Activation of the transcription of Gal4-regulated genes by Physarum 14-3-3 in yeast is related to dimer-binding motif-2 and three phosphorylation sites
The roles of 14-3-3 proteins in the lower eukaryotes are still elusive. We isolated a cDNA encoding the 14-3-3 protein (P14-3-3) from the lower eukaryote Physarum polycephalum. This P14-3-3 gene was then inserted downstream of the Gal4 DNA-binding domain in the yeast expression vector pGBKT7. The re...
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| description | The roles of 14-3-3 proteins in the lower eukaryotes are still elusive. We isolated a cDNA encoding the 14-3-3 protein (P14-3-3) from the lower eukaryote Physarum polycephalum. This P14-3-3 gene was then inserted downstream of the Gal4 DNA-binding domain in the yeast expression vector pGBKT7. The recombinant vector was transformed into auxotrophic AH109 and Y187 yeast cells to detect the activation of Gal4-regulated gene expression mediated by P14-3-3. The results showed that three reporter genes (ADE2, HIS3, and lacZ) could be normally expressed, indicating that the transcriptional activation function of P14-3-3 was retained. We subsequently used a truncated P14-3-3 peptides and mutant peptides to study the activation of the Gal4-regulated genes ADE2, HIS3, and lacZ. We found that deletion of the N-terminal second dimer-binding motif (DBM2) sequence or the C-terminal coil sequence led to the loss of P14-3-3's transcriptional activation function. Specifically, any mutation at the potential phosphorylation sites (Ser62 and Ser67) on DBM2 or at the C-terminal potential phosphorylation site (235ThrSer236) led to the loss of the transcriptional activation function of P14-3-3. Taken together, these observations suggest that the transcriptional activation function of P14-3-3 in lower eukaryotes is related to DBM2 and the C-terminal coil structures. |
| doi_str_mv | 10.1007/s00203-009-0526-3 |
| format | Article |
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We isolated a cDNA encoding the 14-3-3 protein (P14-3-3) from the lower eukaryote Physarum polycephalum. This P14-3-3 gene was then inserted downstream of the Gal4 DNA-binding domain in the yeast expression vector pGBKT7. The recombinant vector was transformed into auxotrophic AH109 and Y187 yeast cells to detect the activation of Gal4-regulated gene expression mediated by P14-3-3. The results showed that three reporter genes (ADE2, HIS3, and lacZ) could be normally expressed, indicating that the transcriptional activation function of P14-3-3 was retained. We subsequently used a truncated P14-3-3 peptides and mutant peptides to study the activation of the Gal4-regulated genes ADE2, HIS3, and lacZ. We found that deletion of the N-terminal second dimer-binding motif (DBM2) sequence or the C-terminal coil sequence led to the loss of P14-3-3's transcriptional activation function. Specifically, any mutation at the potential phosphorylation sites (Ser62 and Ser67) on DBM2 or at the C-terminal potential phosphorylation site (235ThrSer236) led to the loss of the transcriptional activation function of P14-3-3. Taken together, these observations suggest that the transcriptional activation function of P14-3-3 in lower eukaryotes is related to DBM2 and the C-terminal coil structures.</description><identifier>ISSN: 0302-8933</identifier><identifier>EISSN: 1432-072X</identifier><identifier>DOI: 10.1007/s00203-009-0526-3</identifier><identifier>PMID: 19936707</identifier><identifier>CODEN: AMICCW</identifier><language>eng</language><publisher>Berlin/Heidelberg: Berlin/Heidelberg : Springer-Verlag</publisher><subject>14-3-3 Proteins - chemistry ; 14-3-3 Proteins - genetics ; 14-3-3 Proteins - metabolism ; Amino Acid Motifs - genetics ; Amino Acid Sequence ; Apoptosis ; Base Sequence ; Binding Sites - genetics ; Biochemistry ; Biological and medical sciences ; Biomedical and Life Sciences ; Biotechnology ; Cell Biology ; Cell cycle ; Cloning ; DNA, Fungal - genetics ; DNA, Fungal - metabolism ; Ecology ; Eukaryotes ; Fundamental and applied biological sciences. Psychology ; Gene Expression Regulation, Fungal ; Genes ; Genetic engineering ; Growth, nutrition, metabolism, transports, enzymes. Molecular biology ; Kinases ; Life Sciences ; Microbial Ecology ; Microbiology ; Molecular Sequence Data ; Mutation ; Mycology ; Nuclear Proteins - genetics ; Nuclear Proteins - metabolism ; Original Paper ; Peptides ; Phosphorylation ; Physarum polycephalum - genetics ; Physarum polycephalum - metabolism ; Plasmids ; Promoter Regions, Genetic ; Protein Structure, Tertiary - genetics ; Proteins ; Protozoan Proteins - chemistry ; Protozoan Proteins - genetics ; Protozoan Proteins - metabolism ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Repressor Proteins - genetics ; Repressor Proteins - metabolism ; Sequence Deletion ; Signal transduction ; Transcription Factors - metabolism ; Transcriptional Activation ; Yeast ; Yeasts ; Yeasts - genetics ; Yeasts - metabolism</subject><ispartof>Archives of microbiology, 2010, Vol.192 (1), p.33-40</ispartof><rights>Springer-Verlag 2009</rights><rights>2015 INIST-CNRS</rights><rights>Springer-Verlag 2010</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c424t-6ef3d40f320468ef031804ce44972a8d9cc9dc94ff6b9742ac0877b5f3338e2b3</citedby><cites>FETCH-LOGICAL-c424t-6ef3d40f320468ef031804ce44972a8d9cc9dc94ff6b9742ac0877b5f3338e2b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00203-009-0526-3$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00203-009-0526-3$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,4009,27902,27903,27904,41467,42536,51297</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=22382500$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19936707$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Liu, Shide</creatorcontrib><creatorcontrib>Li, Minghua</creatorcontrib><creatorcontrib>Zhang, Jianhua</creatorcontrib><creatorcontrib>Kang, Kang</creatorcontrib><creatorcontrib>Tian, Shengli</creatorcontrib><creatorcontrib>Wang, Yisi</creatorcontrib><creatorcontrib>Xing, Miao</creatorcontrib><title>Activation of the transcription of Gal4-regulated genes by Physarum 14-3-3 in yeast is related to dimer-binding motif-2 and three phosphorylation sites</title><title>Archives of microbiology</title><addtitle>Arch Microbiol</addtitle><addtitle>Arch Microbiol</addtitle><description>The roles of 14-3-3 proteins in the lower eukaryotes are still elusive. We isolated a cDNA encoding the 14-3-3 protein (P14-3-3) from the lower eukaryote Physarum polycephalum. This P14-3-3 gene was then inserted downstream of the Gal4 DNA-binding domain in the yeast expression vector pGBKT7. The recombinant vector was transformed into auxotrophic AH109 and Y187 yeast cells to detect the activation of Gal4-regulated gene expression mediated by P14-3-3. The results showed that three reporter genes (ADE2, HIS3, and lacZ) could be normally expressed, indicating that the transcriptional activation function of P14-3-3 was retained. We subsequently used a truncated P14-3-3 peptides and mutant peptides to study the activation of the Gal4-regulated genes ADE2, HIS3, and lacZ. We found that deletion of the N-terminal second dimer-binding motif (DBM2) sequence or the C-terminal coil sequence led to the loss of P14-3-3's transcriptional activation function. Specifically, any mutation at the potential phosphorylation sites (Ser62 and Ser67) on DBM2 or at the C-terminal potential phosphorylation site (235ThrSer236) led to the loss of the transcriptional activation function of P14-3-3. Taken together, these observations suggest that the transcriptional activation function of P14-3-3 in lower eukaryotes is related to DBM2 and the C-terminal coil structures.</description><subject>14-3-3 Proteins - chemistry</subject><subject>14-3-3 Proteins - genetics</subject><subject>14-3-3 Proteins - metabolism</subject><subject>Amino Acid Motifs - genetics</subject><subject>Amino Acid Sequence</subject><subject>Apoptosis</subject><subject>Base Sequence</subject><subject>Binding Sites - genetics</subject><subject>Biochemistry</subject><subject>Biological and medical sciences</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnology</subject><subject>Cell Biology</subject><subject>Cell cycle</subject><subject>Cloning</subject><subject>DNA, Fungal - genetics</subject><subject>DNA, Fungal - metabolism</subject><subject>Ecology</subject><subject>Eukaryotes</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression Regulation, Fungal</subject><subject>Genes</subject><subject>Genetic engineering</subject><subject>Growth, nutrition, metabolism, transports, enzymes. Molecular biology</subject><subject>Kinases</subject><subject>Life Sciences</subject><subject>Microbial Ecology</subject><subject>Microbiology</subject><subject>Molecular Sequence Data</subject><subject>Mutation</subject><subject>Mycology</subject><subject>Nuclear Proteins - genetics</subject><subject>Nuclear Proteins - metabolism</subject><subject>Original