Recombinant Mouse Serine/threonine-protein kinase BRSK2 (Brsk2)

What is the molecular structure of BRSK2 and which domains are critical for its function?

BRSK2 is a serine/threonine protein kinase belonging to the AMP-activated protein kinase (AMPK) subfamily. Its structure includes several functional domains that regulate its activity and interactions:

• N-terminal protein kinase domain: Contains critical residues for catalytic activity, including Lys48 which is essential for ATP binding

• UBA (ubiquitin-associated) domain

• AIS (auto-inhibitory sequence) domain

• C-terminal KA1 (kinase-associated 1) domain
  The activation loop contains Thr174, which requires phosphorylation for kinase activation. Recent studies have identified important redox-sensitive cysteine residues within the kinase domain, including a T-Loop +2 Cys that communicates with a BRSK-specific Cys residue in the CPE motif (replacing the typical APE motif in other kinases) . These cysteines form disulfide bonds and are involved in redox regulation of BRSK2 activity.
  The UBA and AIS regions interact with the catalytic fold of the kinase domain, maintaining BRSK2 in an inactive state until it's phosphorylated by upstream regulators, bound by interaction partners, or recruited to the plasma membrane. Mutations in the UBA domain or loss of the AIS or KA1 domain results in a constitutively active kinase .

What is the tissue expression pattern of BRSK2 in humans and mice?

Despite its name as "brain-selective kinase," BRSK2 actually shows a broader expression pattern that differs between species:
In humans:

• High expression in brain

• Even higher expression in pancreatic tissue, particularly in islets and ducts

• Specifically co-localized with insulin, but not glucagon, in pancreatic cells
  In mice and rats:

• Primarily detected in brain

• Low levels in testis

• Highly expressed in MIN6 β-cell line and isolated mouse islets
  Immunohistochemical analysis of human pancreatic tissue with BRSK2 antibody reveals abundant staining in pancreatic islets and ducts. Northern blot analysis shows that BRSK2 mRNA is expressed at an even higher level in pancreas than in brain, which has been confirmed at the protein level by Western blot analysis .
  Interestingly, BRSK2 expression is regulated by glucose levels in β-cells. When MIN6 cells are treated with varying concentrations of glucose for 5 hours, BRSK2 expression is down-regulated in a dose-dependent manner, which is accompanied by decreases in the phosphorylation of CDC25C .

How is BRSK2 activity regulated at the molecular level?

BRSK2 activity is subject to multiple layers of regulation:
1. Phosphorylation:

• BRSK2 is activated by phosphorylation of Thr174 within its T-loop

• LKB1/STRADα/MO25α holoenzyme complex phosphorylates and activates BRSK2

• Bacterially expressed BRSK2 is inactive unless phosphorylated by LKB1 complex
  2. Redox regulation:

• BRSK2 activity is fine-tuned through oxidative modification of conserved cysteine residues

• The catalytic activities of both BRSK1 and BRSK2 are regulated through oxidation of the T-Loop +2 Cys residue

• This residue forms disulfide bonds with the 'CPE' motif Cys within the activation segment

• Treatment with oxidants like H₂O₂ or pervanadate inhibits BRSK2 activity

• This inhibition is reversible by treatment with reducing agents like DTT or GSH
  3. Conformational regulation:

• Binding of the UBA and AIS regions to the catalytic fold maintains auto-inhibition

• Removal of the KA1 domain results in constitutive activation
  4. Protein-protein interactions:

• Binding partners like PCTAIRE1 influence BRSK2 activity and substrate specificity

• Interaction with other proteins may regulate its subcellular localization
  5. Transcriptional regulation:

• Glucose levels negatively regulate BRSK2 expression in pancreatic β-cells

• BRSK2 is down-regulated in a dose-dependent manner in response to glucose
  The interplay between these regulatory mechanisms allows for precise control of BRSK2 activity in different cellular contexts.

