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 (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.
Department of Biology, University of California, San Diego (UCSD), La Jolla, California 92093-0322 USA.
E S
ES
Knudsen
C
C
Buckmaster
T T
TT
Chen
J R
JR
Feramisco
J Y
JY
Wang
D017500:Adenovirus E1A Proteins / Q000494:pharmacology; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D019925:Cyclin A / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis*; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D016193:G1 Phase / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D051381:Rats; D016160:Retinoblastoma Protein / Q000235:genetics / Q000502:physiology*; D056465:Retinoblastoma-Binding Protein 1; D016196:S Phase / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D017500:Adenovirus E1A Proteins; D000952:Antigens, Polyomavirus Transforming; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019925:Cyclin A; D019927:Cyclin E; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493755:E2f1 protein, rat; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA