Phospho-CDK1 (Tyr15) Antibody

Basic Research Questions

• What is the biological significance of CDK1 phosphorylation at Tyr15?

  CDK1 (Cyclin-Dependent Kinase 1) phosphorylation at Tyr15 serves as a critical inhibitory mechanism that regulates cell cycle progression. This post-translational modification is essential for maintaining genome integrity and preventing DNA damage during the G2-M phase transition. When phosphorylated at Tyr15, CDK1 remains inactive, preventing premature entry into mitosis and allowing time for DNA repair processes to complete .

  The inhibitory phosphorylation at Tyr15 is particularly important during interphase, where it maintains CDK1 in an inactive state until conditions are appropriate for mitotic entry. This phosphorylation event is part of the G2-M cell-cycle checkpoint arrest mechanism that ensures DNA repair occurs before mitotic entry, thereby preserving genomic stability .

• How is CDK1 Tyr15 phosphorylation regulated during the cell cycle?

  CDK1 Tyr15 phosphorylation is tightly regulated throughout the cell cycle through a balance of kinase and phosphatase activities:

  • Interphase: CDK1 is maintained in an inactive state through phosphorylation at Tyr15 (and Thr14) by the kinases WEE1 and MYT1 .

  • G2/M transition: Dephosphorylation of Tyr15 by the CDC25 family of phosphatases activates CDK1, allowing cell cycle progression into mitosis .

  • Mitotic entry: Once dephosphorylated at Tyr15, and phosphorylated at Thr161 by CDK-activating kinase (CAK), CDK1 becomes fully active and promotes entry into mitosis .

  This regulatory cycle ensures proper timing of mitotic events and prevents premature chromosome condensation and nuclear envelope breakdown .

• What kinases and phosphatases are involved in CDK1 Tyr15 phosphorylation/dephosphorylation?

  The regulation of CDK1 phosphorylation at Tyr15 involves several key enzymes:

  Kinases that phosphorylate Tyr15 (inhibitory):

  • WEE1: Specifically phosphorylates CDK1 at Tyr15

  • MYT1: Dual-specificity kinase that preferentially phosphorylates Thr14 but can also phosphorylate Tyr15

  • c-Abl: For CDK5, rather than WEE family members, c-Abl kinase phosphorylates Tyr15

  Phosphatases that dephosphorylate Tyr15 (activating):

  • CDC25 family phosphatases: Particularly CDC25C, which removes phosphates from Thr14 and Tyr15, triggering CDK1 activation

  Additional regulatory proteins affecting this process include:

  • PTEN: Downregulation increases phospho-WEE1 (Ser642) and decreases phospho-CDK1 (Tyr15)

  • DYRK1A: Mediates phosphorylation of Tyr15 and Thr161 in certain cell types

  • CHEK1: Prevents mitotic entry by inhibiting CDC25 and CDK1 activity

• What detection methods are available for phospho-CDK1 (Tyr15)?

  Several technologies are available for detecting and quantifying phospho-CDK1 (Tyr15) in research settings:

  Detection Method	Sample Type	Sample Volume	Advantages	Applications
  HTRF Kits	Cell-based	16 μL	No-wash assay format, quantitative	Cell signaling studies
  AlphaLISA SureFire Ultra	Cellular lysates	10 μL	Plate-based format, no gels or transfers required	Quantitative detection, compatible with therapeutic antibody research
  Western Blot	Cellular lysates	Varies	Well-established, widely used	Protein expression and phosphorylation status
  Immunohistochemistry (IHC)	Tissue sections	N/A	In situ detection, spatial information	Tissue localization studies
  Immunocytochemistry (ICC/IF)	Fixed cells	N/A	Subcellular localization	Localization studies
  Immunoprecipitation (IP)	Cellular lysates	Varies	Enrichment of target protein	Protein-protein interaction studies
  In vitro kinase assay with MS	Purified proteins	Varies	Direct identification of phosphorylation sites	Mechanistic studies

  For antibody-based detection methods, typical dilutions range from 1:50-1:1,000 depending on the application and specific antibody used .