What are the most effective methods for studying BRSK2 phosphorylation activity in vitro?

Several complementary approaches allow for robust analysis of BRSK2 activity:
1. Recombinant protein expression and purification:

• Express human or mouse BRSK2 in BL21 (DE3) pLysS E. coli cells

• Purify under reducing or non-reducing conditions depending on experimental goals

• Activate by incubation with LKB1/STRADα/MO25α complex in the presence of ATP and MgCl₂

• Verify phosphorylation by mass spectrometry or Western blotting with pThr172 AMPKα antibody
  2. Microfluidic mobility shift-based kinase assays:

• Use fluorescent-tagged peptide substrates (e.g., AMARA: 5-FAM-AMARAASAAALARRR-COOH)

• Monitor generation of phosphopeptide in real-time

• Establish optimal pressure and voltage settings to improve separation of phosphorylated and non-phosphorylated peptides

• Perform assays in 50 mM HEPES (pH 7.4), 0.015% (v/v) Brij-35, and 5 mM MgCl₂

• Calculate degree of peptide phosphorylation by differentiating the ratio of phosphopeptide:peptide
  3. Redox regulation studies:

• Incubate recombinant BRSK1/2 with oxidants (H₂O₂) or redox reagents (GSH/GSSG)

• Assess recovery from oxidative inhibition by adding reducing agents

• Detect glutathione-protein complexes by immunoblotting after non-reducing SDS-PAGE
  4. Structure-function analysis:

• Generate point mutations (kinase-dead: K48A, D141N; redox-insensitive: Cys-to-Ala mutants)

• Create truncation mutants to remove regulatory domains (ΔKA1, ΔAIS)

• Compare wild-type and mutant activity under various conditions
  For accurate comparisons, kinase activity rates should be normalized to activation site phosphorylation signal established with pThr172 AMPKα antibodies and quantified by densitometry .

How does BRSK2 regulate insulin secretion in pancreatic β-cells?

BRSK2 functions as a negative regulator of insulin secretion through several mechanisms:
1. PCTAIRE1-dependent mechanism:

• BRSK2 interacts with PCTAIRE1 (a CDK-related protein kinase) in β-cells

• BRSK2 phosphorylates PCTAIRE1 at Ser-12

• This phosphorylation reduces glucose-stimulated insulin secretion (GSIS) in MIN6 cells
  2. Experimental evidence:

• Knockdown of BRSK2 by siRNA increases serum insulin levels in mice

• BRSK2-RNAi mice exhibit altered islet morphology

• In MIN6 cells, Exendin-4-potentiated insulin secretion is absent in cells overexpressing wild-type BRSK2

• Conversely, interfering with BRSK2 significantly enhances Exendin-4- and Forskolin-potentiated insulin secretion
  3. Glucose-dependent regulation:

• BRSK2 expression is down-regulated in response to increasing glucose concentrations

• This suggests a feedback mechanism where glucose regulates BRSK2 levels to fine-tune insulin secretion
  4. Kinase activity requirement:

• The kinase activity of BRSK2 is required for this effect

• Kinase-dead mutants (k48m) do not inhibit insulin secretion

• Stably overexpressing wild-type BRSK2 MIN6 cells show increased basal insulin secretion but impaired glucose-stimulated secretion compared to control and kinase-dead BRSK2 cells
  This data suggests that BRSK2 is an attractive target for developing novel diabetes treatments, as its inhibition could potentially enhance insulin secretion .

What cellular pathways does BRSK2 interact with and what are their functional implications?

