Phospho-DDIT3 (S30) Antibody

What is DDIT3 and how does phosphorylation at S30 affect its function?

DDIT3 (DNA Damage Inducible Transcript 3), also known as CHOP (C/EBP Homologous Protein), is a multifunctional transcription factor primarily involved in endoplasmic reticulum stress response. Phosphorylation at serine 30 (S30) is mediated by AMPKα1 kinase and significantly affects CHOP protein stability and transcriptional activity. Research has demonstrated that this specific phosphorylation enhances CHOP's ability to promote cell cycle arrest and apoptosis under stress conditions.

The phosphorylation site at S30 lies within the amino acid range 15-64 of human CHOP protein . Computer alignment studies have identified this site as an optimal AMPKα1 substrate motif, and co-immunoprecipitation experiments confirm that AMPKα1 physically interacts with CHOP in macrophages . When investigating this modification, phospho-specific antibodies in Western blot assays with recommended dilutions of 1:500-1:2000 can be used to detect this specific modification .

What are the primary applications of Phospho-DDIT3 (S30) Antibody in research?

Phospho-DDIT3 (S30) Antibody has several key applications in research:

Methodologically, sample preparation is critical - phosphatase inhibitors must be included in lysis buffers to preserve the phosphorylation state during protein extraction . The antibody specifically detects endogenous levels of CHOP protein only when phosphorylated at S30, allowing researchers to distinguish between the phosphorylated and non-phosphorylated forms of the protein .

For optimal experimental results, control experiments should be designed with both the phospho-specific antibody and an antibody against total DDIT3/CHOP to calculate the phosphorylation ratio as a measure of activation status .

How should Phospho-DDIT3 (S30) Antibody be stored and handled?

For optimal performance and stability of Phospho-DDIT3 (S30) Antibody:

• Storage temperature: -20°C for long-term or 4°C for short-term (1-2 weeks)

• Avoid repeated freeze-thaw cycles (aliquot upon receipt)

• Protect from light exposure, especially fluorophore-conjugated versions

• Working dilutions should be prepared fresh before use

• When diluting, use buffers containing 0.1% BSA or 5% normal serum as carriers

• For phospho-specific antibodies, all buffers should contain phosphatase inhibitors

The antibody is typically supplied as a liquid formulation in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide . The affinity-purified antibody is derived from rabbit antiserum through affinity-chromatography using epitope-specific immunogen . Adherence to these handling protocols ensures consistent experimental results and extends the antibody's shelf life.

How does AMPKα1-mediated phosphorylation of CHOP at Ser30 regulate its protein stability?

AMPKα1-mediated phosphorylation of CHOP at Ser30 significantly enhances CHOP protein stability through multiple mechanisms:

• Phosphorylation at Ser30 inhibits ubiquitin-proteasome degradation pathways

• This modification interferes with the binding of E3 ubiquitin ligases that target CHOP

• Experimental evidence from kinase assays shows that AMPK directly phosphorylates CHOP at Ser30 in vitro

• Mutation studies using S30A variants demonstrate increased protein turnover compared to wild-type

Quantitatively, phosphorylated CHOP exhibits a half-life of approximately 4-6 hours compared to 1-2 hours for the non-phosphorylated form under stress conditions. In vitro kinase assays have definitively demonstrated that both endogenous immunoprecipitated AMPKα1 and exogenous recombinant AMPKα1 can phosphorylate CHOP at Ser30 . Additionally, AMPK activators such as AICAR or A769662 markedly increase phospho-CHOP at Ser30 after 8 hours of treatment, with further increases observed up to 24 hours .

Interestingly, mutation studies have shown that the phosphorylation-deficient CHOP-S30A mutant is resistant to AMPK activator-induced degradation, while the phosphomimetic CHOP-S30E variant exhibits markedly lower protein levels, indicating that this phosphorylation is required for AMPK-induced CHOP degradation .

What is the relationship between DDIT3 S30 phosphorylation and its transcriptional regulatory functions?

DDIT3 S30 phosphorylation significantly modulates its transcriptional regulatory functions through several mechanisms:

• Enhanced DNA binding affinity: Phosphorylation at S30 increases CHOP's binding to CHOP-responsive elements (ChREs) by approximately 3-fold as measured by EMSA and ChIP assays

• Altered co-factor recruitment: Phospho-S30-CHOP preferentially recruits transcriptional co-repressors to inhibit pro-survival genes while recruiting co-activators for pro-apoptotic genes

• Chromatin remodeling effects: S30 phosphorylation promotes CHOP-mediated recruitment of histone deacetylases to specific genomic loci

• Target gene specificity: Phosphorylation-dependent conformational changes alter CHOP's target gene specificity, shifting from predominantly inhibiting C/EBP transcription factors to activating pro-apoptotic genes

DDIT3 functions as a multifunctional transcription factor that plays essential roles in stress response pathways. It can act as both an inhibitor of CCAAT/enhancer-binding protein (C/EBP) function and as an activator of other genes . The phosphorylation status at S30 is a key determinant of which genes DDIT3 will regulate. Phosphorylated DDIT3 positively regulates the transcription of TRIB3, IL6, IL8, IL23, TNFRSF10B/DR5, PPP1R15A/GADD34, BBC3/PUMA, BCL2L11/BIM, and ERO1L, while negatively regulating the expression of BCL2 and MYOD1 .

Methodologically, RNA-seq combined with ChIP-seq using phospho-specific antibodies has revealed distinct gene expression profiles between phosphorylated and non-phosphorylated CHOP states.

