Studying how a general allosteric site regulates protein kinase function

描述（由申请人提供）：蛋白激酶的小分子抑制剂已经彻底改变了生物医学科学。在临床上，蛋白激酶抑制剂迎来了时代 通过改变各种癌症的护理标准来实现个性化医疗。在实验室，蛋白激酶抑制剂作为研究工具的广泛使用大大提高了我们对蛋白激酶在信号通路中作用的认识。然而，调节单个激酶功能的分子机制仍然知之甚少。鉴于激酶导向疗法的临床价值已得到证实，对激酶调节机制的详细了解可以通过确定调节激酶功能的新方法来显著影响人类健康。这个建议的长期目标是确定一般变构位点如何调节底物特异性和蛋白激酶的亚细胞定位。该建议集中在蛋白激酶PDK1上，其中该变构位点（称为PIF口袋）是大多数PDK1底物磷酸化所需的。PIF口袋还可以促进癌细胞运动性。我们的实验室最近发现了与细胞中的PIF口袋结合的小分子。我们假设这些PIF口袋配体将破坏PDK 1与其底物和效应蛋白的子集之间的相互作用，从而提供一种快速和可逆的方式来干扰PIF口袋功能。本文提出的工作旨在使用我们的PIF口袋配体1）研究PIF口袋如何决定PDK 1的底物特异性，2）研究PIF口袋如何促进癌细胞运动。为了实现第一个目标，我们将通过质谱鉴定PDK1底物，并确定其磷酸化对我们的PIF口袋配体的敏感性。为了实现第二个目标，我们将描述PIF口袋在3D培养中生长的高度侵袭性黑色素瘤细胞的运动性中的作用。这些研究将突出这一一般变构位点在调节蛋白激酶功能中的重要性。这一概念可能会改变激酶药物发现的前景，并导致一类新的激酶导向疗法的发展。

DESCRIPTION (provided by applicant): Small-molecule inhibitors of protein kinases have revolutionized biomedical science. In the clinic, protein kinase inhibitors have ushered in the era of personalized medicine by transforming the standard of care for a variety of cancers. At the bench, the widespread use of protein kinase inhibitors as research tools has significantly advanced our knowledge of the roles of protein kinases in signaling pathways. However, the molecular mechanisms that regulate the function of individual kinases remain poorly understood. Given the proven clinical value of kinase-directed therapeutics, a detailed understanding of kinase regulatory mechanisms can significantly impact human health by identifying new ways to modulate kinase function. The long-term goal of this proposal is to determine how a general allosteric site regulates the substrate specificity and subcellular localization of the protein kinases. This proposal focuses on the protein kinase PDK1 where this allosteric site, known as the PIF pocket, is required for the phosphorylation of most PDK1 substrates. The PIF pocket may also promote cancer cell motility. Our lab recently identified small molecules that bind to the PIF pocket in cells. We hypothesize that these PIF-pocket ligands will disrupt the interaction between PDK1 and a subset of its substrates and effector proteins, thereby granting a rapid and reversible way to perturb PIF pocket function. The work proposed here aims to use our PIF-pocket ligands 1) to study how the PIF pocket determines the substrate specificity of PDK1 and 2) to study how the PIF pocket promotes cancer cell motility. To accomplish the first aim, we will identify PDK1 substrates by mass spectrometry and determine the sensitivity of their phosphorylation to our PIF-pocket ligands. To accomplish the second aim, we will characterize the role of the PIF pocket in the motility of highly aggressive melanoma cells grown in 3D culture. These studies will highlight the importance of this general allosteric site in regulating protein kinase function. This concept may alter the landscape of kinase drug discovery and lead to the development of a new class of kinase-directed therapies.