Targeted ubiquitination and degradation of G-protein-coupled receptor kinase 5 by the DDB1-CUL4 ubiquitin ligase complex
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...
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| Veröffentlicht in: | PloS one 2012-08, Vol.7 (8), p.e43997-e43997 |
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| creator | Wu, Ziyan Chen, Yuejun Yang, Tong Gao, Qinqin Yuan, Man Ma, Lan |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0043997 |
| format | Article |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0043997</identifier><identifier>PMID: 22952844</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adapter proteins ; Adapters ; Animals ; Biology ; Brain research ; Carrier Proteins - metabolism ; Cattle ; Cell cycle ; Cullin Proteins - metabolism ; Degradation ; Deoxyribonucleic acid ; Desensitization ; Destabilization ; DNA ; DNA-binding protein ; DNA-Binding Proteins - metabolism ; G protein-coupled receptors ; G-Protein-Coupled Receptor Kinase 5 - metabolism ; Gene expression ; Heat shock proteins ; HEK293 Cells ; Hsp90 protein ; Humans ; Immunoprecipitation ; Irradiation ; Kinases ; Laboratories ; Mass Spectrometry ; Mass spectroscopy ; Neurobiology ; Neurosciences ; Pharmacology ; Phosphorylation ; Proteins ; Proteolysis ; Proteolysis - radiation effects ; Proteomics ; Receptor mechanisms ; Receptors ; RNA polymerase ; Science ; Scientific imaging ; Signaling ; U.V. radiation ; Ubiquitin ; Ubiquitin-protein ligase ; Ubiquitination ; Ubiquitination - radiation effects ; Ultraviolet radiation ; Ultraviolet Rays</subject><ispartof>PloS one, 2012-08, Vol.7 (8), p.e43997-e43997</ispartof><rights>Wu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2012 Wu et al 2012 Wu et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c592t-ab6986ca2c79b8b188e662a4bf77b6e8cf9b8683ebcb4259440c5047014b81243</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428324/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428324/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79600,79601</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22952844$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Hess, Sonja</contributor><creatorcontrib>Wu, Ziyan</creatorcontrib><creatorcontrib>Chen, Yuejun</creatorcontrib><creatorcontrib>Yang, Tong</creatorcontrib><creatorcontrib>Gao, Qinqin</creatorcontrib><creatorcontrib>Yuan, Man</creatorcontrib><creatorcontrib>Ma, Lan</creatorcontrib><title>Targeted ubiquitination and degradation of G-protein-coupled receptor kinase 5 by the DDB1-CUL4 ubiquitin ligase complex</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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.</description><subject>Adapter proteins</subject><subject>Adapters</subject><subject>Animals</subject><subject>Biology</subject><subject>Brain research</subject><subject>Carrier Proteins - metabolism</subject><subject>Cattle</subject><subject>Cell cycle</subject><subject>Cullin Proteins - metabolism</subject><subject>Degradation</subject><subject>Deoxyribonucleic acid</subject><subject>Desensitization</subject><subject>Destabilization</subject><subject>DNA</subject><subject>DNA-binding protein</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>G protein-coupled receptors</subject><subject>G-Protein-Coupled Receptor Kinase 5 - metabolism</subject><subject>Gene expression</subject><subject>Heat shock proteins</subject><subject>HEK293 Cells</subject><subject>Hsp90 protein</subject><subject>Humans</subject><subject>Immunoprecipitation</subject><subject>Irradiation</subject><subject>Kinases</subject><subject>Laboratories</subject><subject>Mass Spectrometry</subject><subject>Mass spectroscopy</subject><subject>Neurobiology</subject><subject>Neurosciences</subject><subject>Pharmacology</subject><subject>Phosphorylation</subject><subject>Proteins</subject><subject>Proteolysis</subject><subject>Proteolysis - radiation effects</subject><subject>Proteomics</subject><subject>Receptor mechanisms</subject><subject>Receptors</subject><subject>RNA polymerase</subject><subject>Science</subject><subject>Scientific imaging</subject><subject>Signaling</subject><subject>U.V. radiation</subject><subject>Ubiquitin</subject><subject>Ubiquitin-protein ligase</subject><subject>Ubiquitination</subject><subject>Ubiquitination - radiation effects</subject><subject>Ultraviolet radiation</subject><subject>Ultraviolet