Src kinase phosphoregulation of the human mitotic kinesin, Eg5

描述（由申请人提供）：本提案基于Src家族激酶（SFKs）磷酸化人Eg 5（一种必需的有丝分裂驱动蛋白家族马达蛋白）的新发现。SFK是原始的典型癌基因，Eg 5对纺锤体极分离和有丝分裂纺锤体的稳定至关重要。这两种蛋白质都是抗有丝分裂药物的靶点，抑制剂在I期和II期试验中。我们的初步数据表明Src在体外和细胞中磷酸化酶Eg 5的三个酪氨酸。这些酪氨酸在结构上非常接近Eg 5抑制剂的结合位点。我们的初步数据还表明，磷酸化模拟突变抑制Eg 5的活性，并阻止Eg 5抑制剂，STLC的结合。我们推测，SFK磷酸化的Eg 5头改变Eg 5的活动，本地化，并采取行动，在双极纺锤体组装和维护。我们进一步假设SFK磷酸化阻断了几种Eg 5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。我们将开发功能磷酸化模拟突变体和磷酸化特异性抗体的SFK网站在Eg 5头。接下来，我们将研究SFK磷酸化调节Eg 5对固定和活LLC-PK 1细胞中有丝分裂进程的影响。我们将开发新的方法和细胞系，用于成像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Eg 5抑制剂在体外和细胞中的结合和功效。这些实验将是评估靶向SFKs和Eg 5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.