Catalytic Domain Dynamics in Protein Kinases

描述（由申请人提供）：人类癌症宿主的进展与酪氨酸激酶（SFK）Src家族的表达水平和催化活性升高相关，使其成为关键治疗靶标。即使有多种活性和非活性形式的SFK催化结构域的晶体结构的可用性，也无法对其催化调节进行全面了解。被认为导致催化活性显著增加的中心步骤是“活化环”中调节酪氨酸残基（Tyract）的磷酸化。据推测，这种化学修饰会引起局部和长程相互作用的变化，并改变催化结构域内的调节动力学。虽然其中一些变化是从晶体结构中推断出来的，但缺乏直接的证据。溶液NMR，最适合解决这个问题的生物物理方法，以前受到细菌表达和纯化足够数量的可溶性，正确折叠的蛋白质的困难的阻碍，用于经济上可行的NMR活性同位素标记。我们通过选择最佳的构建体，与分子伴侣共表达和优化纯化方案，实现了细菌产生大量同位素标记的c-Src催化结构域，原型SFK及其Tyract磷酸化形式的能力。这一点，再加上配备最新一代低温探头的超高场NMR仪器（900 MHz）的可用性和初始NMR光谱的高质量，使得SFKs催化域的详细NMR研究首次可行。我们将利用新的NMR方法充分表征的c-Src催化结构域的动力学，其修改后Tyract磷酸化，它们对酶活性的调节和它们的扰动机制的影响，由三个特定类别的小分子抑制剂。SFK使用额外的非催化结构域来调节催化活性，而其他蛋白激酶如胞外信号调节激酶（ERK）类丝氨酸/苏氨酸激酶使用催化结构域本身内的插入来代替外部结构域。值得注意的是，催化结构域的整体结构和关键调控元件在蛋白激酶中高度保守。因此，预计某些功能动力学模式将是保守的，而其他模式将根据激酶的类别而变化。我们将通过确定ERK 2（一种原型ERK）的功能动力学来研究这些效应，它们在正调控激活环Thr-X-Tyr基序的双磷酸化后的修饰，与c-Src进行比较。我们还将研究与调节磷酸酶的对接相互作用（目前在SFKs中未鉴定）对ERK 2功能动力学的修饰作用。了解激酶激活的动态基础将可能允许改善当前的治疗剂，并开发新的治疗剂，用于干预激酶相关疾病，特别是癌症和自身免疫疾病。公共卫生相关性：该项目涉及阐明两个关键细胞信号分子（即c-Src和ERK 2）催化活化所涉及的多个空间和时间过程。这两种分子的催化活性在健康细胞中受到严格调节。然而，这种控制在多种人类癌症和增殖性疾病中丧失。因此，清楚地了解这些分子在空间和时间上的功能将改善目前的抗癌疗法，同时帮助设计针对这种致命疾病的新策略。

DESCRIPTION (provided by applicant): Progression of a host of human cancers is associated with elevated levels of expression and catalytic activity of the Src family of tyrosine kinases (SFKs) making them key therapeutic targets. Even with the availability of multiple crystal structures of active and inactive forms of the SFK catalytic domain, a complete understanding of its catalytic regulation is unavailable. A central step recognized to lead to a dramatic increase in catalytic activity is the phosphorylation of a regulatory tyrosine residue (Tyract) in the "activation loop". This chemical modification is presumed to cause changes in local and long-range interactions and modification of the regulatory dynamics within the catalytic domain. Though some of these changes are inferred from crystal structures, direct evidence is lacking. Solution NMR, the biophysical method best suited to tackle this problem, was previously hindered by difficulties in bacterial expression and purification of sufficient quantities of soluble, properly folded protein for economically viable labeling with NMR-active isotopes. We have through a choice of optimal constructs, co-expression with chaperones and optimization of the purification protocol, achieved the ability to bacterially produce large quantities of the isotopically-labeled catalytic domain of c-Src, the prototypical SFK, and of its Tyract phosphorylated form. This, together with the availability of ultra-high field NMR instrumentation (900 MHz) equipped with the latest generation cryogenic probes and the high-quality of the initial NMR spectra, make the detailed NMR studies of the catalytic domain of the SFKs viable for the first time. We will utilize novel NMR methodology to fully characterize the dynamics of the c-Src catalytic domain, their modifications upon Tyract phosphorylation, their influence on the regulation of enzymatic activity and the mechanism of their perturbation by each of three specific classes of small molecule inhibitors. The SFKs use additional non-catalytic domains to modulate catalytic activity while other protein kinases such as the extracellular signal-regulated kinase (ERK) class of serine/threonine kinases use insertions within the catalytic domain itself in lieu of external domains. Notably, the overall structure and key regulatory elements of the catalytic domain are highly conserved amongst protein kinases. It is thus expected that certain modes of functional dynamics would be conserved while others would vary depending on the class of kinase. We will investigate these effects by ascertaining the functional dynamics in ERK2 (a prototypical ERK), their modification upon dual-phosphorylation of a positive-regulatory activation-loop Thr-X-Tyr motif, for comparison with c-Src. We will also investigate the modifying effects of docking interactions (currently unidentified in SFKs) with regulatory phosphatases, on the functional dynamics in ERK2. Understanding the dynamic underpinnings of kinase activation will likely permit the improvement of current, and the development of new, therapeutic agents for intervention in kinase-associated disorders, especially in cancer and auto-immune diseases. PUBLIC HEALTH RELEVANCE: This project is involved with elucidating the multiple spatial as well as temporal processes involved in the catalytic activation of two key cell signaling molecules namely, c-Src and ERK2. The catalytic activity of these two molecules is tightly regulated in healthy cells. However, this control is lost in a variety of human cancers and proliferative diseases. Thus, a clear understanding of the functioning of these molecules in space and time will improve current anticancer therapies while helping the design of novel strategies targeting this deadly disease.