Phospho-SIRT1 (S47) Antibody

What is SIRT1 and what cellular processes does it regulate?

SIRT1 is the mammalian ortholog of the yeast SIR2 protein, functioning as a nuclear deacetylase involved in regulating numerous cellular processes. SIRT1 plays crucial roles in apoptosis, cellular senescence, endocrine signaling, glucose homeostasis, aging, and longevity. It exerts its effects by deacetylating multiple target proteins including p53, p300, Ku70, forkhead (FoxO) transcription factors, PPARγ, and the PPARγ coactivator-1α (PGC-1α) protein . The deacetylation of p53 and FoxO transcription factors inhibits apoptosis and promotes cell survival, while deacetylation of PPARγ and PGC-1α regulates gluconeogenic/glycolytic pathways in the liver and fat mobilization in white adipocytes during fasting .

What is the significance of SIRT1 phosphorylation and specifically S47 phosphorylation?

SIRT1 undergoes post-translational regulation through phosphorylation at multiple sites, including Serine 47 (S47). While SIRT1 is known to be phosphorylated at both Ser27 and Ser47 in vivo, the S47 phosphorylation site has been identified as particularly important in the context of endothelial senescence . Research has demonstrated that S47 phosphorylation of SIRT1 is significantly enhanced in senescent endothelial cells, with the phosphorylation ratio increasing over 9-fold in late-passage cells compared to early-passage cells . This phosphorylation appears to modulate SIRT1's activity, subcellular localization, and protein-protein interactions, thereby affecting its ability to regulate cellular aging processes.

What are the key differences between phosphorylated and non-phosphorylated SIRT1?

Phosphorylation at S47 critically impacts SIRT1's function and cellular interactions. Non-phosphorylated SIRT1 (or S47A mutant) demonstrates enhanced antisenescent and growth-promoting properties in endothelial cells, whereas the phospho-mimetic form (S47D) loses these protective functions . Additionally, S47 phosphorylation affects SIRT1's nuclear retention and alters its association with telomeric repeat-binding factor 2-interacting protein 1 . In terms of enzymatic activity, the S47D phospho-mimetic mutation significantly decreases both basal deacetylase activity and the stimulated activity in response to activators like resveratrol , suggesting that phosphorylation at this site serves as a negative regulatory mechanism for SIRT1 function.

What are the recommended applications for Phospho-SIRT1 (S47) Antibody?

The Phospho-SIRT1 (S47) Antibody can be utilized in multiple experimental applications with the following recommended dilutions:

This antibody has been validated for detection of endogenous levels of SIRT1 when phosphorylated at S47, with confirmed specificity for human samples . When planning experiments, it's advisable to optimize these dilutions based on your specific experimental conditions and sample types.

What are the best methodologies for detecting changes in SIRT1 S47 phosphorylation?

To effectively detect changes in SIRT1 S47 phosphorylation, researchers should consider multiple complementary approaches:

• Western blotting using the specific Phospho-SIRT1 (S47) antibody is the primary method for quantifying changes in phosphorylation levels. Always normalize phospho-SIRT1 signal to total SIRT1 to accurately assess phosphorylation status.

• Immunoprecipitation followed by western blotting can enhance detection sensitivity, particularly when working with limited samples or low expression levels.

• For cellular localization studies, immunofluorescence staining can visualize the distribution of phosphorylated SIRT1 within cells and track changes in subcellular localization.

• Flow cytometry provides quantitative analysis of phosphorylated SIRT1 levels at the single-cell level and enables population analysis.

• Mass spectrometry offers precise identification and confirmation of phosphorylation sites, as demonstrated in studies identifying T344 phosphorylation by AMPK .

When studying dynamic changes in phosphorylation, time-course experiments with appropriate controls (such as phosphatase inhibitors in lysates) are essential for capturing the temporal regulation of this post-translational modification.

How can researchers effectively validate the specificity of Phospho-SIRT1 (S47) antibody in their experimental systems?

To validate antibody specificity, implement the following rigorous controls:

• Dephosphorylation controls: Treat sample aliquots with lambda phosphatase to remove phosphorylation and confirm the signal disappears.

