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The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function
Q Sheng1, D Denis, M Ratnofsky
1Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Abstract:
Tumor suppressors of the retinoblastoma susceptibility gene family regulate cell growth and differentiation. Polyomavirus large T antigens (large T) bind Rb family members and block their function. Mutations of large T sequences conserved with the DnaJ family affect large T binding to a cellular DnaK, heat shock protein 70. The same mutations abolish large T activation of E2F-containing promoters and Rb binding-dependent large T activation of cell cycle progression. Cotransfection of a cellular DnaJ domain blocks wild-type large T action, showing that the connection between the chaperone system and tumor suppressors is direct. Although they are inactive in assays dependent on Rb family binding, mutants in the J region retain the ability to associate with pRb, p107, and p130. This suggests that binding of Rb family members by large T is not sufficient for their inactivation and that a functional J domain is required as well. This work connects the DnaJ and DnaK molecular chaperones to regulation of tumor suppressors by polyomavirus large T.
Insights
Polyomavirus large T antigens disrupt tumor suppressor proteins. A functional J domain is crucial for this disruption, linking molecular chaperones like DnaJ and DnaK to tumor suppressor regulation.
Area of Science:
- Molecular biology
- Virology
- Cell cycle regulation
Background:
- Tumor suppressors, including the retinoblastoma susceptibility gene family, control cell growth and differentiation.
- Polyomavirus large T antigens (large T) interfere with tumor suppressor function by binding to Rb family members.
Purpose of the Study:
- To investigate the role of conserved DnaJ family sequences in large T antigen function.
- To elucidate the connection between molecular chaperones (DnaJ, DnaK) and the regulation of tumor suppressors by polyomavirus large T.
Main Methods:
- Mutational analysis of polyomavirus large T antigen sequences.
- Cotransfection assays to assess large T antigen activity and binding.
- Examination of interactions with Rb family proteins (pRb, p107, p130) and molecular chaperones (DnaK).
Main Results:
- Mutations in large T antigen sequences conserved with DnaJ affected binding to DnaK and abolished E2F promoter activation.
- These mutations also abrogated large T-dependent cell cycle progression.
- While mutants retained binding to Rb family members, they were inactive in assays dependent on Rb binding, indicating a functional J domain is required for inactivation.
Conclusions:
- The DnaJ functional domain of polyomavirus large T antigen is essential for inactivating Rb family tumor suppressors.
- This study directly links the DnaJ and DnaK molecular chaperone system to the regulation of tumor suppressors by polyomavirus.
- Rb binding alone is insufficient for tumor suppressor inactivation; a functional J domain is also necessary.