An E2F dominant negative mutant blocks E1A induced cell cycle progression.
E2F is a cellular transcription factor that is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene (RB1) and the pRb-like p107 and p130 proteins. Analysis of mutations within both adenovirus E1A and pRb, which affected their ability to regulate cellular proliferation and alter E2F activity, suggested that E2F may play a role in cell cycle progression. Microinjection of a GST-E2F-1 fusion protein into quiescent Balb/c 3T3 cells induced DNA synthesis whereas co-injection of GST-E2F-1 and GST-E2F(95-191) protein, encoding only the DNA binding domain of E2F-1, blocked the induction of S-phase. While E1A likely targets multiple cellular pathways, co-injection of the GST-E2F(95-191) dominant inhibitory protein with 12S E1A protein blocked E1A-mediated induction of DNA synthesis, suggesting that the E2F-dependent pathway is dominant. Analysis of the interval required for microinjected quiescent cells to enter S-phase indicated that E2F-1 acted faster than either E1A or serum.
Dobrowolski
S F
SF
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195-5178.
Stacey
D W
DW
Harter
M L
ML
Stine
J T
JT
Hiebert
S W
SW
D016475:3T3 Cells; D017500:Adenovirus E1A Proteins / Q000502:physiology*; D000818:Animals; D002352:Carrier Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D016259:Genes, myc; D005982:Glutathione Transferase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*
D017500:Adenovirus E1A Proteins; C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA; D005982:Glutathione Transferase