Phospho-CDK2 (Thr160) Antibody

What is the biological significance of CDK2 Thr160 phosphorylation in cell cycle regulation?

Phosphorylation of CDK2 at Thr160 is essential for its kinase activity and proper cell cycle progression. Research demonstrates that replacing T160 with alanine completely abolishes CDK2 activity, confirming this phosphorylation is absolutely required for enzyme function .

CDK2 contains three major regulatory phosphorylation sites:

• Thr160: Activating phosphorylation (required for activity)

• Tyr15: Inhibitory phosphorylation

• Thr14: Inhibitory phosphorylation

During the cell cycle, phosphorylation at Thr160 increases during S phase and G2, coinciding with maximal CDK2 activity periods . CDK2 forms sequential complexes with different cyclins - initially with cyclin E during early stages of DNA synthesis (G1-S transition), and later with cyclin A during late stages of DNA replication (transition to G2 phase) .

What applications are most suitable for Phospho-CDK2 (Thr160) Antibody detection?

Based on validated research applications, Phospho-CDK2 (Thr160) Antibody is most frequently used in:

For Western blotting, the expected molecular weight of phosphorylated CDK2 is approximately 33 kDa . Cell-based ELISA kits offer advantages for detecting dynamic changes in phosphorylation levels across different cell lines or treatment conditions .

Immunohistochemistry studies show that phosphorylated CDK2 (Thr160) localizes to both cytoplasm and nucleus in human colon tissues, with distinct patterns between normal and cancerous samples .

What controls are essential when validating Phospho-CDK2 (Thr160) Antibody specificity?

Rigorous validation is critical for phospho-specific antibodies. Research-validated controls include:

• Genetic Controls:

  • CDK2 knockout samples completely eliminate signal (as demonstrated with WT vs. KO 293T cells)

  • T160A mutant proteins that cannot be phosphorylated at this position

  • Wild-type vs. CDK2 mutant cell comparison

• Treatment Controls:

  • Cell cycle synchronization (e.g., G0-arrested vs. cycling cells)

  • Treatments that alter phosphorylation status (hydroxyurea treatment for 18hr)

  • Phosphatase treatment to eliminate phosphorylation signals

• Specific Recognition Validation:

  • Many commercial antibodies undergo purification against phosphopeptide matrices

  • Non-phospho-specific antibodies are removed by chromatography using non-phosphopeptides

  • The specific immunogen sequence T-Y-T(p)-H-E ensures target specificity

• Technical Controls:

  • Total CDK2 detection in parallel samples

  • GAPDH as internal positive control for normalization

  • Cell density normalization for cell-based assays using Crystal Violet staining

The most convincing validation combines multiple approaches – genetic manipulation, phosphorylation state manipulation, and careful technical controls.

How should I optimize sample preparation to preserve CDK2 Thr160 phosphorylation status?

Phosphorylation states are notoriously labile and require specific precautions:

• Rapid Processing:

  • Work quickly and maintain cold temperatures throughout sample processing

  • Avoid repeated freeze-thaw cycles of samples

• Buffer Formulation:

  • Include phosphatase inhibitors in all lysis buffers

  • For research-grade results, use freshly prepared buffers

  • Standard formulation includes phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, with 150mM NaCl

• Cell Harvesting Considerations:

  • Direct lysis in hot SDS buffer can better preserve phosphorylation

  • For adherent cells, avoid extended trypsinization which may activate signaling cascades

  • Cell synchronization improves detection by enriching for specific cycle phases

• Storage Precautions:

  • For lysates, add 50% glycerol for long-term storage at -20°C

  • For antibodies, store at -20°C for long-term preservation and at 4°C for short-term use

  • Aliquot antibodies to minimize freeze-thaw cycles

• Tissue Sample Handling:

  • For IHC applications, optimal fixation is critical (typically formalin)

  • Antigen retrieval using citrate buffer (pH 6.0, 15 min) significantly improves detection

  • Process tissues rapidly after collection to minimize phosphorylation loss

Following these guidelines will significantly improve detection consistency and reproducibility when working with phosphorylation-specific antibodies.

How can I distinguish contradictory results in Phospho-CDK2 (Thr160) detection across different experimental conditions?

Contradictory results with phospho-specific antibodies are common and can be systematically addressed:

By systematically analyzing these factors, researchers can reconcile apparently contradictory results and extract meaningful biological insights from variable phosphorylation data.

What is the relationship between cyclin binding, CDK2 Thr160 phosphorylation, and kinase activation?

The interrelationship between these events is complex and bidirectional:

• Sequential Activation Process:

  • Research demonstrates that cyclin binding and Thr160 phosphorylation are interdependent events

  • Defects in cyclin A-binding coincide with reduced T-loop phosphorylation

  • In molecular detail, CDK2 phosphorylation on T160 increases during S phase and G2 when CDK2-cyclin binding is maximal

• Experimental Evidence of Interdependence:

  • Studies with Cdk2as/as cells show impaired T-loop phosphorylation correlates with reduced cyclin A binding

  • Treatment with specific inhibitors (3-MB-PP1) restored both Thr160 phosphorylation and cyclin binding in a concentration-dependent manner

  • This rescue also restored binding to the CDK inhibitor p21Cip1, which preferentially binds CDK/cyclin complexes

• Cyclin-Specific Interactions:

  • CDK2 forms sequential complexes:

    • Cyclin E-CDK2: Active during early stages of DNA synthesis (G1-S transition)

    • Cyclin A-CDK2: Predominates during late stages of DNA replication (S-G2 transition)

  • The normal ratio of CDK2 to CDK1 in cyclin A complexes is approximately 2:1

• Regulatory Hierarchy:

  • Cyclin binding induces conformational changes that facilitate Thr160 phosphorylation by CAK

  • Thr160 phosphorylation stabilizes the cyclin-CDK2 complex

  • Inhibitory phosphorylations at Tyr15 and Thr14 can override Thr160 activation

This complex interplay explains why researchers must examine both phosphorylation status and binding partners when studying CDK2 regulatory mechanisms.

How does CDK2 Thr160 phosphorylation connect to DNA damage response and replication stress?

CDK2 regulation is intimately linked to genome integrity and replication stress response:

• Replication Stress Response:

  • Research with CDK2 AF/AF cells (unable to undergo inhibitory phosphorylation) reveals they rapidly accumulate DNA damage during replication arrest

  • These cells show high levels of γH2AX (marker of DNA double-strand breaks) after hydroxyurea (HU) treatment

  • Similar damage occurs during aphidicolin (APH) treatment, confirming this is a general replication stress response

• Aberrant Replication Dynamics:

  • CDK2 has established roles in both replication initiation and elongation

  • Proper regulation of CDK2 through inhibitory phosphorylation is essential during replication stress

  • Microfluidics-assisted replication track analysis reveals that dysregulated CDK2 alters replication dynamics

• Checkpoint Function Integration:

  • The balance between activating (Thr160) and inhibitory (Tyr15/Thr14) phosphorylations likely serves as a molecular checkpoint

  • This mechanism prevents cells with DNA damage from progressing through the cell cycle

  • CDC25 phosphatase, which removes inhibitory phosphorylations, activates CDK2 in vitro

• Therapeutic Implications:

  • Cancer cells often experience elevated replication stress

  • Understanding how CDK2 phosphorylation status affects DNA damage accumulation could inform therapeutic strategies

  • Small molecule manipulation of CDK2 activity shows promise for research and potential therapeutic applications

This connection between CDK2 regulation and genome integrity makes Phospho-CDK2 (Thr160) detection a valuable tool for studying DNA damage response pathways.

What emerging technologies enhance detection and quantification of Phospho-CDK2 (Thr160)?

Several advanced technologies are improving phosphorylation detection beyond traditional Western blotting:

• AlphaLISA SureFire Ultra Technology:

  • This sandwich immunoassay enables quantitative detection of phospho-CDK2 (Thr160) in cellular lysates

  • Requires minimal sample volume (10 μL)

  • Offers improved sensitivity and dynamic range compared to traditional methods

  • Particularly valuable for high-throughput screening applications

• Cell-Based ELISA Systems:

  • Allows determination of CDK2 phosphorylation directly in cultured cells

  • Multiple normalization options:

    • GAPDH serves as internal positive control

    • Crystal Violet whole-cell staining for cell density normalization

    • Parallel total CDK2 detection for phospho/total ratio determination

  • Facilitates treatment effect studies across different cell lines

• Multi-Parameter Flow Cytometry:

  • Combines DNA content analysis with phospho-specific antibody staining

  • Enables single-cell resolution of phosphorylation status across cell cycle phases

