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The viral oncoprotein E1A blocks transforming growth factor beta-mediated induction of p21/WAF1/Cip1 and p15/INK4B
M B Datto1, P P Hu, T F Kowalik
1Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
The adenovirus early gene product E1A is a potent stimulator of cellular proliferation, which when overexpressed can overcome the growth-inhibitory effects of the polypeptide hormone transforming growth factor beta (TGF-beta). The ability of TGF-beta to arrest cell growth in G1 correlates with the transcriptional induction of the cyclin-dependent kinase inhibitors, p15/INK4B and p21/WAF1/Cip1; an inhibition of the G1 cyclin-Cdk complexes; and a maintenance of the retinoblastoma susceptibility gene product, Rb, in a hypophosphorylated state. The ability of E1A to overcome TGF-beta-mediated growth inhibition derives, in part, from its ability to sequester Rb and Rb family members. We report here that E1A also acts upstream of Rb by blocking the TGF-beta-mediated induction of p15 and p21. Consistent with these findings, E1A expression also blocks the ability of TGF-beta to inhibit Cdk2 kinase activity, as well as its ability to hold Rb in a hypophosphorylated state. The effect of E1A on the induction of p15 and p21 is independent of E1A's Rb binding activity. The E1A-mediated decrease in p15 levels is primarily the result of a block at the level of transcriptional activation by TGF-beta. This effect is dependent on E1A's ability to bind p300, one of E1A's target proteins. Thus, the ability of E1A to affect p15 and p21 expression represents an additional possible mechanism by which E1A can circumvent the negative regulation of cell cycle progression.
Insights
Adenovirus E1A protein overcomes transforming growth factor beta (TGF-beta) growth inhibition by blocking the induction of p15 and p21 cell cycle regulators. This mechanism involves E1A binding to p300, impacting cell proliferation control.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Virology
Background:
- Transforming growth factor beta (TGF-beta) inhibits cell proliferation by inducing cell cycle arrest in G1.
- Key mediators of TGF-beta's growth-inhibitory effects include p15/INK4B and p21/WAF1/Cip1, inhibition of cyclin-dependent kinase (Cdk) complexes, and hypophosphorylation of the retinoblastoma protein (Rb).
- Adenovirus early gene product E1A can override TGF-beta's growth suppression, partly by sequestering Rb and its family members.
Purpose of the Study:
- To investigate the mechanism by which adenovirus E1A overcomes TGF-beta-mediated growth inhibition.
- To determine if E1A acts upstream of Rb in blocking TGF-beta signaling.
- To elucidate the role of E1A's interaction with p300 in regulating p15 and p21 expression.
Main Methods:
- Cell-based assays to assess cell proliferation and cell cycle progression.
- Western blotting and quantitative PCR to analyze protein and gene expression levels of p15, p21, and Rb.
- Co-immunoprecipitation assays to study protein-protein interactions between E1A, Rb, and p300.
Main Results:
- E1A expression blocks the TGF-beta-induced upregulation of p15 and p21.
- E1A inhibits TGF-beta's effects on Cdk2 kinase activity and Rb hypophosphorylation.
- The E1A-mediated suppression of p15 induction is independent of Rb binding but dependent on E1A's interaction with p300, indicating a transcriptional block.
- E1A binding to p300 is crucial for blocking TGF-beta-mediated transcriptional activation of p15.
Conclusions:
- Adenovirus E1A circumvents TGF-beta-induced cell cycle arrest by acting upstream of Rb.
- E1A inhibits the TGF-beta-mediated induction of p15 and p21 at the transcriptional level.
- E1A's interaction with p300 is essential for its ability to block p15 expression, providing a novel mechanism for E1A to promote cell proliferation.