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Cumulative effect of phosphorylation of pRB on regulation of E2F activity
V D Brown1, R A Phillips, B L Gallie
1Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1X8, Canada.
Abstract:
The product of the retinoblastoma susceptibility gene, pRB, is a nuclear phosphoprotein that controls cell growth by binding to and suppressing the activities of transcription factors such as the E2F family. Transactivation activity is inhibited when E2F is bound to hypophosphorylated pRB and released when pRB is phosphorylated by cyclin-dependent kinases (CDKs). To determine which of 16 potential CDK phosphorylation sites regulated the pRB-E2F interaction, mutant pRB proteins produced by site-directed mutagenesis were tested for the ability to suppress E2F-mediated transcription in a reporter chloramphenicol acetyltransferase assay. Surprisingly, no one CDK site regulated the interaction of pRB with E2F when E2F was bound to DNA. Instead, disruption of transcriptional repression resulted from accumulation of phosphate groups on the RB molecule.
Insights
The retinoblastoma protein (pRB) controls cell growth by interacting with E2F transcription factors. This study found that disrupting pRB phosphorylation sites did not affect the pRB-E2F interaction, but rather phosphate accumulation on pRB disrupted transcriptional repression.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Cancer Biology
Background:
- The retinoblastoma susceptibility gene product (pRB) is a key nuclear phosphoprotein regulating cell growth.
- pRB suppresses transcription by binding to E2F family proteins, and this interaction is modulated by pRB phosphorylation by cyclin-dependent kinases (CDKs).
Purpose of the Study:
- To identify specific cyclin-dependent kinase (CDK) phosphorylation sites on pRB that regulate its interaction with E2F.
- To understand the mechanism by which CDK phosphorylation controls pRB's transcriptional repressive function.
Main Methods:
- Site-directed mutagenesis was used to create mutant pRB proteins with alterations at 16 potential CDK phosphorylation sites.
- A reporter chloramphenicol acetyltransferase (CAT) assay was employed to assess the ability of mutant pRB proteins to suppress E2F-mediated transcription.
Main Results:
- No single CDK phosphorylation site mutation individually regulated the interaction between pRB and E2F when E2F was bound to DNA.
- Disruption of transcriptional repression was observed to correlate with the overall accumulation of phosphate groups on the pRB molecule, rather than specific site modifications.
Conclusions:
- The regulation of pRB's interaction with E2F is not dependent on individual CDK phosphorylation sites.
- Transcriptional repression by pRB is disrupted by the overall phosphorylation status of the protein, suggesting a more complex regulatory mechanism than previously understood.