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

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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.