Phospho-PRKAA2 (S491) Recombinant Monoclonal Antibody

What is the significance of PRKAA2 S491 phosphorylation in cellular metabolism?

Phosphorylation of PRKAA2 at S491 represents a crucial regulatory event in the AMPK signaling pathway, enabling cells to adapt to energy fluctuations and maintain energy homeostasis. Unlike the activating phosphorylation at T172, S491 phosphorylation serves as an inhibitory mechanism that reduces AMPK activity. This site plays a particularly important role in skeletal muscle metabolism, where its phosphorylation status directly impacts systemic glucose regulation and insulin sensitivity. Dysregulation of this phosphorylation event has significant implications for cellular metabolism and is implicated in various metabolic disorders and cancers .

Recent research has demonstrated that PAK4 (p21-activated kinase 4) promotes insulin resistance specifically by phosphorylating AMPKα2 at Ser491, thereby inhibiting AMPK activity. This inhibition subsequently affects downstream targets involved in glucose uptake and metabolism .

How is the Phospho-PRKAA2 (S491) Recombinant Monoclonal Antibody produced?

The production of Phospho-PRKAA2 (S491) recombinant monoclonal antibody follows a sophisticated multi-step process:

• Initial isolation of genes encoding the PRKAA2 antibody from rabbits immunized with a synthesized peptide derived from human PRKAA2 protein phosphorylated at S491

• Cloning of these antibody genes into expression vectors

• Transfection of the modified vectors into host suspension cells

• Cultivation of positive cells to facilitate antibody expression and secretion

• Purification of the antibody from cell culture supernatant using affinity chromatography

• Rigorous assessment of antibody activity through ELISA and Western Blot tests

This recombinant approach ensures high specificity for the phosphorylated S491 site on human PRKAA2 protein, making it a valuable tool for investigating the regulatory mechanisms of AMPK signaling .

What are the recommended applications and dilutions for this antibody?

The Phospho-PRKAA2 (S491) Recombinant Monoclonal Antibody is primarily validated for Western Blot (WB) applications, with recommended dilutions ranging from 1:500 for detecting low abundance targets to 1:5000 for highly expressed proteins . When optimizing dilutions, researchers should consider:

• Target protein abundance in the sample

• Background signal levels

• Antibody affinity and specificity

• Detection method sensitivity (chemiluminescence, fluorescence, etc.)

Starting with a mid-range dilution (1:1000) is often recommended, followed by optimization based on signal strength and background levels. While Western blotting is the primary validated application, researchers have also successfully used similar phospho-specific antibodies for immunoprecipitation and proximity ligation assays to study protein-protein interactions, as demonstrated in studies examining PAK4 interaction with AMPKα2 .

How do different kinases contribute to PRKAA2 S491 phosphorylation and what are the methodological approaches to distinguish between them?

Multiple kinases can phosphorylate the S491 site on PRKAA2, including:

• Protein Kinase A (PKA)

• p70S6 Kinase (p70S6K)

• Protein Kinase B (Akt) (at the homologous S485 site in α1)

• PAK4 (recently identified)

• Autophosphorylation mechanisms

To distinguish between these kinase contributions, researchers can employ several methodological approaches:

• Pharmacological inhibitors: Selective inhibitors of each kinase pathway (e.g., H89 for PKA, rapamycin for mTOR/p70S6K pathway) can help determine which kinase is responsible for S491 phosphorylation in a given context.

• Genetic approaches: siRNA knockdown or CRISPR-Cas9 knockout of candidate kinases, followed by assessment of S491 phosphorylation status.

• Kinase assays: In vitro kinase assays using purified kinases and AMPKα2 as substrate, with subsequent detection using the Phospho-PRKAA2 (S491) antibody.

• Phospho-mimetic and phospho-deficient mutants: Expression of AMPKα2 S491D (phospho-mimetic) or S491A (phospho-deficient) mutants to understand functional consequences of phosphorylation by different kinases .

Recent studies have demonstrated that PAK4 directly interacts with and phosphorylates AMPKα2 at S491, as confirmed through co-immunoprecipitation experiments and proximity ligation assays. This phosphorylation inhibits the activating phosphorylation at T172 and subsequently reduces AMPK activity .

What are the challenges in detecting PRKAA2 S491 phosphorylation using mass spectrometry, and how can antibody-based methods complement this approach?

Mass spectrometry (MS) analysis of PRKAA2 S491 phosphorylation faces several technical challenges:

• Impeded tryptic cleavage: The S491 site may reside in a sequence context that is resistant to complete tryptic digestion, affecting peptide generation.

