Regulation of the E2F /DP family of heterodimeric transcription factors
Guida, Peter M., Jr.
MetadataShow full item record
Cyclin dependent kinases (Cdks), together with their cyclin subunits, are critical regulators of the mammalian cell cycle. However, the mechanisms by which Cdks specifically recognize their substrates remain poorly understood. We investigated whether the nature of adaptor proteins, which facilitate indirect associations between Cdks and their potential substrates, determined Cdk substrate specificity. We also explored the relationship between the stability of the kinase/substrate interaction and the efficiency of phosphorylation. Multimeric complexes containing cyclin A/Cdk2 and DP1 were assembled in vivo through transient transfection. DP1 was poorly phosphorylated despite being strongly tethered to cyclin A/Cdk2 by p107 and E2F4. However, DP1 was hyper-phosphorylated when weakly connected by E2F4 with a fused cyclin A binding domain of E2F1 (E2F4+A). Thus, the substrate specificity of cyclin A/Cdk2 was determined by the adaptors that bridged the kinase/substrate interaction. In addition, a lower stability interaction between cyclin A/Cdk2 and DP1 produced a more efficient phosphorylation. DP1 hyper-phosphorylation by Cdk2 had functional consequences: it down-regulated the transactivation activity of E2F4+A/DP1 A phosphorylation-deficient DP1 mutant was not similarly down-regulated. Unlike DP1, DP2 was not hyper-phosphorylated by cyclin A/Cdk2 in vivo. Replacing DP2's N-terminus with the first 105 amino acids of DP1 resulted in efficient hyper-phosphorylation of the resulting chimeric protein. Similar to DP1, the transactivation activity of this chimera was also down-regulated by co-expression of cyclin A/Cdk2. Therefore, DP1's first 105 amino acids were sufficient to confer cyclin A/Cdk2 mediated hyper-phosphorylation and functional down-regulation onto DP2. Since the first 91 residues of DP1 could not produce either of these effects, we identified amino acids 92--105 as a region of DP1 required for regulation by cyclin A/Cdk2. Finally, we created stable cell lines that expressed different E2F family members under inducible control. Induced expression of EM2F2, but not E2F4, led to S phase promotion and cell death. Interestingly, E2F4+A acted just like E2F2, not E2F4, in the same assays. Therefore, the induced expression of different E2F family members produced different biological phenotypes.
- Theses and Dissertations