BRSK2 Antibody

What is BRSK2 and where is it primarily expressed?

BRSK2 (Brain-selective Kinase 2) is a serine/threonine kinase belonging to the AMPK-related family of kinases. Despite its name suggesting brain-specific expression, BRSK2 shows surprisingly high expression in pancreatic tissue. Northern blot analysis has revealed that BRSK2 mRNA is expressed at even higher levels in the pancreas than in the brain . Additionally, BRSK2 protein has been detected in pancreatic islets and specifically co-localizes with insulin but not glucagon in these tissues . The protein is also expressed at lower levels in testis tissue. Notably, BRSK2 expression is nearly absent in other tissues beyond these three primary locations, making it a relatively tissue-specific kinase .

What is the molecular structure of BRSK2?

BRSK2 features a characteristic domain organization consisting of:

• N-terminal kinase domain (responsible for phosphorylation activity)

• Ubiquitin-associated domain (UBA)

• Proline-rich region (PRR)

• Kinase-associated domain (KA1) with an auto-inhibitory sequence (AIS) at the C-terminus

The protein has several isoforms produced by alternative splicing with molecular weights ranging from 68-84 kDa, with the primary form observed at 80-88 kDa in Western blots . The gene encoding BRSK2 is highly conserved from invertebrates to humans, suggesting evolutionary importance of its function .

What are the optimal conditions for BRSK2 antibody application in Western blotting?

For optimal Western blotting results with BRSK2 antibodies:

Researchers should note that multiple bands may be observed due to alternative splicing or post-translational modifications. Validation experiments using knockdown or knockout controls are recommended to confirm antibody specificity .

What immunohistochemistry protocols work best for BRSK2 detection in tissue sections?

Based on published methodologies, successful immunohistochemistry for BRSK2 in tissue sections has been achieved using the following approach:

• Fix tissues with 4% paraformaldehyde and section at 10 μm thickness

• Perform antigen retrieval (if needed)

• Block with appropriate serum (typically 5-10% normal goat or donkey serum)

• Incubate with primary BRSK2 antibody (typically 1:50-1:500 dilution)

• For colorimetric detection: use biotin-labeled secondary antibody with DAB/H₂O₂

• For fluorescent detection: use fluorescein isothiocyanate-conjugated secondary antibodies (Alexa Fluor 488 or 555)

• Counterstain nucleus with DAPI

• Wash and mount for confocal microscopy

This approach has successfully demonstrated BRSK2 co-localization with insulin in pancreatic β-cells and neuronal expression patterns .

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

BRSK2 functions as a negative regulator of glucose-stimulated insulin secretion (GSIS) through a complex molecular mechanism:

• BRSK2 interacts with and binds to PCTAIRE1, a CDK-related protein kinase involved in neurite outgrowth and neurotransmitter release

• This interaction leads to phosphorylation of PCTAIRE1 at Ser-12 by BRSK2

• Phosphorylated PCTAIRE1 subsequently reduces GSIS in pancreatic β-cells

• When BRSK2 is knocked down by siRNA in vivo, serum insulin levels increase in mice

This regulatory pathway reveals BRSK2 as a potential therapeutic target for diabetes, as its inhibition could potentially increase insulin secretion. The unique expression of BRSK2 in pancreatic β-cells provides specificity that could be advantageous for targeted therapeutics .

What experimental approaches can be used to study BRSK2's role in insulin secretion?

To investigate BRSK2's function in insulin secretion, researchers have employed several methodological approaches:

• Insulin secretion assays: Transfect MIN6 cells (a murine β-cell line) with plasmids encoding wild-type or mutant BRSK2, pre-incubate in glucose-free medium, then stimulate with glucose (typically 3mM for low and 25mM for high glucose) and measure insulin release by radioimmunoassay .

• In vivo knockdown: Deliver siRNA against BRSK2 via tail-vein injection in mice for 1-3 consecutive days, followed by blood sampling and ELISA assays to measure serum insulin levels .

• Phosphorylation studies: Perform in vitro phosphorylation assays using immunoprecipitated BRSK2 and recombinant PCTAIRE1 substrates in the presence of [γ-³²P]ATP, with subsequent SDS-PAGE separation and autoradiography visualization .

