Inhibition of cyclin A/Cdk2 phosphorylation impairs B-Myb transactivation function without affecting interactions with DNA or the CBP coactivator

Expression of the B-Myb transcription factor is directed by an E2F-dependent transcriptional mechanism to late G1 and S phases of the cell cycle, where its transactivation properties are enhanced post-translationally by cyclin A/Cdk2-mediated phosphorylation, Other experiments have shown that removal of the B-Myb C-terminus constitutively activates both transactivation and DNA-binding activities, suggesting that autoregulation by this inhibitory domain is counteracted by phosphorylation. We report here on further experiments to examine this hypothesis. The importance of this We report here on further experiments to examine this hypothesis. The importance of this modification was first emphasized by showing that co-transfected dominant-negative Cdk2 (Cdk2DN) substantially reduced B-Myb transactivation activity. We then attempted to map the autoregulatory domain by analysing a series of progressively deleted C-terminal B-Myb mutants. Removal of just 29 C-terminal aa increased transactivation appreciably, however, maximal activity required removal of 143 amino acids (as in B-Myb+561). Enhanced B-Myb+561 function correlated with the acquisition of DNA binding activity to a single Myb binding site (MBS) oligonucleotide as determined by bandshift assays, however, further assays showed that even wt B-Myb could bind a DNA fragment containing three MBS. Although transactivation by B-Myb was severely dependent on hyperphosphorylation, neither inhibiting this activity by co-transfecting Cdk2DN nor augmenting it with cyclin A resulted in significant effects on DNA-binding. We also found that B-Myb could synergize with the CBP coactivator and that this cooperativity was cyclin A/Cdk2-dependent. Despite this, the physical association between these proteins was not influenced by the B-Myb phosphorylation status. We discuss these findings in relation to the autoregulation of B-Myb by the C-terminal domain.