  • Can be integrated with other markers (γH2AX, cyclins) for comprehensive signaling analysis

• Quantitative Phosphoproteomics:

  • Mass spectrometry-based approaches can quantify multiple phosphorylation sites simultaneously

  • Provides unbiased detection of all CDK2 phosphorylation sites

  • Can reveal novel regulatory mechanisms and unexpected phosphorylation events

• Microscopy-Based Approaches:

  • Immunofluorescence with phospho-specific antibodies reveals subcellular localization

  • CDK2 (phospho Thr160) localizes to both cytoplasm and nucleus in human tissues

  • Advanced imaging platforms can quantify phosphorylation intensity at single-cell resolution

These technologies offer complementary approaches to standard Western blotting, enabling more comprehensive analysis of CDK2 phosphorylation dynamics.

How is Phospho-CDK2 (Thr160) implicated in cancer biology and potential therapeutic approaches?

CDK2 activation through Thr160 phosphorylation has significant implications for cancer research:

• Cancer-Specific CDK2 Dependency:

  • Research indicates that "CDK2 activity is largely dispensable for normal development, but it is critically associated with tumor growth in multiple cancer types"

  • This differential requirement creates a potential therapeutic window

• Cell Cycle Dysregulation Patterns:

  • Immunohistochemical analysis reveals distinctive Phospho-CDK2 (Thr160) patterns in cancer tissues

  • In colon cancer samples, phosphorylated CDK2 shows altered localization and expression compared to normal tissue

  • These changes likely contribute to aberrant proliferation

• Therapeutic Targeting Strategies:

  • Small molecule manipulation of CDK2 activation shows research promise

  • Compounds that affect T-loop phosphorylation could provide new therapeutic avenues

  • Specific inhibitors like 3-MB-PP1 can restore normal CDK2 regulatory patterns in experimental systems

• Replication Stress Vulnerability:

  • Cancer cells often experience elevated replication stress

  • The requirement for proper CDK2 regulation during replication stress suggests a vulnerability

  • CDK2 AF/AF cells (lacking inhibitory phosphorylation) accumulate DNA damage during replication arrest

• Pathway Integration:

  • Research demonstrates cross-talk between Akt and CDK2 signaling pathways

  • Akt can phosphorylate CDK2 at T39, providing another regulatory mechanism

  • This interconnection might explain how multiple oncogenic pathways converge on cell cycle dysregulation

Understanding the precise regulation of CDK2 through Thr160 phosphorylation provides insights into cancer biology and highlights potential therapeutic vulnerabilities that could be exploited for targeted treatment approaches.

What are the most effective experimental designs for studying temporal dynamics of CDK2 Thr160 phosphorylation?

To effectively capture the dynamic nature of CDK2 phosphorylation:

• Synchronization Strategies:

  • Serum starvation/release protocols synchronize cells at G0/G1 boundary

  • Hydroxyurea treatment (10 mM for 18 hr) creates S-phase arrested populations

  • Double thymidine block provides tight synchronization at G1/S boundary

  • Research shows phosphorylation on T160 increases during S phase and G2

• Time-Course Sampling Design:

  • Collect samples at strategic time points (typical intervals: 0, 2, 4, 8, 12, 16, 20, 24 hours post-release)

  • For cell cycle studies, include parallel samples for flow cytometry to confirm cell cycle distribution

  • Process all samples identically to maintain phosphorylation comparability

• Multi-Parameter Analysis:

  • Simultaneously track:

    • Thr160 phosphorylation (activation)

    • Tyr15/Thr14 phosphorylation (inhibition)

    • Cyclin binding patterns (E vs. A)

    • DNA synthesis (EdU/BrdU incorporation)

  • Research demonstrates all these parameters change throughout the cell cycle

• Visualization Techniques:

  • Pulse labeling with thymidine analogs tracks replication dynamics

  • Microfluidics-assisted replication track analysis reveals how CDK2 regulation affects DNA synthesis

  • Immunofluorescence microscopy captures spatial dynamics of phosphorylated CDK2

• Quantification Methods:

  • For Western blotting: normalize phospho-CDK2 to total CDK2 for each time point

  • For cell-based assays: multiple normalization options (GAPDH, total protein, cell number)

  • For flow cytometry: calculate mean fluorescence intensity across cell cycle phases