Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase
The ubiquitin ligase SCFCdc4 (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2012-02, Vol.109 (9), p.3287-3292 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Tang, Xiaojing Orlicky, Stephen Mittag, Tanja Csizmok, Veronika Pawson, Tony Forman-Kay, Julie D Sicheri, Frank Tyers, Mike |
| description | The ubiquitin ligase SCFCdc4 (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. Composite dynamic interactions of low affinity sites may be a general mechanism to establish phosphorylation thresholds in biological responses. |
| doi_str_mv | 10.1073/pnas.1116455109 |
| format | Article |
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Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. 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Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. Composite dynamic interactions of low affinity sites may be a general mechanism to establish phosphorylation thresholds in biological responses.</description><subject>Amino Acid Motifs</subject><subject>Amino Acid Sequence</subject><subject>Biological Sciences</subject><subject>Cell Cycle Proteins - chemistry</subject><subject>Cell Cycle Proteins - genetics</subject><subject>Cell Cycle Proteins - metabolism</subject><subject>Consensus Sequence</subject><subject>cyclin-dependent kinase</subject><subject>Cyclin-Dependent Kinase Inhibitor Proteins - chemistry</subject><subject>Cyclin-Dependent Kinase Inhibitor Proteins - genetics</subject><subject>Cyclin-Dependent Kinase Inhibitor Proteins - metabolism</subject><subject>F-Box Proteins - chemistry</subject><subject>F-Box Proteins - genetics</subject><subject>F-Box Proteins - metabolism</subject><subject>Hydrogen Bonding</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>mutagenesis</subject><subject>peptides</subject><subject>Phosphorylation</subject><subject>Protein Conformation</subject><subject>Protein Interaction Mapping</subject><subject>Protein Processing, Post-Translational</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Saccharomyces cerevisiae - enzymology</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Saccharomyces cerevisiae Proteins - chemistry</subject><subject>Saccharomyces cerevisiae Proteins - genetics</subject><subject>Saccharomyces cerevisiae Proteins - metabolism</subject><subject>Static Electricity</subject><subject>Surface Plasmon Resonance</subject><subject>ubiquitin</subject><subject>Ubiquitin-Protein Ligases - chemistry</subject><subject>Ubiquitin-Protein Ligases - genetics</subject><subject>Ubiquitin-Protein Ligases - metabolism</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkctOwzAQRS0EglJYswP_QMDjR5NskFBEAakSi8I6chynHUjtkLigfgD_jXlVsJmR5p65upoh5ATYObBUXHROD-cAMJFKAct3yChWSCYyZ7tkxBhPk0xyeUAOh-GJMZarjO2TA84Fz0DlI_Je-FXnBwyWtv6N6qZBh2FD0QXbaxPQu4HWaIKORG-NX0Q5DqlvaFhaajamRZfUtrOuti7QZ4yRbNxfYoXB93SOBmi1-aLnxbSojaTrCl_W0cfRFhcRPyJ7jW4He_zTx-Rxev1Q3Caz-5u74mqWNFzykGibW5krninDFJsI4KAAhExrwYFJPUlTWxnDQWguqwpEYwVAnRolQDGdijG5_Pbt1tXK1iYG7nVbdj2udL8pvcbyv-JwWS78ayl4rgQT0eD0r8F28_egf4B4hq0cn1Lm0ST7jHD2DTTal3rR41A-zjkDGb-TQRqzfwD-J44B</recordid><startdate>20120228</startdate><enddate>20120228</enddate><creator>Tang, Xiaojing</creator><creator>Orlicky, Stephen</creator><creator>Mittag, Tanja</creator><creator>Csizmok, Veronika</creator><creator>Pawson, Tony</creator><creator>Forman-Kay, Julie D</creator><creator>Sicheri, Frank</creator><creator>Tyers, Mike</creator><general>National Academy of Sciences</general><general>National Acad Sciences</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>5PM</scope></search><sort><creationdate>20120228</creationdate><title>Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase</title><author>Tang, Xiaojing ; Orlicky, Stephen ; Mittag, Tanja ; Csizmok, Veronika ; Pawson, Tony ; Forman-Kay, Julie D ; Sicheri, Frank ; Tyers, Mike</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-f242t-ae9e495285c05063121511347d32104a677ebcc213a24bb13fe311d7c53150a73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Amino Acid Motifs</topic><topic>Amino Acid Sequence</topic><topic>Biological Sciences</topic><topic>Cell Cycle Proteins - chemistry</topic><topic>Cell Cycle Proteins - genetics</topic><topic>Cell Cycle Proteins - metabolism</topic><topic>Consensus Sequence</topic><topic>cyclin-dependent kinase</topic><topic>Cyclin-Dependent Kinase Inhibitor Proteins - chemistry</topic><topic>Cyclin-Dependent Kinase Inhibitor Proteins - genetics</topic><topic>Cyclin-Dependent Kinase Inhibitor Proteins - metabolism</topic><topic>F-Box Proteins - chemistry</topic><topic>F-Box Proteins - genetics</topic><topic>F-Box Proteins - metabolism</topic><topic>Hydrogen Bonding</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>mutagenesis</topic><topic>peptides</topic><topic>Phosphorylation</topic><topic>Protein Conformation</topic><topic>Protein Interaction Mapping</topic><topic>Protein Processing, Post-Translational</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Saccharomyces cerevisiae - enzymology</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Saccharomyces cerevisiae Proteins - chemistry</topic><topic>Saccharomyces cerevisiae Proteins - genetics</topic><topic>Saccharomyces cerevisiae Proteins - metabolism</topic><topic>Static Electricity</topic><topic>Surface Plasmon Resonance</topic><topic>ubiquitin</topic><topic>Ubiquitin-Protein Ligases - chemistry</topic><topic>Ubiquitin-Protein Ligases - genetics</topic><topic>Ubiquitin-Protein Ligases - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Tang, Xiaojing</creatorcontrib><creatorcontrib>Orlicky, Stephen</creatorcontrib><creatorcontrib>Mittag, Tanja</creatorcontrib><creatorcontrib>Csizmok, Veronika</creatorcontrib><creatorcontrib>Pawson, Tony</creatorcontrib><creatorcontrib>Forman-Kay, Julie D</creatorcontrib><creatorcontrib>Sicheri, Frank</creatorcontrib><creatorcontrib>Tyers, Mike</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Tang, Xiaojing</au><au>Orlicky, Stephen</au><au>Mittag, Tanja</au><au>Csizmok, Veronika</au><au>Pawson, Tony</au><au>Forman-Kay, Julie D</au><au>Sicheri, Frank</au><au>Tyers, Mike</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>2012-02-28</date><risdate>2012</risdate><volume>109</volume><issue>9</issue><spage>3287</spage><epage>3292</epage><pages>3287-3292</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>The ubiquitin ligase SCFCdc4 (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. 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| subjects | Amino Acid Motifs Amino Acid Sequence Biological Sciences Cell Cycle Proteins - chemistry Cell Cycle Proteins - genetics Cell Cycle Proteins - metabolism Consensus Sequence cyclin-dependent kinase Cyclin-Dependent Kinase Inhibitor Proteins - chemistry Cyclin-Dependent Kinase Inhibitor Proteins - genetics Cyclin-Dependent Kinase Inhibitor Proteins - metabolism F-Box Proteins - chemistry F-Box Proteins - genetics F-Box Proteins - metabolism Hydrogen Bonding Models, Molecular Molecular Sequence Data mutagenesis peptides Phosphorylation Protein Conformation Protein Interaction Mapping Protein Processing, Post-Translational Recombinant Fusion Proteins - metabolism Saccharomyces cerevisiae - enzymology Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae Proteins - chemistry Saccharomyces cerevisiae Proteins - genetics Saccharomyces cerevisiae Proteins - metabolism Static Electricity Surface Plasmon Resonance ubiquitin Ubiquitin-Protein Ligases - chemistry Ubiquitin-Protein Ligases - genetics Ubiquitin-Protein Ligases - metabolism |
| title | Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase |
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