• Interfering mass species: Co-eluting peptides with similar mass-to-charge ratios can interfere with detection.

• Flyability issues: The phosphopeptide containing S491 may have poor ionization efficiency or "flyability" in the mass spectrometer.

As evidenced in recent phosphosite profiling studies, MS approaches failed to detect the well-known α2-pS491 site despite successfully identifying numerous other phosphorylation sites on AMPK subunits .

To overcome these limitations, antibody-based methods provide essential complementary approaches:

• Western blotting with phospho-specific antibodies: This remains the gold standard for detecting specific phosphorylation events, especially when MS detection is challenging.

• Immunoprecipitation followed by MS: Enriching for the protein of interest using antibodies before MS analysis can improve detection sensitivity.

• Proximity ligation assays (PLA): These can confirm direct interactions between kinases and AMPK while verifying phosphorylation status.

The following table shows phosphosites that were successfully detected by MS versus those requiring antibody-based detection:

Adapted from phosphosite profiling data

How does PRKAA2 S491 phosphorylation status impact experimental design for studying AMPK signaling in metabolic disorders?

When designing experiments to study AMPK signaling in metabolic disorders, researchers must carefully consider the PRKAA2 S491 phosphorylation status as it significantly impacts AMPK activity and downstream metabolic processes. Several methodological considerations include:

• Tissue-specific expression systems: Since S491 phosphorylation effects may vary by tissue type, experimental design should account for tissue-specific contexts. For example, skeletal muscle-specific expression of phospho-mimetic mutant AMPKα2 S491D worsens glucose tolerance, while phospho-deficient mutant AMPKα2 S491A improves it .

• Temporal dynamics: Monitoring the temporal relationship between S491 phosphorylation and other AMPK regulatory events (particularly T172 phosphorylation) is crucial, as S491 phosphorylation can inhibit T172 phosphorylation.

• Upstream kinase modulation: Experiments that modulate upstream kinases (PAK4, PKA, p70S6K) should include assessment of S491 phosphorylation as a mechanistic readout.

• Downstream readouts: Include measurement of key downstream AMPK targets such as:

  • Acetyl-CoA Carboxylase (ACC) phosphorylation

  • TBC1D1 and TBC1D4 phosphorylation (GLUT4 trafficking regulators)

  • Raptor phosphorylation (mTORC1 regulation)

  • ULK1 phosphorylation (autophagy regulation)

  • p38 MAPK pathway activation

• Physiological assessments: Include glucose tolerance tests (GTT) and insulin tolerance tests (ITT) to link molecular findings to whole-organism metabolism .

Recent studies have demonstrated that PAK4 knockout mice showed improved insulin sensitivity, accompanied by AMPK activation and GLUT4 upregulation. This phenotype was also replicated through PAK4 inhibitor treatment, indicating that targeting the PAK4-AMPKα2-S491 axis may represent a therapeutic approach for type 2 diabetes .

What controls and validation steps are essential when using Phospho-PRKAA2 (S491) antibody in research applications?

When utilizing the Phospho-PRKAA2 (S491) antibody, implementing rigorous controls and validation steps is critical for ensuring reliable and reproducible results:

• Phosphatase treatment controls:

  • Split your sample and treat one portion with lambda phosphatase to confirm antibody phospho-specificity

  • The signal should significantly decrease or disappear in phosphatase-treated samples

• Genetic controls:

  • Include PRKAA2 knockout/knockdown samples to verify antibody specificity

  • Use phospho-mimetic (S491D) and phospho-deficient (S491A) mutants as positive and negative controls

• Kinase modulation:

  • Treat cells with activators or inhibitors of upstream kinases (PAK4, PKA, p70S6K) to manipulate S491 phosphorylation status

  • PAK4 inhibitor treatment should reduce S491 phosphorylation signal

• Cross-reactivity assessment:

  • Test the antibody against the homologous phospho-site in PRKAA1 (S485) to determine isoform specificity

  • This is particularly important as many antibodies cross-react with both α1-pS485 and α2-pS491

• Correlative validation:

  • Compare phosphorylation at S491 with functional readouts of AMPK activity, such as reduced T172 phosphorylation and decreased phosphorylation of downstream targets

• Loading controls:

  • Always include total PRKAA2 antibody detection on the same or parallel blots

  • Calculate the phospho-to-total ratio for accurate quantification

• AMPK activity assays:

  • Correlate S491 phosphorylation status with direct measurements of AMPK activity using kinase assays

  • This confirms the functional significance of observed phosphorylation changes

Recent studies have validated that increased S491 phosphorylation correlates with decreased T172 phosphorylation and reduced phosphorylation of AMPK substrates including ACC, Raptor, TBC1D1, ULK1, and p38 MAPK, confirming the inhibitory effect of S491 phosphorylation on AMPK activity .