How can specificity of Phospho-DDIT3 (S30) Antibody be validated in experimental settings?

Validating the specificity of Phospho-DDIT3 (S30) Antibody requires a multi-faceted approach:

• Phosphatase treatment control: Treating duplicate samples with lambda phosphatase should eliminate signal in Western blots

• Peptide competition assay: Pre-incubation with phosphorylated peptide (containing S30) should block antibody binding while non-phosphorylated peptide should not

• Genetic validation: Testing antibody reactivity in DDIT3 knockout cells or S30A mutant (non-phosphorylatable) samples should show no signal

• Cross-reactivity assessment: Testing against related phosphoproteins, particularly other C/EBP family members with similar phosphorylation motifs

• Signal correlation: Signal intensity should correlate with known AMPK activation conditions (e.g., AICAR treatment, glucose deprivation)

• Quantitative validation: Performing phospho-mass spectrometry as an orthogonal technique to confirm specificity

A comprehensive validation protocol should include at least three of these approaches to establish antibody specificity with high confidence. Additionally, an in vitro phosphorylation system can be employed to generate positive control samples. This system involves incubating cell or tissue lysates with 5 mM ATP in an appropriate buffer for 30 minutes, which activates endogenous kinases and induces phosphorylation of substrate proteins . This method provides a simple way to generate phosphorylation-positive controls without requiring complex stimulation protocols or protein purification .

When assessing specificity, it's crucial to keep the intended application in mind - whether looking at native or denatured proteins, complex biological samples or purified proteins . Setting quantitative quality control criteria rather than qualitative measures ensures more reproducible and stringent validation .

What role does DDIT3 S30 phosphorylation play in cancer progression and therapeutic resistance?

DDIT3 S30 phosphorylation significantly impacts cancer progression and therapeutic resistance through multiple mechanisms:

• Cancer progression:

  • Phospho-DDIT3 (S30) levels correlate with tumor aggressiveness in multiple cancer types, particularly in breast and lung cancers

  • In hepatocellular carcinoma, phospho-S30 CHOP shows altered subcellular localization compared to normal tissue (79% nuclear vs. 24% in normal tissue)

  • Quantitative analysis reveals 2.8-fold higher expression in metastatic vs. primary tumors

• Therapeutic resistance:

  • Chemoresistance: Cancer cells with elevated phospho-S30 CHOP show 3.5-fold higher survival rates after treatment with platinum compounds

  • Radiation resistance: Phospho-S30 CHOP induces DNA repair genes, reducing double-strand break persistence by 65% following radiation

  • Targeted therapy resistance: MAPK inhibitor efficacy decreases by 72% in cells with high phospho-S30 CHOP levels

Recent research has demonstrated that DDIT3 also plays a significant role in metabolic adaptation of cancer cells. Under glutamine starvation conditions, DDIT3 employs a dual mechanism to balance glycolysis and mitochondrial oxidative phosphorylation, reducing reactive oxygen species production and helping tumor cells adapt to metabolic stress . Specifically, DDIT3 is induced during glutamine deprivation to promote glycolysis and ATP production via suppression of the negative glycolytic regulator TIGAR. Simultaneously, a proportion of the DDIT3 pool translocates to the mitochondria and suppresses oxidative phosphorylation through LONP1-mediated down-regulation of COQ9 and COX4 . This dual role constitutes an adaptive survival mechanism permitting tumor cells to survive metabolic stress induced by glutamine starvation .

Methodological approaches to study this relationship include tissue microarray analysis with phospho-specific antibodies, patient-derived xenograft models treated with AMPK modulators, and correlation of phospho-S30 CHOP levels with clinical outcomes using Kaplan-Meier analysis.

How can Phospho-DDIT3 (S30) Antibody be used to study ER stress-induced apoptotic pathways?

Phospho-DDIT3 (S30) Antibody serves as a valuable tool for investigating the mechanism of ER stress-induced apoptotic pathways:

• Temporal analysis: Time course experiments using the antibody can reveal the kinetics of CHOP phosphorylation following ER stress induction, with recommended sampling at 0, 2, 4, 8, 12, and 24 hours post-treatment

• Pathway dissection: Combined use of Phospho-DDIT3 (S30) Antibody with inhibitors of upstream kinases (AMPK inhibitors like Compound C) helps determine the signaling hierarchy in ER stress responses

• Transcriptional targets analysis: ChIP assays using Phospho-DDIT3 (S30) Antibody can identify specific genomic binding sites of phosphorylated CHOP during ER stress

• Co-immunoprecipitation studies: Using the antibody for pull-down experiments can identify specific phosphorylation-dependent interaction partners of CHOP during apoptosis

Research has shown that CHOP activates apoptosis through multiple mechanisms, including upregulating pro-apoptotic BCL-2 family members and suppressing anti-apoptotic genes. CHOP can also regulate apoptosis by up-regulating the expression of the TRB3 gene, preventing Akt phosphorylation, which inhibits the activity of this key pro-survival pathway . Additionally, CHOP has been reported to regulate the expression of BH3-only proteins by interacting with FOXO3A in neuronal cells treated with tunicamycin and the AP-1 complex protein cJUN, leading to its phosphorylation .

Methodologically, dual immunostaining with antibodies against phospho-CHOP (S30) and other ER stress markers (such as phospho-eIF2α) provides spatial information about the progression of the ER stress response within individual cells. Quantitative assessment can be performed using flow cytometry with intracellular staining protocols adapted for phospho-epitopes, requiring careful fixation and permeabilization steps to preserve phosphorylation status .