1. Computational and Systems Biology

Protein Kinases: Redox takes control

A study of two enzymes in the brain reveals new insights into how redox reactions regulate the activity of protein kinases.
https://doi.org/10.7554/eLife.99765
  • Download
  • Cite
  • CommentOpen annotations (there are currently 0 annotations on this page).
Share this article
Cite this article
  1. Iván Plaza-Menacho
(2024)
Protein Kinases: Redox takes control
eLife 13:e99765.
https://doi.org/10.7554/eLife.99765
  2. Download BibTeX
  3. Download .RIS
  1. Iván Plaza-Menacho  Is a corresponding author
  1. Kinases, Protein Phosphorylation and Cancer Group, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Spain

For decades reactive oxygen species were considered to be the by-products of cell damage. However, more recently these unstable molecules have been shown to play a role in cell signaling as secondary messengers that transfer electrons between proteins.

This exchange of electrons, known as a redox modification, typically takes place on a specific set of amino acids within the protein. Previous work showed that the removal of an electron (known as oxidation) from the amino acid cysteine can alter the structure and function of protein kinases (enzymes that phosphorylate molecules and have an essential role in cell signaling; Garrido Ruiz et al., 2022). As well as adding phosphoryl groups to other proteins, some kinases need to be phosphorylated themselves to switch on their catalytic activity (Cuesta-Hernández et al., 2023). However, much less is known about how cysteine oxidation modulates the activity and structure of protein kinases, and the systematic effect this has on cell signaling.

Now, in eLife, Natarajan Kannan and colleagues – including George Bendzunas and Dominic Byrne as joint first authors – report how cysteine oxidation controls two kinases found in the brain (Bendzunas et al., 2024). The team showed that the catalytic activity of these two enzymes – called AMPK-related Brain-selective kinases 1 (BRSK1) and 2 (BRSK2) – is directly regulated through reversible oxidation. This redox reaction takes place at cysteine residues situated at key structural and functional sites within the catalytic domain (the part of the protein that causes the enzymatic reaction).

In particular, Bendzunas et al. (who are based at the University of Georgia and the University of Liverpool) identified two pairs of cysteine residues in both BRSK1 and BRSK2, which form a bridge between sulfur atoms when oxidized. These connections, known as disulfide bonds, help maintain the structure and shape of proteins. When the team mutated the cysteine residues in vitro, this increased the catalytic activity of the kinases. It also led to higher amounts of phosphorylated Tau, the primary substrate of BRSK1 and BRSK2, in cells.

Molecular modelling and simulations revealed that oxidation of one of the four identified cysteines (which resides on a motif that defines the end of the activation loop) destabilizes bonds required to allosterically activate the catalytic domain. Taken together, these findings suggest that oxidation of the four cysteines, and subsequent formation of the two intramolecular disulfide bridges, represses the activity of BRSK1 and BRSK2.

Many other protein kinases have cysteine residues situated in similar locations within their structure. It is therefore possible that redox regulation of disulfide bridges may be a widespread mechanism for controlling protein kinase activity and signaling.

In a previous study led by Kannan, roughly 10% of protein kinases present in humans were hypothesized to be subjected to redox-dependent regulation (Byrne et al., 2020). This includes key members of the CAMK, AGC, and AGC-like families, which each contain a cysteine residue that lies adjacent to the phosphorylation site in the activation loop. The transmembrane receptor EGFR also has a cysteine residue in another nearby location, which enhances catalytic activity when oxidized (Truong et al., 2016). However, it is unclear how the redox state of these cysteine residues modulates the activity of kinases. As well as altering the conformational landscape of the activation loop itself, redox modifications may enhance the non-catalytic properties of the enzyme, or regulate intermolecular interactions and oligomerization (Cuesta-Hernández et al., 2023).

Cysteine residues have also been found in other parts of the kinase structure. Comprehensive mapping revealed a group of cysteine residues that lie in or adjacent to the binding site for ATP, which are collectively referred to as the ‘cysteinome’ (Chaikuad et al., 2018). Furthermore, the protein kinases cAPK, c-Src and LRRK2 among others, have all been shown to contain two cysteine residues which alter catalytic activity following redox modifications (Humphries et al., 2005; Humphries et al., 2002; Heppner et al., 2018; Trilling et al., 2024). Many of the motifs that contain cysteine residues also have a phosphorylation site, but it is poorly understood how phosphorylation impacts the oxidation of cysteines (Kemper et al., 2022).

These redox-dependent molecular switches provide therapeutic advantages as they can be used to block the activity of kinases. Drugs targeting cysteine residues, known as covalent inhibitors, were initially designed for members of the EGFR family (Tsou et al., 2005). Since then, more than forty covalent inhibitors have been approved by the US Food and Drug Administration (FDA). Most of these target residues in the cysteinome, particularly a cysteine at the front site of the kinase in the F2 position (Chaikuad et al., 2018). However, other cysteine hotspots are yet to be fully explored (Yen-Pon et al., 2018; Chen et al., 2022; Zhang et al., 2016), including the cysteine disulfide bonds identified by Bendzunas et al. which could be important therapeutic targets.

