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Inactivation of the cdk inhibitor p27KIP1 by the human papillomavirus type 16 E7 oncoprotein
K Zerfass-Thome1, W Zwerschke, B Mannhardt
1Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Heidelberg, Germany.
Abstract:
Expression of the E7 oncogene of HPV-16 induces S phase entry of mammalian cells in the presence of antiproliferative signals. In particular, E7 can bypass G0/G1 arrest in response to both serum withdrawal and loss of cell adhesion, two experimental conditions in which cell cycle progression is accompanied by elevated levels of the cdk inhibitor p27KIP1. We show here that E7 can antagonize the ability of p27KIP1 to block cyclin E-associated kinase in vitro and to inhibit transcription from the cyclin A gene in transfection experiments. E7 associates with p27KIP1 both in a reconstituted in vitro system and in extracts of mammalian cells, and association requires the C-terminal part of E7. The interaction between p27KIP1 and E7 can also be demonstrated in a yeast two hybrid system. The data suggest that the ability of E7 to override certain forms of G0/G1 arrest is mediated in part by binding to and subsequent inactivation of the cdk inhibitor p27KIP1.
Insights
Human papillomavirus type 16 (HPV-16) E7 oncogene overrides cell cycle arrest by inactivating the p27KIP1 protein. This interaction allows cells to enter the S phase, bypassing normal antiproliferative signals.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The E7 oncogene of human papillomavirus type 16 (HPV-16) promotes cell cycle progression.
- E7 can overcome antiproliferative signals, including serum withdrawal and loss of cell adhesion.
- These conditions typically lead to increased levels of the cyclin-dependent kinase (cdk) inhibitor p27KIP1.
Purpose of the Study:
- To investigate the mechanism by which HPV-16 E7 overrides G0/G1 cell cycle arrest.
- To determine if E7 interacts with and antagonizes the function of p27KIP1.
Main Methods:
- In vitro kinase assays to assess cyclin E-associated kinase activity.
- Transfection experiments to evaluate inhibition of the cyclin A gene.
- Co-immunoprecipitation assays in vitro and in mammalian cell extracts.
- Yeast two-hybrid system to confirm protein-protein interaction.
Main Results:
- HPV-16 E7 antagonizes p27KIP1's ability to inhibit cyclin E-associated kinase activity in vitro.
- E7 inhibits p27KIP1's function in blocking cyclin A gene transcription.
- E7 physically associates with p27KIP1 in vitro, in mammalian cells, and in yeast, requiring E7's C-terminal region.
- This binding interaction was observed under conditions of G0/G1 arrest.
Conclusions:
- HPV-16 E7 inactivates the cdk inhibitor p27KIP1 through direct binding.
- This inactivation mechanism contributes to E7's ability to override specific forms of G0/G1 cell cycle arrest.
- The findings elucidate a key molecular interaction driving HPV-induced cellular transformation.