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An E2F dominant negative mutant blocks E1A induced cell cycle progression
S F Dobrowolski1, D W Stacey, M L Harter
1Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195-5178.
Abstract:
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.
Insights
The transcription factor E2F-1 (E2F) drives cell cycle progression by inducing DNA synthesis. This E2F-dependent pathway is dominant and acts rapidly, even faster than serum stimulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- E2F is a key transcription factor regulating the cell cycle.
- E2F activity is controlled by interactions with retinoblastoma susceptibility gene product (RB1) and related proteins (p107, p130).
- Mutational analysis of adenovirus E1A and RB1 suggested a role for E2F in cell cycle progression.
Purpose of the Study:
- To investigate the role of E2F in cell cycle progression.
- To determine if the E2F pathway is dominant in cellular proliferation.
- To compare the kinetics of E2F-1, E1A, and serum in inducing S-phase entry.
Main Methods:
- Microinjection of GST-E2F-1 fusion protein into quiescent Balb/c 3T3 cells.
- Co-injection experiments using GST-E2F-1 and a dominant inhibitory E2F DNA-binding domain protein (GST-E2F(95-191)).
- Co-injection of dominant inhibitory E2F protein with 12S E1A protein.
- Analysis of the time interval for quiescent cells to enter S-phase post-microinjection.
Main Results:
- Microinjected GST-E2F-1 induced DNA synthesis in quiescent cells.
- Co-injection of GST-E2F-1 and GST-E2F(95-191) blocked S-phase induction.
- Co-injection of dominant inhibitory E2F with 12S E1A blocked E1A-mediated DNA synthesis, indicating E2F pathway dominance.
- E2F-1 induced S-phase entry more rapidly than E1A or serum.
Conclusions:
- E2F-1 is a potent inducer of DNA synthesis and cell cycle progression.
- The E2F-dependent pathway plays a dominant role in mediating proliferation signals.
- E2F-1 acts as a rapid regulator of the G1/S transition.