BRSK1 Antibody

What is BRSK1 and what are its primary biological functions?

BRSK1 (also known as SAD1, SADB, or hSAD1) is a serine/threonine-protein kinase that plays multiple critical roles in cellular function. BRSK1 primarily functions in neuronal polarization and centrosome duplication, making it essential for proper neural development and function . As a kinase, BRSK1 phosphorylates several important substrates including CDC25B, CDC25C, MAPT/TAU, RIMS1, TUBG1, TUBG2, and WEE1 .

Following phosphorylation and activation by STK11/LKB1, BRSK1 acts as a key regulator of polarization of cortical neurons, likely by mediating phosphorylation of microtubule-associated proteins such as MAPT/TAU at specific residues (Thr-529 and Ser-579) . Within neurons, BRSK1 localizes to synaptic vesicles and regulates neurotransmitter release, possibly through phosphorylation of RIMS1 .

Additionally, BRSK1 functions as a positive regulator of centrosome duplication by mediating phosphorylation of gamma-tubulin (TUBG1 and TUBG2) at Ser-131, which leads to translocation of gamma-tubulin and its associated proteins to the centrosome . Beyond these neuronal functions, BRSK1 is also involved in the UV-induced DNA damage checkpoint response, likely by inhibiting CDK1 activity through phosphorylation and activation of WEE1, and inhibition of CDC25B and CDC25C .

What types of BRSK1 antibodies are commercially available for research?

Researchers have access to several types of BRSK1 antibodies with distinct properties and applications:

• Rabbit Polyclonal antibodies: Examples include ab115475 from Abcam, which is suitable for immunohistochemistry on paraffin-embedded sections (IHC-P) and Western blotting (WB), with demonstrated reactivity against human samples . Similarly, Thermo Fisher Scientific offers PA5-20353, a polyclonal antibody that can be used with the blocking peptide PEP-0473 for specificity validation .

• Rabbit Recombinant Monoclonal antibodies: Abcam's ab206298 (clone EPR18190) is suitable for Western blotting (WB) and immunocytochemistry/immunofluorescence (ICC/IF), with reactivity against mouse, rat, and human samples .

When selecting between polyclonal and monoclonal antibodies, researchers should consider their experimental needs: polyclonal antibodies recognize multiple epitopes and may provide stronger signals, while monoclonal antibodies offer greater specificity but potentially lower sensitivity.

What experimental applications are BRSK1 antibodies validated for?

BRSK1 antibodies have been validated for several common laboratory applications, though the appropriate technique depends on the specific antibody:

• Western Blotting (WB): Most commercial BRSK1 antibodies are validated for Western blotting, making this a reliable method for detecting BRSK1 protein expression levels in tissue or cell lysates .

• Immunohistochemistry on paraffin-embedded sections (IHC-P): Some antibodies like ab115475 are suitable for examining BRSK1 expression and localization in tissue sections .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Monoclonal antibodies such as ab206298 are validated for visualizing BRSK1 subcellular localization through immunofluorescence techniques .

When designing experiments, researchers should consider using human brain tissue lysate as a positive control, as it has been suggested for antibodies like PA5-20353 . Additionally, for ICC/IF applications, neuronal cultures are appropriate given BRSK1's high expression in brain tissue.

What is the tissue distribution of BRSK1 expression?

BRSK1 displays a distinctive expression pattern that researchers should consider when designing experiments:

BRSK1 is ubiquitously expressed throughout the body, but shows notably higher expression levels in specific tissues. The highest levels of BRSK1 expression are found in the brain and testes . This expression pattern has important implications for experimental design, particularly when selecting appropriate positive control tissues or cell lines.

For antibody validation experiments, human brain tissue lysate is frequently recommended as a positive control . The brain-selective nature of BRSK1 expression also explains its critical role in neuronal polarization and function, as demonstrated in knockout studies where BRSK1/BRSK2 double-mutant mice died within two hours of birth due to severe neuronal defects .

How is BRSK1 activity regulated by redox mechanisms?

Recent research has uncovered fascinating insights into the redox regulation of BRSK1, representing an emerging area of interest for researchers:

BRSK1 activity is tightly regulated by reversible cysteine oxidation and reduction, with specific reactive cysteine residues within the kinase domain serving as redox-sensitive switches . These unique reactive cysteine residues are sites of covalent-oxidative modification that can significantly alter BRSK1's enzymatic activity.

Experimentally, researchers have demonstrated that oxidizing agents such as hydrogen peroxide (H₂O₂) and oxidized glutathione (GSSG) can dramatically inhibit BRSK1 kinase activity, while reducing agents like dithiothreitol (DTT) and reduced glutathione (GSH) can restore this activity . This reversible regulation suggests BRSK1 may function as a redox sensor in cells, particularly neurons.

