Phospho-ATXN1 (Ser776) Antibody

What is ATXN1 and why is phosphorylation at Ser776 significant?

ATXN1 is a chromatin-binding factor that functions as a repressor of Notch signaling and plays important roles in brain development. It is primarily known for its association with SCA1, an inherited neurodegenerative disorder caused by the expansion of a polyglutamine tract in the ATXN1 protein .

Phosphorylation of ATXN1 at serine 776 (Ser776) plays a crucial role in SCA1 pathogenesis. Research has demonstrated that this post-translational modification significantly affects protein stability, with phosphorylated ATXN1 being less susceptible to degradation . Studies in cerebellar Purkinje cells, a prominent site of SCA1 pathology, have confirmed that phosphorylation at Ser776 stabilizes the protein . Furthermore, this phosphorylation affects the pathogenicity of proteins with expanded polyglutamine tracts, directly influencing disease progression mechanisms .

What are the key characteristics of Phospho-ATXN1 (Ser776) antibodies?

Phospho-ATXN1 (Ser776) antibodies are specifically designed to detect ATXN1 protein only when phosphorylated at the Ser776 residue . These antibodies are available in multiple formats:

• Rabbit polyclonal antibodies targeting regions around the phosphorylation site (typically amino acids 742-791)

• Mouse monoclonal antibodies with similar specificity

Key specifications include:

• Applications: Western blot (1:500-1:2000 dilution), immunohistochemistry (1:100-1:300), immunofluorescence (1:200-1:1000), and ELISA (1:10000)

• Reactivity: Typically reactive with human and mouse ATXN1

• Formulation: Usually supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Storage: Recommended at -20°C with avoidance of repeated freeze-thaw cycles

Importantly, these antibodies are designed to detect endogenous levels of ATXN1 protein only when phosphorylated at Ser776, making them valuable tools for studying this specific post-translational modification .

How can researchers validate the specificity of Phospho-ATXN1 (Ser776) antibodies?

Validating antibody specificity is crucial for generating reliable research data. For Phospho-ATXN1 (Ser776) antibodies, several approaches are recommended:

• Peptide competition assays: Blocking with the phospho-peptide used as the immunogen should eliminate specific antibody binding. Western blot and immunofluorescence data show that signal disappears when the antibody is pre-incubated with the phosphopeptide, confirming phospho-specificity .

• Phospho-resistant mutants: Using ATXN1 constructs with serine-to-alanine mutations at position 776 (S776A) provides an excellent negative control. These constructs cannot be phosphorylated at this position and should not be detected by phospho-specific antibodies .

• Multiple detection methods: Validating antibody specificity across different applications (Western blot, immunofluorescence, immunohistochemistry) strengthens confidence in antibody performance .

• Treatment conditions: Using samples with known phosphorylation status, such as HepG2 cells treated with Adriamycin (0.5μM for 5 hours), which has been shown to affect ATXN1 phosphorylation, provides positive controls .

These validation steps ensure that the observed signals truly represent phosphorylated ATXN1 rather than non-specific interactions or detection of non-phosphorylated forms.

What are the recommended experimental conditions for using these antibodies?

Optimal experimental conditions vary by application but generally include:

• Western blot:

  • Protein loading: 20-50 μg of total protein per lane

  • Dilution range: 1:500-1:2000

  • Blocking: 5% BSA in TBST (BSA is often preferred over milk for phospho-epitopes)

  • Incubation: Overnight at 4°C for primary antibody

• Immunohistochemistry:

  • Dilution range: 1:100-1:300

  • Antigen retrieval: Typically required for formalin-fixed tissues

  • Detection system: Biotin-streptavidin or polymer-based systems

• Immunofluorescence:

  • Dilution range: 1:200-1:1000

  • Fixation: 4% paraformaldehyde recommended

  • Permeabilization: 0.1-0.5% Triton X-100

• Critical factors across applications:

  • Sample preparation: Include phosphatase inhibitors in all extraction buffers

  • Controls: Include both positive controls (known phosphorylated samples) and negative controls

  • Storage: Maintain antibodies at -20°C and avoid repeated freeze-thaw cycles

Following these recommendations will help maximize signal specificity while minimizing background and non-specific binding.

Which kinase phosphorylates ATXN1 at Ser776 in cerebellar Purkinje cells?

