Phospho-CDC25A (Thr507) Antibody

What is the biological significance of CDC25A Thr507 phosphorylation?

CDC25A Thr507 phosphorylation represents a critical regulatory mechanism within the cell cycle and DNA damage response pathways. This specific phosphorylation site is located within the C-terminal tail of CDC25A and serves as one of the key CHK1 phosphorylation sites (alongside Ser178 in the N-terminal region) . Phosphorylation at Thr507 mediates 14-3-3 protein binding, which plays a crucial role in regulating CDC25A stability and activity . When cells experience DNA damage, this phosphorylation contributes to CDC25A degradation, preventing cells with chromosomal abnormalities from progressing through cell division . Methodologically, researchers can use Phospho-CDC25A (Thr507) antibodies to monitor this phosphorylation status as an indicator of checkpoint activation and cell cycle arrest.

How does CDC25A function within the cell cycle regulatory network?

CDC25A functions as a dual-specificity protein phosphatase that activates cyclin/cyclin-dependent kinase (Cdk) complexes by removing inhibitory phosphates from conserved threonine and tyrosine residues in Cdks . It is specifically required for progression through G1 and S phases of the cell cycle . CDC25A directly dephosphorylates CDK1 and stimulates its kinase activity, while also dephosphorylating CDK2 in complex with cyclin-E in vitro . As a dosage-dependent inducer of mitotic progression, CDC25A acts as a critical regulatory node connecting checkpoint signaling with cell cycle machinery . To study these interactions experimentally, researchers should consider using Phospho-CDC25A (Thr507) antibodies in combination with other cell cycle markers to establish temporal relationships between CDC25A activation and cell cycle progression.

What are the differences between the three human CDC25 isoforms?

The human genome encodes three CDC25 isoforms: CDC25A, CDC25B, and CDC25C, which function at different phases of the cell cycle . While all three isoforms share structural similarities in their catalytic domains, they exhibit distinct temporal activity patterns and regulatory mechanisms:

For experimental design, researchers should carefully select the appropriate CDC25 isoform-specific antibody based on the cell cycle phase under investigation.

How does Thr507 dephosphorylation of CDC25A contribute to apoptotic signaling?

The dephosphorylation of Thr507 in CDC25A represents a molecular switch that redirects CDC25A function from cell cycle regulation to apoptotic signaling. During apoptosis induced by stimuli such as staurosporine, CDC25A undergoes caspase cleavage at Asp-223, generating a C-terminal 37-kDa fragment (C37) . Concurrent with this cleavage, Thr507 becomes dephosphorylated, which prevents 14-3-3 binding as previously demonstrated . This dephosphorylation appears to expose the Cdc2/Cdk2-docking site in C37, allowing enhanced interaction with and activation of cyclin B1/Cdc2 complexes . Experimental data indicates that C37 with alanine substitution for Thr507 (C37/T507A) exhibits markedly higher phosphatase activity than full-length CDC25A and promotes apoptosis through cyclin B1/Cdc2 activation rather than Cdk2 activation . This mechanism establishes CDC25A as a pro-apoptotic protein that amplifies staurosporine-induced apoptosis through the activation of cyclin B1/Cdc2 by its C-terminal domain . To investigate this phenomenon, researchers should employ both phospho-specific antibodies and apoptotic markers in time-course experiments.

What structural insights have emerged from recent Cryo-EM studies of CDC25A interactions with CDK-cyclin complexes?

Recent Cryo-EM studies have provided unprecedented structural insights into the CDC25A-CDK-cyclin interaction. The Cryo-EM structure of the CDK2-cyclin A-CDC25A complex reveals that the CDC25A catalytic domain bridges the bi-lobal structure of CDK2, binding on the opposite face to cyclin A . This arrangement forms an extensive but discontinuous interface between CDC25A and the CDK2-cyclin A complex . The CDC25A catalytic domain adopts an α/β-domain structure with a central 5-stranded parallel β-sheet enclosed by 5 α-helices, consistent with previously determined structures (RMSD of aligned residues = 0.9 Å) .