• What are the common experimental controls when working with phospho-CDK1 (Tyr15) antibodies?

  To ensure reliable results when working with phospho-CDK1 (Tyr15) antibodies, several controls should be implemented:

  • Phosphatase treatment: Treating samples with alkaline phosphatase to remove phosphate groups should eliminate antibody detection, confirming phospho-specificity

  • Cell cycle synchronization: Comparing samples from synchronized cells at different cell cycle stages (e.g., G1/S vs. G2/M) to demonstrate cell cycle-dependent phosphorylation patterns

  • WEE1/MYT1 inhibition: Using specific inhibitors of WEE1 or MYT1 kinases should reduce phospho-CDK1 (Tyr15) signal

  • CDC25 activation/inhibition: Manipulating CDC25 phosphatase activity should inversely correlate with phospho-CDK1 (Tyr15) levels

  • Isotype controls: Using appropriate isotype control antibodies (e.g., Rabbit IgG) to assess non-specific binding

  • Cross-reactivity assessment: Testing for potential cross-reactivity with other phosphorylated CDKs, as some antibodies may detect Tyr15 phosphorylation in related CDKs (CDK2, CDK3, CDK5, and CDK6)

Advanced Research Questions

• How can I optimize phospho-CDK1 (Tyr15) detection in different cellular compartments?

  Optimizing detection of phospho-CDK1 (Tyr15) in different cellular compartments requires careful consideration of several factors:

  For nuclear vs. cytoplasmic phospho-CDK1 detection:

  • Implement subcellular fractionation protocols to separate nuclear and cytoplasmic extracts

  • Use phosphatase inhibitors during sample preparation to preserve phosphorylation status

  • Include compartment-specific markers (e.g., lamin for nuclear fraction, tubulin for cytoplasmic fraction) to validate fractionation quality

  Immunofluorescence optimization:

  • Fix cells with paraformaldehyde (typically 4%) to preserve phosphoepitopes

  • Test multiple permeabilization conditions (e.g., 0.1-0.5% Triton X-100, methanol) as they can affect epitope accessibility

  • Use image-based quantification to compare phospho-CDK1 (Tyr15) levels between compartments

  Research has shown that the phosphorylation status of Tyr15 can differ significantly between cellular compartments, with lower phosphorylation in the cytoplasm compared to the nucleus in certain cancer cells, indicating higher cytoplasmic CDK1 activity . This differential phosphorylation may be critical for understanding CDK1's role in cancer progression.

• What are the challenges in distinguishing between phospho-CDK1 (Tyr15) and other phosphorylated CDKs?

  Distinguishing between phospho-CDK1 (Tyr15) and other phosphorylated CDKs presents several challenges:

  Sequence homology challenges:

  • The region surrounding Tyr15 is highly conserved among CDK family members (CDK1, CDK2, CDK3, CDK5, and CDK6)

  • Some antibodies, like clone M231, detect a phosphopeptide sequence that is conserved across multiple CDKs

  Methodological approaches to improve specificity:

  1. Immunodepletion: Sequentially deplete specific CDKs from lysates to determine contribution to signal

  2. CDK knockdown/knockout validation: Use siRNA or CRISPR to reduce specific CDK expression and assess signal reduction

  3. Mass spectrometry: Use phospho-proteomics to directly identify the specific phosphorylated protein

  4. Combination with CDK-specific antibodies: Use total CDK1 antibodies in combination with phospho-antibodies

  5. In vitro dephosphorylation/phosphorylation: Treat samples with phosphatases followed by specific kinases to verify signal specificity

  Researchers should be aware that antibodies like clone M231 may detect phospho-Tyr15 in multiple CDKs and validate their findings using complementary approaches .

• How does the phosphorylation status of CDK1 at Tyr15 correlate with cancer progression and treatment response?

  The phosphorylation status of CDK1 at Tyr15 has significant implications for cancer biology and therapeutics:

  Cancer progression correlations:

  • Lower phosphorylation at Tyr15 in cancer cells indicates higher CDK1 activity, which is associated with uncontrolled cell division

  • In ovarian cancer, significantly lower phosphorylation of Tyr15 in the cytoplasm compared to the nucleus indicates high cytoplasmic CDK1 activity correlating with cancer growth

  • Dysregulated activation of CDK1 due to altered Tyr15 phosphorylation is considered a hallmark of cancer

  Therapeutic targeting and response:

  • The Wee1/Cdc25A axis regulating CDK1 Tyr15 phosphorylation represents an attractive target for cancer therapy

  • CDK1 knockdown or inhibitor treatment results in inhibition of cell growth via G2/M arrest and apoptosis in ovarian cancer cell lines

  • Monitoring phospho-CDK1 (Tyr15) levels can serve as a biomarker for response to certain cell cycle-targeting therapies

  • Novel approaches involving PROTACs (Proteolysis Targeting Chimeras) targeting CDK1 are being developed

  Understanding the phosphorylation status of CDK1 at Tyr15 in patient samples may help stratify patients for specific therapeutic approaches and predict treatment responses.

• What are the most effective experimental designs for studying CDK1 Tyr15 phosphorylation dynamics during cell cycle progression?

  To effectively study CDK1 Tyr15 phosphorylation dynamics throughout the cell cycle, consider these experimental approaches:

  Cell synchronization strategies:

  • Double thymidine block for G1/S boundary synchronization

  • Nocodazole treatment for mitotic arrest

  • Serum starvation-release for G0/G1 synchronization

  • Time-course sampling after synchronization release to capture dynamics

  Real-time monitoring approaches:

  • Phospho-specific FRET-based biosensors for live-cell imaging

  • Time-lapse immunofluorescence with phospho-specific antibodies

  • Flow cytometry with dual staining for DNA content and phospho-CDK1 (Tyr15)

  Quantitative analysis methods:

  • Western blotting with phospho-CDK1 (Tyr15) antibodies at defined time points

  • AlphaLISA or HTRF assays for high-throughput, quantitative measurement of phosphorylation levels

  • Mass spectrometry-based phosphoproteomics with SILAC or TMT labeling for temporal dynamics

  Perturbation approaches:

  • Chemical inhibition of WEE1/MYT1 kinases or CDC25 phosphatases

  • Genetic manipulation (CRISPR, siRNA) of regulatory proteins

  • DNA damage induction to trigger checkpoint activation and observe effects on CDK1 Tyr15 phosphorylation

  Combining multiple approaches provides the most comprehensive view of CDK1 Tyr15 phosphorylation dynamics during cell cycle progression.

• How can I validate the specificity and sensitivity of phospho-CDK1 (Tyr15) antibodies for my particular experimental setup?

  A comprehensive validation strategy for phospho-CDK1 (Tyr15) antibodies should include:

  Specificity validation:

  1. Phosphatase treatment: Treat samples with alkaline phosphatase to remove the phosphate group at Tyr15; this should eliminate antibody recognition if the antibody is truly phospho-specific

  2. Peptide competition: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides; only the phosphorylated peptide should block antibody binding

  3. CDK1 knockdown/knockout: Reduce CDK1 expression using siRNA or CRISPR and verify signal reduction

  4. WEE1/MYT1 inhibition or overexpression: Manipulate the kinases that phosphorylate CDK1 at Tyr15 and observe expected changes in signal

  Sensitivity assessment:

  1. Dilution series: Create a standard curve using purified phosphorylated and non-phosphorylated CDK1 proteins

  2. Cell cycle manipulation: Compare signals across synchronized cell populations at different cell cycle stages (G1 vs. G2/M)

  3. Detection method comparison: Compare sensitivity across methods (Western blot, ELISA, AlphaLISA, HTRF)

  Cross-reactivity evaluation:

  1. Test on multiple CDK family members: Some antibodies detect phospho-Tyr15 in multiple CDKs (CDK2, CDK3, CDK5, CDK6)

  2. Immunoprecipitation followed by mass spectrometry: Identify all proteins captured by the antibody

  Document all validation steps meticulously to establish confidence in your experimental results.

• What are the current approaches for targeting CDK1 Tyr15 phosphorylation in cancer therapy?

  Several approaches target CDK1 Tyr15 phosphorylation for cancer therapy:

  Direct CDK1 inhibition:

  • Small molecule inhibitors of CDK1 kinase activity

  • PROTACs (Proteolysis Targeting Chimeras) designed to degrade CDK1 protein

  Targeting regulatory enzymes:

  • WEE1 inhibitors: Prevent inhibitory phosphorylation of CDK1 at Tyr15, causing premature mitotic entry and mitotic catastrophe in cancer cells with defective G1 checkpoints

  • Myt1 inhibitors: Block phosphorylation at both Thr14 and Tyr15

  • CDC25 activators/inhibitors: Modulate the phosphatase that removes inhibitory phosphorylation

  Combination approaches:

  • WEE1 inhibitors combined with DNA-damaging agents to exploit synthetic lethality

  • Targeting the entire Wee1/Cdc25A axis for enhanced anti-cancer effects

  • CDK1 inhibition combined with other cell cycle checkpoint inhibitors

  Emerging strategies:

  • Gene therapy approaches to modulate WEE1 or CDC25 expression

  • miRNA-based therapies targeting CDK1 regulation

  • Sensitizing cancer cells with hyperactive CDK1 to conventional therapies by modulating Tyr15 phosphorylation

  These approaches aim to exploit the role of CDK1 Tyr15 phosphorylation in maintaining genome integrity and preventing DNA damage during the cell cycle .

• How can I design in vitro kinase assays to identify novel CDK1 substrates regulated by Tyr15 phosphorylation?

  Designing effective in vitro kinase assays to identify CDK1 substrates affected by Tyr15 phosphorylation requires careful consideration of multiple factors:

  Assay Components and Setup:

  1. Recombinant CDK1 preparation:

     • Use commercially available human Cdk1/cyclin B complex

     • Alternatively, prepare CDK1 variants mimicking phosphorylation states (Y15F vs. wild-type)

  2. Substrate preparation:

     • Purify candidate proteins to homogeneity for in vitro phosphorylation

     • Use protein expression systems that yield non-phosphorylated substrates

  3. Reaction conditions:

     • Buffer: Typically contains Tris-HCl, MgCl₂, DTT, and ATP

     • Include γ-³²P-ATP or non-radioactive ATP for detection

     • Conduct time-course experiments to determine optimal reaction time

  Phosphorylation Site Identification:

  1. SDS-PAGE analysis:

     • Confirm successful phosphorylation by mobility shift

     • Use Phos-tag gels for enhanced separation of phosphorylated proteins

  2. Mass spectrometry analysis:

     • Submit phosphorylated proteins for LC-MS/MS analysis

     • Use phospho-enrichment strategies if needed

  3. Bioinformatic analysis:

     • Compare identified sites with CDK1 consensus motif (Ser/Thr-Pro-X-Lys/Arg)

     • Verify the presence of proline at +1 position and basic residues at +3 position

  Validation Approaches:

  1. Site-directed mutagenesis:

     • Create non-phosphorylatable mutants (S/T to A)

     • Create phosphomimetic mutants (S/T to D/E)

  2. Functional assays:

     • Compare wild-type and mutant proteins in relevant functional assays

     • For example, binding assays for nuclear transport receptors if studying NLS-containing proteins

  Comparing CDK1 with Different Phosphorylation States:

  • Use CDK1 preparations with different Tyr15 phosphorylation states to assess how this modification affects substrate selection and phosphorylation efficiency

  • Compare kinase activity of CDK1 wild-type vs. Y15F mutant (non-phosphorylatable) vs. Y15E mutant (phosphomimetic)

  This comprehensive approach enables identification of bona fide CDK1 substrates and elucidates how Tyr15 phosphorylation affects CDK1's substrate specificity and activity .