• Phosphorylation inducer/inhibitor controls: Compare samples treated with known modulators of S47 phosphorylation. For instance, treatment with roscovitine (CDK5 inhibitor) should reduce S47 phosphorylation by approximately 70% .

• Genetic controls: Express wild-type SIRT1 alongside S47A and S47D mutants. The antibody should detect wild-type SIRT1 (when phosphorylated) but not the S47A mutant.

• Peptide competition assay: Pre-incubate the antibody with a phospho-S47 peptide prior to immunoblotting to block specific binding.

• Cellular context validation: Compare senescent versus young endothelial cells, as senescent cells should show significantly higher levels of S47 phosphorylation (approximately 9-fold increase has been reported) .

Documentation of these validation steps is essential for ensuring experimental rigor and reproducibility in phospho-specific antibody applications.

Which kinase is primarily responsible for phosphorylating SIRT1 at S47, and how was this determined?

Cyclin-dependent kinase 5 (CDK5) has been identified as the primary kinase responsible for phosphorylating SIRT1 at S47. This identification involved multiple complementary approaches:

• Bioinformatic prediction: The NetPhosK 1.0 program initially predicted CDK5 and glycogen synthase kinase 3 as potential upstream kinases for S47 phosphorylation .

• Pharmacological inhibition: Treatment with roscovitine (a CDK5 inhibitor) significantly reduced S47 phosphorylation by more than 70%, while inhibitors of other potential kinases (LY294002 for PI3K, lithium chloride for glycogen synthase kinase 3, and SP600125 for c-Jun N-terminal kinase) showed no significant effect .

• Genetic validation: siRNA-mediated knockdown of CDK5 demonstrated an inhibitory effect on S47 phosphorylation similar to roscovitine treatment, confirming CDK5's role .

• Functional correlation: Endothelial cell cultures with reduced CDK5 expression or decreased CDK5 activity contained significantly fewer senescent cells, establishing a functional link between CDK5 activity, S47 phosphorylation, and cellular senescence .

These multiple lines of evidence collectively establish CDK5 as the key kinase mediating SIRT1 S47 phosphorylation in the context of endothelial senescence.

How does AMPK-mediated phosphorylation of SIRT1 differ from CDK5-mediated phosphorylation?

AMPK and CDK5 phosphorylate SIRT1 at different sites, leading to distinct functional outcomes:

Notably, roscovitine treatment (CDK5 inhibition) also inhibited AMPK-induced phosphorylation of SIRT1, suggesting a complex interplay between these regulatory pathways . This indicates that AMPK may indirectly enhance CDK5-mediated SIRT1 phosphorylation through mechanisms that require further investigation.

What is the relationship between CDK5 activity, SIRT1 phosphorylation, and cellular senescence?

Research has established a clear regulatory axis connecting CDK5 activity, SIRT1 S47 phosphorylation, and the development of cellular senescence:

• CDK5 activity increases during cellular aging, with accumulation of its truncated regulatory subunit P25 in senescent endothelial cells and atherosclerotic aortas .

• Enhanced CDK5 activity leads to hyperphosphorylation of SIRT1 at S47, which was found to increase progressively from early passage (P1) to late passage (P4) endothelial cells, with phosphorylation barely detectable in young cells but substantially elevated (>9-fold) in senescent cells .

• S47 phosphorylation impairs SIRT1's deacetylase activity and alters its subcellular localization, preventing its nuclear export and interaction with telomeric repeat-binding factor 2–interacting protein 1 .

• The compromised SIRT1 function results in increased acetylation of its targets (including histone H4) and upregulation of LKB1, promoting senescence development .

• Intervention at any point in this pathway can disrupt the senescence program:

  • Inhibition of CDK5 with roscovitine reduces S47 phosphorylation and senescent cell numbers

  • Expression of non-phosphorylatable SIRT1 (S47A) enhances antisenescent effects

  • Long-term treatment with roscovitine blocks the development of cellular senescence and atherosclerosis in hypercholesterolemic mice

This regulatory cascade represents a potential therapeutic target for age-related vascular disorders.

How can researchers effectively employ site-directed mutagenesis to study S47 phosphorylation effects?

