Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression
The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated...
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Veröffentlicht in: | Genes & development 1998-08, Vol.12 (15), p.2278-2292 |
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description | The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G1 restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication. |
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Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G1 restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication.</description><identifier>ISSN: 0890-9369</identifier><identifier>DOI: 10.1101/gad.12.15.2278</identifier><identifier>PMID: 9694794</identifier><language>eng</language><publisher>United States: Cold Spring Harbor Laboratory Press</publisher><subject>Adenovirus E1A Proteins - pharmacology ; Animals ; Antigens, Polyomavirus Transforming - genetics ; Antigens, Polyomavirus Transforming - pharmacology ; Base Sequence ; Binding Sites - genetics ; Carrier Proteins ; Cell Cycle Proteins ; Cell Line ; Cyclin A - genetics ; Cyclin A - metabolism ; Cyclin A - pharmacology ; Cyclin E - genetics ; Cyclin E - metabolism ; Cyclin E - pharmacology ; DNA - biosynthesis ; DNA Primers - genetics ; DNA-Binding Proteins ; E2F Transcription Factors ; E2F1 Transcription Factor ; G1 Phase - genetics ; G1 Phase - physiology ; Gene Expression ; Mutagenesis, Site-Directed ; Mutation ; Phosphorylation ; Polymerase Chain Reaction ; Rats ; Research Paper ; Retinoblastoma Protein - genetics ; Retinoblastoma Protein - physiology ; Retinoblastoma-Binding Protein 1 ; S Phase - drug effects ; S Phase - genetics ; S Phase - physiology ; Transcription Factor DP1 ; Transcription Factors - genetics ; Transcription Factors - metabolism</subject><ispartof>Genes & development, 1998-08, Vol.12 (15), p.2278-2292</ispartof><rights>Copyright © 1998, Cold Spring Harbor Laboratory Press 1998</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC317048/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC317048/$$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/9694794$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Knudsen, E S</creatorcontrib><creatorcontrib>Buckmaster, C</creatorcontrib><creatorcontrib>Chen, T T</creatorcontrib><creatorcontrib>Feramisco, J R</creatorcontrib><creatorcontrib>Wang, J Y</creatorcontrib><title>Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression</title><title>Genes & development</title><addtitle>Genes Dev</addtitle><description>The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G1 restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication.</description><subject>Adenovirus E1A Proteins - pharmacology</subject><subject>Animals</subject><subject>Antigens, Polyomavirus Transforming - genetics</subject><subject>Antigens, Polyomavirus Transforming - pharmacology</subject><subject>Base Sequence</subject><subject>Binding Sites - genetics</subject><subject>Carrier Proteins</subject><subject>Cell Cycle Proteins</subject><subject>Cell Line</subject><subject>Cyclin A - genetics</subject><subject>Cyclin A - metabolism</subject><subject>Cyclin A - pharmacology</subject><subject>Cyclin E - genetics</subject><subject>Cyclin E - metabolism</subject><subject>Cyclin E - pharmacology</subject><subject>DNA - biosynthesis</subject><subject>DNA Primers - genetics</subject><subject>DNA-Binding Proteins</subject><subject>E2F Transcription Factors</subject><subject>E2F1 Transcription Factor</subject><subject>G1 Phase - genetics</subject><subject>G1 Phase - physiology</subject><subject>Gene Expression</subject><subject>Mutagenesis, Site-Directed</subject><subject>Mutation</subject><subject>Phosphorylation</subject><subject>Polymerase Chain Reaction</subject><subject>Rats</subject><subject>Research Paper</subject><subject>Retinoblastoma Protein - genetics</subject><subject>Retinoblastoma Protein - physiology</subject><subject>Retinoblastoma-Binding Protein 1</subject><subject>S Phase - drug effects</subject><subject>S Phase - genetics</subject><subject>S Phase - physiology</subject><subject>Transcription Factor DP1</subject><subject>Transcription Factors - genetics</subject><subject>Transcription Factors - metabolism</subject><issn>0890-9369</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkDFPwzAQhT2ASimsbEie2JKe7cSJkRigQKlUgUTpHDmO3RildohTpP57IrVCMN3p3vvunQ6hKwIxIUCmG1nFhMYkjSnN8hM0hlxAJBgXZ-g8hE8A4MD5CI0EF0kmkjFaL1xtS9tb77A3-PH1Hoe962sdbMDlHr8_3GJtjFZ9wINlTqYr3HfShQMiXYVXUVvLoHHb-U2nQxjmF-jUyCboy2OdoPXz08fsJVq-zRez-2XUUpLmkUlNaVJBSs5JzoQGAjLLgdPEAKFaqVINrWRlpiTVCQNQDIwRSjLBeWXYBN0d9ra7cqsrpd1wW1O0nd3Kbl94aYv_irN1sfHfBSMZJPnA3xz5zn_tdOiLrQ1KN4102u9CkQOkPOHpYLz-G_SbcPwj-wHaBHOt</recordid><startdate>19980801</startdate><enddate>19980801</enddate><creator>Knudsen, E S</creator><creator>Buckmaster, C</creator><creator>Chen, T T</creator><creator>Feramisco, J R</creator><creator>Wang, J Y</creator><general>Cold Spring Harbor Laboratory Press</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19980801</creationdate><title>Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression</title><author>Knudsen, E S ; Buckmaster, C ; Chen, T T ; Feramisco, J R ; Wang, J Y</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p2158-f5fbf591b661839e010a780624f012eccbc24fa3b7ca2e4300c30ff9ca3966df3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Adenovirus E1A Proteins - pharmacology</topic><topic>Animals</topic><topic>Antigens, Polyomavirus Transforming - genetics</topic><topic>Antigens, Polyomavirus Transforming - pharmacology</topic><topic>Base Sequence</topic><topic>Binding Sites - genetics</topic><topic>Carrier Proteins</topic><topic>Cell Cycle Proteins</topic><topic>Cell Line</topic><topic>Cyclin A - genetics</topic><topic>Cyclin A - metabolism</topic><topic>Cyclin A - pharmacology</topic><topic>Cyclin E - genetics</topic><topic>Cyclin E - metabolism</topic><topic>Cyclin E - pharmacology</topic><topic>DNA - biosynthesis</topic><topic>DNA Primers - genetics</topic><topic>DNA-Binding Proteins</topic><topic>E2F Transcription Factors</topic><topic>E2F1 Transcription Factor</topic><topic>G1 Phase - genetics</topic><topic>G1 Phase - physiology</topic><topic>Gene Expression</topic><topic>Mutagenesis, Site-Directed</topic><topic>Mutation</topic><topic>Phosphorylation</topic><topic>Polymerase Chain Reaction</topic><topic>Rats</topic><topic>Research Paper</topic><topic>Retinoblastoma Protein - genetics</topic><topic>Retinoblastoma Protein - physiology</topic><topic>Retinoblastoma-Binding Protein 1</topic><topic>S Phase - drug effects</topic><topic>S Phase - genetics</topic><topic>S Phase - physiology</topic><topic>Transcription Factor DP1</topic><topic>Transcription Factors - genetics</topic><topic>Transcription Factors - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Knudsen, E S</creatorcontrib><creatorcontrib>Buckmaster, C</creatorcontrib><creatorcontrib>Chen, T T</creatorcontrib><creatorcontrib>Feramisco, J R</creatorcontrib><creatorcontrib>Wang, J Y</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Genes & development</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Knudsen, E S</au><au>Buckmaster, C</au><au>Chen, T T</au><au>Feramisco, J R</au><au>Wang, J Y</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression</atitle><jtitle>Genes & development</jtitle><addtitle>Genes Dev</addtitle><date>1998-08-01</date><risdate>1998</risdate><volume>12</volume><issue>15</issue><spage>2278</spage><epage>2292</epage><pages>2278-2292</pages><issn>0890-9369</issn><abstract>The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G1 restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication.</abstract><cop>United States</cop><pub>Cold Spring Harbor Laboratory Press</pub><pmid>9694794</pmid><doi>10.1101/gad.12.15.2278</doi><tpages>15</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Adenovirus E1A Proteins - pharmacology Animals Antigens, Polyomavirus Transforming - genetics Antigens, Polyomavirus Transforming - pharmacology Base Sequence Binding Sites - genetics Carrier Proteins Cell Cycle Proteins Cell Line Cyclin A - genetics Cyclin A - metabolism Cyclin A - pharmacology Cyclin E - genetics Cyclin E - metabolism Cyclin E - pharmacology DNA - biosynthesis DNA Primers - genetics DNA-Binding Proteins E2F Transcription Factors E2F1 Transcription Factor G1 Phase - genetics G1 Phase - physiology Gene Expression Mutagenesis, Site-Directed Mutation Phosphorylation Polymerase Chain Reaction Rats Research Paper Retinoblastoma Protein - genetics Retinoblastoma Protein - physiology Retinoblastoma-Binding Protein 1 S Phase - drug effects S Phase - genetics S Phase - physiology Transcription Factor DP1 Transcription Factors - genetics Transcription Factors - metabolism |
title | Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression |
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