Phospho-CDKN1B (Thr187) Antibody

What is the biological significance of p27 Kip1 phosphorylation at Thr187?

Phosphorylation of p27 Kip1 at Thr187 serves as a critical regulatory mechanism that marks the protein for degradation, which is essential for cell cycle progression. P27 Kip1 functions as a negative regulator of G1 progression and has been proposed to function as a possible mediator of TGFβ-induced G1 arrest . As a potent inhibitor of cyclin E- and cyclin A-CDK2 complexes, p27 Kip1 prevents cell cycle advancement until appropriate signals are received .

The phosphorylation at Thr187 is catalyzed by CDK2 and CDK1, which leads to protein ubiquitination and proteasomal degradation . This degradation, triggered by CDK-dependent phosphorylation and subsequent ubiquitination by SCF complexes, is required for the cellular transition from quiescence to the proliferative state . The removal of p27 Kip1's inhibitory effect on CDK2 allows cells to progress through the S phase of the cell cycle.

Interestingly, p27 Kip1 has dual functionality: it can act either as an inhibitor or an activator of cyclin type D-CDK4 complexes, depending on its phosphorylation state and/or stoichiometry . This dual role highlights the complexity of p27 Kip1 regulation and its impact on cell cycle control.

How do Phospho-CDKN1B (Thr187) antibodies differ from regular CDKN1B antibodies?

Phospho-CDKN1B (Thr187) antibodies are specifically designed to detect p27 Kip1 only when it is phosphorylated at the threonine 187 residue, providing a crucial tool for studying this regulatory modification. Unlike regular CDKN1B antibodies that recognize total p27 Kip1 regardless of its phosphorylation status, these phospho-specific antibodies bind exclusively to the phosphorylated epitope around Thr187 .

The specificity of these antibodies is achieved through careful immunization and purification strategies. For example, many phospho-Thr187 antibodies are produced by immunizing animals with synthetic phosphopeptides that encompass the phosphorylated Thr187 residue of human p27 Kip1 . The resulting antibodies are then purified using affinity chromatography with epitope-specific phosphopeptides, and non-phospho-specific antibodies are removed through additional chromatography steps using non-phosphopeptides .

This rigorous production process ensures that the antibodies will only bind to p27 Kip1 when Thr187 is phosphorylated, making them invaluable tools for tracking cell cycle progression, as Thr187 phosphorylation predominantly occurs during late G1 and S phases when p27 Kip1 is targeted for degradation .

What are the standard applications for Phospho-CDKN1B (Thr187) antibodies?

Phospho-CDKN1B (Thr187) antibodies are utilized in several standard research applications:

• Western Blot (WB): This is one of the most common applications, allowing researchers to detect and quantify phospho-Thr187 p27 Kip1 in cell or tissue lysates. Recommended dilutions typically range from 1:500-1:5000, depending on the specific antibody and experimental conditions .

• Immunohistochemistry (IHC): Phospho-Thr187 antibodies can be used to visualize the spatial distribution of phosphorylated p27 Kip1 in tissue sections, providing insights into in vivo regulation. Typical dilutions range from 1:50-1:300 for optimal staining .

• Immunofluorescence (IF): Similar to IHC but utilizing fluorescent detection, IF allows for more sensitive visualization and potential co-localization studies with other proteins. Recommended dilutions usually fall between 1:20-1:200 .

• ELISA (Enzyme-Linked Immunosorbent Assay): Phospho-CDKN1B (Thr187) antibodies can be employed in ELISA formats for quantitative measurement of phosphorylated p27 Kip1 levels in biological samples .

• Flow Cytometry: Although less common, specialized phospho-Thr187 antibodies can be used for flow cytometry analysis to correlate phosphorylation status with cell cycle phases at the single-cell level .

Each application requires specific optimization procedures to ensure accurate and reliable detection of the phosphorylated form, including appropriate controls to validate specificity.

How does the phosphorylation of p27 Kip1 at different sites interact to regulate its function?

P27 Kip1 regulation involves a complex interplay of multiple phosphorylation sites that collectively determine its stability, localization, and activity. This multi-site phosphorylation creates a sophisticated regulatory network:

Thr187 Phosphorylation: Catalyzed primarily by CDK1 and CDK2, Thr187 phosphorylation targets p27 Kip1 for ubiquitination and proteasomal degradation . This modification is critical for cell cycle progression from G1 to S phase.

Ser10 Phosphorylation: This is the major site of phosphorylation in resting cells, occurring at the G0-G1 phase and leading to protein stability . Interestingly, Ser10 phosphorylation is predominant in G0/G1 cells and declines as cells advance to S phase, showing a reciprocal pattern to Thr187 phosphorylation .

Thr198 Phosphorylation: Required for interaction with 14-3-3 proteins, affecting protein localization and stability .

Tyrosine Phosphorylation (Tyr88/Tyr89): Plays a crucial role in binding preferences to CDKs. Research has shown that tyrosine-phosphorylated p27 Kip1 preferentially binds to CDK4, whereas unphosphorylated protein preferentially associates with CDK2 . Additionally, phosphorylation of Tyr88 and Tyr89 has been linked to strong nuclear translocation of p27 Kip1 .

The interaction between these phosphorylation events creates a temporal and spatial regulation mechanism. For example, studies have demonstrated that p27 Ser10 mutants are phosphorylated on Thr187 to the same extent as the wild-type protein, indicating independent regulation of these sites . Similarly, experimental evidence shows that in response to FGF-2 stimulation, pp27Thr187 was detected at 16 hours but not at a 4-hour timepoint, while pp27Ser10 showed the opposite pattern (detected at 4 hours but not at 16 hours) , highlighting the sequential nature of these modifications during cell cycle progression.

What are the key methodological considerations for in vitro phosphorylation assays using Phospho-CDKN1B (Thr187) antibodies?

In vitro phosphorylation assays using Phospho-CDKN1B (Thr187) antibodies require careful methodological consideration to ensure reliable and reproducible results:

• Protein Source and Preparation:

  • Use recombinant p27 Kip1 protein or immunoprecipitated p27 from cell lysates

  • Consider partial purification through heat treatment (90°C for 2 minutes) as p27 is thermostable

  • Filter and dilute using centrifugal units (10K cutoff) to remove small proteins and molecules including nucleotides

• Kinase Selection:

  • Use recombinant Cyclin E/CDK2-GST for Thr187 phosphorylation

  • Ensure kinase activity through parallel assays with known substrates (e.g., histone H1)

• Reaction Conditions:

  • Standard kinase buffer composition: 50 mM Tris/HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, with protease and phosphatase inhibitor cocktails

  • ATP concentration: typically 150 μM

  • Incubation conditions: 30°C for 30 minutes

• Controls:

  • Negative control: reaction without enzyme

  • Positive control: known CDK2 substrate

  • Specificity control: phospho-deficient mutant (T187A)

  • Validation control: lambda phosphatase treatment to remove phosphorylation

• Detection Methods:

  • Western blotting using anti-(pT187)p27 antibody after SDS-PAGE

  • Two-dimensional SDS-PAGE/immunoblotting for resolving different phospho-isoforms

  • Quantification by comparing the phospho-isoforms ratio to the control without enzyme

• Technical Considerations:

  • Stop the reaction by adding SDS-PAGE loading buffer for immediate analysis

  • For 2D analysis, consider stopping the reaction with 6M urea

  • Ensure antibody specificity through appropriate controls (the antibody should not recognize p27 when phosphorylated by other kinases such as ERK2 that target different sites)

By adhering to these methodological considerations, researchers can effectively use in vitro phosphorylation assays to study p27 Thr187 phosphorylation dynamics and regulation.

How can researchers differentiate between cell cycle-dependent changes in phospho-Thr187 levels versus changes in total p27 Kip1 expression?

Distinguishing between cell cycle-dependent changes in phospho-Thr187 levels and changes in total p27 Kip1 expression requires carefully designed experimental approaches:

• Dual Detection Analysis:

  • Perform parallel Western blots using both phospho-Thr187-specific and total p27 Kip1 antibodies

  • Calculate the ratio of phospho-Thr187 to total p27 Kip1 to normalize for expression changes

  • Consider using the same membrane with sequential probing (stripping and reprobing) to ensure accurate comparison

• Cell Cycle Synchronization and Monitoring:

  • Synchronize cells using established methods (serum starvation/restimulation, thymidine block)

  • Verify synchronization by flow cytometry analysis of DNA content

  • Collect samples at defined timepoints throughout the cell cycle

  • Correlate phosphorylation patterns with cell cycle markers

• In Situ Analysis Techniques:

  • Perform dual immunofluorescence staining for both phospho-Thr187 and total p27

  • Combine with cell cycle markers (e.g., PCNA, cyclin A, BrdU incorporation)

  • Analyze at the single-cell level to correlate phosphorylation with cell cycle stage

• Kinase Activity Assessment:

  • Monitor CDK2 activity (responsible for Thr187 phosphorylation) in parallel

  • Use immune complex kinase assays with histone H1 as substrate

  • Compare patterns of CDK2 activity with phospho-Thr187 levels

• Temporal Studies:

  • As demonstrated in research with FGF-2 stimulation, pp27Thr187 was detected at 16 hours but not at 4 hours post-stimulation

  • This time-dependent pattern should be considered when designing experiments

• Quantitative Controls:

  • Use phospho-deficient (T187A) and phospho-mimetic (T187D/E) mutants as controls

  • Include samples treated with proteasome inhibitors to accumulate phosphorylated p27

  • Compare results with established cell cycle models

When analyzing results, researchers should consider that in G0/G1 cells, p27 levels are high but Thr187 phosphorylation is low, while during G1/S transition, Thr187 phosphorylation increases followed by a decrease in total p27 levels due to degradation . This inverse relationship is characteristic of the cell cycle-dependent regulation of p27.

Why might researchers observe weak or no signal when using Phospho-CDKN1B (Thr187) antibodies in Western blots?

Several factors can lead to weak or absent signals when using Phospho-CDKN1B (Thr187) antibodies in Western blots:

• Low Phosphorylation Levels:

  • Thr187 phosphorylation is cell cycle-dependent, with peak levels at G1/S transition

  • Asynchronous cell populations may have too few cells with phosphorylated p27

  • Solution: Synchronize cells or use treatments that promote phosphorylation (e.g., FGF-2)

• Rapid Protein Degradation:

  • Phospho-Thr187 p27 is rapidly targeted for ubiquitination and degradation

  • The protein may be degraded during sample preparation

  • Solution: Treat cells with proteasome inhibitors (e.g., MG132) prior to lysis

• Phosphatase Activity:

  • Phosphate groups can be removed by phosphatases during cell lysis and sample preparation

  • Solution: Include comprehensive phosphatase inhibitor cocktails in all buffers

• Suboptimal Antibody Conditions:

  • Antibody concentration: Recommended dilutions range from 1:500-1:5000 depending on the antibody

  • Incubation conditions: Optimize temperature and duration

  • Blocking agents: Some blocking agents may mask the epitope

  • Solution: Perform a dilution series and test different blocking agents

• Epitope Accessibility Issues:

  • The phospho-epitope may be masked by protein interactions or conformational changes

  • Solution: Test different denaturing conditions or perform immunoprecipitation before Western blotting

• Sample Preparation Problems:

  • Heat stability: p27 is thermostable and can be partially purified by heat treatment (90°C for 2 min)

  • Protein extraction method: Different lysis buffers may affect phospho-epitope preservation

  • Solution: Compare different extraction methods and consider heat purification

• Technical Issues:

  • Transfer efficiency: Phosphorylated proteins may transfer differently

  • Membrane type: PVDF membranes are often preferred for phospho-epitopes

  • Solution: Verify transfer with reversible protein stains and optimize transfer conditions

• Antibody Specificity:

  • The antibody may recognize p27 only when phosphorylated at threonine 187

  • Solution: Include positive controls such as in vitro phosphorylated p27 or lysates from cells treated to enhance Thr187 phosphorylation

If troubleshooting efforts don't improve signal detection, researchers should consider validating the antibody using alternative sources or detection methods.

What controls are essential when using Phospho-CDKN1B (Thr187) antibodies for immunohistochemistry or immunofluorescence?

When using Phospho-CDKN1B (Thr187) antibodies for immunohistochemistry (IHC) or immunofluorescence (IF), the following controls are essential to ensure reliable and interpretable results:

• Antibody Specificity Controls:

  • Phospho-deficient control: Use samples expressing T187A mutant p27 (unable to be phosphorylated at this site)

  • Phosphatase treatment control: Treat duplicate sections with lambda phosphatase to remove phosphorylation

  • Peptide competition: Pre-incubate antibody with the phospho-peptide immunogen to block specific binding

• Biological Controls:

  • Positive tissue/cell control: Include samples known to contain high levels of phospho-Thr187 p27 (e.g., proliferating tissues, cells in S phase)

