Bessa M, Saville M K, Watson R J
Section of Virology and Cell Biology, Imperial College School of Medicine, St Mary's Campus, London W2 1PG, UK.
Oncogene. 2001 Jun 7;20(26):3376-86. doi: 10.1038/sj.onc.1204439.
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 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.
B-Myb转录因子的表达受E2F依赖的转录机制调控，在细胞周期的G1晚期和S期表达，其反式激活特性在翻译后通过细胞周期蛋白A/Cdk2介导的磷酸化作用得到增强。其他实验表明，去除B-Myb的C末端可组成性激活反式激活和DNA结合活性，这表明该抑制结构域的自动调节作用被磷酸化所抵消。我们在此报告进一步的实验以检验这一假设。通过共转染显性负性Cdk2（Cdk2DN）可显著降低B-Myb反式激活活性，首次强调了这种修饰的重要性。然后，我们试图通过分析一系列逐步缺失C末端的B-Myb突变体来定位自动调节结构域。仅去除29个C末端氨基酸就可明显增加反式激活活性，然而，最大活性需要去除143个氨基酸（如在B-Myb + 561中）。通过凝胶迁移试验确定，增强的B-Myb + 561功能与获得对单个Myb结合位点（MBS）寡核苷酸的DNA结合活性相关，然而，进一步的试验表明，即使是野生型B-Myb也能结合含有三个MBS的DNA片段。尽管B-Myb的反式激活严重依赖于过度磷酸化，但共转染Cdk2DN抑制该活性或用细胞周期蛋白A增强该活性均未对DNA结合产生显著影响。我们还发现B-Myb可与CBP共激活因子协同作用，且这种协同作用依赖于细胞周期蛋白A/Cdk2。尽管如此，这些蛋白质之间的物理结合不受B-Myb磷酸化状态的影响。我们结合C末端结构域对B-Myb的自动调节来讨论这些发现。
