Phospho-ACLY (S455) Antibody

What is ACLY and why is its phosphorylation at S455 significant?

ATP-citrate lyase (ACLY) is a key metabolic enzyme that catalyzes the conversion of citrate to acetyl-CoA, providing the primary substrate for fatty acid synthesis and histone acetylation. S455 phosphorylation has been implicated in regulating ACLY activity, with kinases such as Akt and protein kinase A (PKA) mediating this post-translational modification . This phosphorylation site has traditionally been considered a mechanism to enhance ACLY enzymatic activity, though recent research has challenged this notion in some cellular contexts .

What are the validated applications for Phospho-ACLY (S455) antibodies?

Phospho-ACLY (S455) antibodies have been validated for several research applications:

• Western blotting (WB): Used to detect phosphorylated ACLY in cell and tissue lysates

• Immunohistochemistry-paraffin (IHC-P): For detection in fixed tissue sections

• Immunoprecipitation (IP): For isolating phosphorylated ACLY from complex samples

• ELISA: For quantitative measurement of phospho-ACLY levels

The recommended dilutions vary by application: WB (1:100-1:500), IHC-P (1:50-1:100), and IP (0.5μg-4μg antibody for 200μg-400μg extracts) .

What cell types and tissues show detectable phospho-ACLY (S455)?

Phospho-ACLY (S455) has been detected in multiple cell types and tissues including:

• Human cancer cell lines (HeLa)

• Mouse cell lines (C6, NIH/3T3)

• Rat tissues

• Monocytic cells (THP-1)

• Vascular smooth muscle cells (PASMCs, CoASMCs)

• Brown adipocytes

• Various human tissues

Notably, expression levels vary significantly between cell types and are often elevated in cancer cells and during cellular proliferation.

How should I address the conflicting data regarding S455 phosphorylation's impact on ACLY activity?

Recent research has yielded contradictory findings regarding whether S455 phosphorylation directly affects ACLY enzymatic activity:

To address this conflict:

• Perform parallel experiments with phospho-mimetic (S455D) and phospho-deficient (S455A) mutants alongside wild-type ACLY

• Include both enzymatic activity assays and functional readouts (histone acetylation, lipid synthesis)

• Consider cell-type specific factors that might influence the impact of phosphorylation

• Assess subcellular localization, as nuclear versus cytoplasmic ACLY may be differently regulated

What controls are essential when using phospho-ACLY (S455) antibodies in experimental designs?

For rigorous experimental design:

• Specificity controls:

  • Use ACLY knockout cells as negative controls

  • Include phosphatase treatment of samples

  • Compare with total ACLY antibody signals

  • Test with recombinant wild-type and S455A mutant proteins

• Treatment conditions:

  • Include Akt inhibitors (e.g., MK2206) to reduce phosphorylation

  • Add growth factors or insulin to stimulate phosphorylation

  • Compare serum-starved versus stimulated conditions

• Cell-specific validation:

  • Verify antibody performance in your specific cell type

  • Consider tissue-specific expression patterns

  • Compare with other known Akt substrates (e.g., PRAS40) as positive controls

How can I optimize detection of nuclear phospho-ACLY for DNA damage response studies?

For nuclear phospho-ACLY detection in DNA damage research:

• Use subcellular fractionation protocols optimized for nuclear proteins

• Include ATM inhibitors as controls, as ATM is upstream of ACLY phosphorylation after DNA damage

• Employ cell-cycle synchronization, as ACLY nuclear localization is highest during S and G2 phases

• For immunofluorescence:

  • Use confocal microscopy with Z-stacking

  • Co-stain with DNA damage markers (γH2AX, 53BP1)

  • Compare staining patterns before and after ionizing radiation

  • Include BRCA1 co-staining to assess functional correlations

How does ACLY S455 phosphorylation functionally impact epigenetic regulation through histone acetylation?

