Phospho-MAPT (Ser214) Antibody

What is the biological significance of tau phosphorylation at Ser214?

Phosphorylation of tau at Serine 214 (pTAU-S214) plays a crucial role in regulating tau's interaction with microtubules and its tendency to aggregate. Research demonstrates that phosphorylation at this site reduces the pathological assembly of the protein. Specifically, pTAU-S214 detaches tau from microtubules while simultaneously protecting it against aggregation into Alzheimer's paired helical filaments (PHFs) .

This protective mechanism appears to work by preventing the phosphorylation of other sites. For instance, priming phosphorylation at Ser214 by PKA protects other sites of tau (including Thr212 and ser-pro motifs around residue 200) from phosphorylation by glycogen synthase kinase 3β, thus preventing the PHF-like conformation of tau .

Which kinases are responsible for phosphorylating tau at Ser214?

Multiple kinases have been identified that can phosphorylate tau at Ser214:

Experimental studies have shown that activation of adenylyl cyclase increases pTAU-S214 levels, indicating that the cAMP/PKA-activated cascade is a primary pathway leading to this phosphorylation event .

How does phosphorylation at Ser214 differ from other tau phosphorylation sites?

Phosphorylation at Ser214 has distinct effects compared to other tau phosphorylation sites:

• Protection against aggregation: Unlike many phosphorylation sites that promote tau aggregation, Ser214 phosphorylation generally protects against PHF formation .

• Unique regulation: pTAU-S214 is specifically enhanced by cAMP elevation, whereas other sites may be regulated by different signaling pathways .

• Interaction with other sites: Phosphorylation at Ser214 influences the phosphorylation state of other sites, particularly preventing phosphorylation at sites that promote PHF formation .

• Microtubule binding: While many phosphorylation events reduce tau's ability to bind microtubules, Ser214 phosphorylation does so while simultaneously protecting against pathological aggregation—a unique dual effect .

What are the validated applications for Phospho-MAPT (Ser214) antibodies?

Based on the available information, Phospho-MAPT (Ser214) antibodies have been validated for several experimental applications:

When selecting an antibody, researchers should consider the specific clone and validation data for their intended application. For example, Cell Signaling's Phospho-Tau (Ser214) (D1Q2X) Rabbit mAb has been validated for both Western blotting (1:1000) and immunoprecipitation (1:50) .

How can researchers validate the specificity of Phospho-MAPT (Ser214) antibodies?

Validating antibody specificity is critical for reliable research outcomes. Recommended approaches include:

• Peptide competition assays: Using synthesized phosphorylated peptides containing the Ser214 site (such as peptides with the sequence T-P-S(p)-L-P) to compete with endogenous protein binding .

• Phosphatase treatment controls: Treating samples with lambda phosphatase to remove phosphate groups should eliminate signal from phospho-specific antibodies.

• Kinase activation/inhibition: Treating cells with cAMP enhancers like forskolin or GEBR-7b should increase Ser214 phosphorylation, while PKA inhibitors should decrease it. These treatments serve as positive and negative controls, respectively .

• Genetic approaches: Using MAPT knockout models or MAPT siRNA knockdown as negative controls, or complementation with phospho-mutants (S214A/S214D) to confirm specificity.

• Cross-reactivity testing: Assessing reactivity against other phosphorylation sites, particularly the closely related Thr212 site, which can sometimes be recognized by the same antibodies .

What are the optimal experimental conditions for detecting Phospho-MAPT (Ser214) in Western blotting?

For optimal Western blotting results with Phospho-MAPT (Ser214) antibodies:

• Sample preparation:

  • Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) in lysis buffers

  • Process samples quickly and keep on ice to prevent dephosphorylation

  • For brain tissue samples, rapid post-mortem processing is crucial

• Protein separation:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Expected molecular weight range: 50-80 kDa (varies by isoform and phosphorylation state)

• Transfer and blocking:

  • PVDF membranes often provide better results than nitrocellulose

  • Block with 5% BSA in TBST rather than milk (milk contains phosphatases)

• Antibody incubation:

  • Primary antibody dilution: typically 1:1000 for commercial antibodies

  • Overnight incubation at 4°C gives optimal signal-to-noise ratio

  • Washing with TBST containing 0.05% Tween-20 minimizes background

• Detection:

  • Enhanced chemiluminescence (ECL) detection systems are suitable

  • Fluorescent secondary antibodies allow for multiplexing with total tau detection

How does cAMP signaling regulate tau phosphorylation at Ser214?

The cAMP signaling pathway has been demonstrated to regulate tau phosphorylation at Ser214 through several mechanisms:

• Direct activation of PKA: Elevation of intracellular cAMP activates PKA, which directly phosphorylates tau at Ser214. Research shows that activation of adenylyl cyclase with forskolin (FSK) induces significant increases in pTAU-S214 levels in both N2a cells (16.03-fold increase) and rat hippocampal slices (3.31-fold increase) .