BRSK2 participates in multiple signaling networks with diverse functional implications:
1. AMPK signaling pathway:

• BRSK2 overexpression upregulates phosphorylation of AMPK substrates (proteins with LxRxx(pS/pT) motifs)

• Phosphoproteomics analysis confirms that BRSK2 positively regulates AMPK signaling

• This may influence cellular energy homeostasis and metabolism
  2. mTOR pathway:

• BRSK2 negatively impacts mTOR signaling

• Expression of BRSK2 decreases phosphorylation of mTOR substrates (S6K and 4EBP1)

• Total protein levels of 4EBP1 increase following BRSK2 expression

• This suppression of mTOR could affect protein synthesis, cell growth, and autophagy
  3. Cell cycle regulation:

• BRSK2 suppresses signaling through CDK1, CDK2, and CDC7 pathways

• Phosphorylation of CDC25C is affected by BRSK2 expression

• This suggests potential roles in cell cycle control
  4. NRF2 antioxidant pathway:

• BRSK2 functions as an inhibitor of the NRF2 transcription factor

• All BRSK2 variants suppress NRF2 transcriptional activity under both basal and induced conditions

• BRSK2 overexpression downregulates NRF2 target genes (HMOX1, GCLM, SLC7A11)

• BRSK2 can repress both wild-type NRF2 and constitutively active NRF2 mutants (ΔETGE)

• This inhibition modulates cellular responses to oxidative stress
  5. Neuronal development pathways:

• BRSK2 plays essential roles in neuronal polarization and axonogenesis

• It likely functions by phosphorylating cytoskeletal and polarity-related proteins

• One potential substrate is Tau protein, which affects microtubule dynamics
  The involvement of BRSK2 in these diverse pathways explains its multifaceted roles in different tissues and cellular contexts.

What is the evidence for BRSK2's involvement in autism spectrum disorder and neurodevelopmental disorders?

Multiple lines of evidence implicate BRSK2 in neurodevelopmental disorders:
1. Genetic evidence:

• De novo variants in BRSK2 have been identified in individuals with developmental delay, intellectual disability, and autism spectrum disorder

• The observed rate of de novo variation in affected cohorts is significantly higher than the background mutation rate (p = 2.46 × 10⁻⁶)

• Both loss-of-function variants (nonsense, frameshift, splice site) and deleterious missense variants have been identified

• SFARI Gene has classified BRSK2 as a high-confidence ASD risk gene with a score of 1S
  2. Statistical significance in large-scale studies:

• Autism Sequencing Consortium identified de novo loss-of-function variants

• Simons Simplex Collection found de novo in-frame deletion variants

• SPARK cohort study identified additional de novo loss-of-function variants

• Meta-analysis of de novo variants in thousands of ASD trios identified BRSK2 as an ASD candidate gene

• Two-stage analysis of rare variants in 42,607 ASD cases identified BRSK2 as reaching exome-wide significance (P < 2.5E-06)
  3. Clinical phenotypes:

• Common features include speech delay (present in all reported cases), intellectual disability, motor delay, behavioral issues, and autism

• Additional features may include auditory hallucinations, limb tremor, abnormal brain electrical activity, sleep disorders, and mild gait ataxia

• There is notable phenotypic variability even among patients with mutations in the same domain
  4. Functional evidence:

• BRSK2 plays an essential role in neuronal polarization and axonogenesis

• Animal models show neurodevelopmental disruptions including aberrant thin cortex and disturbed specification of axons and dendrites

• BRSK1/BRSK2 null mutant mice display severe dyskinesia

• Zebrafish models with BRSK2 deficiency display morphological and neurobehavioral features resembling human conditions
  5. Variant characteristics:

• BRSK2 is relatively intolerant to protein-altering variation in humans

• All identified pathogenic variants are absent from large population databases

• Computational modeling predicts that missense variants are damaging to BRSK2 structure and function
  A recent case report of a novel frameshift variant (c.442del, p.L148Cfs*39) in a 16-year-old boy with ASD further expanded the phenotypic spectrum, reporting abnormal brain electrical activity mapping and acousma for the first time .

How can oxidative modifications of BRSK2 be studied and what are their functional implications?

