Phospho-HDAC1 (Ser421) Antibody

What is the functional significance of HDAC1 phosphorylation at Serine 421?

HDAC1 phosphorylation at Serine 421 is a critical post-translational modification that significantly enhances its enzymatic activity and modulates its interactions with key protein complexes. Mass spectrometry analysis has confirmed that Serine 421 is the predominant phosphorylation site with >92% site probability according to Mascot scoring . This phosphorylation promotes HDAC1's enzymatic activity and facilitates interactions with both NuRD and SIN3 complexes, which are essential for chromatin remodeling and transcriptional regulation . Functionally, phosphorylated HDAC1 at Ser421 contributes to gene repression and plays regulatory roles in diverse cellular processes including cell cycle progression, developmental events, and specific signaling pathways such as Wnt signaling .

Which kinases are responsible for phosphorylating HDAC1 at Serine 421?

Two primary kinases have been identified as responsible for HDAC1 Ser421 phosphorylation:

Experimental validation generally relies on in vitro kinase assays coupled with western blot analysis using phospho-specific antibodies or mass spectrometry to confirm the specific phosphorylation site . The phosphorylation status can be experimentally modulated by serum addition, which increases phosphorylation levels at Ser421 .

How should researchers select and validate a Phospho-HDAC1 (Ser421) antibody for their experiments?

When selecting a Phospho-HDAC1 (Ser421) antibody, researchers should consider:

• Specificity verification: Choose antibodies that demonstrate specificity for detecting endogenous HDAC1 only when phosphorylated at Serine 421. Antibodies should be validated to distinguish between non-phosphorylated HDAC1 and the phosphorylated form .

• Purification method: Optimal antibodies are typically affinity-purified using epitope-specific phosphopeptides. High-quality products undergo additional purification steps to remove non-phospho-specific antibodies via chromatography using non-phosphopeptides .

• Validation in multiple applications: Confirm the antibody has been validated for your intended applications (Western Blot, Immunohistochemistry, Immunofluorescence, ELISA) .

• Species reactivity: Verify reactivity with your experimental model (human, mouse, rat) .

• Positive controls: Use known conditions that induce HDAC1 Ser421 phosphorylation, such as serum stimulation after serum starvation, to validate antibody performance .

Methodologically, western blot validation should include both phosphorylated and non-phosphorylated controls, and potentially phosphatase treatment to confirm specificity .

What are the differences between HDAC1 phosphorylation at Ser421 versus Ser423?

Distinguishing between phosphorylation at Ser421 and Ser423 requires careful consideration:

Research approaches should employ phospho-specific antibodies that can differentiate between these sites when possible, or mass spectrometry-based approaches for definitive site identification . Some studies use antibodies that recognize both phosphorylation sites (Ser421/423) .

What experimental conditions affect HDAC1 Ser421 phosphorylation status?

Several experimental conditions have been demonstrated to modulate HDAC1 Ser421 phosphorylation:

For experimental manipulation, researchers can induce phosphorylation changes via serum manipulation protocols, mechanical stimulation (flow systems), or pharmacological agents (kinase inhibitors or phosphatase inhibitors like okadaic acid) .

How does the phosphorylation of HDAC1 at Ser421 regulate its subcellular localization?

HDAC1 phosphorylation at Ser421 critically influences its subcellular distribution through a complex regulatory mechanism:

Phosphorylated HDAC1 (Ser421) demonstrates enhanced nuclear retention, while dephosphorylation promotes nuclear export and cytoplasmic accumulation . In endothelial cells subjected to interstitial flow, researchers observed that flow increases phosphorylation at Ser421 and subsequently affects HDAC1 localization . Conversely, AT2R stimulation in human aortic endothelial cells leads to dephosphorylation of Ser421/423-HDAC1 and decreased nuclear localization .

The experimental approach to track this dynamic localization involves:

• Subcellular fractionation followed by western blotting with phospho-specific antibodies

• Immunofluorescence microscopy with dual staining for total HDAC1 and phospho-HDAC1

• Live-cell imaging using fluorescently tagged HDAC1 with phospho-mimetic or phospho-deficient mutations (S421E or S421A)

Leptomycin B (LMB), which inhibits nuclear export, can be used to determine whether cytoplasmic accumulation of dephosphorylated HDAC1 is due to enhanced nuclear export rather than altered import . Researchers have demonstrated that inhibition of HDAC1 export with LMB affects downstream targets, suggesting that the cytoplasmic pool of HDAC1 has distinct functions from its nuclear counterpart .

What is the mechanism by which AT2R-stimulation leads to HDAC1 dephosphorylation?

AT2R (Angiotensin II type 2 receptor) stimulation induces a signaling cascade that results in HDAC1 dephosphorylation through the following mechanistic pathway:

• Receptor activation: Treatment with the AT2R agonist C21 (1 μmol/L) activates AT2R in human aortic endothelial cells (HAECs) .

• Phosphatase recruitment: AT2R activation leads to the activation of serine/threonine phosphatases, as demonstrated by the complete prevention of HDAC1 dephosphorylation when cells are pretreated with okadaic acid (0.1 μmol/L), a serine/threonine phosphatase inhibitor .

• Temporal dynamics: Dephosphorylation of Ser421/423-HDAC1 occurs rapidly, with significant effects observed after 5 and 20 minutes of C21 stimulation .

• Receptor specificity: The effect is AT2R-specific, as preincubation with the AT2R antagonist PD123319 (10 μmol/L) partially inhibits C21-induced HDAC1 dephosphorylation .

• Downstream effects: HDAC1 dephosphorylation is associated with decreased nuclear localization and inhibition of its deacetylating activity .

Methodologically, researchers can track this process using western blot analysis with phospho-specific antibodies after pharmacological manipulation of the AT2R pathway. The AT2R specificity can be validated using cell lines that overexpress AT2R compared to non-transfected control cells .

How does HDAC1 Ser421 phosphorylation interact with the Wnt signaling pathway?

HDAC1 phosphorylation at Ser421 plays a critical role in regulating Wnt signaling through a complex interplay with Nemo-like kinase (NLK):

• Signaling integration: NLK-mediated phosphorylation of HDAC1 at Ser421 occurs during active Wnt signaling . This phosphorylation is elevated in the presence of full-length NLK but not with catalytically inactive NLK mutants .

• Regulatory mechanism: Phosphorylated HDAC1 negatively regulates Wnt signaling via Tcf/Lef transcription repression . This represents a feedback control mechanism within the pathway.

• Cellular consequences: This regulatory axis prevents aberrant proliferation of fibroblasts, suggesting a role in cell cycle control and potentially tumor suppression .

• Experimental evidence: Mass spectrometry analysis has confirmed phosphorylation at Ser421 during NLK activation, with site probability >92% . In vitro kinase assays demonstrate direct phosphorylation by NLK.

• Context-dependent regulation: Serum readdition increases both total HDAC1 levels and its phosphorylation status, suggesting coordination with growth factor signaling .

To study this interaction experimentally, researchers employ co-immunoprecipitation of HDAC1 with Wnt pathway components, reporter gene assays using TOPFlash/FOPFlash systems, and genetic approaches with phospho-mimetic or phospho-deficient HDAC1 mutants to assess functional outcomes on Wnt target gene expression .

What role does HDAC1 Ser421 phosphorylation play in endothelial morphogenesis and angiogenesis?

Phosphorylation of HDAC1 at Ser421 serves as a critical mechanosensory regulator in endothelial morphogenesis and angiogenesis:

• Flow-responsive regulation: Interstitial flow increases HDAC1 phosphorylation at Ser421 in endothelial cells, but only in the presence of VEGF, indicating integration of mechanical and biochemical signals . Flow induces a 67% increase in HDAC1 activity following phosphorylation .

• VEGF dependency: The phosphorylation requires VEGFR2 activation, as demonstrated by the absence of flow-induced HDAC1 phosphorylation when VEGF is not present .

• Functional significance: Inhibition of HDAC1 suppresses endothelial morphogenesis under flow conditions but not under static conditions, highlighting the specific role of HDAC1 in flow-mediated angiogenic responses .

• Downstream effectors: Phosphorylated HDAC1 regulates MMP14 (matrix metalloproteinase-14) expression, which is essential for endothelial cell invasion and capillary formation . Inhibition of HDAC1 decreases MMP14 expression, linking HDAC1 activity to extracellular matrix remodeling necessary for angiogenesis .

• Clinical implications: This mechanism is particularly relevant in pathological contexts such as tumor microenvironments, where both interstitial flow and VEGF levels are elevated .

Experimental approaches to study this process include microfluidic devices that simulate interstitial flow, 3D morphogenesis assays, and macroscale transwell systems to assess endothelial migration and tube formation in response to flow . Researchers can manipulate HDAC1 phosphorylation using specific inhibitors such as 4-(dimethylamino)-N-[6-(hydroxyamino)-6-oxohexyl] or genetic approaches with phospho-site mutants .

How do HDAC1 Ser421 phosphorylation levels correlate with checkpoint kinase signaling during DNA damage response?

HDAC1 phosphorylation at Ser421 integrates with checkpoint kinase signaling during DNA damage response through a regulatory network:

• Bidirectional regulation: Class I HDACs (including HDAC1) are required to sustain checkpoint kinase phosphorylation in response to replicative stress . Conversely, inhibition of HDAC1/2 suppresses the phosphorylation of ATM, CHK1, and CHK2 in response to various DNA-damaging agents .

• Stress response integration: Treatment with DNA-damaging agents such as hydroxyurea, ultraviolet light, or 5-fluorouracil activates a stress response pathway that involves both HDAC1 and checkpoint kinases .

• Phosphatase regulation: HDAC1 and HDAC2 are required to repress the expression of PR130 (a regulatory subunit of protein phosphatase 2A), which dephosphorylates checkpoint kinases . Inhibition of HDAC1/2 leads to increased PR130 expression and subsequent dephosphorylation of checkpoint kinases .

• Experimental evidence: When HDAC inhibitors (such as MS-275) are applied to cells, the phosphorylation of checkpoint kinases is diminished. This effect can be partially rescued by phosphatase inhibitors like okadaic acid and cantharidin, confirming the involvement of phosphatases in this regulation .

• Differential regulation: Interestingly, different checkpoint kinases show varying sensitivity to HDAC1-regulated phosphatases. For example, okadaic acid strongly reduces the inhibition of ATM phosphorylation by MS-275 but barely prevents the dephosphorylation of CHK1 .

Methodologically, researchers investigate this relationship using western blot analysis of phosphorylated checkpoint kinases after treatment with HDAC inhibitors, coupled with phosphatase inhibitors to dissect the mechanism. Ectopic expression of phosphatase regulatory subunits (such as PR130) can be used to validate their role in the pathway .

What are the best experimental approaches to differentiate between Ser421 and Ser423 phosphorylation effects on HDAC1 function?

Differentiating between the specific effects of Ser421 and Ser423 phosphorylation on HDAC1 function requires sophisticated experimental strategies:

• Site-specific phospho-antibodies: Develop and validate antibodies that specifically recognize either phospho-Ser421 or phospho-Ser423, but not both. This requires rigorous validation using phosphopeptide competition assays and site-directed mutants .

• Site-directed mutagenesis approach:

  • Generate single mutants (S421A and S423A) and double mutant (S421A/S423A)

  • Create phospho-mimetic mutants (S421E, S423E, S421E/S423E)

  • Compare these mutants in functional assays to isolate site-specific effects

• Mass spectrometry with phospho-site mapping:

  • Employ high-resolution MS/MS with electron transfer dissociation (ETD) or electron capture dissociation (ECD) fragmentation methods that better preserve labile phosphorylations

  • Quantify the relative abundance of singly phosphorylated peptides (either at Ser421 or Ser423) versus doubly phosphorylated peptides under different conditions

• Temporal analysis of phosphorylation dynamics:

  • Use pulse-chase phosphorylation assays to determine if one site is phosphorylated before the other

  • Apply selective kinase inhibitors to determine if different kinases target each site

• Structural studies:

  • Conduct X-ray crystallography or NMR studies of HDAC1 with either Ser421 or Ser423 phosphorylated

  • Employ molecular dynamics simulations to predict conformational changes induced by phosphorylation at each site

• Interaction partner screening:

  • Conduct differential interactome analysis using BioID or APEX proximity labeling with phospho-site mutants

  • Identify proteins that preferentially interact with HDAC1 phosphorylated at specific sites

When interpreting results, researchers should consider that most published studies have not conclusively separated the effects of these two phosphorylation sites, often treating them as a functional unit (Ser421/423) .

How can researchers accurately quantify changes in HDAC1 Ser421 phosphorylation in complex tissue samples?

