Investigating the Functions and Regulation of Non-Proline Directed CDK1 Phosphorylation

在我们的一生中，我们身体中的细胞需要在正确的时间增殖。例如，我们皮肤中的细胞必须对伤口做出反应以愈合，免疫细胞的数量迅速增加以对抗感染。一旦细胞接收到增殖的信号，称为细胞周期蛋白依赖性激酶（CDK）的蛋白质就会变得活跃，并在称为磷酸化的过程中修饰细胞中的数百种蛋白质。蛋白质磷酸化是协调细胞内容物的复制和分裂所必需的。蛋白质磷酸化的顺序至关重要。例如，基因组复制必须在细胞分裂之前发生，以确保基因组信息从亲本细胞到后代细胞的高保真传输。改变蛋白质磷酸化的顺序会导致基因组不稳定，这被认为是导致衰老和癌症的细胞损伤的来源。CDK如何以正确的顺序选择底物已经在包括酵母在内的模式生物中进行了研究。然而，人类基因组编码CDK和相关蛋白的扩展网络。目前尚不清楚在酵母中发现的机制是否适用于人类细胞。我们开发了一种新的方法来研究CDK家族的一个成员CDK1如何选择蛋白质进行磷酸化。我们首先在试管中（体外）通过加入CDK1和相关蛋白质（称为细胞周期蛋白和Cks 1）来磷酸化细胞蛋白质，这些蛋白质已知会改变CDK1的活性和特异性。然后，我们使用最先进的蛋白质组学技术分析磷酸化的程度。这种方法使我们能够同时测量细胞内数千种蛋白质的CDK1磷酸化。使用这种方法，我们发现许多位点的磷酸化CDK1有一个不寻常的序列，是非典型的CDK1。我们继续表明，这些非典型网站的频率是由细胞周期蛋白和Cks1相互作用的CDK1的合作伙伴改变。拟议的研究旨在了解这些相互作用伙伴如何使用涉及基因组编辑，细胞生物学，生物化学和蛋白质组学的多学科方法在体外和活细胞中改变CDK1的磷酸化。我们的目标是开发CDK1如何选择磷酸化底物的规则，以便我们可以设计在人类细胞中以特定顺序磷酸化的蛋白质。

Throughout our life, the cells in our body need to proliferate at the right time. For example, cells in our skin must respond to wounds to heal, and immune cells quickly increase in number to fight off infections. Once a cell receives the signals to proliferate, proteins called cyclin-dependent kinases (CDKs) become active and modify hundreds proteins in the cell in a process called phosphorylation. Protein phosphorylation is required to orchestrate the duplication and division of cellular contents. The order in which proteins are phosphorylated is crucial. For example, genome duplication must occur before cells divide to ensure high fidelity transmission of genomic information from parental to progeny cells. Changing the order in which proteins are phosphorylated leads to genome instability, which is thought to be a source of cellular damage leading to aging and cancer. How CDKs choose substrates in the right order has been investigated in model organisms, including yeast. However, the human genome encodes an expanded network of CDKs and related proteins. It is unknown if the mechanisms discovered in yeast will apply to human cells. We developed a new approach to investigate how one member of the CDK family, CDK1, chooses proteins for phosphorylation. We first phosphorylate cellular proteins 'in a test tube' (in vitro) by adding CDK1 and related proteins, called cyclins and Cks1, which are known to alter CDK1 activity and specificity. We then analyze the extent of phosphorylation using state-of-the-art technology called proteomics. This approach allows us to measure phosphorylation by CDK1 for thousands of proteins within the cell simultaneously. Using this approach, we showed that many sites phosphorylated by CDK1 have an unusual sequence that is atypical for CDK1. We go on to show that the frequency of these atypical sites is altered by the cyclin and Cks1 interaction partners of CDK1. The proposed research aims to understand how these interaction partners alter the phosphorylation of CDK1 in vitro and in living cells using a multidisiciplinary approach involving genome editing, cell biology, biochemistry, and proteomics. The objective is to develop rules for how CDK1 chooses substrates for phosphorylation so that we can design proteins that are phosphorylated in human cells in a specific order.