Phospho-PARK2 (S131) Antibody

What is the biological significance of Parkin phosphorylation at S131?

Parkin (PARK2/PRKN) phosphorylation at S131 is primarily mediated by cyclin-dependent kinase 5 (cdk5) and plays a crucial role in regulating Parkin's function as an E3 ubiquitin ligase. This phosphorylation site influences subsequent phosphorylation by other kinases, particularly casein kinase I, creating a hierarchical phosphorylation cascade. Phosphorylation at S131 can modulate Parkin's activity, solubility, and tendency to form aggregates, all of which have implications for mitochondrial quality control and the pathophysiology of Parkinson's disease .

Research indicates that S131 phosphorylation serves as a regulatory switch that can significantly impact downstream phosphorylation events. When S131 is phosphorylated (or when phosphorylation is mimicked through an S131E mutation), it enhances casein kinase I-mediated phosphorylation of Parkin at other sites including S101, S127, and S378 . This interconnected phosphorylation network highlights the complex post-translational regulation of Parkin.

What are the expression patterns of Parkin in different tissues?

Parkin shows tissue-specific expression patterns that are relevant when designing experiments:

Parkin is highly expressed in the brain, particularly in the substantia nigra, which is affected in Parkinson's disease. Notably, Parkin expression is down-regulated or absent in tumor biopsies and is absent in the brains of PARK2 patients . Understanding these expression patterns is critical when selecting appropriate experimental models and interpreting antibody staining results.

What are the key structural and functional domains of Parkin affected by S131 phosphorylation?

Functionally, Parkin acts as an E3 ubiquitin ligase, mediating the targeting of substrate proteins for proteasomal degradation. When this ubiquitin ligase activity is compromised, it contributes to the pathogenesis of PARK2-associated Parkinson's disease . Phosphorylation at S131 by cdk5 influences this activity, with S131A (non-phosphorylatable) mutants showing slightly enhanced ubiquitylation activity compared to wild-type Parkin .

What are the recommended applications for Phospho-PARK2 (S131) antibodies?

Phospho-PARK2 (S131) antibodies can be utilized in multiple experimental approaches:

When performing Western blot analysis, cell lysates should be prepared in modified RIPA buffer (containing 150 mmol/L NaCl, 0.1% SDS, 1% Nonidet P40, 1% sodium deoxycholate, and 10 mmol/L sodium phosphate, pH 7.2), supplemented with protease and phosphatase inhibitors . This formulation is crucial to preserve the phosphorylation state of Parkin during sample preparation.

How should I prepare samples to accurately detect Phospho-PARK2 (S131)?

For optimal detection of phosphorylated Parkin at S131:

• Include phosphatase inhibitors in all buffers (sodium vanadate and commercial phosphatase inhibitor cocktails are recommended)

• Maintain samples at 4°C throughout preparation to minimize dephosphorylation

• Use freshly prepared lysates whenever possible

• Include positive controls (e.g., cells treated with cdk5 activators) and negative controls (e.g., samples treated with phosphatases or cdk5 inhibitors like roscovitine)

• For immunoprecipitation experiments, use 400μg of protein lysate with 1μg of antibody to ensure sufficient sensitivity

The antibodies are highly specific and detect Parkin protein only when phosphorylated at S131, not the non-phosphorylated form . This specificity makes them valuable tools for studying the dynamics of Parkin phosphorylation under various experimental conditions.

What are the optimal storage conditions for Phospho-PARK2 (S131) antibodies?

Most commercially available Phospho-PARK2 (S131) antibodies are supplied in liquid form containing buffer components such as:

• 50% glycerol

• 0.5% BSA

• 0.02% sodium azide

• PBS or phosphate buffer

For long-term storage:

• Store antibodies at -20°C for up to one year

• Avoid repeated freeze-thaw cycles by aliquoting upon first thaw

• For short-term use (within two weeks), store at 4°C

• Protect from light, especially FITC-conjugated antibodies

How does S131 phosphorylation interact with other Parkin phosphorylation sites?