The identity of the kinase responsible for ATXN1 phosphorylation at Ser776 has been a subject of investigation with evolving understanding. While earlier studies using transfected cell lines and Drosophila models suggested Akt (protein kinase B) as the responsible kinase, more recent evidence from cerebellar Purkinje cells points to cyclic AMP-dependent protein kinase (PKA) as the primary kinase .

Evidence supporting PKA as the ATXN1-S776 kinase includes:

• Immunodepletion studies: Depletion of Akt from cerebellar extracts did not significantly affect phosphorylation of GST-ATXN1 at S776, whereas PKA immunodepletion substantially reduced this phosphorylation .

• Fractionation experiments: PKA co-fractionated with ATXN1-S776 kinase activity in cerebellar cytosol under both ammonium sulfate precipitation (50-90% fraction) and hydroxyapatite chromatography conditions .

• Inhibitor studies: PKA inhibitors (staurosporine and a 17-residue PKA inhibitor peptide) significantly reduced S776 phosphorylation in a dose-dependent manner, while an Akt inhibitor had no significant effect .

• Subcellular localization: The kinase activity was found to be enriched in the cytoplasmic fraction, while phosphorylated ATXN1 accumulated in the nuclear fraction .

These findings highlight the importance of studying kinase-substrate relationships in physiologically relevant contexts, as results may differ between model systems.

How can researchers establish an in vitro ATXN1 phosphorylation assay?

An in vitro phosphorylation assay for ATXN1 can be established following these methodological steps:

• Substrate preparation:

  • Generate recombinant GST-ATXN1 fusion proteins, including both wild-type (S776) and phospho-resistant mutant (A776) versions

  • The A776 mutant serves as an excellent negative control to confirm phosphorylation specificity

• Kinase source preparation:

  • Prepare cerebellar extracts as a source of native kinase activity

  • For subcellular fractionation, the cytoplasmic fraction contains higher kinase activity than the nuclear fraction

  • Further fractionation can be performed using ammonium sulfate precipitation (50-90% fraction) followed by hydroxyapatite column chromatography with a sodium phosphate gradient

• Phosphorylation reaction:

  • Incubate GST-ATXN1 with cerebellar extract under appropriate buffer conditions

  • The phosphorylation signal increases with increasing amounts of cerebellar extract, confirming enzyme-dependent phosphorylation

  • Include appropriate cofactors and ATP in the reaction buffer

• Detection methods:

  • Western blotting with phospho-Ser776-specific antibodies is the primary detection method

  • The signal should increase with wild-type ATXN1 but remain minimal with the A776 mutant

• Validation approaches:

  • Immunodepletion: Remove specific kinases (e.g., PKA) from the extracts to confirm their role

  • Inhibitor studies: Add kinase inhibitors to test their effects on phosphorylation

  • Peptide competition: Use phospho-peptides to validate antibody specificity

This assay system provides a powerful tool for mechanistic studies of ATXN1 phosphorylation and potential therapeutic interventions.

How does phosphorylation at Ser776 affect ATXN1 protein stability and function?

Phosphorylation of ATXN1 at Ser776 has profound effects on protein properties:

• Protein stability: Research has demonstrated that phosphorylation at S776 stabilizes ATXN1 protein. Studies using phospho-resistant alanine mutations at residue 776 (S776A) show that these mutants are destabilized in Purkinje cells compared to wild-type (S776) ATXN1 . This stabilization effect may contribute to pathology by increasing the cellular burden of mutant ATXN1 protein.

• Subcellular localization: While the kinase activity responsible for S776 phosphorylation is enriched in the cytoplasmic fraction, phosphorylated ATXN1 preferentially accumulates in the nuclear fraction . This suggests that phosphorylation may affect nucleocytoplasmic trafficking or nuclear retention of ATXN1.

• Interaction with other post-translational modifications: Phosphorylation at S776 influences other modifications of ATXN1. For example:

  • Sumoylation of ATXN1 depends on both nuclear localization and phosphorylation at S776

  • Ubiquitination and subsequent proteasomal degradation of ATXN1, mediated by UBE3A, appears to be affected by phosphorylation status

• Polyglutamine expansion effects: In SCA1 patients with expanded polyglutamine repeats in ATXN1, phosphorylation at S776 increases the pathogenicity of these expanded proteins . Additionally, the presence of expanded polyglutamine repeats impairs ubiquitination and degradation, leading to accumulation of ATXN1 in neurons and subsequent toxicity .

These findings highlight the multifaceted role of S776 phosphorylation in determining ATXN1 protein fate and function, particularly in the context of SCA1 pathogenesis.

What experimental challenges exist when studying ATXN1 phosphorylation?

Researchers face several technical and biological challenges when investigating ATXN1 phosphorylation:

• Kinase identification discrepancies: Different experimental systems have yielded contradictory results regarding the responsible kinase. While Akt was implicated in cell lines and Drosophila models, PKA appears to be the primary kinase in cerebellar Purkinje cells . This highlights the importance of validating findings across different model systems.

• Compartmentalization challenges: The finding that kinase activity is enriched in cytoplasmic fractions while phosphorylated ATXN1 accumulates in nuclear fractions complicates the study of the phosphorylation process . This spatial separation makes real-time visualization of the phosphorylation event challenging.

• Technical considerations:

  • Preserving phosphorylation during sample processing requires careful use of phosphatase inhibitors

  • Antibody specificity requires thorough validation through multiple approaches

  • Quantification of phosphorylation stoichiometry remains technically demanding

• Polyglutamine length variations: Different polyglutamine repeat lengths in ATXN1 (e.g., ATXN1[30Q] vs. ATXN1[82Q]) may exhibit different phosphorylation patterns or responses to manipulation , making cross-study comparisons complex.

• Model system relevance: Translation between in vitro systems, cell cultures, and in vivo models requires careful consideration of the biological context and limitations of each approach.

Understanding these challenges is essential for designing robust experimental approaches and correctly interpreting results in ATXN1 phosphorylation studies.

How might targeting ATXN1 phosphorylation lead to therapeutic approaches for SCA1?

Understanding ATXN1 phosphorylation provides several potential therapeutic strategies for SCA1:

• PKA inhibition: Since PKA appears to be the primary kinase responsible for ATXN1 phosphorylation at S776 in cerebellar Purkinje cells , selective PKA inhibitors could potentially reduce phosphorylation. By decreasing phosphorylation, ATXN1 stability might be reduced, potentially lowering levels of toxic expanded polyglutamine ATXN1.

• Phosphatase activation: Enhancing the activity of phosphatases that remove the phosphate group from S776 could increase ATXN1 turnover and reduce toxicity by promoting protein degradation.

• Disruption of phosphorylation-dependent interactions: Phosphorylation at S776 likely mediates specific protein-protein interactions. Compounds that interfere with these interactions could mitigate downstream pathological effects without broadly affecting phosphorylation mechanisms.

• Combined approaches: Since phosphorylation affects multiple aspects of ATXN1 biology, including stability, localization, and interactions with other proteins, combination therapies targeting multiple aspects might be more effective than single-target approaches.

• Biomarker applications: Phospho-ATXN1 (S776) levels could potentially serve as biomarkers for disease progression or treatment response, allowing for pharmacodynamic monitoring during clinical trials.

The development of these approaches faces challenges including achieving sufficient specificity to avoid off-target effects and ensuring adequate delivery to cerebellar Purkinje cells, the primary site of SCA1 pathology.

What methodologies can assess compounds affecting ATXN1 phosphorylation?

Researchers evaluating compounds that modulate ATXN1 phosphorylation can employ several complementary methodological approaches:

• In vitro screening systems:

  • Cerebellar extract-based phosphorylation assays using recombinant GST-ATXN1 substrates

  • Purified PKA-based assays to test direct effects on kinase activity

  • High-throughput screening platforms with phospho-specific antibody readouts

• Cellular validation approaches:

  • Western blot analysis of phospho-ATXN1 (S776) levels in treated cells

  • Immunofluorescence to assess effects on subcellular localization

  • Protein stability assays to determine impact on ATXN1 turnover

  • Cell models expressing either wild-type (30Q) or expanded (82Q) ATXN1

• In vivo evaluation:

  • Transgenic mouse models (e.g., ATXN1[82Q]-S776 mice)

  • Behavioral assessments (e.g., rotarod performance)

  • Biochemical analysis of tissue extracts for phospho-ATXN1 levels

  • Histological examination of cerebellar Purkinje cells

• Target validation approaches:

  • Comparison with genetic models (e.g., ATXN1-A776 phospho-resistant mutations)

  • Epistasis experiments with kinase modulators and phosphatase activators

  • Evaluation of effects on downstream pathways affected by phosphorylated ATXN1

These methodologies provide a comprehensive framework for evaluating compound effects on ATXN1 phosphorylation from initial screening through in vivo validation.

How can phospho-specific antibodies be used to monitor disease progression or treatment response?

