Redox signalling through Ser/Thr protein kinase networks

The growth and survival of organisms depends upon an ability of different cell types to sense, process and respond to a huge number of chemical signals. These include reactive oxygen species (ROS, such as hydrogen peroxide), which is continuously generated in various sub-cellular environments. One way that 'redox-sensing' can be achieved is through rapid, regulated, modifications that take place in chemically-reactive hot-spots found in some proteins. These changes, which are called post-translational modifications (PTMs), alter protein function and create new 'signalling networks', which control how information is relayed within cells. Our study is particularly interested in how PTMs on, and adjacent to, the relatively rare amino acid called Cysteine (Cys) control the behaviour of a family of enzymes termed protein kinases, which are important for controlling the flow of signalling information by the regulated addition of phosphate groups to their target proteins. Because ROS can be detected by a limited number of proteins in cells, they act as specificity 'switches' that modulate information exchange, leading to different sets of biological responses, such as cellular adaption to stress, cell growth and survival. In other circumstances, Cys modifications may become irreversible, leading to the switching 'on' of oxidant responses that promote survival during normal processes such as ageing, or lead to irreversible diseases associated with neurodegeneration. Our preliminary work has shown that Cys residues are found in exactly the same 3D location in ~100 members of the same enzyme family, and these Cys-containing enzymes are found throughout the tree of life, from yeasts to humans. This points to a fundamental importance of this amino acid being in the right place at the right time in order to sense and control redox signalling. Our proposal focusses on the 'redox control' of ~100 Ser/Thr protein kinases that all contain the same conserved Cys residue in a region of the protein called the 'kinase activation segment'. We want to know how these amino acids sense ROS, and how they pass this information along pathways to form signalling networks in cells, acting conceptually like a series of dynamic traffic lights that regulate the flow of traffic in a road network.

To rapidly advance our understanding of redox control of protein kinases, our objectives will be achieved through 4 distinct, but highly complementary, work packages (WPs), undertaken by an experienced team of scientists. Alongside broad initial efforts, our studies will also focus on two specific signalling 'pathways' in detail:

WP1: Cellular redox signaling: Cys modifications, protein complexes and interactomes
Objectives: Endogenous and Bio-ID-based mapping of 100 Cys-containing Ser/Thr protein kinases
Outcomes: Dynamic redox mapping for CAMK and AGC kinases in human cells.

WP2: Structural analysis, AlphaFold2 (AF2) database mining and Cys-residue using computational molecular dynamics.
Objectives: Define published and predicted folds for ~100 full-length Ser/Thr protein kinases.
Outcomes: Modelling of Cys residue interactions within the redox-sensitive Ser/Thr kinome

WP3.1: Quantitative analysis of chemical Cys modifications in kinases in vitro
Objectives: Enzymatic and MS-based approaches to study redox modifications, focussing initially on 'AGC' and 'CAMK' kinase families.
Outcomes: Biochemical and cellular analysis of redox regulation in Ser/Thr kinases

WP3.2: Focused analysis of AGC kinase-based redox signaling mechanisms in cells
Objectives: Evaluating redox and phospho-regulation in three AKT signalling enzymes
Outcomes: Define redox signalling in the model AGC kinase sub-family

WP4: Focused analysis of BRSK1/2-based signalling to Nrf2 during the redox response
Objectives: Analysis of upstream mechanisms contributing to cellular Nrf2 regulation.
Outcomes: Define redox signalling pathways linking BRSK1/2, mTOR and KEAP1/Nrf2.

Cells sense and respond to reactive oxygen species (ROS) via changes in the properties of intracellular signalling proteins, notably protein kinases and phosphatases. This study focuses on how redox-active Cys residues in Ser/Thr kinases assemble signalling networks and dictate cellular behaviour. Cys oxidation to sulfenic, sulfinic and sulfonic species and intra/intermolecular disulfide bonds are poorly-understood kinase redox switches, which diversify protein interactomes and regulate fundamental biological events. Ser/Thr kinase regulation has been studied intensively for 50 years, and kinase analysis is now entering a new phase in the context of redox signalling. Our integrated proposal will employ mass spectrometry and AI to focus on redox control within ~100 Ser/Thr protein kinases, including dozens about which little is known. Through proteomic, biochemical and structure-prediction approaches, we will redefine how many Ser/Thr kinase signalling networks operate.

WP1: Cellular redox signaling: Cys modifications, protein complexes and interactomes
Objectives: Endogenous and Bio-ID-based oxi-Cys mapping of 100 Cys-containing protein kinases
Outcomes: Dynamic redox mapping for CAMK and AGC kinases in human cells.

WP2: Structural analysis, AlphaFold2 (AF2) database mining and Cys-residue modelling by molecular dynamics.
Objectives: Define published and predicted folds for ~100 full-length Ser/Thr protein kinases.
Outcomes: Modelling of Cys residues within the redox-sensitive Ser/Thr kinome

WP3: Focused analysis of AGC kinase-based redox signalling mechanisms
Objectives: Evaluating redox and phospho-regulation in AGC and mTOR signalling kinases
Outcomes: New redox links in canonical AGC kinase signalling networks

WP4: Focused analysis of BRSK1/2-depend signalling to the Nrf2 transcription factor
Objectives: Define redox signalling linking AMPK, BRSK1/2, mTOR and Nrf2
Outcomes: New kinase effectors lying upstream in the Nrf2 stress response