Src kinase phosphoregulation of the human mitotic kinesin, Eg5

描述(申请人提供)：这项建议是基于一项新的发现，即Src家族激酶(SFK)使人EG5磷酸化，EG5是一种重要的有丝分裂蛋白家族运动蛋白。SFK是原始的、典型的癌基因，而EG5对纺锤体极点的分离和有丝分裂纺锤体的稳定至关重要。这两种蛋白质都是抗有丝分裂的药物靶点，其抑制剂正在进行I期和II期试验。我们的初步数据表明，在体外和细胞内，Src以三种酪氨酸对酶促EG5头部进行磷酸化。这些酪氨酸在结构上非常接近EG5抑制剂的结合部位。我们的初步数据还表明，仿磷突变抑制了EG5的活性，并阻止了EG5抑制剂STLC的结合。我们假设，EG5头部的SFK磷酸化改变了EG5的活性、定位和在双极纺锤体组装和维护中的作用。我们进一步假设，SFK的磷酸化阻止了几个EG5靶向抑制物的结合。为了开始我们的研究，我们将在体外确定SFK磷酸化的机制效应。我们将开发功能性仿磷突变体和针对EG5头部SFK位点的磷酸特异性抗体。接下来，我们将研究SFK对EG5的磷酸化调节对固定和活的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点的影响，这将使研究人员能够解决被忽视的问题，即在有丝分裂中任何选择的靶点的SFK活性。本研究的最终目的是测试SFK的磷酸化是否在体外和细胞内直接影响EG5抑制剂的结合和疗效。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。

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.