Notably, hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis identified a region of CDC25A (residues 445-463, VRERDRLGNEYPKLHYPEL) that becomes significantly protected upon complex formation . This region starts in helix α4 C-terminal to the catalytic residue and continues into the succeeding loop, providing valuable information about the molecular interface critical for CDC25A function . For researchers investigating structure-based drug design targeting CDC25A, these detailed structural insights provide crucial molecular coordinates for rational inhibitor development.

How can researchers distinguish between phosphorylation-dependent and independent functions of CDC25A?

Distinguishing between phosphorylation-dependent and independent functions of CDC25A requires sophisticated experimental approaches. Researchers should consider:

• Phosphorylation-site mutant studies: Generate and express CDC25A constructs with alanine substitutions at key phosphorylation sites, particularly Thr507 and Ser178, to create phospho-deficient mutants . Compare these with phosphomimetic mutants (using aspartic or glutamic acid substitutions) to elucidate phosphorylation-dependent functions.

• Phosphatase treatment experiments: Treat immunoprecipitated CDC25A with lambda phosphatase before functional assays to remove all phosphorylations and assess phosphorylation-independent activities.

• Temporal correlation analysis: Use time-course experiments with Phospho-CDC25A (Thr507) antibodies alongside functional readouts to establish temporal relationships between phosphorylation events and functional outcomes.

• Protein-protein interaction studies: Compare the interactome of wild-type CDC25A versus phospho-mutants using co-immunoprecipitation followed by mass spectrometry to identify phosphorylation-dependent binding partners.

• Domain-specific analysis: Employ the C37 fragment (residues 224-524) containing the catalytic domain but lacking the N-terminal regulatory region to study functions potentially independent of N-terminal phosphorylation events .

These approaches collectively provide a methodological framework for dissecting the complex regulatory mechanisms governing CDC25A function.

What are the optimal conditions for using Phospho-CDC25A (Thr507) antibody in different experimental applications?

Based on validated protocols, researchers should follow these application-specific recommendations:

For experimental troubleshooting, researchers should verify antibody specificity using phosphatase treatment or phospho-deficient mutants (T507A). The antibody storage recommendations include short-term storage at 4°C and long-term storage at -20°C, avoiding freeze/thaw cycles .

How should researchers assess CDC25A phosphorylation status in response to DNA damage and cell cycle perturbations?

To rigorously assess CDC25A phosphorylation status in response to experimental perturbations, researchers should implement the following methodological approach:

• Time-course analysis: Collect samples at multiple timepoints after treatment to capture dynamic phosphorylation changes. For DNA damage studies, collect samples from 15 minutes to 24 hours post-treatment.

• Phosphatase inhibitor optimization: Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers to preserve phosphorylation status.

• Multiple detection methods: Combine Western blotting using Phospho-CDC25A (Thr507) antibody (1:1,000 dilution) with other techniques such as Phos-tag gel electrophoresis for mobility shift detection of phosphorylated species.

• Normalizing controls: Always normalize phospho-signal to total CDC25A protein levels using a pan-CDC25A antibody in parallel samples.

• Biological context controls: Include checkpoint kinase inhibitors (CHK1/CHK2 inhibitors) in parallel samples to verify the kinase dependency of observed phosphorylation.

• Cellular fractionation: Separate nuclear and cytoplasmic fractions to assess compartment-specific phosphorylation changes, as CDC25A localization is functionally significant.

For investigating Thr507 phosphorylation specifically in response to staurosporine-induced apoptosis, electroporate cells with pcDNA3–3×FLAG Cdc25A/224–524, treat with vehicle or 300 nm staurosporine for 2 hours, and analyze by immunoblotting using both Phospho-CDC25A (Thr507) and total CDC25A antibodies .

What are the key considerations for interpreting Phospho-CDC25A (Thr507) antibody results in complex biological samples?

Interpreting Phospho-CDC25A (Thr507) antibody results requires careful consideration of several technical and biological factors:

• Antibody specificity: The Phospho-CDC25A (Thr507) antibody detects CDC25A only when phosphorylated at Threonine 507 . Researchers should verify this specificity using phospho-deficient controls (T507A mutants).

• Cell cycle dependency: CDC25A expression and phosphorylation fluctuate throughout the cell cycle. Cell synchronization or cell cycle analysis (e.g., flow cytometry) should accompany phosphorylation assessments.

• Species considerations: While the antibody is validated for human samples, it is predicted to react with bovine, mouse, and rat based on sequence homology . When working with non-human samples, preliminary validation is necessary.

• Sensitivity thresholds: For endogenous CDC25A detection, enhanced chemiluminescence or fluorescent secondary antibody detection systems may be necessary due to generally low expression levels.

• Phosphorylation stoichiometry: Consider that only a fraction of total CDC25A may be phosphorylated at Thr507 at any given time. Quantify the ratio of phosphorylated to total CDC25A rather than absolute signal intensity.

• Context-dependent controls: Include appropriate positive controls based on experimental context - calyculin A treatment for phosphatase inhibition studies or checkpoint kinase activators for DNA damage response studies.

• Tissue-specific expression: CDC25A expression varies across tissues and cancer types. In IHC applications, tissue-specific validation and optimization are essential.

How can Phospho-CDC25A (Thr507) antibody contribute to cancer research and potential therapeutic approaches?

The Phospho-CDC25A (Thr507) antibody offers valuable research applications in cancer biology, particularly given that CDC25A is an oncogene with altered expression in various cancers . Key research applications include:

• Biomarker development: CDC25A phosphorylation status at Thr507 could serve as a potential biomarker for checkpoint activation or dysfunction in tumors. Researchers can use the antibody in IHC studies of tumor tissue microarrays to correlate phosphorylation patterns with clinical outcomes.

• Drug screening platforms: The antibody can be integrated into high-throughput screening assays to identify compounds that modulate CDC25A phosphorylation, potentially disrupting cancer cell cycle progression.

• Mechanism-based combination therapies: Understanding how cancer cells regulate CDC25A Thr507 phosphorylation may reveal synthetic lethal interactions with existing therapies. For example, investigating how CDC25A phosphorylation status affects sensitivity to checkpoint kinase inhibitors or conventional chemotherapeutics.

• Structure-guided drug design: The recent Cryo-EM structure of the CDK2-cyclin A-CDC25A complex provides molecular targets for rational drug design. The antibody can validate whether candidate compounds affect Thr507 phosphorylation in cellular contexts.

• Resistance mechanism studies: Researchers can investigate whether altered CDC25A phosphorylation contributes to therapy resistance by comparing Thr507 phosphorylation patterns in sensitive versus resistant cancer cell populations.

From a therapeutic perspective, small molecule inhibitors targeting CDC25 active sites have been reported, and alternative allosteric approaches targeting CDC25-protein interactions are being considered . The detailed structural information on CDC25A binding with CDK-cyclin substrates provides new opportunities for developing CDC25-targeting anticancer strategies .

What is the relationship between CDC25A Thr507 phosphorylation and apoptotic pathways in different cellular contexts?

The relationship between CDC25A Thr507 phosphorylation and apoptotic pathways represents a complex and context-dependent regulatory mechanism. Research findings indicate:

• Pro-apoptotic signaling: Dephosphorylation of Thr507 within the C37 fragment of CDC25A (generated by caspase cleavage at Asp-223) exposes the Cdc2/Cdk2-docking site, leading to enhanced activation of cyclin B1/Cdc2 complexes and subsequent apoptosis induction .

• Cell type specificity: In Jurkat and K562 cells, C37 with alanine substitution for Thr507 (C37/T507A) induced apoptosis through activation of cyclin B1/Cdc2 but not Cdk2 , suggesting cell type-dependent apoptotic mechanisms.

• Stress-response integration: Staurosporine treatment causes both caspase-mediated cleavage of CDC25A and dephosphorylation of Thr507, establishing a novel pathway of staurosporine-induced apoptosis .

To investigate these relationships across cellular contexts, researchers should:

• Perform comparative analysis of CDC25A Thr507 phosphorylation status during apoptosis induced by diverse stimuli (DNA damage, death receptor activation, metabolic stress)

• Use site-specific phospho-mutants (T507A and T507D/E) to dissect phosphorylation-dependent apoptotic mechanisms

• Employ real-time imaging with phospho-specific antibodies to track CDC25A phosphorylation dynamics during apoptosis progression

• Conduct epistasis experiments using CDC25A phospho-mutants in cells with modulated expression of apoptotic regulators

These approaches will help elucidate how CDC25A Thr507 phosphorylation serves as a molecular switch between cell cycle progression and apoptotic cell death in different cellular contexts.