Phospho-PRKAA1/PRKAA2 (Ser487) Antibody

What is AMPK and what are its primary physiological functions?

AMPK (AMP-activated protein kinase) is a highly conserved serine/threonine kinase that functions as a cellular energy sensor expressed in virtually all eukaryotic cells, from protists to humans. It exists as a heterotrimeric complex consisting of a catalytic α subunit and regulatory β and γ subunits . AMPK's primary functions include:

• Regulation of fatty acid synthesis through phosphorylation of acetyl-CoA carboxylase (ACC)

• Regulation of cholesterol synthesis via phosphorylation and inactivation of hormone-sensitive lipase and hydroxymethylglutaryl-CoA reductase

• Acting as a metabolic stress-sensing protein kinase that switches off biosynthetic pathways when cellular ATP levels are depleted

• Responding to increased 5'-AMP levels during fuel limitation and/or hypoxia

AMPK stimulates glucose uptake in muscle by increasing the translocation of the glucose transporter SLC2A4/GLUT4 to the plasma membrane. It also regulates transcription and chromatin structure by phosphorylating various transcription regulators involved in energy metabolism .

What is the significance of phosphorylation at Ser487 in PRKAA1 and Ser491 in PRKAA2?

Phosphorylation at Ser487 in AMPK-α1 (PRKAA1) and the equivalent Ser491 in AMPK-α2 (PRKAA2) represents a critical regulatory mechanism:

• This phosphorylation inhibits subsequent phosphorylation at Thr172, which is essential for AMPK activation by upstream kinases like LKB1

• It serves as a negative regulatory mechanism, particularly in cancer contexts where AMPK may function as a tumor suppressor

• Specifically, phosphorylation of AMPK-α1 at Ser487 by Akt inhibits its subsequent phosphorylation at Thr172 and activation by LKB1

• This represents a significant cross-talk mechanism between the PI3K/Akt and AMPK signaling pathways

Understanding this phosphorylation site is essential for investigating the complex interplay between cellular energy sensing and growth factor signaling networks.

What sample preparation methods optimize detection of phosphorylated AMPK?

Proper sample preparation is crucial for detecting phosphorylated AMPK:

• Harvest cells or tissues rapidly to minimize postmortem changes in phosphorylation state

• Use lysis buffers containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, and sodium pyrophosphate)

• Maintain cold temperatures throughout sample preparation

• For tissue samples, snap-freeze in liquid nitrogen immediately after collection

• Process tissues in buffer containing 1% SDS and quickly heat to 95°C to inactivate phosphatases

• When appropriate, treat positive control samples with phosphatase inhibitors and negative controls with lambda phosphatase

The quality of phospho-specific detection is highly dependent on sample handling and preparation techniques that preserve the native phosphorylation state.

What are the recommended applications and protocols for Phospho-PRKAA1/PRKAA2 (Ser487) Antibody?

Based on vendor specifications and research protocols, the following applications are recommended:

Protocol recommendations:

• For Western blotting: Block with 5% BSA in TBST, incubate with primary antibody overnight at 4°C

• For IHC: Use antigen retrieval methods (typically citrate buffer pH 6.0), detect with appropriate visualization systems

• Always include positive controls (e.g., tissues or cells with known phosphorylation of AMPK at Ser487/491)

• Include negative controls (untreated samples or samples where phosphorylation is expected to be absent)

How can researchers validate the specificity of Phospho-PRKAA1/PRKAA2 (Ser487) Antibody?

Validating antibody specificity is essential for reliable research results:

• Positive and negative controls:

  • Use cell lines with known AMPK activation states

  • Include phosphatase-treated samples as negative controls

  • Use cells treated with AMPK activators or inhibitors

• Peptide competition assays:

  • Pre-incubate antibody with phospho-peptide used as immunogen

  • Signal should be blocked by phospho-peptide but not by non-phosphorylated peptide

• Genetic validation:

  • Use CRISPR/Cas9 knockout models lacking PRKAA1/PRKAA2

  • Utilize cell lines where Ser487 has been mutated to alanine

• Cross-validation:

  • Compare results with alternative antibodies targeting the same phosphorylation site

  • Confirm with mass spectrometry analysis of immunoprecipitated AMPK

Commercial antibodies are typically validated using affinity-chromatography with epitope-specific phosphopeptides, with non-phospho specific antibodies removed by chromatography using non-phosphopeptide .

How do different AMPK isoform combinations affect experimental design and interpretation?

