Investigation of novel interaction partners of the transcription factor B-Myb and the role of B-Myb in the DNA-damage response

MYB蛋白是一个转录因子家族，其成员(A-Myb、B-Myb和c-Myb)在调节细胞增殖和分化中起关键作用。虽然AMyb和c-Myb的表达仅限于特定的细胞类型，但B-Myb几乎在所有增殖细胞中都有表达，并被认为在细胞周期中发挥普遍作用。作为一种转录因子，B-Myb的活性受细胞周期蛋白A/CDK2依赖的磷酸化以及与其他蛋白质相互作用的调节，在细胞周期的S期达到最大值。虽然B-Myb的几个靶基因已经被确定，但B-Myb的功能还不是很清楚。我们最近的工作为B-Myb功能的两个新方面提供了洞察力。基因敲除研究表明，B-Myb除了在细胞周期中发挥作用外，还参与细胞对DNA损伤的反应。此外，为了寻找新的B-Myb蛋白相互作用伙伴，我们发现B-Myb与cullin3和几个BTB结构域蛋白相互作用。CULLINS是一类泛素连接酶复合体的支架蛋白，BTB蛋白被认为是底物特异性的适配器，招募底物蛋白被泛素化，随后降解为基于CULLIN3的泛素连接酶。这些复合体是如何作用的，底物蛋白是如何选择的，人们才刚刚开始了解。因此，这些蛋白与B-Myb的相互作用是非常有趣的，因为它可以进一步深入了解Cullins的功能，以及B-Myb在细胞周期进程和DNA损伤反应中的作用。这里描述的项目解决了B-Myb功能的两个新方面。我们计划详细研究B-Myb、Cullins和BTB-蛋白之间的相互作用，并解决我们的发现提出的几个关键问题。B-Myb本身是以CuIIinS为基础的泛素连接酶复合体的底物，还是受B-Myb调控的该复合体对其他蛋白质的泛素化？B-Myb与某些BTB-蛋白的相互作用是否与功能相关？这种作用不依赖于culin介导的泛素化？最后，B-Myb、cullin3和BTB蛋白相互作用的蛋白结构域是什么？这些结构域的鉴定将为这些蛋白质相互作用的结构分析提供基础。除了蛋白质相互作用的研究外，我们还计划确定在DNA损伤后激活B-Myb的信号转导途径，并确定DNA损伤后B-Myb的下游靶点。我们认为，这里描述的工作将为B-Myb的作用和基于库林斯的泛素连接酶的功能提供有趣的新见解。

Myb proteins constitute a family of transcription factors whose members (A-Myb, B-Myb and c- Myb) play key roles in regulating cell proliferation and differentiation. While the expression of AMyb and c-Myb is restricted to particular cell types, B-Myb is expressed in virtually all proliferating cells and is thought to play a general role during the cell cycle. The activity of B-Myb as a transcription factor is regulated by cyclinA/Cdk2-dependent phosphorylation as well as by interaction with other proteins and reaches a maximum during the S-phase of the cell cycle. Although several target genes for B-Myb have been identified the function of B-Myb is not yet well understood. Our recent work has provided insight into two novel aspects of B-Myb function. Knock-out studies have shown that B-Myb,, in addition to its role in the cell cycle, is also involved in the cellular response to DNA-damage. Furthermore, in an attempt to identify novel protein interaction partners of B-Myb we have found that B-Myb interacts with cullin3 and several BTBdomain proteins. CullinS is a scaffold protein of a class of ubiquitin ligase complexes and BTBproteins are thought to be substrate-specific adaptors that recruit substrate proteins to be ubiquitinated and subsequently degraded to the cullin3-based ubiquitin ligase. How these complexes act and how substrate proteins are selected is only beginning to be understood. The interaction of these proteins with B-Myb is therefore very interesting as it may provide further insight into the function of cullinS as well as the role of B-Myb in cell cycle progression and the DNA-damage response. The project described here addresses both novel aspects of B-Myb function. We plan to study the interaction of B-Myb, cullinS and BTB-proteins in detail and address several key issues raised by our findings. Is B-Myb itself a substrate of a cuIIinS-based ubiquitin ligase complex or is the ubiquitination of other proteins by this complex regulated by B-Myb? Is there a functional relevance to the interaction of B-Myb with certain BTB-proteins which is independent of cullinSmediated ubiquitination? Finally, what are the protein domains of B-Myb, cullin3 and the BTBproteins that interact with each other? Identification of these domains will provide the basis for the structural analysis of the interaction of these proteins. In addition to the protein interaction studies, we plan to characterize the signal transduction pathway that activates B-Myb in response to DNA-damage and identify downstream targets of B-Myb following DNA-damage. We think that the work described here will provide interesting novel insight into the role of B-Myb and the function of the cullinS-based ubiquitin ligase.