• PDE4D inhibition: Inhibition of phosphodiesterase 4D (PDE4D), which hydrolyzes cAMP, by compounds like GEBR-7b increases pTAU-S214 levels. This effect is enhanced when combined with forskolin treatment, suggesting a synergistic effect .

• Independent of Aβ peptide: Research indicates that cAMP-mediated phosphorylation of tau at Ser214 occurs independently of Aβ peptide production. γ-secretase inhibition, which effectively prevents Aβ production, does not affect cAMP-induced elevation of pTAU-S214 .

These findings suggest that pharmacological interventions targeting the cAMP signaling pathway may have therapeutic potential in reducing tau aggregation and its neurotoxic effects .

What is the relationship between Ser214 phosphorylation and tau's involvement in neurodegenerative diseases?

The relationship between Ser214 phosphorylation and neurodegenerative diseases is complex:

Understanding this relationship provides insights for developing therapeutic strategies targeting tau phosphorylation in neurodegenerative diseases.

How do researchers distinguish between single-site phosphorylation at Ser214 and dual phosphorylation at Thr212/Ser214?

Distinguishing between these phosphorylation states is critical as they have opposite effects on tau aggregation. Researchers employ several approaches:

• Site-specific antibodies:

  • Single phospho-specific antibodies targeting only pSer214 (e.g., Cell Signaling's D1Q2X Rabbit mAb)

  • Dual phospho-specific antibodies recognizing both pThr212 and pSer214 simultaneously (e.g., Cell Signaling's F2A4N Rabbit mAb)

• Mass spectrometry-based approaches:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can identify specific phosphopeptides and distinguish single from multiple phosphorylation events

  • Phosphopeptide mapping can quantify the relative abundance of different phosphorylation patterns

• NMR spectroscopy:

  • 1H, 15N-HSQC spectra of MAP2c fragments and tau mutants can be used to assign peaks of phosphorylated residues

  • 2D FOSY NMR spectra can confirm phosphorylation at specific sites

• Functional assays:

  • Microtubule binding assays to differentiate functional effects of different phosphorylation states

  • PHF formation assays to assess aggregation propensity

For optimal experimental design, researchers should consider using both single and dual phospho-specific antibodies as complementary approaches, alongside functional assays to confirm the biological relevance of their findings.

How can phosphorylation at Ser214 be manipulated in experimental models?

Researchers have several methods to experimentally manipulate Ser214 phosphorylation:

• Pharmacological approaches:

  • cAMP enhancers: Forskolin (adenylyl cyclase activator) significantly increases pTAU-S214 levels (16.03-fold in N2a cells) .

  • PDE4D inhibitors: GEBR-7b enhances cAMP levels and increases pTAU-S214 .

  • Kinase activators/inhibitors: PKA activators increase Ser214 phosphorylation, while PKA inhibitors decrease it.

• Genetic approaches:

  • Phosphomimetic mutations: S214D mutants mimic constitutive phosphorylation

  • Phosphodeficient mutations: S214A mutations prevent phosphorylation at this site

  • Kinase overexpression/knockdown: Modulating PKA, CDK5, GSK3β, or SGK1 expression affects Ser214 phosphorylation levels

• Model systems:

  • Cell culture: N2a neuroblastoma cells and primary neuronal cultures show robust phosphorylation responses to cAMP enhancers .

  • Tissue ex vivo: Rat hippocampal slices maintain the regulatory mechanisms for Ser214 phosphorylation .

  • Animal models: Transgenic mice expressing human tau can be treated with cAMP-enhancing drugs to modulate Ser214 phosphorylation.

These approaches can be used individually or in combination for comprehensive studies of Ser214 phosphorylation.

What is the interplay between Ser214 phosphorylation and other post-translational modifications of tau?

The interplay between Ser214 phosphorylation and other post-translational modifications reveals complex regulatory networks:

• Interactions with other phosphorylation sites:

  • Phosphorylation at Ser214 by PKA protects Thr212 and ser-pro motifs around residue 200 from phosphorylation by GSK3β .

  • Dual phosphorylation at Thr212/Ser214 has different effects than Ser214 phosphorylation alone, promoting rather than preventing aggregation .

  • Ser214 phosphorylation occurs independently of phosphorylation at Ser202, as cAMP elevation increases pTAU-S214 while leaving Ser202 unaffected .

• Crosstalk with O-GlcNAcylation:

  • There is a reciprocal down-regulation between phosphorylation and O-GlcNAcylation of tau .

  • Phosphorylation at certain sites can reduce glycosylation by varying factors, and vice versa.

• Relationship with ubiquitination:

  • Phosphorylation states may influence tau's susceptibility to ubiquitination and subsequent degradation.

  • Tau can be modified by three different forms of polyubiquitination (Lys-48, Lys-6, and Lys-11 linked) .

Understanding these interactions is critical for developing effective therapeutic strategies targeting tau modifications in neurodegenerative diseases.