The redox regulation of BRSK2 can be studied through multiple complementary approaches:
1. Analysis of oxidative modifications:

• Biochemical detection:

  • Incubate recombinant BRSK2 with oxidants (H₂O₂) or glutathione disulfide (GSSG)

  • Perform non-reducing SDS-PAGE followed by immunoblotting

  • Detect glutathione-protein complexes using anti-glutathione antibodies

• Mass spectrometry approaches:

  • Purify BRSK1/2 under non-reducing conditions

  • Treat with iodoacetamide to alkylate free thiols

  • Perform trypsin digestion with or without prior reduction

  • Use LC-MS/MS to identify oxidized cysteine residues and disulfide bonds
    2. Functional impact assessment:

• In vitro activity assays:

  • Monitor BRSK2 kinase activity with fluorescent peptide substrates

  • Compare activity under oxidizing vs. reducing conditions

  • Assess recovery from oxidative inhibition by adding reducing agents like DTT or GSH

• Cellular studies:

  • Express BRSK1/2 with redox-sensitive substrates (e.g., Tau) in cells

  • Treat with H₂O₂ or glucose oxidase to generate steady-state H₂O₂

  • Assess substrate phosphorylation by Western blotting

  • Perform recovery experiments with GSH to demonstrate reversibility
    3. Structure-function analysis:

• Site-directed mutagenesis:

  • Create Cys-to-Ala mutants to prevent disulfide bond formation

  • Compare wild-type and mutant BRSK2 activity under oxidizing conditions

  • Demonstrate that mutating the CPE-Cys to alanine increases BRSK activity relative to wild-type enzyme
    Functional implications:

• Oxidative stress inhibits BRSK2 activity through formation of disulfide bonds

• H₂O₂ treatment leads to dose-dependent decrease in BRSK-mediated Tau phosphorylation

• This inhibition is reversible by treatment with reducing agents

• Chronic oxidative stress causes time-dependent depletion of BRSK1/2-associated Tau phosphorylation

• At high concentrations of peroxide, total BRSK protein levels decrease, suggesting reduced stability
  These findings demonstrate that BRSK1/2 activity is fine-tuned through oxidative modification of conserved cysteine residues, providing a novel mechanism for regulation of the eukaryotic AMPK family through Cys-based redox mechanisms .

What are the best approaches for validating BRSK2 substrates in experimental systems?

Validating authentic BRSK2 substrates requires a comprehensive, multi-tiered approach:
1. Initial substrate identification:

• Phosphoproteomics:

  • Perform TMT-based quantitative phosphoproteomics comparing samples from BRSK2 overexpressing vs. control cells

  • Analyze ~10,000 phosphosites to identify differentially phosphorylated peptides

  • Look for enrichment in specific signaling pathways

• Consensus motif analysis:

  • Identify proteins containing BRSK2 phosphorylation motifs

  • Focus on sites matching the AMPK-like kinase motif (LxRxx(S/T))

• Candidate approach:

  • Test known substrates of related kinases (e.g., AMPK substrates)

  • Focus on proteins in pathways known to involve BRSK2
    2. In vitro validation:

• Direct kinase assays:

  • Incubate purified recombinant BRSK2 with candidate substrates

  • Use purified LKB1-activated BRSK2 to ensure kinase activity

  • Include kinase-dead BRSK2 (K48A or D141N) as negative control

  • Detect phosphorylation by autoradiography or phospho-specific antibodies

• Phosphorylation site mapping:

  • Use mass spectrometry to identify exact phosphorylation sites

  • Confirm with site-directed mutagenesis of putative phosphorylation sites
    3. Cellular validation:

• Modulation of BRSK2 expression/activity:

  • Compare wild-type vs. kinase-dead BRSK2 expression

  • Use siRNA knockdown of BRSK2

  • Apply BRSK2 inhibitors (e.g., ROCK inhibitors Y-27632 and H-1152 also inhibit BRSK2)

• Substrate phosphorylation analysis:

  • Monitor changes in phosphorylation of candidate substrates

  • Use phospho-specific antibodies when available

  • Employ phospho-motif antibodies (e.g., LxRxx(pS/pT)) for broader detection
    4. Physiological significance:

• Functional assays:

  • Assess biological consequences of substrate phosphorylation

  • For example, PCTAIRE1 phosphorylation by BRSK2 reduces glucose-stimulated insulin secretion

• Phenotypic rescue:

  • Express phospho-mimetic or phospho-deficient substrate mutants

  • Determine if they recapitulate or rescue BRSK2-dependent phenotypes
    5. In vivo confirmation:

• Animal models:

  • Analyze substrate phosphorylation in BRSK2 knockout/knockdown models

  • Compare tissue samples from wild-type and BRSK2-deficient animals

  • Correlate substrate phosphorylation with physiological outcomes
    The example of PCTAIRE1 validation demonstrates this approach: researchers used yeast two-hybrid screening to identify the interaction, confirmed binding by multiple methods, demonstrated co-localization in MIN6 cells, identified the phosphorylation site as Ser-12, and showed functional consequences on insulin secretion .

How can CRISPR/Cas9 be utilized to generate and characterize BRSK2 mutations in model organisms?

CRISPR/Cas9 offers powerful approaches for studying BRSK2 function through genetic manipulation:
1. Generating knockout models:

• Design strategies:

  • Target early exons to maximize disruption

  • Use multiple sgRNAs to increase efficiency

  • Screen for frameshift mutations causing premature stop codons

• Validated examples:

  • brsk2b-/- zebrafish with 2-bp insertion resulting in p.K117Rfs*27

  • brsk2a-/- zebrafish with 23-bp deletion causing p.V284Qfs*5

  • Double knockouts generated by crossing single mutant lines

• Verification methods:

  • Genotyping by PCR and sequencing

  • qRT-PCR to confirm reduction in mRNA expression levels

  • Western blotting to verify protein loss
    2. Introducing specific mutations:

• Structure-function studies:

  • Kinase-dead mutations (K48A, D141N)

  • Redox-insensitive mutations (Cys-to-Ala substitutions)

  • Deletion of regulatory domains (ΔKA1, ΔAIS)

• Disease-associated variants:

  • Patient-derived mutations to model neurodevelopmental disorders

  • Example mutations from research include:

    • p.Arg65Gln: Disrupts salt bridge with Glu330 and MAPK docking motif

    • p.Gly212Glu: May disrupt secondary structure in flexible linker region

    • p.Arg620/621 mutations: Affect di-arginine ER retrieval-and-retention motif
      3. Tissue-specific approaches:

• Conditional systems:

  • Cre-loxP for tissue-specific knockout in mice

  • Inducible promoters for temporal control

  • Cell-type specific promoters for spatial control

• Mosaic analysis:

  • Generate chimeric animals with BRSK2 mutations in specific tissues

  • Compare mutant and wild-type cells within the same organism
    4. Phenotypic characterization:

• Neuronal development:

  • Analysis of neuronal polarization, axon specification, and dendrite development

  • Assessment of brain morphology and neuronal connectivity

  • Electrophysiological studies of synaptic function

• Metabolic function:

  • Glucose tolerance tests and insulin measurements

  • Islet morphology and β-cell size studies

  • In vitro insulin secretion assays with isolated islets

• Behavioral assessment:

  • Social interaction tests for autism-like behaviors

  • Motor function assessment for coordination and movement disorders

  • Cognitive testing for learning and memory deficits
    5. Dynamic developmental analysis:

• Multiple timepoint assessment:

  • Study BRSK2 function at different developmental stages

  • Evaluate trajectory and changes in individual development

  • Assess social and motor development processes
    CRISPR/Cas9-generated models provide valuable tools for understanding BRSK2 function in development and disease. The zebrafish models mentioned in the research have already contributed to our understanding of BRSK2's role in neurodevelopment and provide platforms for future mechanistic studies and therapeutic testing .