The complexity of AMPK isoforms presents significant considerations for experimental design:

• In mammals, there are two α subunits (α1, α2), two β subunits (β1, β2), and three γ subunits (γ1, γ2, γ3) encoded by separate genes (PRKAA1/A2, PRKAB1/B2, and PRKAG1/G2/G3), potentially forming up to 12 different heterotrimeric combinations

• These isoform combinations may have different:

  • Subcellular locations

  • Regulatory inputs

  • Substrate preferences

  • Tissue-specific distributions

Experimental design implications:

• Identify which isoforms are predominantly expressed in your experimental system

• Consider using isoform-specific antibodies when examining specific complexes

• Recognize that compensatory upregulation of one isoform upon knockout of another may not fully restore AMPK function

• Appreciate that different tissues exhibit unique patterns of AMPK complex formation that cannot be fully reconstituted when altered

Studies have shown that "even when compensatory increase in expression of one subunit occurs due to knockout of another subunit, AMPK complex formation and activity still remains uncompensated" .

What is the evolutionary significance of AMPK isoform diversity?

The evolutionary history of AMPK subunits provides insight into their functional specialization:

• The multiple isoforms of each subunit in vertebrates are 2R-ohnologues generated by two rounds of whole genome duplication at the evolutionary origin of vertebrates

• Evolutionary analysis reveals that the β subunit evolved 1.65 times faster than the α subunit (mutation rate of 1.8183 vs. 1.1024 substitutions per site)

• Drosophila melanogaster contains single genes encoding each subunit, whereas mammals have multiple isoforms encoded by distinct genes

This evolutionary divergence suggests functional specialization of different isoforms, which may be particularly relevant in complex tissues and in pathological conditions like cancer, where certain isoform genes (e.g., PRKAA1 and PRKAB2) are frequently amplified while others (e.g., PRKAA2) may be downregulated .

What are the primary mechanisms of AMPK activation and inhibition?

AMPK regulation occurs through multiple mechanisms:

Activation mechanisms:

• Nucleotide binding to γ subunit:

  • AMP/ADP binding to the γ subunit activates AMPK through three mechanisms:
    a) Allosteric activation
    b) Promotion of Thr172 phosphorylation by upstream kinases (e.g., LKB1)
    c) Inhibition of Thr172 dephosphorylation by protein phosphatases

  • Site 3 on the γ subunit appears critical for mechanisms (a) and (c)

• Upstream kinases:

  • LKB1 (Liver kinase B1/STK11)

  • CaMKKβ (Calcium/calmodulin-dependent kinase kinase β)

  • TAK1 (TGFβ-activated kinase)

Inhibition mechanisms:

• Phosphorylation at inhibitory sites:

  • Phosphorylation of AMPK-α1 at Ser487 by Akt inhibits subsequent phosphorylation at Thr172

  • This represents cross-talk between growth factor signaling and energy sensing

• Degradation mechanisms:

  • Polyubiquitylation of AMPK-α1 by the E3 ligase TRIM28 (targeted by MAGE-A3/A6)

  • This degradation pathway is particularly relevant in cancer contexts

• High ATP:AMP ratio:

  • High cellular energy status (high ATP, low AMP) prevents AMPK activation

How does the structural conformation of AMPK change upon activation?

AMPK undergoes significant conformational changes upon activation:

• The γ subunit contains three AMP-binding sites (sites 1, 3, and 4; site 2 is non-functional)

• In active human heterotrimers, α-RIM1 interacts with the surface of CBS2 close to the unoccupied site 2, whereas α-RIM2 interacts with the surface of CBS3 via residues that also interact with AMP bound in site 3

• ATP binding causes conformational changes that disrupt these interactions, promoting the release of the α-linker from the γ subunit

• This partial separation of the catalytic and nucleotide-binding modules may make Thr172 more accessible to protein phosphatases, explaining how ATP binding relieves the protective effect of AMP on Thr172 dephosphorylation

These structural insights are crucial for understanding AMPK regulation and for developing targeted therapeutic approaches.

What is the role of AMPK in cancer progression and therapeutic potential?

AMPK plays complex, context-dependent roles in cancer:

• Tumor suppressor functions:

  • Inhibits cell growth and proliferation in normal cells

  • Activates p53 and other tumor suppressors

  • Phosphorylation of AMPK-α1 at Ser487 by Akt inhibits AMPK activation, representing a mechanism of AMPK down-regulation in cancers

  • Loss of LKB1 (a major AMPK activator) occurs in multiple cancer types

• Pro-tumorigenic functions:

  • Complete loss of AMPK function may limit viability of solid tumors by reducing tolerance to stresses such as hypoxia, glucose deprivation, or oxidative stress

  • AMPK activation can provide metabolic adaptations that allow cancer cells to survive nutrient limitation

• Isoform-specific considerations:

  • Genes encoding some isoforms like PRKAA1 (α1) and PRKAB2 (β2) are frequently amplified in tumor cells

  • Genes encoding other isoforms like PRKAA2 (α2) may be downregulated in certain cancers

This complexity suggests that therapeutic approaches targeting AMPK must be carefully tailored to specific cancer contexts and genetic backgrounds.