How can researchers address technical challenges when working with Phospho-MAPT (Ser214) antibodies?

Researchers face several technical challenges when working with phospho-specific antibodies that can be addressed through methodological optimizations:

• Phosphorylation stability issues:

  • Use phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) in all buffers

  • Process samples rapidly and maintain at cold temperatures

  • Consider chemical fixation methods that preserve phosphorylation states for microscopy

• Antibody cross-reactivity:

  • Validate antibody specificity using phospho-peptide competition assays

  • Include phospho-mutant (S214A) samples as negative controls

  • Test multiple antibody clones from different vendors to confirm results

• Quantification challenges:

  • Normalize phospho-tau signals to total tau levels rather than housekeeping proteins

  • Use recombinant phospho-proteins as standards for absolute quantification

  • Consider using quantitative techniques like ELISA for more precise measurements

• Reproducibility concerns:

  • Document lot numbers of antibodies as there can be lot-to-lot variation

  • Use recombinant antibodies where available for better consistency

  • Standardize protocols across experiments, particularly sample preparation methods

• Multi-site phosphorylation detection:

  • Use sequential immunoprecipitation with different phospho-specific antibodies

  • Consider mass spectrometry approaches for comprehensive phosphorylation profiling

  • Use antibodies specifically designed to detect dual phosphorylation (e.g., Thr212/Ser214)

By implementing these strategies, researchers can increase the reliability and reproducibility of their phospho-tau research.

What therapeutic strategies are being explored based on tau phosphorylation at Ser214?

Several therapeutic approaches are being investigated based on the understanding of tau phosphorylation at Ser214:

These therapeutic strategies highlight the importance of understanding the complex role of specific phosphorylation sites in tau pathology.

How do different neuronal populations exhibit variation in tau phosphorylation at Ser214?

Research indicates significant heterogeneity in tau phosphorylation patterns across different brain regions and neuronal populations:

• Regional variation in tau expression and phosphorylation:

  • Significant regional variation in mRNA expression and splicing of MAPT has been observed within the human brain .

  • The cerebellum consistently shows the highest MAPT expression, while white matter shows the lowest .

  • These regional differences in expression may influence the phosphorylation patterns, including at Ser214.

• Cell-type specific phosphorylation patterns:

  • Phosphorylated map kinase (ERK1, ERK2) expression is associated with early tau deposition in both neurons and glial cells .

  • Different neuronal populations may have varying kinase and phosphatase activities, affecting the equilibrium of Ser214 phosphorylation.

• Disease-specific patterns:

  • In Alzheimer's disease, certain neuronal populations are more vulnerable to tau pathology.

  • Phosphorylation at Ser214 has been detected in both Alzheimer's disease and dementia with Lewy bodies .

  • The protective effect of Ser214 phosphorylation may vary across different neuronal populations, potentially explaining differential vulnerability to tau pathology.

• Developmental considerations:

  • Tau phosphorylation patterns, including at Ser214, change during development and aging.

  • The expression of different tau isoforms across brain regions during development may influence phosphorylation patterns.

Understanding these variations is crucial for developing targeted therapeutic approaches for neurodegenerative diseases.

What are the latest methodological advances in studying site-specific tau phosphorylation?

Recent technological advances have enhanced our ability to study site-specific tau phosphorylation:

• Advanced microscopy techniques:

  • Super-resolution microscopy (e.g., STORM, STED) allows visualization of tau phosphorylation in subcellular compartments.

  • Expansion microscopy combined with phospho-specific antibodies provides enhanced spatial resolution.

  • Live-cell imaging using phospho-specific sensors enables real-time monitoring of phosphorylation dynamics.

• Mass spectrometry innovations:

  • Targeted MS approaches like parallel reaction monitoring (PRM) provide quantitative measurement of specific phosphorylation sites.

  • Crosslinking mass spectrometry reveals structural changes induced by phosphorylation.

  • Top-down proteomics approaches allow analysis of intact tau proteoforms with different combinations of modifications.

• Genetic tools:

  • CRISPR-Cas9 gene editing to introduce phosphomimetic or phosphodeficient mutations.

  • Inducible expression systems to control timing of mutant tau expression.

  • AAV-based approaches for neuronal-specific expression of tau variants.

• Phospho-specific probes and sensors:

  • Development of recombinant antibody fragments (Fabs) with enhanced specificity.

  • Phospho-specific intrabodies for live-cell imaging of tau phosphorylation.

  • Biosensors that report on kinase activity toward specific tau residues.

• Computational approaches:

  • Molecular dynamics simulations to predict structural changes induced by specific phosphorylation events.

  • Machine learning algorithms to identify patterns in multiparametric phosphorylation data.

  • Systems biology approaches to model kinase-phosphatase networks regulating tau phosphorylation.

These methodological advances provide researchers with unprecedented tools to investigate the complex regulation and consequences of site-specific tau phosphorylation, including at Ser214.