• Isolated islet studies: Isolate pancreatic islets by collagenase perfusion in situ and culture in appropriate medium for ex vivo experiments measuring insulin secretion and β-cell size .

What role does BRSK2 play in cell cycle progression and cancer?

BRSK2 exhibits dynamic regulation during the cell cycle and has emerging roles in cancer biology:

• Cell cycle dynamics: BRSK2 protein levels fluctuate throughout the cell cycle, peaking during mitosis and declining in G1 phase. The protein co-localizes with centrosomes during mitosis .

• Degradation mechanism: BRSK2 is targeted for degradation by the anaphase-promoting complex/cyclosome-Cdh1 (APC/C^Cdh1) during G1 phase via its conserved KEN box motif. This occurs through the ubiquitin-proteasome pathway .

• Mitotic regulation: Overexpression of wild-type BRSK2 or KEN box mutants increases the percentage of cells in G2/M phase, suggesting a role in mitotic progression .

• Cancer implications: BRSK2 is highly elevated in breast cancer tumors compared to control breast tissue, with particularly high expression in stage IV patients. Expression analysis of TCGA and METABRIC cohort data revealed significant upregulation (p<0.001) in advanced disease .

• Metastasis association: RNA sequencing analysis identified BRSK2 as one of the top genes highly expressed in primary breast cancer tissues that subsequently developed bone metastasis compared to non-metastatic primary tumors .

How does BRSK2 contribute to cancer cell survival under nutrient stress?

BRSK2 has been found to promote cancer cell survival during nutrient limitation through several mechanisms:

• Autophagy regulation: BRSK2 augments nutrient starvation-mediated autophagic flux in both triple-negative and ER/PR-positive breast cancer cell lines .

• Survival signaling: BRSK2 expression correlates with elevated AKT, phosphorylated NF-κB, and STAT3 signaling pathways, all of which promote cell survival under stress conditions .

• AMPK connection: As an AMPK-related kinase, BRSK2 functions downstream of LKB1 signaling in nutrient sensing. It activates WIPI proteins that function as building blocks for autophagosome formation .

• Cancer-specific expression: BRSK2 shows differential expression across various breast cancer cell lines but is not expressed in non-malignant breast epithelial cells (MCF10A), suggesting cancer-specific functions .

These findings suggest BRSK2 as a potential therapeutic target in cancer, particularly for tumors that rely on autophagy for survival under metabolic stress conditions.

How is BRSK2 involved in neurodevelopmental disorders?

Deleterious variations in BRSK2 have been strongly associated with neurodevelopmental disorders:

• Through GeneMatcher, researchers identified nine probands with rare, heterozygous variants in the BRSK2 gene who presented with developmental delays and/or intellectual disability .

• Common clinical features among these individuals included:

  • Speech delay (all probands)

  • Intellectual disability (majority)

  • Motor delay

  • Behavioral issues

  • Autism spectrum features

• Six of the nine variants were predicted to result in loss of function, while computational modeling indicated the remaining three missense variants were damaging to BRSK2 structure and function .

• In all six probands for whom parents were available, the mutations were found to have arisen de novo, strengthening the causative relationship .

• Statistical analysis showed a significantly higher rate of de novo variation in affected individuals compared to the background mutation rate (p = 2.46 × 10^-6) .

This evidence strongly implicates BRSK2 in normal neurodevelopment and suggests that BRSK2 dysfunction contributes to developmental delays and intellectual disability.

What is the connection between BRSK2 and paraneoplastic neurological syndromes?

BRSK2 has been identified as an autoantigen in paraneoplastic limbic encephalitis:

• Anti-BRSK2 antibodies were discovered in the serum of a patient with limbic encephalitis and small-cell lung cancer (SCLC) .

• The patient's serum immunolabeled neuronal cytoplasm and, to a lesser extent, the neuropil of rat brain, but did not react with other rat tissues except testis .

• Immunoblots of rat brain homogenate identified several immunoreactive bands in the 88-82 kDa range and a weaker broad band of 47-43 kDa .

• BRSK2 antibodies were found to react with two SCLC samples from patients without paraneoplastic neurological syndromes, suggesting expression of BRSK2 in these tumors .