The work of Bendzunas et al. and others highlights how significant redox biology is for understanding protein kinase function and pharmacology. However, the regulatory power of cysteine oxidation is often underappreciated, and the effect it has on most human kinases remains unknown. Further exploration of the cysteines in protein kinases, together with more high-resolution structural data, will help to bridge this gap and may lead to the discovery of more drug targets for covalent inhibitors.

References

Article and author information

Author details

  1. Iván Plaza-Menacho

    Iván Plaza-Menacho leads the Kinases, Protein Phosphorylation and Cancer Group in the Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
    For correspondence
    iplaza@cnio.es
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4666-9431

Publication history

  1. Version of Record published:

Copyright

© 2024, Plaza-Menacho

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 584
    views
  • 62
    downloads
  • 0
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

Categories and tags

Research organisms

    1. Related to

    Redox regulation and dynamic control of brain-selective kinases BRSK1/2 in the AMPK family through cysteine-based mechanisms

    George N Bendzunas, Dominic P Byrne ... Natarajan Kannan
  2. Further reading

Further reading

    1. Computational and Systems Biology

    Redox regulation and dynamic control of brain-selective kinases BRSK1/2 in the AMPK family through cysteine-based mechanisms

    George N Bendzunas, Dominic P Byrne ... Natarajan Kannan
    Research Article

    In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications, including phosphorylation of Ser/Thr residues and oxidation of cysteine (Cys) residues. While regulation by activation segment phosphorylation of Ser/Thr residues is well understood, relatively little is known about how oxidation of cysteine residues modulate catalysis. In this study, we investigate redox regulation of the AMPK-related brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intramolecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-loop Cys with a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide-mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.

    1. Computational and Systems Biology
    2. Genetics and Genomics

    Loss of CTRP10 results in female obesity with preserved metabolic health

    Fangluo Chen, Dylan C Sarver ... G William Wong
    Research Article

    Obesity is a major risk factor for type 2 diabetes, dyslipidemia, cardiovascular disease, and hypertension. Intriguingly, there is a subset of metabolically healthy obese (MHO) individuals who are seemingly able to maintain a healthy metabolic profile free of metabolic syndrome. The molecular underpinnings of MHO, however, are not well understood. Here, we report that CTRP10/C1QL2-deficient mice represent a unique female model of MHO. CTRP10 modulates weight gain in a striking and sexually dimorphic manner. Female, but not male, mice lacking CTRP10 develop obesity with age on a low-fat diet while maintaining an otherwise healthy metabolic profile. When fed an obesogenic diet, female Ctrp10 knockout (KO) mice show rapid weight gain. Despite pronounced obesity, Ctrp10 KO female mice do not develop steatosis, dyslipidemia, glucose intolerance, insulin resistance, oxidative stress, or low-grade inflammation. Obesity is largely uncoupled from metabolic dysregulation in female KO mice. Multi-tissue transcriptomic analyses highlighted gene expression changes and pathways associated with insulin-sensitive obesity. Transcriptional correlation of the differentially expressed gene (DEG) orthologs in humans also shows sex differences in gene connectivity within and across metabolic tissues, underscoring the conserved sex-dependent function of CTRP10. Collectively, our findings suggest that CTRP10 negatively regulates body weight in females, and that loss of CTRP10 results in benign obesity with largely preserved insulin sensitivity and metabolic health. This female MHO mouse model is valuable for understanding sex-biased mechanisms that uncouple obesity from metabolic dysfunction.

    1. Computational and Systems Biology

    Untargeted pixel-by-pixel metabolite ratio imaging as a novel tool for biomedical discovery in mass spectrometry imaging

    Huiyong Cheng, Dawson Miller ... Qiuying Chen
    Research Article

    Mass spectrometry imaging (MSI) is a powerful technology used to define the spatial distribution and relative abundance of metabolites across tissue cryosections. While software packages exist for pixel-by-pixel individual metabolite and limited target pairs of ratio imaging, the research community lacks an easy computing and application tool that images any metabolite abundance ratio pairs. Importantly, recognition of correlated metabolite pairs may contribute to the discovery of unanticipated molecules in shared metabolic pathways. Here, we describe the development and implementation of an untargeted R package workflow for pixel-by-pixel ratio imaging of all metabolites detected in an MSI experiment. Considering untargeted MSI studies of murine brain and embryogenesis, we demonstrate that ratio imaging minimizes systematic data variation introduced by sample handling, markedly enhances spatial image contrast, and reveals previously unrecognized metabotype-distinct tissue regions. Furthermore, ratio imaging facilitates identification of novel regional biomarkers and provides anatomical information regarding spatial distribution of metabolite-linked biochemical pathways. The algorithm described herein is applicable to any MSI dataset containing spatial information for metabolites, peptides or proteins, offering a potent hypothesis generation tool to enhance knowledge obtained from current spatial metabolite profiling technologies.