Methodologically, researchers can assess BRSK1's redox sensitivity through:

• In vitro kinase assays using microfluidic real-time mobility shift-based assays with fluorescent-tagged BRSK1/2 peptide substrates (e.g., AMARA: 5-FAM-AMARAASAAALARRR-COOH) and ATP .

• Cell-based assays where BRSK1 (wild-type or cysteine-to-alanine mutants) is expressed in cell lines, treated with oxidizing or reducing agents, and then activity is assessed via substrate phosphorylation (such as TAU phosphorylation) .

• Recovery experiments where BRSK1 is first inhibited with H₂O₂ (typically 1-10 mM), followed by treatment with reducing agents like DTT (2 mM) or GSH (20 mM) to demonstrate reversibility .

These findings connect BRSK1 to broader cellular redox signaling networks, with emerging evidence suggesting BRSK1 may be part of a multi-protein cysteine-based 'relay' network of ROS-sensitive effectors upstream of NRF2, potentially constituting a previously unrecognized oxidative stress signaling mechanism .

What role does BRSK1 play in neuronal polarization and how can researchers study this function?

BRSK1 serves as a critical regulator of neuronal polarization, with compelling evidence from knockout studies:

BRSK1 and its related protein BRSK2 are essential for proper neuronal polarization in mammals. While individual BRSK1 or BRSK2 knockout mice remain viable, suggesting some functional redundancy, double-mutant mice lacking both proteins die within two hours of birth . Neurons from these double-knockout mice display a striking phenotype: they develop uniformly-sized neurites instead of the typical morphology featuring one long axon and multiple shorter dendrites. Moreover, these abnormal neurites inappropriately express both axonal and dendritic markers, indicating a fundamental failure in establishing proper neuronal polarity .

Mechanistically, BRSK1 regulates neuronal polarization through:

• Phosphorylation of microtubule-associated proteins like MAPT/TAU at specific residues (Thr-529 and Ser-579), which helps establish and maintain the distinct cytoskeletal organization needed for axonal growth .

• Phosphorylation of WEE1 at Ser-642 in postmitotic neurons, which down-regulates WEE1 activity in polarized neurons, contributing to the cytoskeletal remodeling necessary for polarization .

Researchers studying BRSK1's role in neuronal polarization can employ several methodological approaches:

• Immunostaining of primary neuronal cultures with BRSK1 antibodies (such as ab206298) to visualize its subcellular localization during different stages of neuronal development .

• Knockdown or knockout experiments (using siRNA or CRISPR) followed by morphological analysis of neurite development and expression of axonal/dendritic markers.

• Rescue experiments in BRSK1/2-deficient neurons by re-expressing wild-type or mutant forms of BRSK1 to identify critical domains and residues.

• Phosphorylation assays to detect BRSK1-mediated modification of downstream substrates like TAU in neuronal cultures, particularly following treatments that induce polarization.

How does BRSK1 function in the DNA damage response pathway?

BRSK1 plays a significant role in cellular responses to DNA damage, particularly in the context of UV-induced damage:

BRSK1 functions as a UV-induced DNA damage checkpoint kinase, contributing to cell cycle arrest in response to genotoxic stress . Similar to its yeast homolog, BRSK1 appears to be involved in stress-induced cell cycle arrest mechanisms. Experimental evidence demonstrates that overexpression of BRSK1 leads to G2/M arrest in HeLa S2 cells . Conversely, reduced expression of BRSK1 through siRNA can partially abrogate UV-induced G2/M arrest, confirming its functional role in DNA damage checkpoint responses .

At the molecular level, BRSK1 likely contributes to the DNA damage response by inhibiting CDK1 activity through multiple mechanisms:

• Phosphorylation and activation of WEE1, a known negative regulator of CDK1 .

• Inhibition of CDC25B and CDC25C, which normally activate CDK1 by removing inhibitory phosphorylations .

Researchers investigating BRSK1's role in DNA damage responses can utilize several experimental approaches:

• Cell cycle analysis (flow cytometry) in cells with manipulated BRSK1 levels (overexpression or knockdown) following UV or other DNA-damaging treatments.

• Immunoblotting to assess phosphorylation status of BRSK1 targets (WEE1, CDC25B, CDC25C) after DNA damage induction.

• Kinase activity assays to measure changes in BRSK1 enzymatic activity following exposure to DNA-damaging agents.

• Colocalization studies to determine whether BRSK1 relocates to sites of DNA damage or interacts with other DNA damage response proteins following genotoxic stress.

What methodologies are most effective for measuring BRSK1 kinase activity?

