Phospho-CHEK1 (Ser280) Antibody

What is the biological significance of CHEK1 Ser280 phosphorylation in normal cell cycle progression?

Phosphorylation of CHEK1 at Ser280 plays a critical role in cell cycle regulation, particularly during mitosis. Research has demonstrated that CHEK1 is maximally phosphorylated on S280 during mitosis, suggesting this modification has important functions in controlling cellular division . The phosphorylation state of CHEK1 at Ser280 regulates its subcellular localization, with evidence showing that phosphorylated CHEK1 (Ser280) translocates from the cytoplasm to the nucleus in response to serum stimulation .

Experimental evidence using phospho-mutants provides strong support for this regulatory mechanism:

• Nonphosphorylated mutant (S280A) fails to localize to the nucleus

• Phosphomimetic mutant (S280E) shows enhanced nuclear accumulation

This phosphorylation-dependent translocation appears to be a key regulatory mechanism for CHEK1 function across multiple cell lines, including RPE1, U2OS, and HeLa cells .

Which kinases are responsible for phosphorylating CHEK1 at Ser280?

Multiple kinases have been identified as capable of phosphorylating CHEK1 at Ser280, with their activity appearing to be cell-type and context-dependent:

PIM2 has been particularly well-characterized as controlling CHEK1 S280 phosphorylation during mitosis. This creates a regulatory pathway where PIM2 phosphorylates CHEK1 at S280, which then enables CHEK1 to phosphorylate PLK1 at T210, activating PLK1 during mitotic progression .

What detection methods are available for studying Phospho-CHEK1 (Ser280)?

Researchers have multiple validated methods for detecting and studying CHEK1 phosphorylated at Ser280:

• Western Blot Analysis: Using specific antibodies that recognize only the phosphorylated form of CHEK1 at Ser280 . Recommended dilutions range from 1:500-1:2000 .

• Immunofluorescence/Immunocytochemistry: For visualizing subcellular localization of phosphorylated CHEK1. Recommended dilutions are 1:100-1:500 . This method has been crucial in demonstrating nuclear translocation following Ser280 phosphorylation .

• ELISA-Based Detection:

  • Sandwich ELISA kits using anti-pan CHEK1 antibody coated plates with rabbit anti-CHEK1 (Ser280) for detection

  • Allows for semi-quantitative measurement of phosphorylated CHEK1 in cell lysates and tissue samples

• Immunohistochemistry: For tissue section analysis, with recommended dilutions of 1:50-1:500 .

Each method requires proper validation, including appropriate controls. For antibody-based approaches, phospho-peptide competition assays have been used to confirm specificity .

How can researchers validate the specificity of Phospho-CHEK1 (Ser280) antibodies?

Validating phospho-specific antibodies is critical for ensuring reliable experimental results. Based on published methodologies, researchers should consider the following approaches:

• Phosphopeptide Competition Assay: Pre-incubate the antibody with:

  • Phosphopeptide pS280 corresponding to Ser280-phosphorylated CHEK1 (should block signal)

  • Non-phosphorylated peptide S280 (should not block signal)

  • Phosphopeptides for other sites within CHEK1 (should not block signal)

• RNA Interference Control: Deplete endogenous CHEK1 using siRNA to confirm signal specificity:

  • Reduction in both immunoblotting and immunocytochemistry signals should be observed

• Phosphatase Treatment: Treat samples with lambda-phosphatase:

  • Should abolish or significantly reduce phospho-specific signals

• Mutant Expression: Replace endogenous CHEK1 with phospho-mutants:

  • S280A (non-phosphorylatable) should show reduced antibody recognition

  • S280E (phosphomimetic) should maintain recognition

• Inducible Expression Systems: Use Tet-On systems combined with endogenous CHEK1 depletion to evaluate mutant forms in a controlled manner .

How does the PIM2-CHEK1(S280)-PLK1(T210) signaling pathway regulate mitosis?