Phospho-ATXN1 (Ser776) antibodies offer valuable tools for monitoring disease mechanisms and therapeutic interventions:

• Biomarker development:

  • Quantitative measurement of phospho-ATXN1 (S776) levels in accessible biofluids or tissues could potentially track disease progression

  • Longitudinal monitoring during clinical interventions might provide pharmacodynamic evidence of target engagement

• Mechanism of action studies:

  • Determining whether experimental therapeutics affect phosphorylation status, protein levels, or downstream effects

  • Differentiating between compounds that directly affect phosphorylation versus those that act through other mechanisms

• Patient stratification:

  • Potentially identifying patient subgroups with different phosphorylation profiles who might respond differently to specific therapeutic approaches

  • Correlating phosphorylation levels with disease severity or progression rates

• Methodological approaches:

  • ELISA-based quantification using phospho-specific antibodies

  • Immunohistochemical analysis of post-mortem samples or animal models

  • Multiplexed assays combining phospho-ATXN1 with other disease-relevant biomarkers

• Technical considerations:

  • Sample preparation standardization is crucial for reliable quantification

  • Inclusion of appropriate controls for antibody specificity validation

  • Development of robust normalization strategies to account for total ATXN1 levels

These applications demonstrate the translational potential of phospho-specific antibodies beyond basic research contexts.

What controls should be included when studying ATXN1 phosphorylation?

A comprehensive experimental design for studying ATXN1 phosphorylation should include several types of controls:

• Antibody specificity controls:

  • Phospho-peptide blocking: Pre-incubating antibodies with the phosphopeptide used as immunogen should eliminate specific binding

  • Non-phosphorylated control peptides: Should not affect antibody binding

  • Secondary antibody-only controls: To assess background signal

• Genetic controls:

  • Phospho-resistant mutants: ATXN1-A776 constructs serve as negative controls that cannot be phosphorylated at position 776

  • Wild-type ATXN1-S776 constructs: Positive controls that can be phosphorylated

  • Varying polyglutamine lengths: Comparing ATXN1[30Q] versus ATXN1[82Q] can reveal polyglutamine length effects

• Enzymatic controls:

  • Phosphatase treatment: Samples treated with phosphatases should show reduced or eliminated phospho-specific signals

  • Kinase inhibitors: PKA inhibitors like staurosporine should reduce phosphorylation in assay systems

  • Kinase activators: Agents that increase PKA activity can serve as positive controls

• Treatment controls:

  • Known modulators: Adriamycin treatment (0.5μM, 5h) has been shown to affect ATXN1 phosphorylation in certain cell types

  • Time course analyses: To capture dynamic phosphorylation events

  • Dose-response studies: To establish optimal treatment parameters

These controls help establish the specificity of detection methods and provide context for interpreting experimental results in phosphorylation studies.

What are optimal methods for subcellular fractionation when studying ATXN1 phosphorylation?

Subcellular fractionation is particularly important for ATXN1 phosphorylation studies given the finding that kinase activity is enriched in the cytoplasmic fraction while phosphorylated ATXN1 accumulates predominantly in the nuclear fraction . Optimal methods include:

• Fractionation approach:

  • Differential centrifugation: Sequential centrifugation steps at increasing speeds can separate nuclei from cytoplasmic components

  • Density gradient separation: For higher purity fractions when needed

  • Commercial kits: Several validated nuclear/cytoplasmic fractionation kits are available

• Critical considerations:

  • Phosphatase inhibitors: Must be included in all buffers to preserve phosphorylation status

  • Protease inhibitors: Essential to prevent protein degradation

  • Temperature control: Performing all steps at 4°C helps maintain protein integrity and phosphorylation

  • Buffer composition: Hypotonic buffers for cell swelling followed by mechanical disruption are often effective

• Validation of fraction purity:

  • Western blotting for compartment-specific markers:

    • Nuclear markers: Lamin B, histone H3

    • Cytoplasmic markers: GAPDH, α-tubulin

  • Microscopic examination: To confirm nuclear integrity during isolation

• Application to ATXN1 studies:

  • Analysis has shown that pS776-endogenous ATXN1 is enriched in nuclear fractions

  • Kinase activity assays using fractions have demonstrated that S776 kinase activity is predominantly found in cytoplasmic fractions

  • Combining fractionation with immunoprecipitation can enrich for specific ATXN1 populations

These approaches allow researchers to study the subcellular compartmentalization of ATXN1 phosphorylation processes and potentially identify the site of initial phosphorylation versus the site of phosphorylated protein accumulation.