• No anti-BRSK2 antibodies were detected in 50 patients with SCLC without paraneoplastic neurological syndromes, 19 with limbic encephalitis without onconeural antibodies, 50 with anti-Hu antibodies and other paraneoplastic syndromes, or 160 with non-paraneoplastic neurological disorders .

These findings suggest BRSK2 may be an autoantigen involved in the pathogenesis of SCLC-associated limbic encephalitis, though it appears to be a rare phenomenon.

How does BRSK2 interface with the NRF2 oxidative stress response pathway?

BRSK2 has been identified as a negative regulator of NRF2, a master transcription factor controlling cellular antioxidant responses:

• A gain-of-function genetic screen revealed that both BRSK2 and its homolog BRSK1 act as negative regulators of NRF2 signaling .

• Mechanistically, BRSK2 suppresses NRF2 through inhibition of protein synthesis via mTOR pathway downregulation, rather than through the canonical KEAP1-dependent mechanism .

• Integrated phosphoproteomics and RNAseq studies demonstrated that BRSK2 drives AMPK signaling while suppressing the mTOR pathway .

• BRSK2 overexpression decreases NRF2 protein levels and downregulates NRF2 target genes such as HMOX1, GCLM, and SLC7A11 in both basal conditions and after treatment with CDDO-me (an NRF2 activator) .

• The kinase activity of BRSK2 is essential for this function, as kinase-dead mutants fail to suppress NRF2 signaling or target gene expression .

This BRSK2-NRF2 axis represents a novel regulatory mechanism connecting BRSK2 to cellular stress responses and may have implications for diseases involving oxidative stress, including neurodegenerative conditions and cancer.

What experimental approaches are most effective for studying BRSK2 phosphorylation targets?

To effectively study BRSK2's phosphorylation targets and kinase activity, researchers have employed several sophisticated approaches:

• In vitro phosphorylation assays:

  • Immunoprecipitate HA-tagged BRSK2 from transfected 293T cells

  • Incubate with recombinant GST-fusion substrates (full-length, deletion mutants, or site-directed mutants) in kinase buffer containing [γ-³²P]ATP

  • Separate samples by SDS-PAGE and visualize by autoradiography

• Phosphorylation site identification:

  • Generate phosphorylation-deficient mutants of potential substrates by site-directed mutagenesis (typically changing Ser/Thr to Ala)

  • Compare phosphorylation patterns between wild-type and mutant proteins

  • Confirm findings using phospho-specific antibodies if available

• Integrated phosphoproteomics:

  • Perform mass spectrometry-based phosphoproteome analysis after BRSK2 overexpression or knockdown

  • Identify phosphopeptides with significantly altered abundance

  • Conduct motif analysis to identify potential direct BRSK2 substrates

• Functional validation:

  • Express wild-type BRSK2 versus kinase-dead mutants (typically K49A or D143A mutations)

  • Compare downstream effects on cellular processes and signaling pathways

  • Use specific pathway inhibitors to delineate BRSK2-dependent mechanisms

These approaches have successfully identified PCTAIRE1 as a BRSK2 substrate in insulin secretion and characterized the effects of BRSK2 on AMPK and mTOR signaling pathways.

Why might Western blots show multiple bands when using BRSK2 antibodies?

Multiple bands in Western blots with BRSK2 antibodies may occur for several reasons:

• Alternative splicing: BRSK2 has multiple isoforms produced by alternative splicing with molecular masses ranging from 68-84 kDa .

• Post-translational modifications: Phosphorylation of BRSK2 may alter its migration pattern. BRSK2 is activated by phosphorylation at Thr-174 in its activation loop, and may have other modification sites .

• Proteolytic processing: Immunoblots of rat brain homogenate have identified both high molecular weight bands (88-82 kDa) and lower molecular weight bands (47-43 kDa), potentially representing proteolytic fragments .

• Cross-reactivity: Some antibodies may cross-react with the highly homologous BRSK1 protein, which has similar molecular weight and domain structure .

To address these issues, researchers should:

• Use positive controls from tissues known to express BRSK2 (brain, pancreas, testis)

• Include knockdown/knockout controls when possible

• Consider using antibodies targeting different epitopes to confirm findings

• Perform dephosphorylation experiments to identify bands resulting from phosphorylation states

What are the critical controls needed when studying BRSK2 function in various biological contexts?

When investigating BRSK2 function, several critical controls should be included: