Phospho-LIG4 (T650) Antibody

What is the biological significance of DNA Ligase 4 in cellular processes?

DNA Ligase 4 (LIG4) serves as a key factor in the non-homologous end-joining (NHEJ) DNA double-strand break repair pathway. This mechanism is essential for V(D)J recombination during the development of T cell receptors and immunoglobulin molecules. Defects in LIG4 can result in a variable syndrome characterized by growth retardation, pancytopenia, combined immunodeficiency, cellular radiosensitivity, and developmental delay . LIG4 functions in a complex with XRCC4, which enhances its enzymatic activity and stability. The critical role of LIG4 is further highlighted by studies showing that LIG4-deficient mice exhibit embryonic lethality due to defective neurogenesis and neuronal apoptosis, indicating its essential function in development beyond immune system formation .

How does phosphorylation regulate LIG4 activity in the DNA repair pathway?

Phosphorylation of LIG4 represents a post-translational regulatory mechanism that modulates its activity within the NHEJ repair complex. While the search results don't specifically address T650 phosphorylation, research has shown that phosphorylation of repair proteins like XRCC4 and XLF (which form a complex with LIG4) affects their DNA-binding properties and interaction dynamics . Phosphorylation appears to play a role in regulating the dissociation of repair proteins from DNA after the completion of the repair process. Studies using phospho-mimicking mutations (replacing phosphorylation sites with aspartic acid) have demonstrated dramatically altered dissociation rates from DNA and reduced stability of DNA tethering in vitro . This suggests that phosphorylation of LIG4 at sites like T650 may serve as a regulatory switch for modulating repair complex dynamics.

What experimental approaches confirm the T650 site is phosphorylated in vivo?

To confirm T650 phosphorylation in vivo, researchers should employ a multi-faceted approach. Initial identification often comes from phospho-proteomic mass spectrometry, which can detect phosphorylated residues in LIG4 isolated from cells. This should be followed by site-directed mutagenesis, where threonine 650 is replaced with alanine (phospho-blocking) or aspartic acid (phospho-mimicking) to assess functional consequences. Researchers have successfully used this approach for other phosphorylation sites in the NHEJ machinery, demonstrating effects on DNA repair efficiency and cellular survival after exposure to radiomimetic drugs . Additionally, validation requires demonstrating antibody specificity through phosphatase treatment of samples, which should eliminate signal from a true phospho-specific antibody, and comparison of signal between wild-type cells and those expressing T650A mutant LIG4.

What are the optimal conditions for using Phospho-LIG4 (T650) Antibody in Western blotting?

For optimal Western blotting with Phospho-LIG4 (T650) Antibody, researchers should consider the following protocol based on typical practices for phospho-specific antibodies and available data for LIG4 antibodies:

• Sample preparation: Include phosphatase inhibitors in all buffers to preserve phosphorylation status

• Loading controls: Use both total LIG4 and a housekeeping protein

• Recommended dilution: Start with 1:500-1:1000 dilution for Western blot applications

• Blocking: Use 5% BSA in TBST rather than milk (milk contains phosphatases)

• Primary antibody incubation: Overnight at 4°C with gentle rocking

• Controls: Include samples treated with lambda phosphatase to confirm phospho-specificity

The effectiveness of this antibody has been demonstrated across multiple cellular contexts, with positive Western blot detection reported in mouse testis tissue, mouse liver tissue, HepG2 cells, HeLa cells, and rat testis tissue .

How can researchers differentiate between phosphorylated and non-phosphorylated forms of LIG4?

Differentiating between phosphorylated and non-phosphorylated forms of LIG4 requires careful experimental design:

Additionally, researchers should employ phospho-mimetic (Asp) and phospho-ablating (Ala) mutants as controls in cellular experiments. This approach has been successfully used to study the functional impacts of phosphorylation on XRCC4 and XLF, which form complexes with LIG4 during NHEJ .

What methodological approaches best reveal the functional significance of T650 phosphorylation?

To elucidate the functional significance of T650 phosphorylation, researchers should implement a comprehensive experimental strategy:

• Generate cell lines expressing LIG4-T650A (phospho-blocking) and LIG4-T650D (phospho-mimicking) mutants in a LIG4-null background

• Assess DNA repair efficiency using comet assays or γH2AX foci formation/resolution after exposure to ionizing radiation

• Analyze cell survival following DNA damage using clonogenic assays

• Examine protein-protein interactions via co-immunoprecipitation to determine if T650 phosphorylation alters LIG4 interactions with XRCC4, XLF, or other repair factors

• Use fluorescence recovery after photobleaching (FRAP) to measure the dynamics of LIG4 recruitment to and dissociation from DNA damage sites

This multi-faceted approach mirrors successful strategies used to study phosphorylation of other NHEJ components, where phospho-mimicking mutations were shown to dramatically affect XRCC4/XLF dissociation from DNA and impede cellular survival after exposure to radiomimetic drugs .

How does LIG4 T650 phosphorylation status correlate with radiosensitivity in different cell types?

The correlation between LIG4 T650 phosphorylation and radiosensitivity likely varies across cell types based on their DNA repair capacity and reliance on NHEJ. Research on LIG4 syndrome has demonstrated that different mutations in LIG4 can produce varying degrees of radiosensitivity, even within the same family . For example, fibroblasts obtained from a patient with severe LIG4 syndrome showed high radiosensitivity, while T cells from siblings with the same bi-allelic LIG4 mutations exhibited variable responses to irradiation .

To study this correlation systematically, researchers should:

• Quantify baseline T650 phosphorylation levels across a panel of cell lines with known radiosensitivity profiles

• Compare phosphorylation dynamics before and after radiation exposure

• Assess the impact of phosphatase or kinase inhibitors on both T650 phosphorylation and radiosensitivity

• Analyze clinical samples from patients with different radiosensitivity levels

This approach would help establish whether T650 phosphorylation could serve as a biomarker for predicting radiation response in different tissues or tumor types.

What is the relationship between LIG4 phosphorylation and its interaction with other NHEJ pathway proteins?

LIG4 phosphorylation likely modulates its interactions with other NHEJ components, particularly XRCC4 and XLF. While the specific effects of T650 phosphorylation are not detailed in the search results, studies on related proteins offer valuable insights. Research has shown that phospho-mimicking mutations in both XRCC4 and XLF significantly affect their dissociation from DNA and the stability of DNA tethering in vitro, without altering their direct protein-protein interaction affinity .

For LIG4, T650 phosphorylation may similarly regulate its functional interactions with XRCC4 and XLF without necessarily changing the formation of the complex itself. Testing this hypothesis would require comparing wild-type LIG4 with T650D mutants in biochemical assays measuring complex formation, DNA binding, and ligation activity.

How do different assay conditions affect the detection of phosphorylated LIG4 in experimental systems?

Detection of phosphorylated LIG4 is highly sensitive to experimental conditions, requiring careful optimization:

Additionally, researchers should consider that DNA damage type may influence phosphorylation patterns. Studies have shown that phospho-mimicking mutations in NHEJ proteins particularly impede repair of complex DNA lesions , suggesting that LIG4 phosphorylation may be differentially regulated depending on damage complexity.

What controls are essential when interpreting results from Phospho-LIG4 (T650) Antibody experiments?

Proper experimental controls are critical for accurate interpretation of Phospho-LIG4 (T650) Antibody results:

• Phosphatase-treated sample: Treating a duplicate sample with lambda phosphatase should eliminate signal, confirming antibody phospho-specificity

• Total LIG4 detection: Parallel detection with a phosphorylation-independent LIG4 antibody establishes total protein levels

• Positive control: Include samples from cells treated with agents known to induce DNA damage (e.g., etoposide, ionizing radiation)

• Negative control: Include samples from cells with LIG4 knocked down/out or expressing T650A mutant

• Loading control: Use housekeeping proteins (β-actin, GAPDH) to normalize protein loading

• Peptide competition: Pre-incubation of antibody with phospho-T650 peptide should abolish specific signal

Given the importance of phosphorylation in regulating DNA repair proteins, these controls help distinguish genuine phosphorylation-dependent signals from artifacts. Research on related proteins has demonstrated that phosphorylation alters their function in DNA repair, and similar regulatory mechanisms likely apply to LIG4 .

How should researchers address contradictory results when studying LIG4 phosphorylation across different experimental models?

When encountering contradictory results regarding LIG4 phosphorylation across different experimental models, researchers should systematically evaluate several factors:

• Cell type differences: LIG4 function shows significant variability even within related cell types. For example, studies have documented variable severities of radiosensitivity and immune abnormalities among genotypically identical siblings with LIG4 mutations .

• Experimental timing: Phosphorylation events are often transient and occur in specific temporal windows after DNA damage. Comprehensive time-course experiments are essential to capture these dynamics.

• DNA damage context: The nature and complexity of DNA damage influence repair pathway choice and protein modifications. Phospho-mimicking mutations in NHEJ proteins particularly affect repair of complex DNA lesions .

• Antibody validation: Not all phospho-specific antibodies have equal specificity and sensitivity. Cross-validation with multiple techniques is essential:

  • Mass spectrometry to directly detect phosphorylated peptides

  • Site-directed mutagenesis (T650A) to eliminate the phosphorylation site

  • Immunoprecipitation followed by phospho-specific Western blotting

• Technical variables: Standardize protein extraction methods, being particularly careful with phosphatase inhibitor usage, sample handling, and gel systems optimized for phospho-protein detection.

What are the limitations of current methodologies for studying phosphorylated LIG4 in cellular systems?

Current methodologies for studying phosphorylated LIG4 have several important limitations:

How can Phospho-LIG4 (T650) Antibody be used to study the relationship between DNA repair defects and immunodeficiency?

The Phospho-LIG4 (T650) Antibody offers a valuable tool for investigating the mechanistic link between DNA repair and immune system development:

What emerging technologies will enhance the study of LIG4 phosphorylation dynamics in live cells?

Several cutting-edge technologies are poised to revolutionize our understanding of LIG4 phosphorylation:

• CRISPR-based phospho-sensors: Endogenous tagging of LIG4 with fluorescent sensors that undergo conformational changes upon phosphorylation, allowing real-time visualization of phosphorylation events.

• Proximity labeling approaches: BioID or TurboID fused to LIG4 can identify proteins that interact specifically with phosphorylated versus non-phosphorylated forms.

• Mass spectrometry advancements:

  • Selected reaction monitoring (SRM) for targeted quantification of phosphorylated LIG4 peptides

  • Crosslinking mass spectrometry to capture transient interactions dependent on phosphorylation status

  • Single-cell phospho-proteomics to address cellular heterogeneity

• Super-resolution microscopy combined with phospho-specific antibodies: This would enable visualization of the spatial organization of phosphorylated LIG4 at DNA damage sites with unprecedented detail.

• Optogenetic tools: Light-inducible kinases/phosphatases targeting LIG4 would allow precise temporal control over phosphorylation status to study functional consequences.

These technologies would help overcome current limitations in studying the dynamic nature of LIG4 phosphorylation and its impact on DNA repair processes.

How might understanding T650 phosphorylation contribute to radiation sensitization strategies in cancer therapy?

The potential of targeting LIG4 T650 phosphorylation for radiation sensitization in cancer therapy is a promising avenue for research:

• Biomarker development: T650 phosphorylation status could serve as a predictive biomarker for radiation sensitivity. Studies have demonstrated that LIG4 deficiency correlates with increased radiosensitivity , suggesting that abnormal phosphorylation might similarly affect radiation response.

• Small molecule development: Compounds that specifically inhibit the kinase responsible for T650 phosphorylation could sensitize cancer cells to radiation therapy. If phosphorylation at T650 promotes efficient DNA repair, inhibiting this process could enhance the cytotoxic effects of radiation.

• Synthetic lethality approaches: Cancer cells with specific mutations might be particularly vulnerable to combined inhibition of LIG4 phosphorylation and other DNA repair pathways. Research has shown that phospho-mimicking mutations in NHEJ proteins impede cellular survival after exposure to radiomimetic drugs , suggesting that manipulating phosphorylation status could enhance therapeutic efficacy.

• Precision medicine strategies: Genomic profiling of tumors could identify patients most likely to benefit from therapies targeting LIG4 phosphorylation, based on their mutation profiles in DNA repair pathways.

• Combination therapy design: Understanding how T650 phosphorylation affects DNA repair pathway choice could inform rational combinations of radiation with other DNA damage response inhibitors for maximal therapeutic benefit.