Accurately quantifying HDAC1 Ser421 phosphorylation in complex tissue samples requires specialized methodologies to overcome tissue heterogeneity and ensure specific detection:

• Tissue preparation and phosphatase inhibition:

  • Rapid tissue collection and flash-freezing to preserve phosphorylation status

  • Inclusion of multiple phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate, and okadaic acid) in all extraction buffers

  • Homogenization at cold temperatures to minimize enzymatic activity

• Cell-type specific analysis:

  • Laser capture microdissection to isolate specific cell populations from heterogeneous tissue

  • Single-cell phosphoproteomics for high-resolution analysis

  • Flow cytometry with phospho-specific antibodies for cell type identification and quantification

• Quantitative western blotting protocol:

  • Simultaneous detection of phospho-HDAC1 (Ser421) and total HDAC1 using dual-color fluorescent secondary antibodies

  • Include phosphorylation standards for calibration

  • Implement loading controls appropriate for phosphoprotein analysis (not phosphorylation-sensitive proteins)

• Advanced mass spectrometry approaches:

  • Targeted multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) for specific quantification of phosphopeptides

  • SILAC labeling of reference cells to create internal standards for accurate quantification

  • Phosphopeptide enrichment using titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC)

• Validation and controls:

  • Include HDAC1 knockout or knockdown samples as negative controls

  • Use lambda phosphatase-treated samples as dephosphorylation controls

  • Apply samples with known phosphorylation states (e.g., serum-stimulated cells) as positive controls

• Immunohistochemical analysis:

  • Implement antigen retrieval methods optimized for phosphoepitopes

  • Use signal amplification methods (e.g., tyramide signal amplification) for low-abundance detection

  • Apply digital pathology and automated image analysis for unbiased quantification

When reporting results, normalize phospho-HDAC1 (Ser421) levels to total HDAC1 expression, and consider both the percentage of cells showing phosphorylation and the intensity of phosphorylation signal .

What are the implications of HDAC1 Ser421 phosphorylation in disease contexts and potential therapeutic applications?

HDAC1 Ser421 phosphorylation has significant implications in disease pathophysiology and therapeutic targeting:

• Cancer progression and angiogenesis:

  • Elevated interstitial flow and VEGF in tumor microenvironments can enhance HDAC1 Ser421 phosphorylation, promoting angiogenesis through regulation of MMP14 expression

  • Targeting the phosphorylation status of HDAC1 could provide a more selective approach than general HDAC inhibition, potentially reducing side effects while maintaining anti-angiogenic efficacy

• DNA damage response and chemotherapy resistance:

  • HDAC1 phosphorylation status affects checkpoint kinase signaling during DNA damage response

  • Manipulating HDAC1 phosphorylation could potentially sensitize cancer cells to DNA-damaging chemotherapeutics by disrupting checkpoint activation

• Cardiovascular diseases:

  • AT2R-stimulation leads to HDAC1 dephosphorylation at Ser421/423, which may contribute to the anti-proliferative effects of AT2R signaling in vascular cells

  • This mechanism could be relevant for developing treatments for pathologies involving abnormal vascular cell proliferation

• Therapeutic strategies targeting phosphorylation:

  • Development of small molecules that specifically inhibit kinases responsible for HDAC1 Ser421 phosphorylation (e.g., CK2 inhibitors)

  • Creation of phosphorylation-state specific HDAC1 inhibitors that target only the phosphorylated or non-phosphorylated forms

  • Combination therapies that modulate both HDAC1 phosphorylation and its interaction partners

• Biomarker potential:

  • The phosphorylation status of HDAC1 at Ser421 could serve as a biomarker for disease progression or treatment response

  • Immunohistochemical analysis using phospho-specific antibodies could stratify patients for personalized medicine approaches

• Challenges in therapeutic development:

  • Context-dependent functions of HDAC1 phosphorylation across different tissues and disease states

  • Need for specific delivery systems to target relevant cell types

  • Potential compensatory mechanisms through other HDAC family members

Future research directions should focus on developing cell-type specific and phosphorylation-state specific modulators of HDAC1 activity, as well as validating the prognostic and predictive value of HDAC1 Ser421 phosphorylation in clinical samples .

How does HDAC1 Ser421 phosphorylation coordinate with other post-translational modifications to create a regulatory code?

