Engineered mutants of pRB with improved growth suppression potential.
We have constructed a panel of substitution mutants which affect one or more of the putative cdk target sites of the RB protein. We have examined the activity of these mutants relative to wild-type RB by both a transcriptional repression assay and by measuring growth suppression in vitro. We find that some phosphorylation site mutants of pRB can repress E2 transcription more strongly than wild-type RB. These mutants are partially resistant to phosphorylation by cdks and can arrest tumor cells in G1 in vitro. Our results indicate a functional correlation between the ability to repress E2F-dependent transcription and the ability to suppress tumor cell growth in vitro. In addition, we describe two classes of RB mutants: N-terminal truncated p56RB and a novel mutant of RB containing multiple substitutions near its nuclear localization signal. Both classes of RB mutants have greater activity than the wild-type protein. Because RB is a key regulator of cell cycle progression, expression of a more potent, phosphorylation resistant RB may have utility in both RB(-/-) and RB(+/+) tumors as well as in hyperproliferative disorders.
Antelman
D
D
Canji Inc., San Diego, California 92121, USA.
Perry
S
S
Hollingsworth
R
R
Gregory
R J
RJ
Driscoll
B
B
Fung
Y K
YK
Bookstein
R
R
D019943:Amino Acid Substitution / Q000235:genetics*; D000970:Antineoplastic Agents / Q000037:antagonists & inhibitors / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019927:Cyclin E / Q000502:physiology; D018844:Cyclin-Dependent Kinases / Q000502:physiology; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016193:G1 Phase / Q000187:drug effects / Q000235:genetics; D006131:Growth Inhibitors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D012516:Osteosarcoma; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D056465:Retinoblastoma-Binding Protein 1; D017384:Sequence Deletion; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000502:physiology; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019927:Cyclin E; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D006131:Growth Inhibitors; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D018844:Cyclin-Dependent Kinases