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

研究非脯氨酸定向 CDK1 磷酸化的功能和调控

• 批准号: BB/X007057/1
• 负责人: Tony Ly
• 金额: $ 52.57万
• 依托单位: University of Dundee
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX007057%2F1
• 关键词: Investigating; Functions; Regulation; Non; Proline

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.

在我们的一生中，我们体内的细胞需要在适当的时间扩散。例如，我们皮肤中的细胞必须应对伤口的愈合，免疫细胞数量迅速增加以抵抗感染。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）的蛋白质在一个称为磷酸化的过程中变为活性，并在细胞中修饰数百种蛋白质。需要蛋白质磷酸化来协调细胞含量的复制和分裂。蛋白质磷酸化的顺序至关重要。例如，基因组重复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忠诚传播。改变蛋白质是磷酸化的顺序会导致基因组不稳定性，这被认为是导致衰老和癌症的细胞损伤来源。在包括酵母在内的模型生物体中，已经研究了CDKS如何以正确顺序选择底物。但是，人类基因组编码了扩展的CDK和相关蛋白网络。尚不清楚酵母中发现的机制是否适用于人类细胞。我们开发了一种新方法来研究CDK家族CDK1的一个成员如何选择用于磷酸化的蛋白质。我们首先通过添加CDK1和相关蛋白（称为细胞周期蛋白和CKS1）磷酸化细胞蛋白“在测试管中”（在体外），这些蛋白已知会改变CDK1活性和特异性。然后，我们使用称为蛋白质组学的最先进技术分析磷酸化的程度。这种方法使我们能够通过CDK1同时测量成千上万的细胞内蛋白质的磷酸化。使用这种方法，我们表明许多由CDK1磷酸化的位点具有不寻常的序列，对于CDK1而言是非典型的。我们继续表明，这些非典型位点的频率通过CDK1的Cyclin和Cks1相互作用伙伴改变了。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医学方法在体外和活细胞中改变CDK1的磷酸化。目的是制定CDK1如何选择磷酸化底物的规则，以便我们可以设计以特定顺序在人类细胞中磷酸化的蛋白质。