Paper</subject><subject>Peptides</subject><subject>Phosphorylation</subject><subject>Physarum polycephalum - genetics</subject><subject>Physarum polycephalum - metabolism</subject><subject>Plasmids</subject><subject>Promoter Regions, Genetic</subject><subject>Protein Structure, Tertiary - genetics</subject><subject>Proteins</subject><subject>Protozoan Proteins - chemistry</subject><subject>Protozoan Proteins - genetics</subject><subject>Protozoan Proteins - metabolism</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Repressor Proteins - genetics</subject><subject>Repressor Proteins - metabolism</subject><subject>Sequence Deletion</subject><subject>Signal transduction</subject><subject>Transcription Factors - metabolism</subject><subject>Transcriptional Activation</subject><subject>Yeast</subject><subject>Yeasts</subject><subject>Yeasts - genetics</subject><subject>Yeasts - metabolism</subject><issn>0302-8933</issn><issn>1432-072X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp9kduKFDEQhhtR3NnVB_BGg7B4Fa2kMn24XBZdhQUFXfAupNOVmSx9GJO00E_i65qhxwNeeBECqa_-FPUVxTMBrwVA9SYCSEAO0HDYypLjg2IjFEoOlfz6sNgAguR1g3hWnMd4DyBkXdePizPRNFhWUG2KH1c2-e8m-Wlkk2NpTywFM0Yb_OHX443pFQ-0m3uTqGM7GimydmGf9ks0YR6YUBw5Mj-yhUxMzEcWaIXTxDo_UOCtHzs_7tgwJe-4ZGbMxX0gYof9FPMJS7-OEX2i-KR45Ewf6enpviju3r39cv2e3368-XB9dcutkirxkhx2ChxKUGVNDlDUoCwp1VTS1F1jbdPZRjlXtk2lpLFQV1W7dYhYk2zxoni15h7C9G2mmPTgo6W-NyNNc9QVYpm3pjCTL_8h76c5jHk4fVynwG0jMyRWyIYpxkBOH4IfTFi0AH10pldnOjvTR2f6GPz8FDy3A3V_Ok6SMnB5Aky0pnfZj_XxNycl1nILkDm5cjGXxh2Fvyb8z-8v1iZnJm12IQfffZYgEEQlBWwBfwLgZ7fO</recordid><startdate>2010</startdate><enddate>2010</enddate><creator>Liu, Shide</creator><creator>Li, Minghua</creator><creator>Zhang, Jianhua</creator><creator>Kang, Kang</creator><creator>Tian, Shengli</creator><creator>Wang, Yisi</creator><creator>Xing, Miao</creator><general>Berlin/Heidelberg : Springer-Verlag</general><general>Springer-Verlag</general><general>Springer</general><general>Springer Nature B.V</general><scope>FBQ</scope><scope>IQODW</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>3V.</scope><scope>7QL</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</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>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>2010</creationdate><title>Activation of the transcription of Gal4-regulated genes by Physarum 14-3-3 in yeast is related to dimer-binding motif-2 and three phosphorylation sites</title><author>Liu, Shide ; Li, Minghua ; Zhang, Jianhua ; Kang, Kang ; Tian, Shengli ; Wang, Yisi ; Xing, Miao</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c424t-6ef3d40f320468ef031804ce44972a8d9cc9dc94ff6b9742ac0877b5f3338e2b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>14-3-3 Proteins - chemistry</topic><topic>14-3-3 Proteins - genetics</topic><topic>14-3-3 Proteins - metabolism</topic><topic>Amino Acid Motifs - genetics</topic><topic>Amino Acid Sequence</topic><topic>Apoptosis</topic><topic>Base Sequence</topic><topic>Binding Sites - genetics</topic><topic>Biochemistry</topic><topic>Biological and medical sciences</topic><topic>Biomedical and Life Sciences</topic><topic>Biotechnology</topic><topic>Cell Biology</topic><topic>Cell cycle</topic><topic>Cloning</topic><topic>DNA, Fungal - genetics</topic><topic>DNA, Fungal - metabolism</topic><topic>Ecology</topic><topic>Eukaryotes</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression Regulation, Fungal</topic><topic>Genes</topic><topic>Genetic engineering</topic><topic>Growth, nutrition, metabolism, transports, enzymes. Molecular biology</topic><topic>Kinases</topic><topic>Life Sciences</topic><topic>Microbial Ecology</topic><topic>Microbiology</topic><topic>Molecular Sequence Data</topic><topic>Mutation</topic><topic>Mycology</topic><topic>Nuclear Proteins - genetics</topic><topic>Nuclear Proteins - metabolism</topic><topic>Original Paper</topic><topic>Peptides</topic><topic>Phosphorylation</topic><topic>Physarum polycephalum - genetics</topic><topic>Physarum polycephalum - metabolism</topic><topic>Plasmids</topic><topic>Promoter Regions, Genetic</topic><topic>Protein Structure, Tertiary - genetics</topic><topic>Proteins</topic><topic>Protozoan Proteins - chemistry</topic><topic>Protozoan Proteins - genetics</topic><topic>Protozoan Proteins - metabolism</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Repressor Proteins - genetics</topic><topic>Repressor Proteins - metabolism</topic><topic>Sequence Deletion</topic><topic>Signal transduction</topic><topic>Transcription Factors - metabolism</topic><topic>Transcriptional