Accurate measurement of BRSK1 kinase activity is crucial for understanding its function in various cellular contexts. Recent research has employed several sophisticated techniques:

Microfluidic real-time mobility shift-based assays represent the current gold standard for BRSK1 activity measurement . This technique offers several advantages:

• Real-time monitoring of substrate phosphorylation, allowing for dynamic assessment of kinase activity.

• High sensitivity and reproducibility compared to traditional methods.

• Ability to test various conditions (e.g., redox agents) within the same experimental setup.

The experimental protocol typically involves:

• Incubating recombinant BRSK1 (0.5 μg) with a fluorescent-tagged peptide substrate (often AMARA: 5-FAM-AMARAASAAALARRR-COOH) at approximately 2 μM concentration .

• Adding 1 mM ATP and optimizing pressure and voltage settings to improve separation of phosphorylated and non-phosphorylated peptides .

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

• Calculating peptide phosphorylation by differentiating the ratio of phosphopeptide to non-phosphorylated peptide .

• Normalizing data with respect to control assays, typically limiting phosphate incorporation to <20% to prevent ATP depletion and ensure assay linearity .

For activation studies, researchers should account for potential variability in LKB1-dependent phosphorylation of BRSK proteins by normalizing rates of kinase activity (calculated as pmol phosphate incorporation per min) to the activation site phosphorylation signal, established using pThr 172 AMPKα antibodies and quantified through densitometry .

In cellular contexts, researchers can assess BRSK1 activity by:

• Transfecting cells with tagged BRSK1 constructs (Flag, HA, or tandem Strep tag) along with known substrates like EGFP-TAU .

• Treating cells with various conditions (e.g., oxidizing or reducing agents).

• Analyzing substrate phosphorylation via immunoblotting using phospho-specific antibodies.

How does oxidative stress affect BRSK1 function in neuronal cells?

Oxidative stress profoundly impacts BRSK1 function in neuronal cells, revealing a complex relationship between redox signaling and neuronal physiology:

In neuronal cells, BRSK1 activity is highly sensitive to oxidative conditions. Recent research demonstrates that exposure of cells expressing BRSK1 to hydrogen peroxide (typically 10 mM H₂O₂ for 20 minutes) significantly inhibits BRSK1 kinase activity . This inhibition appears to be reversible, as subsequent treatment with reducing agents like reduced glutathione (20 mM GSH for 15 minutes) can restore BRSK1 function .

The redox regulation of BRSK1 has particularly important implications for neuronal cells, which are especially vulnerable to oxidative stress due to their high metabolic activity and limited regenerative capacity. The relationship between BRSK1 and cellular redox status extends beyond simple inhibition, as BRSK1 can indirectly modulate the cellular antioxidant response by orchestrating suppression of NRF2 (a master regulator of antioxidant response) in an mTOR-dependent manner .

Researchers investigating BRSK1's response to oxidative stress in neuronal contexts can employ several methodological approaches:

• Transfection of primary neurons or neuronal cell lines with wild-type or redox-insensitive mutant (Cysteine-to-Alanine) BRSK1 constructs, followed by oxidative stress treatments.

• Analysis of BRSK1-dependent phosphorylation events (such as TAU phosphorylation) under oxidative stress conditions.

• Assessment of neuronal polarization and morphology in the presence of redox-modulating agents, comparing neurons expressing wild-type versus redox-insensitive BRSK1 mutants.

• Investigation of potential protective effects of BRSK1 manipulation against oxidative stress-induced neuronal damage or death.

This emerging understanding of BRSK1 as part of a redox-sensitive signaling network in neurons offers promising avenues for research into neurodegenerative conditions characterized by oxidative stress.

What are the most significant unresolved questions in BRSK1 research?

Despite significant advances in understanding BRSK1 biology, several critical questions remain unresolved and represent fertile ground for future research:

• The precise mechanism by which redox regulation of BRSK1 influences neuronal development and function requires further elucidation. While we know BRSK1 activity is modulated by oxidation and reduction, how this relates to physiological redox fluctuations in neurons during development and in response to stress remains unclear .

• The functional relationship between BRSK1 and BRSK2 needs deeper exploration. Although double knockout studies show they have some redundant functions, the unique contributions of each kinase are not fully understood .

• The therapeutic potential of targeting BRSK1 redox sensitivity in neurological disorders characterized by oxidative stress and abnormal neuronal polarization represents an untapped opportunity. The identification of specific reactive cysteine residues within BRSK1's kinase domain may provide a foundation for developing targeted therapeutic strategies for BRSK1-associated pathologies .

• The complete catalog of BRSK1 substrates and interacting partners in different cellular contexts remains incomplete. While several substrates have been identified (CDC25B, CDC25C, MAPT/TAU, RIMS1, TUBG1, TUBG2, WEE1), others likely exist and may reveal new functions of this kinase .