ATF2 (Ab-69 or 51) Antibody

What is ATF2 and what cellular functions does it regulate?

ATF2 (Activating Transcription Factor 2) is a transcription factor belonging to the leucine zipper family of DNA binding proteins. It regulates the transcription of various genes involved in:

• Anti-apoptosis mechanisms

• Cell growth and proliferation

• DNA damage response

• Cell development and differentiation

ATF2 functions through binding to specific DNA sequences: either CRE (cAMP response element) consensus sequences (5'-TGACGTCA-3') or AP-1 (activator protein 1) consensus sequences (5'-TGACTCA-3'), depending on its binding partner . In the nucleus, ATF2 contributes to global transcription and DNA damage response, while in the cytoplasm, it interacts with mitochondrial proteins affecting membrane potential and promoting cell death under certain conditions . Additionally, ATF2 exhibits histone acetyltransferase (HAT) activity, specifically acetylating histones H2B and H4 in vitro .

What is the significance of phosphorylation at Thr69 and Thr51 sites of ATF2?

Phosphorylation at Thr69 and Thr71 (sometimes referred to as Thr51 depending on the numbering system) is essential for ATF2's transcriptional activation function. These residues are phosphorylated in vivo and can be efficiently phosphorylated in vitro by the JNK/SAPK subgroup of the MAPK family . The transcriptional activity of ATF2 is induced through this phosphorylation in response to:

• Extracellular stresses (UV irradiation, oxidative stress)

• Inflammatory cytokines

• Growth factor stimulation

Research has demonstrated that these phosphorylation sites are critical for mediating transcriptional activation but are distinct from ATF2's role in DNA damage response, which depends on phosphorylation at different sites (S490/S498) by ATM .

What experimental applications is ATF2 (Ab-69 or 51) Antibody suitable for?

ATF2 (Ab-69 or 51) Antibody has been validated for the following applications:

The antibody has been purified by affinity chromatography using epitope-specific peptide, making it a reliable tool for these applications . It detects total ATF2 protein regardless of phosphorylation status, unlike phospho-specific antibodies that only detect specific phosphorylated forms .

What is the specificity of ATF2 (Ab-69 or 51) Antibody across species?

The antibody shows consistent reactivity with multiple mammalian species:

This cross-species reactivity makes the antibody versatile for comparative studies across different model organisms .

How can I validate the specificity of ATF2 (Ab-69 or 51) Antibody in my experimental system?

For rigorous validation of antibody specificity, implement the following approaches:

• Peptide competition assay: Use the immunizing peptide (sequence around aa.67~71 or 49~53, D-Q-T-P-T) to competitively block antibody binding. The signal should be significantly reduced when the specific peptide is present .

• Genetic knockdown/knockout validation: Use ATF2 siRNA, shRNA, or CRISPR/Cas9 to reduce or eliminate ATF2 expression, then confirm signal reduction or elimination. The ATF2 knockdown validation approach was demonstrated in the RCC studies where shRNA was used to suppress ATF2 expression .

• Multiple antibody comparison: Compare results with other commercial antibodies targeting different epitopes of ATF2 to confirm consistent detection patterns.

• Positive and negative control samples: Include cell lines or tissues with known ATF2 expression levels. For instance, the 3T3 cell extracts (untreated vs. anisomycin-treated) that show differential phosphorylation states could serve as valuable controls .

• Western blot analysis: Confirm that the antibody detects a band of the expected molecular weight (55 kDa) .

What experimental conditions affect ATF2 phosphorylation status and antibody detection?

Several experimental conditions can modulate ATF2 phosphorylation:

• Growth factor stimulation: Insulin and EGF activate ATF2 via a two-step mechanism involving its phosphorylation .

• Stress induction: UV irradiation and anisomycin treatment stimulate the JNK/SAPK pathway, leading to ATF2 phosphorylation at Thr69/71 .

• DNA damage inducers: Ionizing radiation (IR) and neocarzinostatin (NCS) induce ATF2 phosphorylation at S490/S498 through ATM activation .

• Chemical inhibitors:

  • JNK-specific inhibitors (e.g., JNK-IN-8) reduce phosphorylation at Thr69/71

  • p38 inhibitors similarly affect ATF2 phosphorylation

• Cell cycle phase: ATF2 phosphorylation varies during different phases of the cell cycle, affecting its role in cell proliferation through regulation of cyclins .

• Zinc chelation: EDTA treatment affects the zinc-finger domain of ATF2, decreasing JNK-ATF2 binding approximately 10-fold, which may indirectly affect phosphorylation .

How can I distinguish between ATF2 phosphorylated at Thr69 versus Thr71 in my experiments?

To differentiate between these closely spaced phosphorylation sites:

• Use site-specific phospho-antibodies: Employ antibodies that specifically recognize phospho-Thr71 versus phospho-Thr69+71 .

• Kinase fractionation: Different kinase fractions show selective phosphorylation of these sites. For example, A14 fractions 12 and 13 were shown to phosphorylate ATF2 Thr71 efficiently but not Thr69, whereas partially purified p38 (fraction 17) phosphorylated both sites .

• In vitro kinase assays: Use purified kinases with ATF2 substrate to assess site-specific phosphorylation patterns, using GST-ATF2 fusion proteins with site-specific mutations .

What kinases regulate ATF2 phosphorylation at Thr69/71 and how can I manipulate these pathways?

Multiple kinases can phosphorylate ATF2 at Thr69/71, each responsive to different stimuli:

Experimental manipulation strategies:

• Chemical activators: Anisomycin treatment can activate both JNK and p38 pathways simultaneously .

• Genetic approaches:

  • Overexpress upstream activators (e.g., MLK3-MKK7 for JNK)

  • Use JNK-/- cells to isolate p38 effects on ATF2 phosphorylation

  • Express constitutively active forms of these kinases

• p38-specific binding region: The research identified a critical sequence (92-FENEF-96) C-terminal to the 69-TPTP-72 phosphoswitch that is required for p38-mediated phosphorylation but not JNK-mediated phosphorylation .

How does ATF2 phosphorylation correlate with its transcriptional activity versus DNA damage response function?

ATF2 exhibits distinct functions based on different phosphorylation patterns:

Transcriptional Activity Pathway:

• Phosphorylation sites: Thr69 and Thr71 (or Thr51/71)

• Responsible kinases: JNK/SAPK, p38

• Function: Activates transcription of genes involved in cell growth, anti-apoptosis

• Experimental evidence: Mutation of these sites (T69A/T71A) abolishes transcriptional activity but does not affect DNA damage response

DNA Damage Response Pathway:

• Phosphorylation sites: Ser490 and Ser498

• Responsible kinase: ATM

• Function: Recruits MRN complex to DNA double-strand breaks, controls IR-induced S phase checkpoint

• Experimental evidence: ATF2 phosphorylated by ATM colocalizes with γ-H2AX in DNA repair foci within 3 minutes after IR

Key findings supporting functional separation:

• ATF2 mutated on ATM phosphoacceptor sites (S490/498A) can still activate an ATF2 reporter to the same level as wild-type ATF2

• Transcriptionally inactive ATF2 (T69A/T71A) still forms repair foci following IR and functions in DNA damage response

• Cells expressing ATF2 with mutations at S490/498 showed radioresistant DNA synthesis (RDS) phenotype, indicating defective checkpoint control

How can I use ATF2 (Ab-69 or 51) Antibody to investigate ATF2's role in cancer models?

For cancer research applications, consider these experimentally validated approaches:

How does ATF2 phosphorylation at Thr69/71 differ from other sites in biological function?

Different phosphorylation sites dictate distinct ATF2 functions:

Thr69/71 Phosphorylation (Transcriptional Regulation):

• Kinases: JNK/SAPK, p38

• Stimuli: UV, stress, inflammatory cytokines, growth factors

• Functions:

  • Activates transcription of genes involved in cell growth and survival

  • Promotes cancer cell proliferation through regulation of cyclins

  • Enhances EMT and metastasis in cancer cells

• Experimental evidence: In RCC, "the transcriptional activity of ATF2 is induced through Thr69- and/or Thr71-phosphorylation by JNK or p38"

Ser490/498 Phosphorylation (DNA Damage Response):

• Kinase: ATM

• Stimuli: Ionizing radiation, DNA damaging agents

• Functions:

  • Localizes to DNA double-strand break sites

  • Recruits MRN repair complex

  • Controls IR-induced S phase checkpoint

  • Promotes radioresistance

• Experimental evidence: "Phosphorylation of ATF2 was observed as soon as 15 min after IR with a peak after 1hr"

Functional independence demonstrated by:

• ATF2 mutated on S490/498 retains normal transcriptional activity

• ATF2 mutated on T69/71 still forms repair foci and functions in DNA damage response

• Cells expressing S490/498 mutant ATF2 show defective checkpoint control despite normal transcriptional activity

What are the optimal storage and handling conditions for ATF2 (Ab-69 or 51) Antibody?

For maximum antibody stability and performance:

These storage conditions ensure maintenance of antibody specificity and activity over time .

What experimental approaches can study ATF2 phosphorylation and its interaction with binding partners?

Several validated techniques can assess ATF2 phosphorylation dynamics and protein interactions:

• Co-immunoprecipitation: Pull down ATF2 protein complexes under different conditions to analyze binding partners.

• NanoBit Protein-Protein Interaction (PPI) assay: This assay revealed increased binding of ATF2 S90N mutant to p38 compared to wild-type ATF2 .

• Isothermal Titration Calorimetry (ITC): Used to measure binding affinity between JNK and ATF2, showing ~10-fold decrease in binding in the presence of EDTA due to disruption of the zinc-finger domain .

• Fractionation studies: Separate nuclear and cytoplasmic fractions to determine subcellular localization of phosphorylated ATF2.

• Immunofluorescence microscopy: Visualize colocalization of ATF2 with other proteins (such as γ-H2AX in DNA damage foci) .

• In vitro kinase assays: Determine phosphorylation kinetics and site preference using purified kinases and ATF2 substrates .

How can I differentiate between ATF2's transcriptional and non-transcriptional functions in my research?

To distinguish between these distinct functions:

• Use mutant constructs:

  • T69A/T71A: Transcriptionally inactive but retains DNA damage response

  • S490A/S498A: Defective in DNA damage response but retains transcriptional activity

• Cell cycle checkpoint analysis: Assess radioresistant DNA synthesis (RDS) phenotype to evaluate DNA damage response function separate from transcriptional activity .

• Subcellular localization: Monitor ATF2 localization to different cellular compartments:

  • Nuclear localization correlates with transcriptional function

  • Localization to DNA damage foci indicates DNA repair function

• Selective pathway inhibition:

  • ATM inhibitors block DNA damage response function

  • JNK/p38 inhibitors block transcriptional activation