Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners
Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six...
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| Veröffentlicht in: | Molecular & cellular proteomics 2017-01, Vol.16 (1), p.73-85 |
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| container_title | Molecular & cellular proteomics |
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| creator | Paul, Florian Zauber, Henrik von Berg, Laura Rocks, Oliver Daumke, Oliver Selbach, Matthias |
| description | Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases. |
| doi_str_mv | 10.1074/mcp.M116.061531 |
| format | Article |
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Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases.</description><identifier>ISSN: 1535-9476</identifier><identifier>EISSN: 1535-9484</identifier><identifier>DOI: 10.1074/mcp.M116.061531</identifier><identifier>PMID: 27852748</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Affinity ; Animals ; Binding ; Brain - metabolism ; Cdc42 protein ; Glycoproteins ; Guanosine triphosphatases ; HEK293 Cells ; HeLa Cells ; Humans ; Lysates ; Mass Spectrometry ; Mice ; Molecular machines ; Protein Interaction Maps ; Protein purification ; Proteomics - methods ; Purification ; Rac1 protein ; rho GTP-Binding Proteins - metabolism ; rhoA GTP-Binding Protein - metabolism ; RhoA protein ; Switches</subject><ispartof>Molecular & cellular proteomics, 2017-01, Vol.16 (1), p.73-85</ispartof><rights>2017 © 2017 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>2017 by The American Society for Biochemistry and Molecular Biology, Inc.</rights><rights>Copyright American Society for Biochemistry and Molecular Biology Jan 2017</rights><rights>2017 by The American Society for Biochemistry and Molecular Biology, Inc. 2017 The American Society for Biochemistry and Molecular Biology, Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c616t-53a95257309dd8ed4eaf9c2f362652922ea96b9368c484add3796ae3c1e0b5a43</citedby><cites>FETCH-LOGICAL-c616t-53a95257309dd8ed4eaf9c2f362652922ea96b9368c484add3796ae3c1e0b5a43</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217783/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217783/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27852748$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Paul, Florian</creatorcontrib><creatorcontrib>Zauber, Henrik</creatorcontrib><creatorcontrib>von Berg, Laura</creatorcontrib><creatorcontrib>Rocks, Oliver</creatorcontrib><creatorcontrib>Daumke, Oliver</creatorcontrib><creatorcontrib>Selbach, Matthias</creatorcontrib><title>Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners</title><title>Molecular & cellular proteomics</title><addtitle>Mol Cell Proteomics</addtitle><description>Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases.</description><subject>Affinity</subject><subject>Animals</subject><subject>Binding</subject><subject>Brain - metabolism</subject><subject>Cdc42 protein</subject><subject>Glycoproteins</subject><subject>Guanosine triphosphatases</subject><subject>HEK293 Cells</subject><subject>HeLa Cells</subject><subject>Humans</subject><subject>Lysates</subject><subject>Mass Spectrometry</subject><subject>Mice</subject><subject>Molecular machines</subject><subject>Protein Interaction Maps</subject><subject>Protein purification</subject><subject>Proteomics - methods</subject><subject>Purification</subject><subject>Rac1 protein</subject><subject>rho GTP-Binding Proteins - metabolism</subject><subject>rhoA GTP-Binding Protein - metabolism</subject><subject>RhoA protein</subject><subject>Switches</subject><issn>1535-9476</issn><issn>1535-9484</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkc1LXDEUxYNYqtWu3ZUHbtzMmI-X5GVTEFErWDoVuyuETHJfjbyXTJO8Af_7Zjp2aAuFrm7g_nI49xyETgieEyzb89Gu5h8JEXMsCGdkDx3WwWeq7dr93VuKA_Qm5yeMKSaSv0YHVHacyrY7RF8_TyYUX0zxa2huHhYmQ3PR9z748twspuR7b-syhubWQSV7D7m5f4zNtRn9UJEUC_i6DQWSsT_JhUklQMrH6FVvhgxvX-YR-nJ99XD5YXb36eb28uJuZgURZcaZUZxyybByrgPXgumVpT0TVHCqKAWjxFIx0dl6l3GOSSUMMEsAL7lp2RF6v9VdTcsRnK0-kxn0KvnRpGcdjdd_boJ_1N_iWnNKpOxYFTh7EUjx-wS56NFnC8NgAsQpa9LxTYyK_A_aElKtclrR07_QpzilUJPQRHVMSCHURvB8S9kUc07Q73wTrDcl61qy3pSstyXXH-9-P3fH_2q1AmoLQA197SHpbD0EC84nsEW76P8p_gPenbbk</recordid><startdate>20170101</startdate><enddate>20170101</enddate><creator>Paul, Florian</creator><creator>Zauber, Henrik</creator><creator>von Berg, Laura</creator><creator>Rocks, Oliver</creator><creator>Daumke, Oliver</creator><creator>Selbach, Matthias</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><general>The American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><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>7QO</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20170101</creationdate><title>Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners</title><author>Paul, Florian ; Zauber, Henrik ; von Berg, Laura ; Rocks, Oliver ; Daumke, Oliver ; Selbach, Matthias</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c616t-53a95257309dd8ed4eaf9c2f362652922ea96b9368c484add3796ae3c1e0b5a43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Affinity</topic><topic>Animals</topic><topic>Binding</topic><topic>Brain - metabolism</topic><topic>Cdc42 protein</topic><topic>Glycoproteins</topic><topic>Guanosine triphosphatases</topic><topic>HEK293 Cells</topic><topic>HeLa Cells</topic><topic>Humans</topic><topic>Lysates</topic><topic>Mass Spectrometry</topic><topic>Mice</topic><topic>Molecular machines</topic><topic>Protein Interaction Maps</topic><topic>Protein purification</topic><topic>Proteomics - methods</topic><topic>Purification</topic><topic>Rac1 protein</topic><topic>rho GTP-Binding Proteins - metabolism</topic><topic>rhoA GTP-Binding Protein - metabolism</topic><topic>RhoA protein</topic><topic>Switches</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Paul, Florian</creatorcontrib><creatorcontrib>Zauber, Henrik</creatorcontrib><creatorcontrib>von Berg, Laura</creatorcontrib><creatorcontrib>Rocks, Oliver</creatorcontrib><creatorcontrib>Daumke, Oliver</creatorcontrib><creatorcontrib>Selbach, Matthias</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Molecular & cellular proteomics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Paul, Florian</au><au>Zauber, Henrik</au><au>von Berg, Laura</au><au>Rocks, Oliver</au><au>Daumke, Oliver</au><au>Selbach, Matthias</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners</atitle><jtitle>Molecular & cellular proteomics</jtitle><addtitle>Mol Cell Proteomics</addtitle><date>2017-01-01</date><risdate>2017</risdate><volume>16</volume><issue>1</issue><spage>73</spage><epage>85</epage><pages>73-85</pages><issn>1535-9476</issn><eissn>1535-9484</eissn><abstract>Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>27852748</pmid><doi>10.1074/mcp.M116.061531</doi><tpages>13</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Affinity Animals Binding Brain - metabolism Cdc42 protein Glycoproteins Guanosine triphosphatases HEK293 Cells HeLa Cells Humans Lysates Mass Spectrometry Mice Molecular machines Protein Interaction Maps Protein purification Proteomics - methods Purification Rac1 protein rho GTP-Binding Proteins - metabolism rhoA GTP-Binding Protein - metabolism RhoA protein Switches |
| title | Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners |
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