Complex Dynamics in Multisite Phosphorylation

描述(申请人提供)：多位点蛋白质磷酸化和去磷酸化(P&D)是大多数真核细胞过程的基础。多位点P&D的失调与包括癌症在内的多种人类疾病有关，而激酶和磷酸酶都是临床使用或开发的重要药物的靶点。这项提议的目的是研究当激酶和磷酸酶共同调节细胞磷酸化状态时所产生的动力学复杂性。一个具有n个磷酸化位点的底物分子可能占据2n个态，一群底物分子将具有这种磷酸型的分布，而激酶、磷酸酶和底物的系统通过稳定的ATP供应而保持远离平衡。我们将开发一种质谱学方法来区分所有2n个磷酸形式，并组装一套模型P&D系统，这些系统具有不同数量的磷酸化位点、酶机制和细胞角色，在其上进行系统生化。我们将在数学上确定这类系统表现出复杂行为的条件，如多稳定性和振荡，并将在模型系统上测试这些预测。这种多学科的方法将为这些复杂的、医学上重要的和鲜为人知的系统提供新的见解。公共卫生相关性：蛋白质磷酸化和去磷酸化的失调与包括癌症在内的几种人类疾病有关，所涉及的酶，激酶和磷酸酶，是临床使用和开发的重要药物的靶标。激酶、磷酸酶和底物系统表现出惊人的复杂性，特别是当磷酸化位点的数量增加时。这一建议试图通过多学科的数学和实验方法，使用具有不同细胞角色、酶机制和磷酸化位点数量的几个模型磷酸化和去磷酸化系统来研究这种复杂性。

DESCRIPTION (provided by applicant): Multisite protein phosphorylation and dephosphorylation (P&D) are fundamental to most eukaryotic cellular processes. Disregulation of multisite P&D is implicated in several human diseases, including cancer, and both kinases and phosphatases are targets of important drugs in clinical use or development. The goal of this proposal is to investigate the dynamical complexity that arises when kinases and phosphatases collectively regulate the cellular phosphorylation state. A substrate molecule with n phosphorylation sites may occupy 2n states and a population of substrate molecules will have a distribution of such phospho-forms, with the system of kinase, phosphatase and substrate being maintained away from equilibrium by a steady supply of ATP. We will develop a mass spectrometry protocol to distinguish all 2n phospho-forms and assemble a set of model P&D systems, having varying numbers of phosphorylation sites, enzymatic mechanisms and cellular roles, on which to undertake systems biochemistry. We will determine mathematically the conditions under which such systems exhibit complex behaviors, such as multistability and oscillations, and will test these predictions on the model systems. Such a multidisciplinary approach will provide novel insights into these complex, medically important and poorly-understood systems. PUBLIC HEALTH RELEVANCE: Disregulation of protein phosphorylation and dephosphorylation is implicated in several human diseases, including cancer, and the enzymes involved, kinases and phosphatases, are targets of important drugs in clinical use and development. Systems of kinase, phosphatase and substrate show surprising complexity, especially as the number of phosphorylation sites increases. This proposal seeks to investigate such complexity through a multidisciplinary mathematical and experimental approach, using several model phosphorylation and dephosphorylation systems having varying cellular roles, enzymatic mechanisms and numbers of phosphorylation sites.