Protein Kinases: Redox takes control
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
Publication history
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
-
- 497
- views
-
- 54
- downloads
-
- 0
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
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)
Further reading
-
- Cancer Biology
- Computational and Systems Biology
Assay for Transposase-Accessible Chromatin sequencing (ATAC-Seq) is a widely used technique to explore gene regulatory mechanisms. For most ATAC-Seq data from healthy and diseased tissues such as tumors, chromatin accessibility measurement represents a mixed signal from multiple cell types. In this work, we derive reliable chromatin accessibility marker peaks and reference profiles for most non-malignant cell types frequently observed in the microenvironment of human tumors. We then integrate these data into the EPIC deconvolution framework (Racle et al., 2017) to quantify cell-type heterogeneity in bulk ATAC-Seq data. Our EPIC-ATAC tool accurately predicts non-malignant and malignant cell fractions in tumor samples. When applied to a human breast cancer cohort, EPIC-ATAC accurately infers the immune contexture of the main breast cancer subtypes.
-
- Computational and Systems Biology
- Neuroscience
Diffusional kurtosis imaging (DKI) is a methodology for measuring the extent of non-Gaussian diffusion in biological tissue, which has shown great promise in clinical diagnosis, treatment planning, and monitoring of many neurological diseases and disorders. However, robust, fast, and accurate estimation of kurtosis from clinically feasible data acquisitions remains a challenge. In this study, we first outline a new accurate approach of estimating mean kurtosis via the sub-diffusion mathematical framework. Crucially, this extension of the conventional DKI overcomes the limitation on the maximum b-value of the latter. Kurtosis and diffusivity can now be simply computed as functions of the sub-diffusion model parameters. Second, we propose a new fast and robust fitting procedure to estimate the sub-diffusion model parameters using two diffusion times without increasing acquisition time as for the conventional DKI. Third, our sub-diffusion-based kurtosis mapping method is evaluated using both simulations and the Connectome 1.0 human brain data. Exquisite tissue contrast is achieved even when the diffusion encoded data is collected in only minutes. In summary, our findings suggest robust, fast, and accurate estimation of mean kurtosis can be realised within a clinically feasible diffusion-weighted magnetic resonance imaging data acquisition time.