How can researchers optimize Western blot protocols specifically for detection of PRKAA2 S491 phosphorylation?

Optimizing Western blot protocols for detecting PRKAA2 S491 phosphorylation requires specific considerations to enhance sensitivity and specificity:

• Sample preparation:

  • Use phosphatase inhibitors (sodium fluoride, sodium pyrophosphate, sodium orthovanadate) in lysis buffers

  • Process samples rapidly at 4°C to prevent dephosphorylation

  • Consider using phospho-protein enrichment methods for low-abundance targets

• Gel electrophoresis:

  • Use lower percentage (7.5-10%) acrylamide gels for better resolution of PRKAA2 (~63 kDa)

  • Phostag™ acrylamide gels can provide enhanced separation of phosphorylated vs. non-phosphorylated forms

• Transfer conditions:

  • Optimize transfer time and voltage based on protein size

  • Use PVDF membranes for stronger protein binding and potential stripping/reprobing

• Blocking optimization:

  • Test both BSA and milk-based blocking buffers (note: milk contains phosphatases and may reduce signal)

  • Many phospho-specific antibodies perform better with BSA blocking

• Antibody dilution and incubation:

  • Begin with manufacturer's recommended dilution (1:500-1:5000)

  • Extended incubation periods (overnight at 4°C) may improve signal quality

  • Consider adding 5% BSA to antibody dilution buffer

• Detection system:

  • Enhanced chemiluminescence (ECL) systems with high sensitivity are recommended

  • Avoid overexposure as this can mask differences in phosphorylation levels

• Quantification approach:

  • Always normalize phospho-PRKAA2 (S491) to total PRKAA2

  • Consider using fluorescence-based detection systems for improved quantitative accuracy

• Validation controls:

  • Include phosphatase-treated samples and PAK4-inhibited samples as controls

Studies examining PAK4-mediated regulation of AMPK have successfully employed these optimization strategies to detect subtle changes in S491 phosphorylation status under various treatment conditions .

What are the common pitfalls in interpreting PRKAA2 S491 phosphorylation data, and how can they be avoided?

Interpreting PRKAA2 S491 phosphorylation data presents several challenges that researchers should be aware of:

• Antibody cross-reactivity issues:

  • Many phospho-antibodies cross-react with both α1-pS485 and α2-pS491 due to sequence similarity

  • Solution: Use isoform-specific antibodies or validate with genetic approaches (α1 vs α2 knockouts)

• Temporal dynamics misinterpretation:

  • S491 phosphorylation may show different kinetics than T172 phosphorylation

  • Solution: Perform detailed time-course experiments to capture dynamic relationships

• Tissue-specific variations:

  • Effects of S491 phosphorylation vary between tissues (skeletal muscle vs. adipose vs. liver)

  • Solution: Avoid generalizing findings across tissues without validation

• Upstream kinase ambiguity:

  • Multiple kinases can phosphorylate S491, leading to incorrect pathway attribution

  • Solution: Use specific kinase inhibitors or genetic approaches to confirm the responsible kinase

• Functional significance attribution:

  • Changes in S491 phosphorylation don't always correlate with proportional changes in AMPK activity

  • Solution: Always measure downstream AMPK targets (ACC, Raptor, etc.) alongside S491 phosphorylation

• Quantification limitations:

  • Relying solely on phospho-to-total ratios without considering absolute expression levels

  • Solution: Assess total AMPK levels alongside phosphorylation status

• Contextual influences:

  • Energy status, culture conditions, and cell confluency can all affect baseline phosphorylation

  • Solution: Standardize experimental conditions and include appropriate controls

• Pathophysiological context:

  • Disease states may alter regulatory mechanisms affecting S491 phosphorylation

  • Solution: Include disease-relevant models alongside normal controls

Recent research demonstrates that interpreting S491 phosphorylation data requires consideration of the complete signaling context. For example, studies on metabolic disorders have shown that while S491 phosphorylation inhibits AMPK, the functional consequences depend on the tissue type and metabolic state .

How can researchers integrate phospho-PRKAA2 (S491) antibody data with other methodologies to gain comprehensive insights into AMPK regulation?

