Phospho-PIKFYVE (S307) Antibody

What is PIKFYVE and what biological functions does it serve?

PIKFYVE (phosphoinositide kinase, FYVE finger containing) is a dual specificity kinase implicated in numerous essential cellular processes. It functions as the sole enzyme that catalyzes the phosphorylation of phosphatidylinositol 3-phosphate on the fifth hydroxyl of the myo-inositol ring to form phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2). PIKFYVE plays critical roles in maintenance of endomembrane homeostasis, endocytic-vacuolar pathway regulation, lysosomal trafficking, nuclear transport, stress- or hormone-induced signaling, and cell cycle progression. Additionally, it is essential for systemic glucose homeostasis, mediating insulin-induced signals for endosome/actin remodeling during GLUT4 translocation and glucose uptake activation . As part of the PI(3,5)P2 regulatory complex, it regulates both the synthesis and turnover of PtdIns(3,5)P2 .

What is the significance of the Ser307 phosphorylation site on PIKFYVE?

The Ser307 phosphorylation site represents a key regulatory point for PIKFYVE activity. This specific phosphorylation occurs within the amino acid region 273-322 of the human PIKFYVE protein . Phosphorylation at this site is believed to modulate the enzymatic activity of PIKFYVE, potentially affecting its capacity to generate PtdIns(3,5)P2 and thus influencing downstream cellular processes including membrane dynamics necessary for cell homeostasis and signal transduction . The site-specific phosphorylation may also play a role in PIKFYVE's functions related to glucose metabolism and insulin signaling .

What species reactivity can be expected with Phospho-PIKFYVE (S307) antibodies?

Commercially available Phospho-PIKFYVE (S307) antibodies typically demonstrate reactivity against human, mouse, and rat samples . This cross-species reactivity is particularly valuable for comparative studies across model organisms. It's worth noting that while the human phosphorylation site is at Ser307, the equivalent phosphorylation sites in mouse and rat are at Ser318 and Ser357, respectively . This difference should be considered when designing experiments and interpreting results across different species.

What applications is the Phospho-PIKFYVE (S307) antibody validated for?

The Phospho-PIKFYVE (S307) antibody has been validated for several research applications, including:

• Immunohistochemistry (IHC) at dilutions of 1:100-1:300

• Immunofluorescence (IF) at dilutions of 1:200-1:1000

• Enzyme-Linked Immunosorbent Assay (ELISA) at dilutions of approximately 1:10000

These applications enable researchers to visualize and quantify phosphorylated PIKFYVE in various experimental contexts, from tissue sections to cell cultures and protein extracts.

How does phosphorylation at Ser307 regulate PIKFYVE's role in membrane trafficking?

Phosphorylation at Ser307 likely serves as a molecular switch that modulates PIKFYVE's enzymatic activity in generating PtdIns(3,5)P2, a crucial phosphoinositide for membrane dynamics. Research suggests that this phosphorylation event may regulate the association of PIKFYVE with endosomal membranes, thereby controlling its spatial activity. The generation of PtdIns(3,5)P2 by activated PIKFYVE is essential for proper endosomal maturation, multivesicular body formation, and retrograde trafficking from endosomes to the trans-Golgi network . In the context of membrane trafficking, phosphorylated PIKFYVE at Ser307 may facilitate the recruitment of effector proteins containing PtdIns(3,5)P2-binding domains, orchestrating the assembly of protein complexes required for vesicle budding, fusion, and trafficking events.

What is the relationship between PIKFYVE Ser307 phosphorylation and insulin signaling?

PIKFYVE plays a critical role in insulin-stimulated glucose uptake through its involvement in GLUT4 translocation to the plasma membrane. Phosphorylation at Ser307 appears to be an important regulatory mechanism in this process. Upon insulin stimulation, signaling cascades may lead to increased phosphorylation at Ser307, activating PIKFYVE to facilitate endosome/actin remodeling necessary for GLUT4 translocation . This phosphorylation event potentially integrates insulin receptor signaling with membrane trafficking machinery. Researchers investigating metabolic disorders like type 2 diabetes might find particular value in examining how alterations in Ser307 phosphorylation correlate with insulin resistance or defects in glucose homeostasis.

How do different experimental conditions affect PIKFYVE Ser307 phosphorylation levels?

Several experimental conditions can influence PIKFYVE Ser307 phosphorylation levels:

These conditions should be carefully controlled and documented when designing experiments to study PIKFYVE phosphorylation dynamics.

What are the implications of PIKFYVE Ser307 phosphorylation in neurodegenerative disorders?

