Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

David W. Litchfield

Abstract

Protein functions are regulated by the post-translational addition of covalent modifications on certain amino acids. Depending on their distance within the 3-dimensional structure, addition/removal of individual post translational modifications (PTMs) can be impacted by others. This PTM interplay constitutes an essential regulatory mechanism that interconnects the molecular networks in the cell. Protein CK2, a clinically relevant acidophilic Ser/Thr kinase, may be responsible for 10-20% of the human phosphoproteome. Such estimates agree with the number of known substrates, which continues to expand. Furthermore, the demonstration that CK2 participates in hierarchical phosphorylation and has similar sequence determinants to caspases suggest extensive PTM interplay in CK2-dependent signaling.

In this thesis, we explore the role of lysine acetylation in the vicinity of the phosphorylatable residue(s) as a modulator of phosphorylation by CK2. To explore this association a biochemistry approach was followed to decipher the impact of lysine acetylation in CK2 specificity and a proteomics approach was employed for profiling the affected sites in cells. In solution peptides conforming to the CK2 consensus containing lysine or acetyllysine at positions +1, +2, or +3 downstream the phosphorylatable site, were found only to be phosphorylated by CK2 when either determinant was present at position +2. Linear patterns were generated to reflect this specificity and the PTM databases and the human proteome were searched for substrate hits. The boundaries and cellular conditions of the hits were assessed including conservation, mutations, regulatory role, and enzyme-substrate relationships. Several hits matching the patterns were observed in the cells to be both acetylated and phosphorylated. Since chromatin organization proteins were hits the regulatory role of CK2 in this process was summarized. The data processing and analysis steps followed were incorporated in a data-driven R Shiny web application visualRepo.

Collectively, our work shows how CK2-dependent phosphorylation and lysine acetylation network integration could contribute to the complexity of cellular processes. It also provides a computational and analytical framework for further studies exploring the PTM interplay occurring at a kinase consensus sequence. Finally, given the druggability of CK2, our results offer new molecular insights for exploring the combination of CK2 and lysine deacetylase inhibitors.

Summary for Lay Audience

Proteins are main components of the cell and are subjected to many chemical changes or modifications, affecting their function. Some chemical modifications in proteins have been described to interplay with one another resulting in intricate regulatory networks. If we were to look at a protein as a sequence of characters, where two proximal characters were to be substituted, i.e., modified, an interplay between these can occur preventing or facilitating the addition of the second modification once the first is added. The protein CK2 is one of many proteins that modify other proteins in the cell, referred to as substrates. CK2 prefers modifying characters (amino acids) contained in a negatively charged sequence. In this thesis, we decipher how modification by CK2 is enabled by a pre-existing modification (lysine acetylation) that removes a positive charge two amino acids away. Biological databases were searched for protein sequences conforming to this view and several candidates were identified. The sequence features and cellular functions of the candidates were also mined from the databases and the literatures. Finally, for the first time a list of hits containing both modifications occurring in the cell were identified. All the steps in the processing and analysis were incorporated into a web application, visualRepo, for facilitating the reuse and reanalysis of the data. Overall, our work can be extended to identify other modifying proteins and the interplay that can occur. Given CK2 is a therapeutic target, our results provide us with information that can be used to devise combination therapies involving CK2 in the future.

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