Integrating phospho-PRKAA2 (S491) antibody data with complementary methodologies creates a more comprehensive understanding of AMPK regulation:

Research examining the PAK4-AMPK axis in insulin resistance exemplifies this integrated approach by linking molecular events (S491 phosphorylation) to metabolic outcomes (glucose tolerance) through a combination of biochemical, genetic, and physiological methodologies .

How is PRKAA2 S491 phosphorylation implicated in the development of metabolic disorders and potential therapeutic strategies?

PRKAA2 S491 phosphorylation has emerged as a critical regulatory mechanism in metabolic disorders, particularly insulin resistance and type 2 diabetes:

• Mechanistic role in insulin resistance:

  • S491 phosphorylation inhibits AMPK activity by reducing T172 phosphorylation

  • Inhibited AMPK leads to decreased glucose uptake in skeletal muscle

  • This contributes to systemic insulin resistance and glucose intolerance

• Upstream regulatory kinases as therapeutic targets:

  • PAK4 has been recently identified as a direct kinase for S491 phosphorylation

  • PAK4 knockout or inhibition in diet-induced obese mice preserves insulin sensitivity

  • This is accompanied by increased AMPK activation and GLUT4 upregulation

  • PAK4 inhibitors represent a potential therapeutic avenue for type 2 diabetes

• Genetic evidence from animal models:

  • Skeletal muscle-specific expression of phospho-mimetic mutant AMPKα2 S491D worsens glucose tolerance

  • Conversely, phospho-inactive mutant AMPKα2 S491A improves glucose tolerance

  • These findings establish a causal link between S491 phosphorylation and metabolic dysfunction

• Molecular pathways affected:

  • S491 phosphorylation decreases the activation of downstream AMPK targets:

    • ACC (regulating fatty acid metabolism)

    • Raptor (controlling protein synthesis)

    • TBC1D1 and TBC1D4 (regulating GLUT4 trafficking)

    • ULK1 (mediating autophagy)

    • p38 MAPK (stress response signaling)

• Tissue-specific considerations:

  • The metabolic impact of S491 phosphorylation is particularly pronounced in skeletal muscle

  • This tissue-specific effect makes targeted therapeutic approaches feasible

• Potential intervention strategies:

  • Small molecule inhibitors of PAK4 or other S491-targeting kinases

  • Peptide-based approaches that might block the S491 phosphorylation site

  • Combined approaches targeting both S491 dephosphorylation and T172 phosphorylation

The therapeutic potential of targeting this pathway is supported by research showing that PAK4 inhibition promotes insulin sensitivity through AMPK activation, establishing PAK4 as a promising target for type 2 diabetes treatment .

What role does PRKAA2 S491 phosphorylation play in regulating AMPK heterotrimer composition and subcellular localization?

PRKAA2 S491 phosphorylation influences AMPK heterotrimer composition and subcellular localization through several mechanisms:

• Impact on heterotrimer stability and assembly:

  • Phosphorylation at S491 may alter α2 subunit conformation, affecting its interaction with β and γ subunits

  • Recent phosphosite profiling indicates differential S491 phosphorylation levels across various AMPK heterotrimeric complexes

  • For example, γ3-containing complexes show distinct phosphorylation patterns compared to γ1 or γ2 complexes

• Subcellular localization effects:

  • S491 phosphorylation may influence AMPK trafficking between cellular compartments

  • This affects access to substrates in different subcellular locations

  • For instance, nuclear vs. cytoplasmic distribution of AMPK impacts gene expression regulation versus metabolic enzyme control

• Interplay with γ-subunit regulation:

  • Recent research has shown that γ-subunit N-terminal extensions (NTEs) may protect α-T172 from dephosphorylation

  • S491 phosphorylation could potentially modulate this protective effect

  • γ2-containing complexes show higher α-T172 phosphorylation, suggesting interplay between different regulatory phosphosites

• Isoform-specific consequences:

  • While α1-S485 and α2-S491 are homologous sites, they may have distinct effects on heterotrimer composition

  • Different tissue distribution of α1 vs. α2 creates tissue-specific regulation patterns

  • Skeletal muscle predominantly expresses α2, making S491 phosphorylation particularly important in this tissue

• Methodological approaches to study compositional effects:

  • Co-immunoprecipitation experiments with phospho-specific antibodies

  • Blue native PAGE to analyze intact AMPK complexes

  • Proximity ligation assays to detect interactions between specific subunit isoforms

  • Fluorescence microscopy to track subcellular localization

Mass spectrometry studies have shown that phosphorylation patterns vary significantly across different AMPK heterotrimeric complexes, suggesting that S491 phosphorylation may play a role in determining the functional specificity of particular AMPK complexes in different cellular contexts .