  • Negative tissue/cell control: Include samples expected to have low/no phospho-Thr187 (e.g., quiescent cells, G0/G1 arrested cells)

  • Treatment-induced control: Compare tissues/cells with and without treatments that induce cell cycle progression (e.g., FGF-2 treatment in corneal endothelial cells)

• Technical Controls:

  • Primary antibody omission: Process sections without primary antibody to assess non-specific binding of detection systems

  • Isotype control: Use non-specific antibody of the same isotype and concentration

  • Dilution series: Test multiple antibody dilutions (typical range: 1:50-1:300 for IHC; 1:20-1:200 for IF)

• Validation Controls:

  • Dual labeling: Co-stain with antibodies against total p27 and cell cycle markers

  • Cross-validation: Compare results with another phospho-Thr187 antibody from a different source

  • Correlation with Western blot: Confirm IHC/IF findings with parallel Western blot analysis

• Sample-Specific Controls:

  • Tissue-specific autofluorescence control (for IF)

  • Endogenous peroxidase blocking verification (for IHC with HRP detection)

  • Antigen retrieval optimization: Test multiple methods as phospho-epitopes may require specific retrieval conditions

An excellent example from the literature demonstrates the importance of these controls: when corneal endothelial cells were stained with anti-pp27Thr187 antibody, positive staining was only observed in cells maintained in medium with FGF-2 (which stimulates cell cycle progression), but not in cells maintained without FGF-2 . This biological control confirms the specificity of the antibody for the cell cycle-dependent phosphorylation event.

How can researchers quantitatively assess changes in p27 Thr187 phosphorylation across experimental conditions?

Quantitative assessment of p27 Thr187 phosphorylation changes requires rigorous methodological approaches to ensure accurate and reproducible results:

For example, in studies with FGF-2 stimulation of corneal endothelial cells, researchers observed differential phosphorylation patterns at specific timepoints (pp27Thr187 at 16 hours vs. pp27Ser10 at 4 hours) , demonstrating how quantitative temporal analysis can reveal important regulatory mechanisms.

How can researchers use phospho-mimetic and phospho-deficient p27 Kip1 mutants to complement studies with Phospho-CDKN1B (Thr187) antibodies?

Phospho-mimetic and phospho-deficient p27 Kip1 mutants serve as powerful tools to complement antibody-based detection approaches:

• Types of Mutants and Their Properties:

  • Phospho-deficient (T187A): Threonine replaced with alanine, cannot be phosphorylated

  • Phospho-mimetic (T187D/E): Threonine replaced with aspartic acid or glutamic acid, which mimic the negative charge of phosphorylation

  • These mutants allow researchers to study the functional consequences of permanent "off" or "on" states of Thr187 phosphorylation

• Validation of Antibody Specificity:

  • Phospho-deficient T187A mutant should not be recognized by phospho-Thr187 antibodies

  • This provides a critical negative control to confirm antibody specificity

  • Interestingly, some phospho-mimetic mutants (S10D/E) can be recognized by certain phospho-specific antibodies, indicating successful mimicry of the phosphorylated state

• Functional Studies:

  • Compare cell cycle progression, proliferation rates, and protein stability between wild-type and mutant p27

  • Assess interaction with binding partners (e.g., Skp2) using co-immunoprecipitation

  • Studies have shown that p27 Ser10 mutants are phosphorylated on Thr187 to the same extent as wild-type protein, revealing independent regulation of these sites

• Localization Studies:

  • Use immunofluorescence to determine how phosphorylation affects subcellular localization

  • Research has demonstrated that phosphorylation of tyrosine residues (Y88/Y89) affects nuclear translocation of p27 Kip1

  • Similar approaches can be applied to study Thr187 phosphorylation effects

• Protein-Protein Interaction Analysis:

  • Employ mutations to investigate how phosphorylation affects binding to cyclins, CDKs, and ubiquitin ligase components

  • For example, mutation of Ser10 to Ala or Asp did not affect the ability of p27 to associate with Skp2, while the T187A mutant failed to co-precipitate labeled Skp2

• Rescue Experiments:

  • In cells where endogenous p27 has been knocked down, introduce wild-type or mutant versions to assess functional rescue

  • This approach can reveal the importance of Thr187 phosphorylation in specific cellular contexts

• Combined Approaches:

  • Use phospho-specific antibodies to detect endogenous protein while simultaneously expressing tagged mutant versions

  • This allows simultaneous observation of natural regulation and mutant effects

• Cancer-Associated Mutations:

  • Study cancer-associated p27 mutations that affect phosphorylation sites

  • For example, the G9R mutation creates a new consensus sequence for kinases, causing phosphorylation at S12, which affects protein function

By integrating antibody-based detection with mutational analysis, researchers can gain comprehensive insights into the functional significance of p27 Thr187 phosphorylation in various biological contexts.