The interplay between different Parkin phosphorylation sites reveals a complex regulatory network:

Research demonstrates that phosphorylation of Parkin by cdk5 at S131 enhances its propensity to serve as a substrate for casein kinase I and vice versa . This creates a phosphorylation cascade where modification at one site influences modifications at other sites.

Critically, while individual phosphorylation events (either at S131 by cdk5 or at casein kinase I sites) do not significantly alter Parkin's ubiquitylation activity, compound phosphorylation by both kinases profoundly affects Parkin's tendency to form aggregates. A phospho-mimetic mutant for compound phosphorylation (101E/127E/131E/378E) showed a 6.2 ± 2-fold increase in cells with Parkin inclusions compared to wild-type . This suggests that simultaneous phosphorylation at multiple sites dramatically alters Parkin's biochemical properties and cellular behavior.

What is the relationship between Parkin S131 phosphorylation and mitochondrial quality control?

Parkin plays a crucial role in mitochondrial quality control, particularly in the clearance of damaged mitochondria through mitophagy. Research indicates that Parkin phosphorylation status significantly impacts this process:

• Following mitochondrial depolarization (e.g., with CCCP treatment), Parkin is phosphorylated at various sites including S101

• Studies suggest that phosphorylation at S131 may affect Parkin's translocation to depolarized mitochondria

• Non-phosphorylatable mutations (e.g., S101A) show deficiencies in:

  • Translocation of Parkin to depolarized mitochondria

  • Ubiquitination of outer mitochondrial membrane proteins

  • Recruitment of p62 (an autophagy adaptor)

  • Perinuclear mitochondrial clustering

  • Mitophagy

While S131 and S378 appear to be constitutively phosphorylated in some cell lines, phosphorylation at S101 increases following CCCP treatment . This suggests that different phosphorylation sites may serve distinct functions in the regulation of mitochondrial quality control, with some being dynamically regulated in response to mitochondrial stress.

How does S131 phosphorylation affect Parkin solubility and aggregation?

Parkin aggregation and solubility are significantly influenced by its phosphorylation status:

• Mimicking phosphorylation at only S131 (S131E mutant) or only at casein kinase I sites (101E/127E/378E) does not significantly increase Parkin inclusion formation compared to wild-type

• A Parkin mutant mimicking compound phosphorylation at both cdk5 and casein kinase I sites (101E/127E/131E/378E) displays:

  • 6.2 ± 2-fold increase in cells with Parkin inclusions

  • Decreased solubility in Triton detergent

  • Enhanced aggregation into inclusion bodies

• These effects are not observed with the corresponding non-phosphorylatable mutant (101A/127A/131A/378A)

This suggests that compound phosphorylation at multiple sites, including S131, dramatically increases Parkin's propensity to form insoluble aggregates. This has significant implications for Parkinson's disease pathogenesis, where protein aggregation is a key pathological feature.

What controls should be included when using Phospho-PARK2 (S131) antibodies?

For rigorous experimental design, incorporate the following controls:

Additionally, using cells expressing Parkin phospho-mutants (S131A or S131E) provides excellent controls for antibody specificity . For immunohistochemistry applications, it's advisable to include tissue samples known to be negative for Parkin expression as negative controls.

How can I modulate Parkin S131 phosphorylation in experimental systems?

To manipulate Parkin phosphorylation at S131 for mechanistic studies:

• Pharmacological approaches:

  • Inhibit cdk5 with roscovitine (shown to decrease phosphorylation of Parkin at casein kinase I sites by 47 ± 10%)

  • Modulate casein kinase I activity with IC261 or D4476 (the latter leads to a 44 ± 10% decrease in Parkin phosphorylation)

• Genetic approaches:

  • Express phospho-mimetic mutants (S131E) to simulate constitutive phosphorylation

  • Express non-phosphorylatable mutants (S131A) to prevent phosphorylation

  • Use combined mutants to study interactions between different phosphorylation sites

• Mitochondrial stress induction:

  • Treat cells with CCCP (a mitochondrial uncoupler) to induce changes in Parkin phosphorylation

These approaches can be combined with Phospho-PARK2 (S131) antibodies to monitor the effects of experimental manipulations on Parkin phosphorylation status and downstream functional consequences.

