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
Hauptverfasser: Knudsen, E S, Buckmaster, C, Chen, T T, Feramisco, J R, Wang, J Y
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creator Knudsen, E S
Buckmaster, C
Chen, T T
Feramisco, J R
Wang, J Y
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. 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However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. 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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 &amp; 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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