Emerging research suggests a potential link between dysregulated PIKFYVE activity and neurodegenerative disorders. PIKFYVE's role in endolysosomal trafficking and membrane homeostasis places it at a critical intersection for processes like protein degradation and neuronal maintenance. Abnormal phosphorylation at Ser307 might alter PIKFYVE's capacity to generate PtdIns(3,5)P2, potentially contributing to endolysosomal dysfunction observed in conditions such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis . Researchers investigating these conditions might benefit from examining PIKFYVE Ser307 phosphorylation status in relevant cellular and animal models, potentially identifying novel therapeutic targets or biomarkers.

What are the optimal sample preparation protocols for detecting phosphorylated PIKFYVE (Ser307)?

Optimal sample preparation is crucial for reliable detection of phosphorylated PIKFYVE at Ser307. The following protocol is recommended:

• Cell/Tissue Lysis: Use a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS.

• Phosphatase Inhibitors: Include a cocktail of phosphatase inhibitors (e.g., 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM β-glycerophosphate, and 10 mM sodium pyrophosphate) to prevent dephosphorylation during sample processing.

• Protease Inhibitors: Add protease inhibitors (e.g., 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin) to prevent protein degradation.

• Temperature Control: Maintain samples at 4°C throughout processing to minimize phosphatase activity.

• Rapid Processing: Process samples promptly after collection to preserve phosphorylation status.

• Storage: Store prepared lysates at -80°C and avoid repeated freeze-thaw cycles .

For immunohistochemistry applications, proper fixation is critical. Use 4% paraformaldehyde fixation for 10-15 minutes for cultured cells or 24 hours for tissue sections, followed by careful permeabilization that preserves epitope accessibility while allowing antibody penetration.

What controls should be included when using Phospho-PIKFYVE (S307) antibody?

To ensure reliable and interpretable results, the following controls should be incorporated:

Including these controls will strengthen the validity of experimental findings and aid in troubleshooting if unexpected results occur.

How should researchers optimize immunohistochemistry protocols for Phospho-PIKFYVE (S307) antibody?

Optimization of immunohistochemistry protocols for Phospho-PIKFYVE (S307) antibody requires attention to several key parameters:

• Antigen Retrieval: Test multiple methods, including heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0), to determine optimal conditions for exposing the phospho-epitope.

• Antibody Dilution: Perform a dilution series (starting with recommended 1:100-1:300 range) to identify the optimal concentration that maximizes specific signal while minimizing background .

• Incubation Conditions: Test various incubation times (1 hour at room temperature versus overnight at 4°C) and determine which provides the best signal-to-noise ratio.

• Blocking: Use 5-10% normal serum from the same species as the secondary antibody, supplemented with 1% BSA, to reduce non-specific binding.

• Signal Amplification: Consider using biotin-streptavidin systems or tyramide signal amplification for low-abundance targets.

• Counterstaining: Select appropriate counterstains that won't mask the signal of interest.

• Tissue-Specific Considerations: Adjust protocols based on tissue type, as different tissues may require modified approaches for optimal results.

Titration experiments are essential to determine the optimal antibody concentration for each specific application and sample type.

What are the best approaches for quantifying Phospho-PIKFYVE (S307) levels in experimental samples?

Quantification of Phospho-PIKFYVE (S307) levels can be accomplished through several methodological approaches:

• Immunoblotting Quantification:

  • Normalize phospho-PIKFYVE band intensity to total PIKFYVE

  • Use digital imaging systems with linear detection range

  • Apply appropriate statistical analysis across biological replicates

• Immunofluorescence Quantification:

  • Measure mean fluorescence intensity in defined cellular compartments

  • Use consistent acquisition parameters across all samples

  • Employ z-stack imaging for three-dimensional analysis

  • Analyze at least 50-100 cells per condition for statistical robustness

• ELISA-Based Quantification:

  • Develop standard curves using recombinant phosphorylated protein

  • Apply samples in technical triplicates

  • Calculate phospho-to-total PIKFYVE ratios for accurate comparison

• Flow Cytometry:

  • Optimize fixation and permeabilization for intracellular phospho-epitope detection

  • Include appropriate compensation controls

  • Gate on relevant cell populations before analyzing phospho-signal

Regardless of the method chosen, researchers should report detailed quantification parameters, normalize data appropriately, and apply rigorous statistical analysis to ensure reproducibility and validity of findings.

What factors might contribute to inconsistent detection of Phospho-PIKFYVE (S307)?

Several factors can lead to variability in detecting Phospho-PIKFYVE (S307):

• Phosphorylation Dynamics: PIKFYVE phosphorylation at Ser307 may be transient and context-dependent, fluctuating based on cellular conditions or stimuli. Careful timing of experiments is crucial.