Activation</topic><topic>Yeast</topic><topic>Yeasts</topic><topic>Yeasts - genetics</topic><topic>Yeasts - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Liu, Shide</creatorcontrib><creatorcontrib>Li, Minghua</creatorcontrib><creatorcontrib>Zhang, Jianhua</creatorcontrib><creatorcontrib>Kang, Kang</creatorcontrib><creatorcontrib>Tian, Shengli</creatorcontrib><creatorcontrib>Wang, Yisi</creatorcontrib><creatorcontrib>Xing, Miao</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Archives of microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Liu, Shide</au><au>Li, Minghua</au><au>Zhang, Jianhua</au><au>Kang, Kang</au><au>Tian, Shengli</au><au>Wang, Yisi</au><au>Xing, Miao</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Activation of the transcription of Gal4-regulated genes by Physarum 14-3-3 in yeast is related to dimer-binding motif-2 and three phosphorylation sites</atitle><jtitle>Archives of microbiology</jtitle><stitle>Arch Microbiol</stitle><addtitle>Arch Microbiol</addtitle><date>2010</date><risdate>2010</risdate><volume>192</volume><issue>1</issue><spage>33</spage><epage>40</epage><pages>33-40</pages><issn>0302-8933</issn><eissn>1432-072X</eissn><coden>AMICCW</coden><abstract>The roles of 14-3-3 proteins in the lower eukaryotes are still elusive. We isolated a cDNA encoding the 14-3-3 protein (P14-3-3) from the lower eukaryote Physarum polycephalum. This P14-3-3 gene was then inserted downstream of the Gal4 DNA-binding domain in the yeast expression vector pGBKT7. The recombinant vector was transformed into auxotrophic AH109 and Y187 yeast cells to detect the activation of Gal4-regulated gene expression mediated by P14-3-3. The results showed that three reporter genes (ADE2, HIS3, and lacZ) could be normally expressed, indicating that the transcriptional activation function of P14-3-3 was retained. We subsequently used a truncated P14-3-3 peptides and mutant peptides to study the activation of the Gal4-regulated genes ADE2, HIS3, and lacZ. We found that deletion of the N-terminal second dimer-binding motif (DBM2) sequence or the C-terminal coil sequence led to the loss of P14-3-3's transcriptional activation function. Specifically, any mutation at the potential phosphorylation sites (Ser62 and Ser67) on DBM2 or at the C-terminal potential phosphorylation site (235ThrSer236) led to the loss of the transcriptional activation function of P14-3-3. Taken together, these observations suggest that the transcriptional activation function of P14-3-3 in lower eukaryotes is related to DBM2 and the C-terminal coil structures.</abstract><cop>Berlin/Heidelberg</cop><pub>Berlin/Heidelberg : Springer-Verlag</pub><pmid>19936707</pmid><doi>10.1007/s00203-009-0526-3</doi><tpages>8</tpages></addata></record> |
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| ispartof | Archives of microbiology, 2010, Vol.192 (1), p.33-40 |
| issn | 0302-8933 1432-072X |
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| source | MEDLINE; Springer Nature - Complete Springer Journals |
| subjects | 14-3-3 Proteins - chemistry 14-3-3 Proteins - genetics 14-3-3 Proteins - metabolism Amino Acid Motifs - genetics Amino Acid Sequence Apoptosis Base Sequence Binding Sites - genetics Biochemistry Biological and medical sciences Biomedical and Life Sciences Biotechnology Cell Biology Cell cycle Cloning DNA, Fungal - genetics DNA, Fungal - metabolism Ecology Eukaryotes Fundamental and applied biological sciences. Psychology Gene Expression Regulation, Fungal Genes Genetic engineering Growth, nutrition, metabolism, transports, enzymes. Molecular biology Kinases Life Sciences Microbial Ecology Microbiology Molecular Sequence Data Mutation Mycology Nuclear Proteins - genetics Nuclear Proteins - metabolism Original Paper Peptides Phosphorylation Physarum polycephalum - genetics Physarum polycephalum - metabolism Plasmids Promoter Regions, Genetic Protein Structure, Tertiary - genetics Proteins Protozoan Proteins - chemistry Protozoan Proteins - genetics Protozoan Proteins - metabolism Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Repressor Proteins - genetics Repressor Proteins - metabolism Sequence Deletion Signal transduction Transcription Factors - metabolism Transcriptional Activation Yeast Yeasts Yeasts - genetics Yeasts - metabolism |
| title | Activation of the transcription of Gal4-regulated genes by Physarum 14-3-3 in yeast is related to dimer-binding motif-2 and three phosphorylation sites |
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