Conformational Dynamics of Protein Tyrosine Kinases Src and Abl

描述（由申请人提供）：蛋白激酶介导许多细胞信号事件，它们的严格控制对于调节从细胞分裂到能量代谢的基本过程是必不可少的。因此，毫不奇怪，蛋白激酶直接或间接参与许多疾病，激酶是关键的药物靶点。例如，Src激酶是第一个被发现的原癌基因，而在95%的慢性髓性白血病患者中，去调控Abl融合蛋白（BCR-Abl）的形成是导致疾病的原因。x射线晶体结构表明，相同的激酶可以获得活性和各种非活性构象，这意味着激酶具有固有的柔韧性。活性和非活性状态如何稳定以及状态如何相互转换是理解激酶调控的关键问题。由于x射线晶体结构只提供静态快照，我们将使用核磁共振（NMR）实验来研究Abl和Src激酶结构域结构相互转换的时间尺度和振幅。

DESCRIPTION (provided by applicant): Protein kinases mediate many cell signaling events, and their tight control is essential for regulating essential processes ranging from cell division to energy metabolism. Thus, it is not surprising that protein kinases are directly or indirectly involved in many diseases and that kinases are key drug targets. For example, Src kinase was the first identified proto-oncogene and the formation of a de-regulated Abl fusion protein (BCR-Abl) is the cause of disease in 95% of patients with chronic myeloid leukemia. X-ray crystal structures have shown that the same kinases can obtain an active and various inactive conformations, implying that kinases are inherently flexible. How the active and inactive states are stabilized and how the states interconvert are key questions in understanding kinase regulation. Because X-ray crystal structures provide only static snapshots, we will use nuclear magnetic resonance (NMR) experiments to study the time scales and amplitudes of structural interconversions in Abl and Src kinase domains. BCR-Abl is the target of the clinically highly successful drug imatinib (Gleevec(r), Novartis) in the treatment of chronic myelogenous leukemia (CML). Why does imatinib bind and inhibit c-Abl but not the structurally closely related c-Src kinase? The crystal structure of Src in complex with imatinib shows protein-drug interactions similar to that of Abl, even though the affinity of imatinib for Src is orders of magnitude lower than for Abl. Because imatinib binds only to the inactive conformation of the kinase, drug binding is intimately related to the interconversion between active and inactive states. The goal of this study is to examine whether differences in this interconversion underlie the differential sensitivities for imatinib. Therefore, we will compare the time scales and amplitudes of backbone motions between Src and Abl kinases in the presence of imatinib by NMR experiments. In preparation for these dynamics experiments, we have established expression systems and NMR conditions and will next pursue the assignment of the Src and Abl NMR spectra. Kinase inhibitory drugs such as imatinib have a great therapeutic potential because of the many signaling events that protein kinases mediate. However, these drugs have to be exceptionally specific for their target kinase and resistance mutations can render these drugs ineffective as seen in leukemia patients under imatinib treatment. The proposed experiments will clarify how inhibitors such as imatinib exploit the characteristic movements of kinase proteins, rather than just their structures, to achieve specificity. Furthermore, the results will have broader impact on the understanding of the fundamental mechanisms of kinase regulation and of drug resistance mutations that are known to arise in cancer patients undergoing kinase inhibitory treatment.