How does AMPK signaling intersect with other metabolic and stress-response pathways?

AMPK functions within a complex network of interacting signaling pathways:

• mTOR pathway:

  • AMPK inhibits mTORC1 through phosphorylation of TSC2 and Raptor

  • This coordinates energy status with protein synthesis and cell growth

• Insulin/IGF-1 signaling:

  • Akt phosphorylates AMPK-α1 at Ser487, inhibiting AMPK activation

  • This creates reciprocal regulation between growth factor signaling and energy sensing

• Cell cycle regulation:

  • AMPK phosphorylates the retinoblastoma (RB) protein to allow cell cycle progression in neural progenitor cells

  • High levels of phosphorylated AMPK α2 have been found during mitosis, maintained even at high ATP levels

• Xenobiotic sensing:

  • AMPK is necessary for xenobiotics-induced transcription

  • This may represent an evolutionarily conserved function independent of energy sensing

• Autophagy:

  • AMPK promotes autophagy through direct phosphorylation of ULK1 and indirectly through inhibition of mTORC1

These interactions highlight the importance of considering pathway cross-talk when designing experiments and interpreting results related to AMPK phosphorylation and function.

How can researchers effectively study temporal dynamics of AMPK phosphorylation at different sites?

Studying the temporal dynamics of AMPK phosphorylation requires specific methodological approaches:

• Time-course experiments:

  • Design with appropriate intervals based on expected kinetics (seconds to hours)

  • Include both early time points (seconds to minutes) and later ones (hours to days)

  • Synchronize cells when studying cell-cycle-dependent changes

• Multi-site phosphorylation analysis:

  • Use antibodies against different phosphorylation sites simultaneously:

    • pThr172 (activation site)

    • pSer487/491 (inhibitory sites)

    • Other functional phosphorylation sites

  • Consider using phospho-specific protein arrays for broader coverage

• Technical approaches:

  • Use rapid cell lysis techniques to capture transient phosphorylation events

  • Consider in situ approaches (e.g., proximity ligation assays) to detect phosphorylation events in intact cells

  • Employ phospho-proteomic mass spectrometry for unbiased analysis

  • Develop FRET-based sensors for real-time monitoring in living cells

• Quantification and normalization:

  • Normalize phospho-signals to total AMPK protein

  • Use phosphorylation-independent loading controls

  • Consider using phosphorylation site-specific standards for absolute quantification

What strategies can overcome common challenges in detecting phosphorylated AMPK?

Researchers face several challenges when detecting phosphorylated AMPK:

Additionally, when working with tissue samples where cell type heterogeneity may complicate interpretation, consider:

• Laser capture microdissection to isolate specific cell populations

• Single-cell phospho-flow cytometry

• Spatial approaches like phospho-specific immunohistochemistry

What are the optimal storage and handling procedures for maintaining antibody integrity?

Proper storage and handling are critical for maintaining antibody functionality and specificity:

• Upon receipt, store antibodies at -20°C or -80°C for long-term storage

• For short-term storage (up to 6 months), antibodies can be stored at 4°C

• Avoid repeated freeze-thaw cycles, which can lead to antibody degradation and loss of activity

• Many phospho-specific antibodies are supplied in glycerol (typically 50%), which prevents freezing at -20°C and reduces damage from freeze-thaw cycles

• Aliquot antibodies upon first thaw to minimize freeze-thaw cycles

• Typical shelf life is approximately 12 months when stored properly

Specific storage recommendations from commercial sources include:

• "Store the antibody at 4°C, stable for 6 months. For long-term storage, store at -20°C. Avoid repeated freeze and thaw cycles."

• "Upon receipt, store at -20°C or -80°C. Avoid repeated freeze."

What controls are essential when using Phospho-PRKAA1/PRKAA2 (Ser487) Antibody in different experimental contexts?

Appropriate controls are essential for meaningful interpretation of results:

Western Blotting:

• Positive control: Cells treated with activators of pathways known to phosphorylate AMPK at Ser487 (e.g., insulin for Akt activation)

• Negative control: Cells treated with phosphatase or cells where AMPK phosphorylation at Ser487 is minimized

• Loading control: Total AMPK antibody or housekeeping protein

• Molecular weight marker: To confirm the expected ~62-63 kDa size

Immunohistochemistry:

• Positive control tissue: Tissues known to express phosphorylated AMPK (e.g., breast carcinoma tissue has been validated)

• Negative control: Omission of primary antibody or use of isotype control

• Blocking peptide control: Pre-incubation of antibody with immunizing phosphopeptide

Genetic controls:

• CRISPR/Cas9 knockout of PRKAA1/PRKAA2

• Site-directed mutagenesis (S487A) to prevent phosphorylation

• siRNA knockdown of AMPK to confirm specificity