Site-directed mutagenesis offers powerful insights into S47 phosphorylation functions through precise genetic manipulation:

• Design strategy: Create three parallel constructs for comparative studies:

  • Wild-type SIRT1

  • S47A mutation (non-phosphorylatable)

  • S47D mutation (phospho-mimetic)

• Experimental validation:

  • Confirm mutation success through sequencing and expression verification

  • Compare deacetylase activity using purified proteins (wild-type showed intermediate activity, S47A enhanced activity, S47D significantly decreased activity)

  • Evaluate protein-protein interactions through co-immunoprecipitation assays (S47A showed strongest interaction with LKB1, S47D significantly attenuated binding)

• Cellular phenotype assessment:

  • Senescence markers (SA-β-gal staining showed ~42% reduction with wild-type SIRT1 and ~56% with S47A, but no significant effect with S47D)

  • Proliferation assays (enhanced with wild-type and S47A, but not S47D)

  • Acetylation status of SIRT1 targets (histone H4 acetylation was significantly lower in cells with S47A compared to wild-type)

• Mechanistic investigation:

  • Nuclear-cytoplasmic fractionation to assess subcellular localization changes

  • ChIP assays to evaluate chromatin association

  • Live-cell imaging with fluorescently tagged constructs to monitor real-time localization

This comprehensive mutagenesis approach provides mechanistic insights into how phosphorylation status at a single residue profoundly influences SIRT1 function.

What experimental approaches can resolve contradictory findings regarding SIRT1 phosphorylation at different sites?

To resolve contradictions in phosphorylation studies, implement a multi-faceted experimental strategy:

• Temporal resolution analysis:

  • Conduct precise time-course experiments to determine the sequence and kinetics of phosphorylation events at different sites (S47 vs T344)

  • Use synchronized cell populations to eliminate cell-cycle dependent variations

• Context-dependent phosphorylation assessment:

  • Compare phosphorylation patterns across different cellular contexts (replicative senescence vs. stress-induced senescence vs. normal growth)

  • Evaluate tissue-specific patterns in different vascular beds or organs

• Multi-site phosphorylation analysis:

  • Generate combined mutants (e.g., S47A/T344A double mutants) to assess cooperative or antagonistic relationships

  • Employ mass spectrometry for unbiased detection of all phosphorylation sites simultaneously

  • Utilize Phos-tag gels to separate and visualize multiple phosphorylated species

• Kinase-phosphatase networks:

  • Map the upstream kinases and phosphatases for each site (CDK5 for S47, AMPK for T344)

  • Investigate cross-talk between pathways using specific inhibitors/activators

  • Employ mathematical modeling to predict hierarchical phosphorylation patterns

• Downstream functional readouts:

  • Compare how different phosphorylation sites affect distinct SIRT1 functions (deacetylase activity, protein-protein interactions, subcellular localization)

  • Utilize domain-specific functions to assess which phosphorylation events affect particular SIRT1 activities

This integrated approach can reconcile apparently contradictory findings by revealing how different phosphorylation events function within a coordinated regulatory network.

How can phospho-SIRT1 (S47) be leveraged as a biomarker for vascular aging and atherosclerosis progression?

Developing phospho-SIRT1 (S47) as a biomarker requires rigorous validation across multiple analytical platforms:

• Clinical correlation studies:

  • Compare phospho-SIRT1 (S47) levels in endothelial cells from healthy individuals versus patients with vascular diseases

  • Establish age-dependent reference ranges for phosphorylation levels

  • Correlate phosphorylation status with established biomarkers of vascular aging

• Tissue and circulation analysis methods:

  • Develop immunohistochemistry protocols for tissue sections to visualize phospho-SIRT1 in atherosclerotic plaques

  • Optimize detection in circulating endothelial cells or endothelial microparticles as accessible biomarkers

  • Create sensitive ELISA or other immunoassays for quantitative assessment

• Predictive value determination:

  • Conduct longitudinal studies to determine if phospho-SIRT1 (S47) levels predict future vascular events

  • Establish threshold values that correlate with increased risk

  • Compare prognostic value to established risk markers

• Intervention response monitoring:

  • Evaluate phospho-SIRT1 (S47) dynamics in response to CDK5 inhibitors like roscovitine

  • Assess correlation between phosphorylation reduction and clinical improvement

  • Track phosphorylation changes during lifestyle interventions (exercise, caloric restriction) known to affect SIRT1 activity

• Combined biomarker panels:

  • Integrate phospho-SIRT1 (S47) with other markers (inflammatory cytokines, oxidative stress markers) for improved predictive power

  • Develop algorithms that incorporate phosphorylation status with clinical parameters

Early research indicates that phospho-SIRT1 (S47) levels were significantly elevated in regenerated endothelial cells collected from porcine aortas after balloon denudation injury compared to native cells , suggesting potential utility as a marker of vascular injury and repair processes.