ACLY provides acetyl-CoA for histone acetylation, linking metabolism with gene expression. Research findings on S455 phosphorylation's role:

• In brown preadipocytes, mTORC2-dependent ACLY S455 phosphorylation increases acetyl-CoA levels and histone acetylation, promoting adipogenic gene expression

• In monocytic THP-1 cells, phosphorylation-deficient S455A mutants showed similar histone acetylation patterns to wild-type ACLY, suggesting context-dependent roles

• After DNA damage, nuclear ACLY phosphorylation at S455 facilitates histone acetylation near double-strand breaks, influencing DNA repair pathway choice by promoting BRCA1 recruitment

For experimental assessment:

• Measure global histone acetylation (H3K9ac, H3K27ac)

• Perform ChIP-seq with acetylation marks at specific loci

• Compare histone acetylation in cells expressing wild-type versus S455A ACLY

What is the relationship between ACLY S455 phosphorylation and metabolic disease models?

ACLY S455 phosphorylation has been implicated in several metabolic disease contexts:

• Vascular remodeling diseases:

  • Increased pACLY and ACLY expression in pulmonary arterial hypertension (PAH) tissues

  • Elevated in coronary artery disease (CAD) models

  • ACLY genetic deletion in smooth muscle cells attenuated PAH severity in mouse models

• Cancer metabolism:

  • Alternative splicing of ACLY correlates with cancer phenotypes

  • ACLY phosphorylation may support lipid synthesis in cancer cells

  • No clear functional differences between ACLY isoforms were detected despite clinical correlations

• Inflammatory responses:

  • In THP-1 monocytic cells, ACLY catalytic activity affected pro-inflammatory gene expression

  • S455A mutation did not significantly alter inflammatory responses compared to wild-type ACLY

For studying metabolic disease contexts:

• Consider tissue-specific ACLY knockout models

• Compare pharmacological inhibition versus genetic approaches

• Investigate metabolic flux with isotope tracing (e.g., [U-13C]glucose)

How does ACLY S455 phosphorylation impact T cell differentiation and autoimmunity?

Research has revealed specific roles for ACLY in T cell biology:

• ACLY inhibition enhanced inducible regulatory T (iTreg) cell differentiation

• This effect occurred through:

  • Reduced de novo fatty acid synthesis

  • Increased fatty acid oxidation

  • Altered carnitine palmitoyltransferase 1 (CPT1) activity

• Mechanistically, ACLY inhibition leads to decreased malonyl-CoA levels, relieving CPT1 inhibition and promoting fatty acid oxidation

For experimental approaches:

• Assess how S455 phosphorylation affects these metabolic shifts

• Compare pharmacological inhibition (SB204990) with S455A mutation

• Measure fatty acid synthesis using [U-13C]glucose tracing

• Analyze oxygen consumption rate (OCR) to assess fatty acid oxidation

• Determine the impact on iTreg differentiation through flow cytometry for CD4+CD25+Foxp3+ cells

What are the optimal conditions for detecting phospho-ACLY (S455) in western blotting?

For optimal western blot detection:

• Sample preparation:

  • Use phosphatase inhibitors (sodium fluoride, sodium orthovanadate)

  • Harvest cells rapidly to prevent dephosphorylation

  • Include serum or insulin-stimulated samples as positive controls

• Protocol optimization:

  • Transfer proteins to PVDF membranes (preferred over nitrocellulose)

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Incubate with primary antibody overnight at 4°C

  • Use the recommended dilution (1:100-1:500)

  • Consider enhanced chemiluminescence detection for sensitivity

• Expected results:

  • Observed molecular weight: 125 kDa (calculated: 121 kDa)

  • Always run total ACLY detection in parallel

  • Compare with other Akt substrates (e.g., pPRAS40) as controls

How can I distinguish between mTORC2-dependent and mTORC2-independent ACLY S455 phosphorylation?

