Inhibition of cyclin A/Cdk2 phosphorylation impairs B-Myb transactivation function without affecting interactions with DNA or the CBP coactivator
Inhibition of cyclin A/Cdk2 phosphorylation impairs B-Myb transactivation function without affecting interactions with DNA or the CBP coactivator
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DOI：
10.1038/sj.onc.1204439
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发表时间：
2001-06-07
期刊：
影响因子：
8
通讯作者：
Watson, RJ
中科院分区：
文献类型：
--
作者：
Bessa, M;Saville, MK;Watson, RJ
B-Myb转录因子的表达由E2 F依赖性转录机制引导至细胞周期的晚期G1和S期，其中其反式激活特性在细胞周期后通过细胞周期蛋白A/Cdk 2介导的磷酸化而增强。其它实验已经表明，去除B-Myb C末端组成性地激活反式激活和DNA结合活性，提示这种抑制性结构域的自身调节被磷酸化抵消。我们在这里报告进一步的实验来检验这一假设。我们在这里报告进一步的实验来检验这一假设的重要性。这种修饰的重要性首先通过显示共转染的显性负性Cdk 2（Cdk 2DN）显著降低B-Myb反式激活活性来强调。然后，我们试图通过分析一系列逐渐删除的C-末端B-Myb突变体来绘制自动调节结构域。仅去除29个C-末端氨基酸就明显增加了反式激活，然而，最大活性需要去除143个氨基酸（如在B-Myb+561中）。增强的B-Myb+561功能与获得与单个Myb结合位点（MBS）寡核苷酸的DNA结合活性相关，如通过带移测定所确定的，然而，进一步的测定表明，即使wt B-Myb也可以结合含有三个MBS的DNA片段。虽然B-Myb的反式激活严重依赖于过度磷酸化，但无论是通过共同抑制Cdk 2DN抑制这种活性，还是用细胞周期蛋白A增强这种活性，都不会对DNA结合产生显著影响。我们还发现，B-Myb可以协同CBP辅激活剂，这种协同作用是细胞周期蛋白A/Cdk 2依赖。尽管如此，这些蛋白质之间的物理关联不受B-Myb磷酸化状态的影响。我们讨论了这些发现与B-Myb的C-末端结构域的自动调节。
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.