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.
- 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
-
The cysteinome of protein kinases as a target in drug developmentAngewandte Chemie 57:4372–4385.https://doi.org/10.1002/anie.201707875
-
Structure-based design of a dual-warhead covalent inhibitor of FGFR4Communications Chemistry 5:36.https://doi.org/10.1038/s42004-022-00657-9
-
Direct cysteine sulfenylation drives activation of the Src kinaseNature Communications 9:4522.https://doi.org/10.1038/s41467-018-06790-1
-
Regulation of cAMP-dependent protein kinase activity by glutathionylationThe Journal of Biological Chemistry 277:43505–43511.https://doi.org/10.1074/jbc.M207088200
-
Enhanced dephosphorylation of cAMP-dependent protein kinase by oxidation and thiol modificationThe Journal of Biological Chemistry 280:2750–2758.https://doi.org/10.1074/jbc.M410242200
-
RedOx regulation of LRRK2 kinase activity by active site cysteinesNPJ Parkinson’s Disease 10:75.https://doi.org/10.1038/s41531-024-00683-5
-
Molecular basis for redox activation of epidermal growth factor receptor kinaseCell Chemical Biology 23:837–848.https://doi.org/10.1016/j.chembiol.2016.05.017
-
Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitorsNature Chemical Biology 12:876–884.https://doi.org/10.1038/nchembio.2166
Article and author information
Author details
Iván Plaza-Menacho
Publication history
- Version of Record published: June 20, 2024 (version 1)
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
-
- 39
- views
-
- 3
- downloads
-
- 0
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Protein Kinases: Redox takes control
eLife 13:e99765.
https://doi.org/10.7554/eLife.99765
Further reading
-
- Computational and Systems Biology
- Medicine
Background:
Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Most cases of preterm birth occur spontaneously and result from preterm labor with intact (spontaneous preterm labor [sPTL]) or ruptured (preterm prelabor rupture of membranes [PPROM]) membranes. The prediction of spontaneous preterm birth (sPTB) remains underpowered due to its syndromic nature and the dearth of independent analyses of the vaginal host immune response. Thus, we conducted the largest longitudinal investigation targeting vaginal immune mediators, referred to herein as the immunoproteome, in a population at high risk for sPTB.
Methods:
Vaginal swabs were collected across gestation from pregnant women who ultimately underwent term birth, sPTL, or PPROM. Cytokines, chemokines, growth factors, and antimicrobial peptides in the samples were quantified via specific and sensitive immunoassays. Predictive models were constructed from immune mediator concentrations.
Results:
Throughout uncomplicated gestation, the vaginal immunoproteome harbors a cytokine network with a homeostatic profile. Yet, the vaginal immunoproteome is skewed toward a pro-inflammatory state in pregnant women who ultimately experience sPTL and PPROM. Such an inflammatory profile includes increased monocyte chemoattractants, cytokines indicative of macrophage and T-cell activation, and reduced antimicrobial proteins/peptides. The vaginal immunoproteome has improved predictive value over maternal characteristics alone for identifying women at risk for early (<34 weeks) sPTB.
Conclusions:
The vaginal immunoproteome undergoes homeostatic changes throughout gestation and deviations from this shift are associated with sPTB. Furthermore, the vaginal immunoproteome can be leveraged as a potential biomarker for early sPTB, a subset of sPTB associated with extremely adverse neonatal outcomes.
Funding:
This research was conducted by the Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health and Human Services (NICHD/NIH/DHHS) under contract HHSN275201300006C. ALT, KRT, and NGL were supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health.
-
- Computational and Systems Biology
- Genetics and Genomics
Runs of homozygosity (ROH) segments, contiguous homozygous regions in a genome were traditionally linked to families and inbred populations. However, a growing literature suggests that ROHs are ubiquitous in outbred populations. Still, most existing genetic studies of ROH in populations are limited to aggregated ROH content across the genome, which does not offer the resolution for mapping causal loci. This limitation is mainly due to a lack of methods for the efficient identification of shared ROH diplotypes. Here, we present a new method, ROH-DICE, to find large ROH diplotype clusters, sufficiently long ROHs shared by a sufficient number of individuals, in large cohorts. ROH-DICE identified over 1 million ROH diplotypes that span over 100 SNPs and are shared by more than 100 UK Biobank participants. Moreover, we found significant associations of clustered ROH diplotypes across the genome with various self-reported diseases, with the strongest associations found between the extended HLA region and autoimmune disorders. We found an association between a diplotype covering the HFE gene and hemochromatosis, even though the well-known causal SNP was not directly genotyped or imputed. Using a genome-wide scan, we identified a putative association between carriers of an ROH diplotype in chromosome 4 and an increase in mortality among COVID-19 patients (P-value=1.82×10-11). In summary, our ROH-DICE method, by calling out large ROH diplotypes in a large outbred population, enables further population genetics into the demographic history of large populations. More importantly, our method enables a new genome-wide mapping approach for finding disease-causing loci with multi-marker recessive effects at a population scale.
