Redox-Dependent Regulation of Brain-Selective Kinases BRSK1/2: Implications for Dynamic Control of the AMPK Family through Cysteine-Based Mechanisms

Core Concepts

Reversible oxidation and reduction of conserved cysteine residues within the catalytic domains of BRSK1 and BRSK2 kinases dynamically modulate their catalytic activities.

Abstract

The study investigates the redox regulation of the AMPK-related brain-selective kinases BRSK1 and BRSK2. The key findings are:

The catalytic activities of full-length BRSK1 and BRSK2 are highly sensitive to redox conditions. In the presence of oxidants like H2O2, BRSK activity is inhibited, while reducing agents like DTT strongly activate the kinases.

Mass spectrometry analysis revealed the formation of intramolecular disulfide bonds between conserved cysteine residues within the kinase domains of BRSK1 and BRSK2. These include disulfide bonds between the T-loop +2 cysteine and a cysteine in the CPE motif of the activation segment.

Mutational analysis of the conserved cysteine residues showed that the T-loop +2 cysteine and the HRD-proximal cysteine pairs are critical for redox-dependent regulation of BRSK catalytic activity. Mutation of these cysteine residues to alanine impairs the ability of reducing agents to activate the kinases.

Phylogenetic analysis indicates that the diversification of BRSK kinases from the broader AMPK family is accompanied by the accumulation of conserved cysteine pairs within the kinase domain, suggesting that disulfide-mediated regulation may be a prevalent mechanism for controlling catalytic activity in the AMPK kinase family.

Overall, the study provides mechanistic insights into how reversible oxidation and reduction of cysteine residues dynamically regulate the catalytic output of BRSK1 and BRSK2, with broader implications for understanding redox-based control of the AMPK kinase family.

Stats

The catalytic activities of full-length BRSK1 and BRSK2 purified from Sf21 cells were strongly inhibited by 1 mM H2O2 and could be restored by the addition of 10 mM DTT.
Mutation of the T-loop +2 cysteine (C191A in BRSK1, C176A in BRSK2) or the HRD-proximal cysteine pair (C147/153A in BRSK1, C132/138A in BRSK2) significantly impaired the ability of DTT to activate the kinases.

Quotes

"Reversible oxidation and reduction of conserved cysteine residues within the catalytic domains of BRSK1 and BRSK2 kinases dynamically modulate their catalytic activities."
"The diversification of BRSK kinases from the broader AMPK family is accompanied by the accumulation of conserved cysteine pairs within the kinase domain, suggesting that disulfide-mediated regulation may be a prevalent mechanism for controlling catalytic activity in the AMPK kinase family."

Key Insights Distilled From

Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms

by Bendzunas,G.... at www.biorxiv.org 10-06-2023

https://www.biorxiv.org/content/10.1101/2023.10.05.561145v3

Deeper Inquiries

How might the redox-dependent regulation of BRSK1/2 kinases impact their physiological functions in the brain and central nervous system

The redox-dependent regulation of BRSK1/2 kinases can have significant implications for their physiological functions in the brain and central nervous system. As key regulators of cellular metabolism, growth, differentiation, and polarity, BRSK1/2 play crucial roles in neuronal development and function. The brain-specific expression of BRSK1/2 suggests specialized functions in neural processes, including synaptic plasticity, neuronal migration, and axonal growth. The redox-sensitive nature of BRSK1/2 implies that their activity can be dynamically modulated in response to oxidative stress or changes in the cellular redox environment. This redox regulation may impact the phosphorylation of downstream substrates involved in neuronal signaling pathways, such as Tau protein, which is crucial for microtubule stability and neuronal function. Dysregulation of BRSK1/2 redox signaling could potentially contribute to neurodevelopmental disorders, neurodegenerative diseases, or cognitive impairments by disrupting critical signaling cascades in the brain.

What are the potential implications of BRSK redox regulation for the development of therapeutic interventions targeting this kinase family

The redox regulation of BRSK1/2 presents promising opportunities for the development of therapeutic interventions targeting this kinase family. By understanding the mechanisms by which cysteine residues modulate BRSK activity, researchers can design specific covalent compounds that selectively modulate kinase function through redox-based mechanisms. Targeting the redox-sensitive cysteine residues in BRSK1/2 could offer a novel approach to regulate their activity in disease states characterized by oxidative stress or redox imbalance. Therapeutic strategies aimed at manipulating the redox state of BRSK1/2 could potentially be used to treat neurodevelopmental disorders, neurodegenerative diseases, or other conditions where BRSK dysregulation is implicated. Additionally, the development of small molecules or compounds that target the redox-sensitive sites in BRSK1/2 may provide new avenues for drug discovery and personalized medicine approaches in the treatment of brain-related disorders.

Given the conservation of cysteine-based regulatory mechanisms across diverse protein kinase families, what other signaling pathways or cellular processes might be influenced by similar redox-sensing mechanisms

The conservation of cysteine-based regulatory mechanisms across diverse protein kinase families suggests that similar redox-sensing mechanisms may influence a wide range of signaling pathways and cellular processes. Redox-dependent regulation of protein kinases through cysteine oxidation is likely to impact various cellular functions beyond the AMPK family. For instance, redox-sensitive cysteine residues in other protein kinases, such as CAMK, AGC, and AGC-like families, may also play critical roles in modulating kinase activity in response to oxidative stress. These redox-sensitive mechanisms could regulate cell signaling, gene expression, cell proliferation, and apoptosis in different cell types and tissues. Furthermore, the broader implications of cysteine-based redox regulation extend to other redox-sensitive proteins involved in cellular homeostasis, antioxidant defense, and stress response pathways. Understanding the interplay between redox signaling and protein kinase activity can provide insights into the molecular mechanisms underlying various physiological and pathological processes in cells and tissues.