Bayly Richard, Murase Takayuki, Hyndman Brandy D, Savage Rachel, Nurmohamed Salima, Munro Kim, Casselman Richard, Smith Steven P, LeBrun David P
Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Mol Cell Biol. 2006 Sep;26(17):6442-52. doi: 10.1128/MCB.02025-05.
In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.
在大约5%的急性淋巴细胞白血病病例中，染色体易位会导致致癌蛋白E2A-PBX1的表达。由E2A基因编码的E2A-PBX1的N端部分，其序列与E蛋白E12/E47的相应部分相同，并包含转录激活结构域。C端由大部分与HOX相互作用的转录因子PBX1组成，包括其DNA结合同源结构域。结构-功能相关实验表明，E2A-PBX1致癌需要位于最N端的一个称为AD1的激活结构域。我们最近证明，AD1的一个潜在螺旋部分直接与转录共激活蛋白环磷酸腺苷反应元件结合蛋白（CBP）相互作用，并且这种相互作用在组织培养中原代骨髓细胞的永生化过程中至关重要。在此我们表明，E2A-PBX1与CBP的KIX结构域之间的相互作用需要AD1内一个保守的LXXLL基序。我们通过圆二色光谱表明，AD1中含LXXLL的部分在与KIX结构域相互作用时会发生螺旋转变，并且阻止螺旋形成的氨基酸取代会同时阻止KIX相互作用以及E2A-PBX1介导的细胞永生化。也许最引人注目的是，LXXLL基序内单个保守亮氨酸残基（L20）的取代会损害用表达E2A-PBX1的骨髓进行移植后小鼠的白血病诱导。CBP的KIX结构域介导与几种与白血病诱导相关的转录因子的充分表征的相互作用。间接证据表明，L20的侧链可能与KIX结构域中的一个深疏水口袋相互作用。因此，我们的结果有助于确定一个潜在的新药物靶点。
