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Identification of a viral kinase that phosphorylates specific E2Fs and pocket proteins
S Pajovic1, E L Wong, A R Black
1Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Abstract:
The transcription factor E2F and its regulation by pRB and related pocket proteins are central to cell cycle control in higher eukaryotes. Much of our knowledge of this regulation has come from studies using immediate-early proteins of DNA tumor viruses. Previously, we reported that the 72-kDa immediate-early region 1 gene product of the human cytomegalovirus, IE72, transactivates the dihydrofolate reductase promoter through the E2F site and that it physically interacts with E2F1 (M. J. Margolis, S. Pajovic, E. L. Wong, M. Wade, R. Jupp, J. A. Nelson, and J. C. Azizkhan, J. Virol. 69:7759-7767, 1995). In this study, we further characterized the mechanism by which IE72 modulates E2F-dependent transcription. In vitro phosphorylation reactions using gel-purified bacterially expressed proteins revealed that IE72 is a kinase that autophosphorylates and phosphorylates E2F1, -2, and -3 (but not E2F4 or -5) and the RB-related pocket proteins p130 and p107 (but not pRB). The region of IE72 spanning amino acids 173 to 197 shows a high level of homology to the ATP binding sites in over 500 kinases. The kinase-negative protein IE72deltaATP, from which this region has been deleted, cannot activate E2F-dependent transcription. The kinase activity of IE72 is also required for its ability to reduce the association of E2F4 with p107 and p130. Taken together, these data suggest that the kinase activity of IE72 is required for E2F-dependent transcriptional activation and that this is likely to result from phosphorylation of specific members of the E2F and pocket protein families by IE72.
Insights
Human cytomegalovirus IE72 protein acts as a kinase, phosphorylating E2F and pocket proteins to regulate cell cycle transcription. Its kinase activity is essential for activating E2F-dependent transcription.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- The E2F transcription factor and pocket proteins (like pRB) are crucial for eukaryotic cell cycle control.
- Human cytomegalovirus immediate-early protein IE72 interacts with E2F1 and activates transcription via the E2F site.
Purpose of the Study:
- To elucidate the mechanism by which IE72 modulates E2F-dependent transcription.
- To investigate the role of IE72's kinase activity in this process.
Main Methods:
- In vitro kinase assays using purified proteins.
- Site-directed mutagenesis to create a kinase-negative IE72 mutant (IE72deltaATP).
- Analysis of protein-protein interactions and transcriptional activation.
Main Results:
- IE72 functions as a kinase, autophosphorylating and phosphorylating E2F1, E2F2, E2F3, p107, and p130.
- A conserved ATP-binding motif in IE72 (amino acids 173-197) is essential for its kinase activity.
- The kinase activity of IE72 is required for E2F-dependent transcriptional activation and for reducing E2F4 association with p107 and p130.
Conclusions:
- The kinase activity of IE72 is indispensable for its role in E2F-dependent transcription.
- IE72 likely achieves transcriptional activation by phosphorylating specific E2F and pocket protein family members.