreated with 10 µM MG132 for 4 h, and GRK5-ubiquitin complex was examined as in A.</p>", "links"=>[], "tags"=>["regulates", "grk5"], "article_id"=>256618, "categories"=>["Biochemistry"], "users"=>["Ziyan Wu", "Yuejun Chen", "Tong Yang", "Qinqin Gao", "Man Yuan", "Lan Ma"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043997.g004", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_DDB1_regulates_GRK5_ubiquitination_/256618", "title"=>"DDB1 regulates GRK5 ubiquitination.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-27 01:50:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/586300"], "description"=>"<p>(A) 293 T cells were treated with 20, 100 or 300 J/m<sup>2</sup> of UV-C irradiation. One or two hours after UV treatment, cells were harvested and analyzed by Western blotting with antibodies specific for the indicated proteins. (B) 293 T cells were incubated for 0, 5, 15, 30, 60, or 120 min after 20 J/m<sup>2</sup> UV-C irradiation, and then cells were harvested and analyzed by Western blotting with antibodies specific for the indicated proteins. Quantitative graphs are shown. The amount of GRK5 at 0 h was defined as 100%. Data are the mean±SE from 4 independent experiments. (C) 293 T cells were infected by lentivirus encoding control or DDB1 RNAi. After 72 h, cells were treated with 20 J/m<sup>2</sup> UV-C irradiation, and then incubated for the indicated time. Cells were harvested and analyzed by Western blotting. (D) 293 T cells were pretreated with or without isoproterenol (1 µM) for 20 min before 20 J/m<sup>2</sup> UV-C irradiation, and then incubated in the presence or absence of isoproterenol (1 µM) for the indicated time. Cells were harvested and analyzed by Western blotting.</p>", "links"=>[], "tags"=>["mediates", "uv", "irradiation-induced", "grk5"], "article_id"=>256791, "categories"=>["Biochemistry"], "users"=>["Ziyan Wu", "Yuejun Chen", "Tong Yang", "Qinqin Gao", "Man Yuan", "Lan Ma"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043997.g006", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_DDB1_mediates_UV_irradiation_induced_GRK5_degradation_/256791", "title"=>"DDB1 mediates UV irradiation-induced GRK5 degradation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-27 01:53:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/585740"], "description"=>"<p>(A) Lysates of MDA-MB-231 cells stably expressing GFP or GRK5-Flag were immunoprecipitated with M2 affinity gel. The complexes were separated on SDS-PAGE and stained with Coomassie Blue. Six pieces of gels were excised and labled as line 1-line 6 as indicated. The corresponding control lane was also excised. These gels were analyzed by mass spectrometry. The major associated proteins identified by mass spectrometry were indicated. (B) Lysates of HUVEC cells transiently expressing GFP or GRK5-Flag were treated as in (A). The gels were analyzed by mass spectrometry. The associated proteins identified by mass spectrometry were indicated.</p>", "links"=>[], "tags"=>["grk5", "interacting", "proteins"], "article_id"=>256229, "categories"=>["Biochemistry"], "users"=>["Ziyan Wu", "Yuejun Chen", "Tong Yang", "Qinqin Gao", "Man Yuan", "Lan Ma"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0043997.g001", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Identification_of_GRK5_interacting_proteins_by_mass_spectrometry_/256229", "title"=>"Identification of GRK5 interacting proteins by mass spectrometry.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-27 01:43:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/307928", "https://ndownloader.figshare.com/files/307948", "https://ndownloader.figshare.com/files/307972", "https://ndownloader.figshare.com/files/307995"], "description"=>"<div><p>The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors (GPCRs) and desensitize GPCR-mediated signaling. Recent studies indicate they also function non-catalytically via interaction with other proteins. In this study, a proteomic approach was used to screen interacting proteins of GRK5 in MDA-MB-231 cells and HUVEC cells. Mass spectrometry analysis reveals several proteins in the GRK5 immunocomplex including damaged DNA-binding protein 1 (DDB1), an adaptor subunit of the CUL4-ROC1 E3 ubiquitin ligase complex. Co-immunoprecipitation experiments confirmed the association of GRK5 with DDB1-CUL4 complex, and reveal that DDB1 acts as an adapter to link GRK5 to CUL4 to form the complex. Overexpression of DDB1 promoted, whereas knockdown of DDB1 inhibited the ubiquitination of GRK5, and the degradation of GRK5 was reduced in cells deficient of DDB1. Furthermore, the depletion of DDB1 decreased Hsp90 inhibitor-induced GRK5 destabilization and UV irradiation-induced GRK5 degradation. Thus, our study identified potential GRK5 interacting proteins, and reveals the association of GRK5 with DDB1 in cell and the regulation of GRK5 level by DDB1-CUL4 ubiquitin ligase complex–dependent proteolysis pathway.</p> </div>", "links"=>[], "tags"=>["targeted", "ubiquitination", "degradation", "g-protein-coupled", "receptor", "kinase", "ddb1-cul4", "ubiquitin", "ligase"], "article_id"=>120755, "categories"=>["Biochemistry"], "users"=>["Ziyan Wu", "Yuejun Chen", "Tong Yang", "Qinqin Gao", "Man Yuan", "Lan Ma"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0043997.s001", "https://dx.doi.org/10.1371/journal.pone.0043997.s002", "https://dx.doi.org/10.1371/journal.pone.0043997.s003", "https://dx.doi.org/10.1371/journal.pone.0043997.s004"], "stats"=>{"downloads"=>20, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Targeted_Ubiquitination_and_Degradation_of_G_Protein_Coupled_Receptor_Kinase_5_by_the_DDB1_CUL4_Ubiquitin_Ligase_Complex/120755", "title"=>"Targeted Ubiquitination and Degradation of G-Protein-Coupled Receptor Kinase 5 by the DDB1-CUL4 Ubiquitin Ligase Complex", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-08-27 00:12:35"}

PMC Usage Stats | Further Information

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Relative Metric

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