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
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
-
- 534
- views
-
- 58
- 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
- Neuroscience
Hypothalamic kisspeptin (Kiss1) neurons are vital for pubertal development and reproduction. Arcuate nucleus Kiss1 (Kiss1ARH) neurons are responsible for the pulsatile release of gonadotropin-releasing hormone (GnRH). In females, the behavior of Kiss1ARH neurons, expressing Kiss1, neurokinin B (NKB), and dynorphin (Dyn), varies throughout the ovarian cycle. Studies indicate that 17β-estradiol (E2) reduces peptide expression but increases Slc17a6 (Vglut2) mRNA and glutamate neurotransmission in these neurons, suggesting a shift from peptidergic to glutamatergic signaling. To investigate this shift, we combined transcriptomics, electrophysiology, and mathematical modeling. Our results demonstrate that E2 treatment upregulates the mRNA expression of voltage-activated calcium channels, elevating the whole-cell calcium current that contributes to high-frequency burst firing. Additionally, E2 treatment decreased the mRNA levels of canonical transient receptor potential (TPRC) 5 and G protein-coupled K+ (GIRK) channels. When Trpc5 channels in Kiss1ARH neurons were deleted using CRISPR/SaCas9, the slow excitatory postsynaptic potential was eliminated. Our data enabled us to formulate a biophysically realistic mathematical model of Kiss1ARH neurons, suggesting that E2 modifies ionic conductances in these neurons, enabling the transition from high-frequency synchronous firing through NKB-driven activation of TRPC5 channels to a short bursting mode facilitating glutamate release. In a low E2 milieu, synchronous firing of Kiss1ARH neurons drives pulsatile release of GnRH, while the transition to burst firing with high, preovulatory levels of E2 would facilitate the GnRH surge through its glutamatergic synaptic connection to preoptic Kiss1 neurons.
-
- Computational and Systems Biology
Degree distributions in protein-protein interaction (PPI) networks are believed to follow a power law (PL). However, technical and study bias affect the experimental procedures for detecting PPIs. For instance, cancer-associated proteins have received disproportional attention. Moreover, bait proteins in large-scale experiments tend to have many false-positive interaction partners. Studying the degree distributions of thousands of PPI networks of controlled provenance, we address the question if PL distributions in observed PPI networks could be explained by these biases alone. Our findings are supported by mathematical models and extensive simulations and indicate that study bias and technical bias suffice to produce the observed PL distribution. It is, hence, problematic to derive hypotheses about the topology of the true biological interactome from the PL distributions in observed PPI networks. Our study casts doubt on the use of the PL property of biological networks as a modeling assumption or quality criterion in network biology.
