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: 8744294
• 负责人: Sarah E. Rice
• 金额: $ 35.16万
• 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/8744294
• 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; Drug effect disorder; 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; 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Src家族激酶（SFKs）磷酸化人Eg5的新发现，Eg5是一种必需的有丝分裂运动蛋白家族运动蛋白。sfk是原始的、典型的癌基因，Eg5对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的靶点，在I期和II期试验中都有抑制剂。我们的初步数据表明，Src在体外和细胞中磷酸化酶促的Eg5头部的三个酪氨酸。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，拟磷突变抑制Eg5活性并阻断Eg5抑制剂STLC的结合。我们假设SFK对Eg5头的磷酸化改变了Eg5在双极纺锤体组装和维护中的活性、定位和作用。我们进一步假设SFK磷酸化阻断了几种eg5靶向抑制剂的结合。为了开始我们的研究，我们将在体外确定SFK磷酸化的机制作用。我们将开发Eg5头部SFK位点的功能性拟磷突变体和磷酸化特异性抗体。接下来，我们将研究SFK磷酸化调控Eg5对固定和活的LLC-PK1细胞有丝分裂进程的影响。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标的影响，这将使研究人员能够解决SFK活性在有丝分裂中被忽视的问题。本研究的最终目的是测试SFK磷酸化是否直接影响Eg5抑制剂在体外和细胞内的结合和疗效。这些实验将是评估针对SFKs和Eg5的潜在联合治疗方案的第一步。