This distinction requires careful experimental design:

• Genetic approaches:

  • Use RICTOR knockout cells (RICTOR is an essential mTORC2 component)

  • Reconstitute with wild-type RICTOR to confirm recovery of phosphorylation

  • Compare with AKT inhibitor treatment to distinguish direct mTORC2 effects

• Pathway analysis:

  • Monitor phosphorylation of AKT at S473 (direct mTORC2 target)

  • Compare ACLY S455 with other AKT substrates (pPRAS40 T246 is less mTORC2-dependent)

  • Use phospho-mimetic AKT (S473D) expression to test rescue of ACLY phosphorylation

• Nutritional conditions:

  • Test both serum-stimulated and serum-deprived conditions

  • Compare insulin and growth factor stimulation patterns

  • Analyze timing of phosphorylation events with detailed time courses

How can ACLY S455 phosphorylation be quantitatively analyzed in clinical samples?

For clinical sample analysis:

• Tissue preparation:

  • Use phosphatase inhibitors during tissue collection and processing

  • Prepare parallel samples for total ACLY detection

  • Consider laser capture microdissection for specific cell populations

• Quantification methods:

  • Multiplex immunohistochemistry to correlate with other markers

  • Develop targeted mass spectrometry assays for absolute quantification

  • Use phospho-specific ELISA for high-throughput screening

• Analysis considerations:

  • Always normalize phospho-ACLY to total ACLY levels

  • Compare with known Akt pathway activation markers

  • Correlate with clinical parameters and outcomes

  • Consider cell-type heterogeneity within samples

How do nuclear and cytoplasmic pools of phospho-ACLY (S455) differ in function?

Recent research has revealed distinct roles for nuclear ACLY:

• Nuclear functions:

  • Facilitation of histone acetylation near DNA damage sites

  • Promotion of BRCA1 recruitment for homologous recombination

  • Potential cell cycle-dependent regulation (highest in S and G2 phases)

• Cytoplasmic functions:

  • Support of de novo lipid synthesis

  • Contribution to general histone acetylation through cytosolic acetyl-CoA production

• Experimental approaches:

  • Use nuclear export inhibitors (leptomycin B) to trap ACLY in the nucleus

  • Create nuclear localization or export signal-tagged ACLY variants

  • Perform chromatin immunoprecipitation to identify genomic binding sites

  • Combine with proximity labeling techniques to identify compartment-specific interactors

What are the emerging roles of ACLY S455 phosphorylation in disease beyond metabolism?

Research has uncovered non-metabolic functions of phospho-ACLY:

• DNA damage response:

  • Nuclear ACLY is phosphorylated at S455 following DNA damage

  • This phosphorylation occurs downstream of ATM and AKT

  • It promotes homologous recombination by enabling BRCA1 recruitment

  • ACLY deficiency sensitizes cells to PARP inhibition

• Vascular remodeling:

  • Increased expression of phospho-ACLY in pulmonary arterial hypertension

  • Genetic deletion of ACLY in smooth muscle cells attenuates disease

  • Associated with reduced medial wall thickness and PASMC proliferation

• Alternative splicing:

  • ACLY splicing patterns correlate with cancer phenotypes

  • Regulated by epithelial splicing regulatory protein 1 (ESRP1)

  • Associated with specific immune signatures in tumors

How can multi-omics approaches advance our understanding of ACLY S455 phosphorylation?

Integrative multi-omics strategies offer powerful insights:

• Proteomics approaches:

  • Quantitative phosphoproteomics to identify co-regulated phosphosites

  • Interactome analysis to identify phosphorylation-dependent binding partners

  • Spatial proteomics to determine subcellular distribution changes

• Metabolomics integration:

  • Stable isotope tracing to quantify acetyl-CoA flux

  • Lipidomics to assess impact on specific lipid species

  • Acyl-CoA profiling to determine broader metabolic consequences

• Epigenomics correlation:

  • ChIP-seq for histone acetylation marks

  • ATAC-seq to correlate with chromatin accessibility

  • Integration with transcriptomics to link to gene expression changes

  • Single-cell multi-omics to address cellular heterogeneity