Phospho-CCND2 (T280) Antibody

What is CCND2 and what role does the Thr280 phosphorylation site play?

CCND2 (Cyclin D2) is a G1/S-specific cyclin that forms active complexes with CDK4 and CDK6 to drive the G1-S phase transition of the cell cycle. This complex phosphorylates tumor suppressor "pocket proteins" including retinoblastoma (RB), p107, and p130. The phosphorylation of these proteins leads to their release from E2F transcription factors, allowing the transcription of key proteins required for cell cycle progression and proliferation .

Thr280 is a critical threonine phosphorylation site located in the C-terminal region of CCND2 (within amino acids 240-289). This site functions as a phosphodegron, mediating the degradation of cyclin D2. When Thr280 is phosphorylated by glycogen synthase kinase 3β (GSK-3β), it signals for ubiquitin-mediated proteasomal degradation of CCND2 . Mutations at this site, particularly Thr280Ala, prevent this phosphorylation and lead to CCND2 protein stabilization, resulting in increased levels of the protein and enhanced cell proliferation .

How does Phospho-CCND2 (T280) Antibody differ from other CCND2 antibodies?

Phospho-CCND2 (T280) Antibody specifically detects endogenous levels of Cyclin D2 protein only when phosphorylated at the Thr280 residue . This specificity enables researchers to distinguish between the phosphorylated and non-phosphorylated forms of CCND2, which is crucial for understanding its regulation and function in normal and disease states.

Unlike general CCND2 antibodies that detect the protein regardless of its phosphorylation status, the Phospho-CCND2 (T280) Antibody provides information specifically about the fraction of CCND2 that is targeted for degradation. This allows researchers to gain insights into the dynamics of CCND2 regulation through phosphorylation and subsequent protein turnover .

What are the primary applications for Phospho-CCND2 (T280) Antibody?

The Phospho-CCND2 (T280) Antibody can be used in multiple research applications including:

• Western Blot (WB): For quantitative analysis of phospho-CCND2 levels in cell or tissue lysates

• Immunohistochemistry (IHC): For detecting phosphorylated CCND2 in tissue sections

• Immunofluorescence (IF): For visualizing the subcellular localization of phosphorylated CCND2

• ELISA: For high-throughput quantitative analysis of phospho-CCND2 levels

Recommended dilution ranges for each application are:

• WB: 1:500-1:2000

• IHC: 1:100-1:300

• IF: 1:50-200

• ELISA: 1:10000

What diseases are associated with alterations in CCND2 Thr280 phosphorylation?

Mutations affecting the Thr280 site of CCND2 have been implicated in several pathological conditions:

• Acute Myeloid Leukemia (AML): CCND2 mutations, particularly those affecting Thr280, have been found in approximately 15% of AML patients with t(8;21) translocation, making them the third most frequent mutational events in these patients .

• Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH): De novo mutations in CCND2 clustered around Thr280 have been identified in patients with this neurodevelopmental disorder characterized by brain overgrowth .

These mutations typically result in a more stable CCND2 protein that resists degradation, leading to increased cell proliferation and contributing to disease pathogenesis .

How can Phospho-CCND2 (T280) Antibody be used to study cell cycle dysregulation in cancer?

Phospho-CCND2 (T280) Antibody provides valuable insights into cell cycle dysregulation in cancer through several advanced research approaches:

• Comparative analysis of phospho-CCND2 levels: Researchers can quantify the relative levels of phosphorylated CCND2 in tumor versus normal tissues to understand alterations in CCND2 turnover. Decreased phospho-CCND2 (T280) relative to total CCND2 may indicate impaired degradation mechanisms contributing to oncogenic cell proliferation .

• Monitoring therapy response: Changes in phospho-CCND2 levels can serve as a biomarker for response to therapies targeting cell cycle regulation, particularly those affecting the PI3K-AKT-GSK3β pathway which regulates CCND2 phosphorylation .

• Mutation effect studies: In patients with CCND2 Thr280 mutations (particularly Thr280Ala), using the phospho-specific antibody alongside total CCND2 antibodies can confirm the functional impact of these mutations on phosphorylation status in patient samples .

• Cell cycle checkpoint analysis: Combined with markers of cell cycle progression, phospho-CCND2 antibody can help elucidate how alterations in CCND2 degradation affect G1/S transition rates in cancer cells .