HDAC1 Ser421 phosphorylation operates within a complex post-translational modification (PTM) network that creates a sophisticated regulatory code:

• Multi-site phosphorylation patterns:

  • Ser421 phosphorylation frequently occurs in conjunction with Ser423 phosphorylation

  • Both modifications collectively promote enzymatic activity and interactions with NuRD and SIN3 complexes

  • The temporal sequence and relative stoichiometry of these phosphorylation events may determine specific functional outcomes

• Crosstalk with sumoylation:

  • HDAC1 is sumoylated on Lys-444 and Lys-476, which promotes enzymatic activity

  • Phosphorylation at Ser421 may influence the sumoylation process, creating interdependent regulatory mechanisms

  • SENP1 desumoylates HDAC1, potentially creating a dynamic interplay between phosphorylation and sumoylation states

• Ubiquitination regulation:

  • HDAC1 is ubiquitinated by E3 ligases such as CHFR and KCTD11, leading to proteasomal degradation

  • Phosphorylation status at Ser421 may influence recognition by these ubiquitin ligases, affecting HDAC1 stability and turnover

• Integration with acetylation signaling:

  • While HDAC1 primarily acts as a deacetylase, it may itself be subject to acetylation

  • The interplay between its own acetylation status and phosphorylation at Ser421 could create feedback loops in acetylation signaling networks

• Protein complex assembly regulation:

  • Phosphorylation at Ser421 promotes HDAC1 interactions with specific protein complexes including NuRD and SIN3

  • These interactions may be further modulated by additional PTMs on HDAC1 or its binding partners

  • Complex formation is essential for targeting HDAC1 to specific genomic loci

This sophisticated PTM code can be studied using:

• Multiparameter mass spectrometry to detect co-occurring modifications

• Proximity ligation assays to visualize specific modification patterns in situ

• Sequential chromatin immunoprecipitation (ChIP-reChIP) to identify genomic regions bound by HDAC1 with specific modification signatures

• Proteomic approaches to identify interaction partners specific to particular HDAC1 modification states

Understanding this PTM code is essential for developing targeted therapeutic strategies that modulate specific functions of HDAC1 while preserving others.

What emerging technologies are advancing the study of HDAC1 Ser421 phosphorylation dynamics?

Several cutting-edge technologies are transforming our ability to study HDAC1 Ser421 phosphorylation with unprecedented spatial and temporal resolution:

• Genetically encoded phosphorylation sensors:

  • FRET-based sensors that specifically detect HDAC1 Ser421 phosphorylation in living cells

  • Allows real-time visualization of phosphorylation dynamics in response to stimuli

  • Can be targeted to specific subcellular compartments to track localized phosphorylation events

• Phospho-specific degrons and optogenetic tools:

  • Engineered systems where light activation can trigger dephosphorylation or phosphorylation of HDAC1 at Ser421

  • Enables precise temporal control for studying downstream effects

  • Can be combined with live-cell imaging to correlate phosphorylation changes with functional outcomes

• Single-cell phosphoproteomics:

  • Mass cytometry (CyTOF) with phospho-specific antibodies for high-dimensional analysis

  • SCoPE-MS (Single Cell ProtEomics by Mass Spectrometry) adapted for phosphopeptide analysis

  • Reveals cell-to-cell heterogeneity in HDAC1 phosphorylation status within populations

• Cryo-electron microscopy of HDAC complexes:

  • Structural determination of HDAC1 within native complexes in different phosphorylation states

  • Visualizes conformational changes induced by Ser421 phosphorylation

  • Provides insights into how phosphorylation affects protein-protein interactions

• CRISPR-based phosphorylation site engineering:

  • Base editing or prime editing to create precise phospho-null or phospho-mimetic mutations

  • Avoids overexpression artifacts associated with traditional mutagenesis approaches

  • Can be combined with high-throughput phenotypic screening

• Spatial -omics integration:

  • Combining phosphoproteomics with spatial transcriptomics

  • Maps HDAC1 phosphorylation status to specific tissue regions and correlates with gene expression patterns

  • Particularly valuable for understanding HDAC1 phosphorylation in heterogeneous tissues like tumors

• Machine learning approaches:

  • Predictive models for HDAC1 phosphorylation dynamics based on multiple input signals

  • Integration of phosphoproteomics data with other -omics datasets

  • Identification of context-specific regulators of HDAC1 phosphorylation

These technologies collectively provide a systems-level understanding of how HDAC1 Ser421 phosphorylation is regulated and how it contributes to cellular decision-making in both normal physiology and disease states .