Src kinase phosphoregulation of the human mitotic kinesin, Eg5

人有丝分裂驱动蛋白 Eg5 的 Src 激酶磷酸调节

• 批准号: 8559178
• 负责人: Sarah E. Rice
• 金额: $ 36.85万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8559178
• 关键词: Address; Affect; Antibodies; Antimitotic Agents; Binding; Binding Sites; Breast; Cell Death; Cell Line; Cell Proliferation; Cell division; Cell surface; Cells; Centrosome; Chemicals; Clinical Trials; Combined Modality Therapy; Communication; Cues; Cysteine; Cytokinesis; Cytoskeletal Modeling; Data; Drosophila genus; Drug Targeting; Embryo; Epithelial; Family; Family suidae; Fibroblasts; Future; Goals; Golgi Apparatus; Head; Head and Neck Cancer; Homologous Gene; Human; Image; Imaging Techniques; In Vitro; Kidney; Kinesin; LLC-PK1 Cells; Lead; Life; Link; Maintenance; Metaphase; Methods; Microtubules; Mitosis; Mitotic; Mitotic Activity; Mitotic spindle; Modeling; Motor; Motor Activity; Movement; Mus; Mutation; Oncogenes; Phase; Phase I/II Trial; Phospho-Specific Antibodies; Phosphorylation; Phosphorylation Site; Phosphotransferases; Prophase; Proteins; Reagent; Regimen; Research; Research Personnel; Role; Signal Transduction; Site; Staging; Stomach; Structure; Testing; Tyrosine; Tyrosine Phosphorylation; Vesicle; Work; Yeasts; base; cell motility; cellular imaging; chemical genetics; crosslink; drug action; in vitro activity; inhibitor/antagonist; interest; kidney cell; kinase inhibitor; migration; mutant; neglect; physical process; public health relevance; research study; src-Family Kinases; v-src Oncogenes

DESCRIPTION (provided by applicant): This proposal is based on a new finding that Src family kinases (SFKs) phosphorylate human Eg5, an essential mitotic kinesin family motor protein. SFKs are the original, canonical oncogenes and Eg5 is critical for spindle pole separation and stabilization of the mitotic spindle. Both proteins are anti-mitotic drug targets, with inhibitors in Phase I and II trials. Our preliminary data indicates that Src phosphorylates th enzymatic Eg5 heads at three tyrosines in vitro and in cells. These tyrosines are structurally very near the binding sites for Eg5 inhibitors. Our preliminary data also show that phosphomimetic mutations inhibit Eg5 activity and block the binding of an Eg5 inhibitor, STLC. We hypothesize that SFK phosphorylation of Eg5 heads alters Eg5 activity, localization, and action in bipolar spindle assembly and maintenance. We further hypothesize that SFK phosphorylation blocks the binding of several Eg5-targeted inhibitors. To begin our study, we will determine the mechanistic effects of SFK phosphorylation in vitro. We will develop functional phosphomimetic mutants and phospho-specific antibodies for the SFK sites in Eg5 heads. Next, we will examine the effects of SFK phosphoregulation of Eg5 on the progression of mitosis in fixed and live LLC-PK1 cells. We will develop new methods and cell lines for imaging the effects of SFK phosphorylation on mitotic targets, which will enable researchers to address the neglected issue of SFK activity in mitosis for any target of choice. The final aim of this study isto test whether SFK phosphorylation directly affects Eg5 inhibitor binding and efficacy in vitro and in cells. These experiments will be a first step in evaluating a potential combination therapy regimen targeting SFKs and Eg5.

描述（由申请人提供）：该提案基于一项新发现，即 Src 家族激酶 (SFK) 磷酸化人类 Eg5，Eg5 是一种重要的有丝分裂驱动蛋白家族运动蛋白。 SFK 是原始的、典型的癌基因，Eg5 对于纺锤体极分离和有丝分裂纺锤体的稳定至关重要。这两种蛋白都是抗有丝分裂药物靶标，抑制剂处于 I 期和 II 期试验中。我们的初步数据表明，Src 在体外和细胞内磷酸化酶 Eg5 头的三个酪氨酸。这些酪氨酸在结构上非常接近 Eg5 抑制剂的结合位点。我们的初步数据还表明，拟磷突变会抑制 Eg5 活性并阻断 Eg5 抑制剂 STLC 的结合。我们假设 Eg5 头的 SFK 磷酸化改变了 Eg5 活性、定位以及双极纺锤体组装和维护中的作用。我们进一步假设 SFK 磷酸化会阻断几种 Eg5 靶向抑制剂的结合。为了开始我们的研究，我们将确定 SFK 磷酸化的体外机制效应。我们将为 Eg5 头中的 SFK 位点开发功能性磷酸模拟突变体和磷酸化特异性抗体。接下来，我们将检查 SFK 磷酸化 Eg5 对固定和活 LLC-PK1 细胞有丝分裂进展的影响。我们将开发新的方法和细胞系来成像 SFK 磷酸化对有丝分裂靶点的影响，这将使研究人员能够解决任何选择的有丝分裂中被忽视的 SFK 活性问题。本研究的最终目的是测试 SFK 磷酸化是否直接影响 Eg5 抑制剂的体外和细胞内结合和功效。这些实验将是评估针对 SFK 和 Eg5 的潜在联合治疗方案的第一步。