What molecular mechanisms regulate CCND2 Thr280 phosphorylation?

The phosphorylation of CCND2 at Thr280 is regulated through a complex signaling network:

• GSK-3β-mediated phosphorylation: Glycogen synthase kinase 3β directly phosphorylates CCND2 at Thr280, creating a phosphodegron that marks the protein for proteasomal degradation .

• PI3K-AKT pathway modulation: The PI3K-AKT pathway inhibits GSK-3β activity through phosphorylation. When this pathway is activated (through mutations in PIK3CA, PIK3R2, or AKT3), GSK-3β activity decreases, resulting in reduced CCND2 Thr280 phosphorylation and consequently increased CCND2 stability .

• Cell cycle-dependent regulation: The phosphorylation status of CCND2 Thr280 varies throughout the cell cycle, with mechanisms to ensure appropriate degradation after cells progress through the G1/S transition .

• Feedback mechanisms: CCND2-CDK4/6 complexes participate in feedback loops that can impact CCND2 phosphorylation and stability, creating a dynamic regulatory system .

Understanding these mechanisms provides important insights into how CCND2 levels are precisely controlled during normal cell cycle progression and how dysregulation contributes to disease states.

How does the Thr280Ala mutation affect CCND2 function in experimental models?

Research using experimental models has provided significant insights into the functional consequences of the Thr280Ala mutation in CCND2:

• Enhanced protein stability: The Thr280Ala mutation renders CCND2 resistant to proteasomal degradation compared to wild-type protein in vitro, resulting in accumulation of the mutant protein .

• Increased cell proliferation: In in utero electroporation experiments in mouse embryonic brain, expression of CCND2 Thr280Ala resulted in significantly more proliferating progenitors (6.41±0.22% of cells positive for phosphohistone H3) compared to wild-type CCND2 (2.83±0.37%), demonstrating the mutation's pro-proliferative effect .

• Reduced cell cycle exit: Cells expressing the mutant CCND2 showed a smaller fraction exiting the cell cycle compared to cells expressing wild-type CCND2 .

• Enhanced Rb phosphorylation: CCND2 Thr280Ala mutation leads to increased phosphorylation of the retinoblastoma protein (Rb), a key event promoting cell cycle progression .

These findings demonstrate that the Thr280Ala mutation creates a hyperactive form of CCND2 that drives excessive cell proliferation, providing mechanistic insight into how this mutation contributes to diseases like AML and MPPH syndrome.

What is the relationship between CCND2 Thr280 phosphorylation and other cell cycle regulators?

CCND2 Thr280 phosphorylation status affects and is affected by a network of cell cycle regulators:

Understanding these relationships is crucial for developing targeted therapies for conditions with dysregulated cell cycle control.

What are the optimal sample preparation methods for Phospho-CCND2 (T280) detection?

To achieve optimal detection of phosphorylated CCND2 at Thr280, researchers should consider the following sample preparation guidelines:

• Rapid sample processing: Phosphorylation states can change rapidly after sample collection. Process samples immediately or snap-freeze in liquid nitrogen to preserve phosphorylation status .

• Phosphatase inhibitors: Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, and β-glycerophosphate) in all lysis and extraction buffers to prevent dephosphorylation during sample preparation .

• Protein extraction protocol:

  • For cell lines: Use buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, supplemented with protease and phosphatase inhibitors .

  • For tissue samples: Homogenize in the same buffer using mechanical disruption while keeping samples cold.

• Western blot sample treatment: Heat protein samples to 70°C for 10 minutes with appropriate sample buffer before loading on 4-12% Bis-Tris gels .

• Blocking conditions: Block membranes for one hour at room temperature with 5% dried skimmed milk in PBS with 0.1% Tween-20 .

Following these guidelines will help maintain the integrity of the phosphorylation status and improve detection specificity.

What controls should be included when using Phospho-CCND2 (T280) Antibody?