• Sample Preparation Issues:

  • Inadequate phosphatase inhibition during sample collection and processing

  • Protein degradation due to improper protease inhibition

  • Extended storage or multiple freeze-thaw cycles of samples

• Technical Variables:

  • Batch-to-batch variation in antibody performance

  • Inconsistent blocking or washing procedures

  • Variations in incubation times or temperatures

• Biological Variables:

  • Cell confluence levels affecting signaling pathways

  • Passage number of cultured cells

  • Variations in stimulation protocols or reagent potency

• Detection System Limitations:

  • Suboptimal signal amplification methods

  • Detector sensitivity variations

  • Non-linear response range issues

Maintaining rigorous experimental protocols, including detailed documentation of all variables, can help identify and minimize sources of inconsistency.

How can researchers distinguish between specific and non-specific signals when using Phospho-PIKFYVE (S307) antibody?

Distinguishing specific from non-specific signals requires systematic validation approaches:

• Comparison with Multiple Antibodies: Use antibodies from different suppliers or those recognizing different epitopes of phosphorylated PIKFYVE to confirm signal specificity.

• Phosphatase Treatment Controls: Treat duplicate samples with lambda phosphatase to demonstrate that the signal is phosphorylation-dependent and can be eliminated by phosphatase treatment.

• Peptide Competition Assays: Pre-incubate the antibody with the immunizing phosphopeptide (containing pSer307) versus a non-phosphorylated version of the same peptide. Specific signals should be blocked by the phosphopeptide but not by the non-phosphopeptide.

• Genetic Approaches: Use PIKFYVE knockdown/knockout systems or phospho-mutants (S307A) to validate antibody specificity. The phospho-specific signal should be reduced or eliminated in these systems.

• Expected Cellular Localization: Verify that the detected signal corresponds to the known subcellular distribution of PIKFYVE (primarily endosomal membranes).

• Molecular Weight Verification: Confirm that the detected band in immunoblotting corresponds to the expected molecular weight of PIKFYVE (approximately 235 kDa).

These complementary approaches provide strong evidence for signal specificity when consistently applied.

What emerging technologies might enhance the study of PIKFYVE Ser307 phosphorylation?

Several cutting-edge technologies hold promise for advancing our understanding of PIKFYVE Ser307 phosphorylation:

• Proximity Ligation Assays (PLA): These could reveal spatial relationships between phosphorylated PIKFYVE and its interaction partners or substrates with high sensitivity and specificity.

• CRISPR-Cas9 Gene Editing: Generation of endogenous phospho-mimetic (S307D/E) or phospho-deficient (S307A) PIKFYVE mutants would allow direct assessment of this phosphorylation event's functional significance.

• Phospho-Proteomics: Mass spectrometry-based approaches can identify additional phosphorylation sites on PIKFYVE and their stoichiometric relationships with Ser307 phosphorylation.

• Biosensors: Development of FRET-based biosensors for monitoring PIKFYVE activity or PtdIns(3,5)P2 production in real-time within living cells.

• Super-Resolution Microscopy: Techniques like STORM or PALM could reveal nanoscale spatial organization of phosphorylated PIKFYVE on specific membrane domains.

• Single-Cell Analysis: Examining phospho-PIKFYVE levels at single-cell resolution could uncover population heterogeneity and identify distinct cellular states.

These technologies, when combined with traditional biochemical approaches, will provide more comprehensive insights into PIKFYVE regulation and function.

What are the key knowledge gaps in understanding PIKFYVE Ser307 phosphorylation?

Despite progress in PIKFYVE research, several important questions remain unanswered:

• Kinase Identification: The specific kinase(s) responsible for phosphorylating PIKFYVE at Ser307 under different physiological conditions remain incompletely characterized.

• Phosphorylation Dynamics: The temporal dynamics and stoichiometry of Ser307 phosphorylation in response to various stimuli need further elucidation.

• Structural Consequences: How Ser307 phosphorylation alters PIKFYVE's three-dimensional structure and catalytic properties remains unclear.

• Cross-talk with Other Modifications: The interplay between Ser307 phosphorylation and other post-translational modifications on PIKFYVE requires investigation.

• Cell-Type Specificity: Whether the regulation and function of PIKFYVE Ser307 phosphorylation varies across different cell types and tissues needs clarification.

• Pathological Relevance: The potential dysregulation of this phosphorylation event in disease states, particularly metabolic and neurodegenerative disorders, represents an important area for future research.

Addressing these knowledge gaps will significantly advance our understanding of PIKFYVE regulation and its roles in cellular physiology and pathology.