What cell lines and experimental models are most appropriate for studying Parkin S131 phosphorylation?

Based on the available research, consider the following experimental models:

For studying mitochondrial quality control and Parkin phosphorylation, SH-SY5Y cells stably expressing wild-type Parkin or phosphorylation site mutants (S101A, S101D) have been successfully used . These models allow for detailed investigation of how phosphorylation affects Parkin's role in mitochondrial dynamics and mitophagy.

What are common challenges when detecting Phospho-PARK2 (S131) and how can they be addressed?

Researchers frequently encounter these technical challenges:

• Weak or absent signal:

  • Ensure phosphatase inhibitors are fresh and active in all buffers

  • Optimize antibody concentration (try 1:500 to 1:1000 for WB)

  • Increase protein loading (30-50μg for Western blot)

  • Consider enhanced chemiluminescence (ECL) detection systems for greater sensitivity

• High background:

  • Increase blocking time (5% BSA in TBST recommended)

  • Use more stringent washing conditions

  • Optimize primary antibody dilution

  • Pre-absorb antibody with non-specific proteins

• Non-specific bands:

  • Validate with phospho-mutant controls

  • Perform peptide competition assays

  • Use gradient gels for better resolution around 50-52 kDa

• Inconsistent results between experiments:

  • Standardize lysate preparation protocols

  • Maintain consistent sample handling to preserve phosphorylation

  • Include positive controls in every experiment

  • Consider the influence of cell confluency and passage number

How can I distinguish between physiological and pathological patterns of Parkin S131 phosphorylation?

Interpreting Parkin phosphorylation patterns requires consideration of multiple factors:

• Baseline phosphorylation: Some studies indicate that S131 and S378 may be constitutively phosphorylated in certain cell types , making it important to establish baseline levels in your specific model.

• Stress-induced changes: Look for changes in phosphorylation following treatments that induce mitochondrial stress (e.g., CCCP) or activate relevant kinases.

• Correlation with functional readouts: Connect phosphorylation patterns to functional outcomes such as:

  • Parkin E3 ligase activity

  • Parkin translocation to mitochondria

  • Ubiquitination of mitochondrial proteins

  • Mitophagy progression

• Comparison across models: Compare phosphorylation patterns between:

  • Control and disease models

  • Different cell types (neuronal vs. non-neuronal)

  • Different tissues (brain vs. muscle)

Understanding the physiological regulation of Parkin phosphorylation provides context for interpreting potentially pathological changes in disease models or patient samples.

How should I analyze phosphorylation data in the context of compound phosphorylation events?

Given the complex relationship between different Parkin phosphorylation sites, data analysis should consider:

• Sequential phosphorylation: Evidence suggests a hierarchical relationship where S131 phosphorylation by cdk5 enhances subsequent phosphorylation by casein kinase I .

• Combinatorial effects: The effects of phosphorylation at multiple sites are not simply additive. For example, mimicking phosphorylation at all four sites (S101, S127, S131, and S378) dramatically increases aggregation, while single-site modifications have minimal effects .

• Kinase inhibitor studies: When using kinase inhibitors, consider their specificity and potential off-target effects. Interpret changes in phosphorylation patterns accordingly.

• Time-course experiments: Follow phosphorylation at multiple sites over time to capture the dynamics of the phosphorylation cascade.

• Quantitative analysis: Use densitometry to measure the relative intensity of phospho-specific signals normalized to total Parkin levels, and compare across conditions.