SIGNALING PATHWAY INVOLVING BC12 PHOSPHORYLATION

涉及 BC12 磷酸化的信号通路

基本信息

批准号：
2752192
负责人：
SUBRATA HALDAR
金额：
$ 20.75万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-01-01 至 2003-12-31
项目状态：
已结题

项目摘要

The long term goal of the proposed research is to develop apoptosis inducing therapy that can be targeted for chemoprevention of cancer caused by aberrant expression of the anti-apoptosis oncogene Bc12 ( B cell lymphoma/leukemia-2). Taxol and other microtubule-targeting drugs can trigger Bc12 phosphorylation at G2-M phase of the cell cycle. By site directed mutagenesis, the phosphorylation sites were identified to be Serine-70 & Serine-87 residues of Bc12 protein. Our preliminary studies employing this unique phosphorylation-deficient mutant clearly indicate that Bc12 phosphorylation is detrimental to its anti-apoptotic function. Interestingly, sequences surrounding both Serine-70 and 87 constitute consensus motifs for substrate of MAP/ERK2 kinase. Precisely, the clear understanding of the loss of Be12 function by phosphorylation as well as the identification of the kinase/phosphatase pathway regulating function of Bc12 are crucial at this stage of our investigation. The generation of the phosphorylation-deficient mutant will now be extremely helpful for the accomplishment of five Specific Aims proposed here. In Specific Aim 1, the interdependence of Bc12 phosphorylation and G2-M arrest will be tested in G2-M population of normal dividing cells transfected with wild and phospho-deficient mutant Bc12. Due to phosphorylation, any change in subcellular localization of phospho-Bc12 will be assessed by employing phospho-Bc12 specific antibody. Specific Aim 2 will test the hypothesis that phospho-Bc12 fails to form dimer with its proapoptotic partner Bax by co- immunoprecipitation and protein-protein interaction using GST-fusion column. In Specific Aim 3, the experiments pursuing the characterization of the kinase and phosphatase regulating Bc12 phosphorylation will be undertaken. In order to search for in vivo phospho-deficient form of Bc12, a variety of malignant cells (not responsive to microtubule targeting drug induced Bc12 phosphorylation and apoptosis) will be screened by RT-PCR in Specific Aim 4. The generation of transgenic mice expressing phospho-deficient mutant Bc12 in Specific Aim 5 will be helpful for in depth functional analysis of Bc12 phosphorylation. In all, these studies will offer new insights into the molecular mechanism of Bc12 function as well as provide valuable information for a potential genetic screen of apoptosis related human diseases.

拟议研究的长期目标是开发细胞凋亡可针对癌症化学预防的诱导治疗由抗凋亡癌基因 Bc12 的异常表达引起（B 细胞淋巴瘤/白血病-2)。紫杉醇和其他微管靶向药物可以在细胞周期的 G2-M 期触发 Bc12 磷酸化。经过定点诱变，磷酸化位点被鉴定为是Bc12蛋白的丝氨酸70和丝氨酸87残基。我们的初步研究清楚地利用了这种独特的磷酸化缺陷突变体表明Bc12磷酸化不利于其抗凋亡作用功能。有趣的是，丝氨酸 70 和 87 周围的序列构成 MAP/ERK2 激酶底物的共有基序。准确地说，对 Be12 功能丧失的清晰认识磷酸化以及激酶/磷酸酶的鉴定 Bc12的通路调节功能在我们的现阶段至关重要调查。磷酸化缺陷突变体的产生现在对于实现五个具体目标非常有帮助这里提出的目标。在具体目标 1 中，Bc12 的相互依赖性磷酸化和 G2-M 停滞将在 G2-M 群体中进行测试用野生和磷酸缺陷突变体转染的正常分裂细胞公元前 12 年。由于磷酸化，亚细胞定位发生任何变化磷酸化 Bc12 将通过采用磷酸化 Bc12 特异性进行评估抗体。具体目标 2 将检验磷酸化 Bc12 的假设未能与其促凋亡伙伴 Bax 形成二聚体使用 GST 融合进行免疫沉淀和蛋白质-蛋白质相互作用柱子。在具体目标 3 中，追求表征的实验调节 Bc12 磷酸化的激酶和磷酸酶将是进行。为了寻找体内磷酸缺乏形式 Bc12，多种恶性细胞（对微管无反应）靶向药物诱导的 Bc12 磷酸化和细胞凋亡）将是具体目标4中通过RT-PCR筛选转基因小鼠的产生在特定目标 5 中表达磷酸缺陷突变体 Bc12 将是有助于Bc12磷酸化的深入功能分析。在总之，这些研究将为分子机制提供新的见解 Bc12 功能以及为潜在的提供有价值的信息细胞凋亡相关人类疾病的基因筛查。