Proper experimental controls are essential for accurate interpretation of results when using Phospho-CCND2 (T280) Antibody:

• Positive controls:

  • Cell lines known to express phosphorylated CCND2 (e.g., proliferating hematopoietic cells)

  • Recombinant phosphorylated CCND2 protein

  • Cells treated with phosphatase inhibitors to maximize phosphorylation levels

• Negative controls:

  • Samples treated with lambda phosphatase to remove phosphorylation

  • Cells expressing CCND2 Thr280Ala mutant (which cannot be phosphorylated at this site)

  • Samples from CCND2 knockout models

• Specificity controls:

  • Parallel blots with antibodies against total CCND2 to compare with phospho-specific signal

  • Peptide competition assays using the immunizing phosphopeptide

• Loading controls:

  • β-actin (1:10,000 dilution) or other suitable housekeeping proteins

  • Total protein staining methods for normalization

Including these controls will significantly enhance the reliability and interpretability of experiments using Phospho-CCND2 (T280) Antibody.

How can Phospho-CCND2 (T280) Antibody be validated for specific research applications?

Thorough validation of Phospho-CCND2 (T280) Antibody for specific research applications ensures reliable and interpretable results:

• Western blot validation:

  • Compare signal between wild-type cells and those expressing CCND2 Thr280Ala mutant

  • Verify single band of appropriate molecular weight (~33-36 kDa)

  • Confirm signal reduction after phosphatase treatment

  • Perform siRNA knockdown of CCND2 to confirm specificity

• Immunohistochemistry/Immunofluorescence validation:

  • Compare staining patterns with total CCND2 antibody

  • Verify appropriate subcellular localization (primarily nuclear)

  • Confirm specificity using tissues from CCND2 knockout models

  • Validate signal reduction in phosphatase-treated sections

• Cross-reactivity assessment:

  • Test antibody against other D-type cyclins (CCND1, CCND3) to ensure specificity

  • Evaluate reactivity across relevant species (human, mouse, rat)

• Application-specific validation:

  • For electroporation studies: Confirm expression using immunostaining of nuclear CCND2 in GFP-positive cells compared to untransfected cells

  • For proliferation assays: Validate correlation between antibody signal and other proliferation markers like phosphohistone H3

Proper validation increases confidence in experimental results and facilitates accurate interpretation of biological phenomena.

What are common technical challenges when working with Phospho-CCND2 (T280) Antibody and how can they be addressed?

Researchers may encounter several technical challenges when working with Phospho-CCND2 (T280) Antibody:

• Weak or absent signal:

  • Increase antibody concentration within recommended range (WB: 1:500-1:2000)

  • Extend primary antibody incubation time or temperature

  • Ensure sample contains adequate amounts of phosphorylated protein

  • Verify phosphatase inhibitors were included during sample preparation

• High background:

  • Optimize blocking conditions (5% dried skimmed milk in PBS with 0.1% Tween-20)

  • Increase washing duration and frequency

  • Reduce antibody concentration

  • Try alternative blocking agents (BSA instead of milk)

• Non-specific bands in Western blot:

  • Increase blocking stringency

  • Optimize antibody dilution

  • Consider using gradient gels for better separation

  • Perform peptide competition assays to identify specific bands

• Poor reproducibility:

  • Standardize lysate preparation methods

  • Maintain consistent sample handling time

  • Use freshly prepared buffers with phosphatase inhibitors

  • Document and control for cell culture conditions that affect phosphorylation status

Addressing these challenges through methodical optimization will improve experimental outcomes and data quality.

How should researchers interpret discrepancies between phospho-specific and total CCND2 antibody results?

Discrepancies between phospho-specific and total CCND2 antibody results can provide valuable biological insights if properly interpreted:

• High total CCND2 with low phospho-CCND2:

  • May indicate mutations affecting the Thr280 site (e.g., Thr280Ala)

  • Could suggest activation of pathways that inhibit GSK-3β activity

  • May reflect active PI3K-AKT signaling that stabilizes CCND2

• Temporal discrepancies during cell cycle:

  • Normal as phosphorylation status changes throughout the cell cycle

  • Compare with other cell cycle phase markers to interpret correctly

  • Consider examining multiple timepoints to capture dynamic changes

• Spatial discrepancies in tissue/subcellular localization:

  • Phosphorylation may affect protein localization

  • Different pools of CCND2 may have different phosphorylation status

  • Consider co-localization studies with markers of cellular compartments

• Treatment-induced changes:

  • Discrepancies following drug treatment may indicate mechanism of action

  • Rapid changes in phospho-CCND2 without changes in total CCND2 suggest direct effects on the phosphorylation machinery

  • Delayed changes in both might indicate transcriptional regulation

Understanding these patterns helps researchers distinguish between technical artifacts and meaningful biological phenomena.