What are the potential therapeutic applications of targeting CDK5-mediated SIRT1 phosphorylation?

Targeting the CDK5-SIRT1 phosphorylation pathway offers several promising therapeutic approaches:

• Direct CDK5 inhibition: Long-term treatment with roscovitine, a CDK5 inhibitor, has been shown to block the development of cellular senescence and atherosclerosis in hypercholesterolemic apolipoprotein E-deficient mice . This provides proof-of-concept for CDK5 inhibition as a therapeutic strategy for vascular aging disorders.

• Development of selective CDK5 inhibitors: More selective CDK5 inhibitors could minimize off-target effects while maximizing the beneficial impact on SIRT1 phosphorylation, offering potential treatments for atherosclerosis, endothelial dysfunction, and other age-related vascular diseases.

• SIRT1 phosphorylation-resistant analogs: Creating therapeutic agents that mimic non-phosphorylatable SIRT1 (similar to S47A) could enhance antisenescent effects without requiring direct kinase inhibition.

• Combination therapies: Pairing CDK5 inhibitors with SIRT1 activators (such as resveratrol or newer synthetic activators) could synergistically enhance vascular protection by both preventing inhibitory phosphorylation and directly stimulating SIRT1 activity.

• Targeted delivery systems: Developing endothelial-specific delivery mechanisms for CDK5 inhibitors could maximize therapeutic effects while minimizing systemic exposure and potential side effects.

The significant reduction in atherosclerosis development observed with CDK5 inhibition in animal models suggests these approaches may hold substantial promise for treating vascular aging conditions in humans.

How do different models of induced senescence affect SIRT1 S47 phosphorylation patterns?

Different senescence induction models show distinct but convergent effects on SIRT1 S47 phosphorylation:

These diverse models demonstrate that S47 phosphorylation represents a common endpoint in various senescence pathways, suggesting it may serve as a central regulatory node in age-related endothelial dysfunction regardless of the initiating stimulus.

What are the most promising research directions for understanding the broader implications of SIRT1 phosphorylation in aging and disease?

Future research on SIRT1 phosphorylation should focus on these high-impact directions:

• Comprehensive phosphorylation mapping: Utilize advanced phosphoproteomics to create a complete atlas of SIRT1 phosphorylation sites across tissues, ages, and disease states, establishing how multiple phosphorylation events interact in regulatory networks.

• Tissue-specific phosphorylation patterns: Investigate whether S47 phosphorylation has different significance in non-vascular tissues (neurons, hepatocytes, skeletal muscle) to understand its broader role in organismal aging.

• Interplay with other post-translational modifications: Examine how S47 phosphorylation interacts with other SIRT1 modifications (acetylation, SUMOylation, ubiquitination) to create a comprehensive regulatory code.

• Genetic models with phospho-site mutations: Develop knock-in mouse models expressing S47A or S47D SIRT1 to assess long-term physiological consequences of altered phosphorylation status.

• Systems biology approaches: Apply network analysis to understand how SIRT1 phosphorylation integrates signals from energy status, stress response, and aging pathways to coordinate cellular adaptations.

• Translational biomarker development: Establish whether phospho-SIRT1 (S47) detection in accessible samples (blood cells, plasma exosomes) can serve as non-invasive biomarkers for aging and vascular disease.

• Therapeutic targeting strategies: Design novel compounds that specifically prevent S47 phosphorylation without affecting SIRT1's catalytic domain or other phosphorylation sites.

• Epigenetic consequences: Map how altered S47 phosphorylation affects the genomic binding profile of SIRT1 and subsequent chromatin modifications that regulate age-related gene expression patterns.

These research directions could revolutionize our understanding of how post-translational regulation of SIRT1 contributes to the aging process and age-related diseases.