Rays</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNptUs1u1DAYjBCIlsIbILDEhUsW_8e5IMEWSqWVuLRny3a-pF6ycWo7qH17st20tIiT7fHMeD5riuItwSvCKvJpG6Y4mH41hgFWGHNW19Wz4pjUjJaSYvb80f6oeJXSFmPBlJQviyNKa0EV58fFzYWJHWRo0GT99eSzH0z2YUBmaFADXTTN4RxadFaOMWTwQ-nCNPazJoKDMYeIfs2yBEgge4vyFaDT06-kXF9u-F9b1Ptuz3FhN2tvXhcvWtMneLOsJ8Xl928X6x_l5ufZ-frLpnSiprk0VtZKOkNdVVtliVIgJTXctlVlJSjXzrBUDKyznIqac-wE5hUm3CpCOTsp3h98xz4kvXxa0oRRKTgTNZkZ5wdGE8xWj9HvTLzVwXh9B4TYaROzdz1oYrBsWy6xNJITZRUAbknFGgdSgKlmr8_La5PdwQwPOZr-ienTm8Ff6S781oxTxe7iflwMYrieIGW988lB35sBwjTnxkyJWpBqT_3wD_X_0_EDy8WQUoT2IQzBel-ke5XeF0kvRZpl7x4P8iC6bw77Ax-dxzk</recordid><startdate>20120827</startdate><enddate>20120827</enddate><creator>Wu, Ziyan</creator><creator>Chen, Yuejun</creator><creator>Yang, Tong</creator><creator>Gao, Qinqin</creator><creator>Yuan, Man</creator><creator>Ma, Lan</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20120827</creationdate><title>Targeted ubiquitination and degradation of G-protein-coupled receptor kinase 5 by the DDB1-CUL4 ubiquitin ligase complex</title><author>Wu, Ziyan ; Chen, Yuejun ; Yang, Tong ; Gao, Qinqin ; Yuan, Man ; Ma, Lan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c592t-ab6986ca2c79b8b188e662a4bf77b6e8cf9b8683ebcb4259440c5047014b81243</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Adapter proteins</topic><topic>Adapters</topic><topic>Animals</topic><topic>Biology</topic><topic>Brain research</topic><topic>Carrier Proteins - metabolism</topic><topic>Cattle</topic><topic>Cell cycle</topic><topic>Cullin Proteins - metabolism</topic><topic>Degradation</topic><topic>Deoxyribonucleic acid</topic><topic>Desensitization</topic><topic>Destabilization</topic><topic>DNA</topic><topic>DNA-binding protein</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>G protein-coupled receptors</topic><topic>G-Protein-Coupled Receptor Kinase 5 - metabolism</topic><topic>Gene expression</topic><topic>Heat shock proteins</topic><topic>HEK293 Cells</topic><topic>Hsp90 protein</topic><topic>Humans</topic><topic>Immunoprecipitation</topic><topic>Irradiation</topic><topic>Kinases</topic><topic>Laboratories</topic><topic>Mass Spectrometry</topic><topic>Mass spectroscopy</topic><topic>Neurobiology</topic><topic>Neurosciences</topic><topic>Pharmacology</topic><topic>Phosphorylation</topic><topic>Proteins</topic><topic>Proteolysis</topic><topic>Proteolysis - radiation effects</topic><topic>Proteomics</topic><topic>Receptor mechanisms</topic><topic>Receptors</topic><topic>RNA polymerase</topic><topic>Science</topic><topic>Scientific imaging</topic><topic>Signaling</topic><topic>U.V. radiation</topic><topic>Ubiquitin</topic><topic>Ubiquitin-protein ligase</topic><topic>Ubiquitination</topic><topic>Ubiquitination - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wu, Ziyan</au><au>Chen, Yuejun</au><au>Yang, Tong</au><au>Gao, Qinqin</au><au>Yuan, Man</au><au>Ma, Lan</au><au>Hess, Sonja</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Targeted ubiquitination and degradation of G-protein-coupled receptor kinase 5 by the DDB1-CUL4 ubiquitin ligase complex</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2012-08-27</date><risdate>2012</risdate><volume>7</volume><issue>8</issue><spage>e43997</spage><epage>e43997</epage><pages>e43997-e43997</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>22952844</pmid><doi>10.1371/journal.pone.0043997</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Adapter proteins Adapters Animals Biology Brain research Carrier Proteins - metabolism Cattle Cell cycle Cullin Proteins - metabolism Degradation Deoxyribonucleic acid Desensitization Destabilization DNA DNA-binding protein DNA-Binding Proteins - metabolism G protein-coupled receptors G-Protein-Coupled Receptor Kinase 5 - metabolism Gene expression Heat shock proteins HEK293 Cells Hsp90 protein Humans Immunoprecipitation Irradiation Kinases Laboratories Mass Spectrometry Mass spectroscopy Neurobiology Neurosciences Pharmacology Phosphorylation Proteins Proteolysis Proteolysis - radiation effects Proteomics Receptor mechanisms Receptors RNA polymerase Science Scientific imaging Signaling U.V. radiation Ubiquitin Ubiquitin-protein ligase Ubiquitination Ubiquitination - radiation effects Ultraviolet radiation Ultraviolet Rays |
| title | Targeted ubiquitination and degradation of G-protein-coupled receptor kinase 5 by the DDB1-CUL4 ubiquitin ligase complex |
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