What approaches can be used to study the functional consequences of altered CCND2 Thr280 phosphorylation?

Several experimental approaches can elucidate the functional impact of altered CCND2 Thr280 phosphorylation:

• Mutation studies:

  • Compare effects of wild-type, phosphodeficient (Thr280Ala), and phosphomimetic (Thr280Asp) CCND2 in cellular models

  • Measure proliferation, cell cycle progression, and downstream signaling

• In vivo modeling:

  • In utero electroporation of mutant versus wild-type CCND2 into embryonic mouse brain

  • Analyze effects on neural progenitor proliferation using markers like phosphohistone H3

  • Quantify percentage of cells in M-phase (PH3-positive) to assess proliferative effects

• Cell cycle analysis:

  • Flow cytometry to quantify cell cycle distribution

  • BrdU incorporation to measure S-phase entry

  • Time-lapse imaging to track cell division rates

• Biochemical assays:

  • Co-immunoprecipitation to assess interaction with CDK4/6

  • Kinase assays to measure activity of CCND2-CDK4/6 complexes

  • Quantification of Rb phosphorylation as a downstream readout

• Protein stability measurements:

  • Cycloheximide chase assays to compare degradation rates

  • Ubiquitination assays to assess proteasomal targeting

  • Pulse-chase experiments to measure protein half-life

These approaches provide complementary data on how Thr280 phosphorylation affects CCND2 function in diverse biological contexts.

How can Phospho-CCND2 (T280) Antibody be used in patient sample analysis?

Phospho-CCND2 (T280) Antibody offers valuable applications for analyzing patient samples across several clinical contexts:

• AML patient stratification:

  • Detect phosphorylation status of CCND2 in bone marrow samples from t(8;21) AML patients

  • Correlate with mutational status of CCND2 Thr280 residue

  • Associate with clinical outcomes and treatment response

• Tissue microarray analysis:

  • Quantify phospho-CCND2 levels across tumor and normal tissues

  • Correlate with proliferation markers and patient outcomes

  • Compare with total CCND2 levels to identify potential dysregulation

• Diagnostic applications:

  • Aid in identifying cases with likely CCND2 mutations

  • Screen for potential PI3K-AKT pathway hyperactivation when phospho-CCND2 levels are reduced

  • Monitor disease progression through serial sampling

• Treatment response monitoring:

  • Assess changes in phospho-CCND2 levels following treatment with cell cycle-directed therapies

  • Monitor effects of PI3K inhibitors or other targeted therapies on CCND2 regulation

  • Identify potential resistance mechanisms

Researchers working with patient samples should optimize protocols for specific sample types and validate antibody performance in relevant clinical specimens.

What is the potential of targeting CCND2 phosphorylation in therapeutic development?

The critical role of CCND2 Thr280 phosphorylation in regulating protein stability and cell proliferation presents several therapeutic opportunities:

• Direct targeting strategies:

  • Development of compounds that enhance GSK-3β-mediated phosphorylation of CCND2

  • Small molecules that promote degradation of phosphorylation-resistant CCND2 mutants

  • Proteolysis-targeting chimeras (PROTACs) to induce CCND2 degradation independent of phosphorylation status

• Indirect targeting approaches:

  • Inhibitors of the PI3K-AKT pathway to enhance GSK-3β activity and promote CCND2 degradation

  • CDK4/6 inhibitors to block activity of stabilized CCND2-CDK4/6 complexes

  • Combination therapies targeting multiple nodes in the CCND2 regulatory network

• Biomarker-guided therapy:

  • Use of phospho-CCND2 status to identify patients likely to respond to cell cycle-directed therapy

  • Monitoring phospho-CCND2/total CCND2 ratio as a pharmacodynamic marker for therapy response

  • Identifying synthetic lethal interactions specific to cells with altered CCND2 stability

• Disease-specific considerations:

  • In AML with t(8;21), cell cycle-directed therapy may provide particular benefit given the frequency of CCND2 mutations

  • For MPPH syndrome, early developmental targeting of PI3K-AKT-CCND2 axis might provide therapeutic benefit

These approaches could lead to novel therapeutic strategies for diseases characterized by dysregulated CCND2 activity.