What techniques can be used to study the temporal dynamics of p27 Thr187 phosphorylation during cell cycle progression?

Studying the temporal dynamics of p27 Thr187 phosphorylation during cell cycle progression requires specialized techniques that capture both timing and magnitude of phosphorylation events:

• Synchronized Cell Systems:

  • Serum Starvation/Restimulation: Arrest cells in G0/G1 by serum deprivation, then release with serum addition

  • Thymidine Block: Single or double thymidine block to synchronize cells at G1/S boundary

  • Nocodazole Treatment: Arrest cells in M phase, then release

  • Collect samples at regular intervals post-synchronization release

  • Verify synchronization efficiency using flow cytometry analysis of DNA content

• Live-Cell Imaging Approaches:

  • Develop phospho-sensors using fluorescence resonance energy transfer (FRET) technology

  • Use fluorescently-tagged p27 constructs combined with cell cycle markers

  • Monitor phosphorylation events in real-time at single-cell resolution

• Pulse-Chase Analysis:

  • Metabolically label cells with [32P]orthophosphate

  • Immunoprecipitate p27 and analyze phosphorylation by autoradiography

  • This approach allows detection of newly phosphorylated protein

  • Normalize to the amount of immunoprecipitated protein to account for expression changes

• Sequential Sampling with Growth Factor Stimulation:

  • Treat quiescent cells with growth factors known to promote cell cycle entry

  • Collect samples at defined timepoints (e.g., 4, 8, 12, 16, 20, 24 hours)

  • Studies with FGF-2 have shown that pp27Thr187 was detected at 16 hours but not at 4 hours post-stimulation, while pp27Ser10 showed the opposite pattern

• Phosphorylation Site-Specific Kinase Activity Assays:

  • Extract cellular lysates at different cell cycle phases

  • Test their ability to phosphorylate recombinant p27 in vitro

  • Research has shown that kinase activity for Ser10 phosphorylation is elevated in G0/G1 and declines as cells advance to S phase

• Quantitative Mass Spectrometry:

  • Use stable isotope labeling (SILAC) to quantify phosphopeptides

  • Perform targeted mass spectrometry focusing on the Thr187-containing peptide

  • This approach provides absolute quantification of phosphorylation stoichiometry

• Correlation with CDK2 Activity:

  • Measure CDK2 activity using histone H1 kinase assays

  • Correlate with Thr187 phosphorylation levels

  • This establishes the relationship between kinase activation and substrate phosphorylation

• Mathematical Modeling:

  • Develop computational models based on experimental data

  • Predict phosphorylation dynamics under various conditions

  • Test model predictions experimentally for validation

These approaches collectively provide a comprehensive view of how p27 Thr187 phosphorylation is regulated throughout the cell cycle, revealing the precise timing of this critical regulatory event in relation to other cell cycle milestones.

How can researchers address data variability when measuring phospho-Thr187 levels across different cell types or tissues?

Addressing data variability in phospho-Thr187 measurements across different cellular contexts requires systematic approaches:

Research has demonstrated that different cell types show distinctive patterns of p27 regulation. For example, primary T lymphocytes show different p27 phosphorylation dynamics compared to cancer cell lines , highlighting the importance of considering cellular context when interpreting phosphorylation data.

What are the challenges in interpreting conflicting data on p27 Thr187 phosphorylation in cancer studies?

Interpreting conflicting data on p27 Thr187 phosphorylation in cancer research presents several significant challenges:

A noteworthy example comes from research on a cancer-associated CDKN1B mutation (G9R), which creates a new consensus sequence for basophilic kinases, causing phosphorylation at S12 . This unexpected phosphorylation reduces p27 Kip1-dependent cyclin-dependent kinase inhibition, enhances protein degradation, and reduces its anticancer activities . This illustrates how cancer-specific mutations can create novel regulatory mechanisms that confound standard interpretations of p27 phosphorylation data.