Regulation of E2F by phosphorylation

Morris, Lorna Josephine (2000) Regulation of E2F by phosphorylation. PhD thesis, University of Glasgow.

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Abstract

The E2F proteins are a family of transcription factors that function in regulating the G1 to S phase transition. The activity of E2F is regulated in a cell-cycle dependent manner by its physical interaction with members of the retinoblastoma protein (pRb) family, which are inactivated via phosphorylation by cyclin-dependent kinases (CDK), most notably cyclinD-CDK complexes in Gl. This ensures that the activation of E2F- responsive genes is closely coupled with cell cycle position. In this study, the role of direct phosphorylation of E2F in the regulation of E2F activity is explored. CyclinD-CDK2 can directly phosphorylate E2F-5 in vitro, and phosphorylation is abolished by mutation of threonine 251 to alanine in the transactivation domain of E2F-5 (T251A), whilst mutation of serine 307 to alanine (S307A) did not affect phosphorylation. Evidence is presented that phosphorylation of E2F-5 by cyclinE-CDK2 facilitates transcription and cell cycle progression. Specifically cyclinE-CDK2 causes a significant increase in E2F-5 dependent transcription, which is not observed for T251A. In addition, S307A is compromised in its ability to activate transcription. Taken together the results suggest that phosphorylation of E2F-5 at distinct sites by cyclinD-CDK2 and an as yet unidentified kinase augments E2F-5 dependent transcription. The p300/CBP family of proteins have been implicated as important coactivators for a variety of transcription factors, including E2F-1. Here it is shown that the binding of E2F-5 to p300 is stabilised upon phosphorylation of E2F-5 by cyclinD-CDK2, suggesting enhanced recruitment of p300 is mechanistically important for stimulating E2F-5 dependent transcription. Since cyclinD is a well-known E2F responsive gene and the results imply that E2F activity may be directly regulated by cyclinE-CDK2, an autoregulatory feedback loop may exist between E2F-5 and cyclin E that drives the expression of E2F target genes, thereby promoting irreversible entry into S phase. Overall, these results define a novel level of control in the regulation of cell cycle progression by E2F.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Nick La Thangue
Keywords: Molecular biology
Date of Award: 2000
Depositing User: Enlighten Team
Unique ID: glathesis:2000-73137
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56
URI: http://theses.gla.ac.uk/id/eprint/73137

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