The PIM2-CHEK1(S280)-PLK1(T210) signaling pathway represents a novel regulatory mechanism for mitotic progression. This pathway functions through a sequential phosphorylation cascade:

• PIM2 Kinase Activity: PIM2 phosphorylates CHEK1 at Ser280 during mitosis

• CHEK1 Activation: Phosphorylation at S280 enables CHEK1 to interact with and phosphorylate PLK1

• PLK1 Activation: CHEK1 directly phosphorylates PLK1 at T210, a critical activation site

• Mitotic Regulation: Activated PLK1 then promotes mitotic progression

Key experimental evidence supporting this pathway includes:

• Co-expression of PIM2 and wild-type CHEK1 induces significant increases in PLK1 T210 phosphorylation

• This effect is abolished when S280A mutant CHEK1 is expressed instead of wild-type CHEK1

• In vitro kinase assays demonstrate that recombinant CHEK1 directly phosphorylates PLK1 on T210

• PIM2 alone cannot phosphorylate PLK1 directly

Interestingly, while S280 phosphorylation is necessary for CHEK1's ability to phosphorylate PLK1, it doesn't appear to alter CHEK1's catalytic activity directly. Instead, it likely modifies CHEK1's capacity to interact with PLK1 through changes in localization or protein-protein interactions .

What is the relationship between CHEK1 Ser280 phosphorylation and other CHEK1 phosphorylation sites during DNA damage response?

CHEK1 phosphorylation represents a complex regulatory network with different sites serving distinct functions in the DNA damage response (DDR). The interplay between these phosphorylation events is particularly interesting:

• Primary DDR Sites: ATR primarily phosphorylates CHEK1 at Ser345 and Ser317 during DNA damage response

• Ser280 in Normal Conditions: Under normal cell cycle conditions, Ser280 phosphorylation promotes cell cycle progression through:

  • Interaction with CHEK1-S (a splice variant of CHEK1)

  • CHEK1-S binding inhibits normal CHEK1 activity

• Dissociation Mechanism: During DNA damage:

  • ATR phosphorylates CHEK1 at Ser345/Ser317

  • This phosphorylation disrupts the interaction between CHEK1 and CHEK1-S

  • Dissociation releases CHEK1 from inhibition by CHEK1-S

• Regulation of Dissociation: The CHEK1-CHEK1-S dissociation during DNA damage is dependent on:

  • ATR activity (inhibition of ATR prevents dissociation)

  • Phosphorylation at Ser345/Ser317 (S345A/S317A mutant does not dissociate from CHEK1-S)

This presents a model where Ser280 phosphorylation helps regulate normal cell cycle progression, while ATR-mediated phosphorylation at Ser345/Ser317 serves to override this regulation during DNA damage, allowing CHEK1 activation and cell cycle arrest.

What methodological considerations are important when studying CHEK1 phosphorylation during different cell cycle phases?

Studying CHEK1 phosphorylation across different cell cycle phases requires careful experimental design:

• Cell Synchronization Techniques:

  • Thymidine block after serum starvation for G1/S boundary

  • Nocodazole treatment for mitotic arrest

  • Monitor synchronization efficiency using flow cytometry

• Temporal Resolution:

  • Use time-course experiments following release from synchronization

  • Collect samples at regular intervals to capture dynamic phosphorylation changes

• Phosphorylation Site Specificity:

  • Use phospho-specific antibodies validated for the site of interest

  • Confirm antibody specificity using phospho-mutants (S280A) and phosphatase treatment

• Context-Dependent Regulation:

  • Different kinases may phosphorylate Ser280 in different contexts:

    • PIM kinases during mitosis

    • p90RSK during serum stimulation

    • AKT in some cancer cells

• Subcellular Localization Analysis:

  • Combine fractionation with western blotting

  • Use immunofluorescence to track CHEK1 localization changes

  • Consider live-cell imaging with fluorescently tagged CHEK1

• Pathway Integration:

  • Inhibit upstream kinases (PIM2, AKT, p90RSK) to assess contribution

  • Use phospho-mutants in rescue experiments

Understanding that CHEK1 is maximally phosphorylated on S280 during mitosis informs proper experimental timing and the selection of appropriate cell synchronization methods.

How can researchers utilize sequence specificity information to identify potential CHEK1 substrates?

Identifying CHEK1 substrates can be approached through predictive methods based on sequence specificity combined with experimental validation:

• CHEK1 Consensus Sequence Characteristics:

  • Strong bias towards basic residues amino-terminal to the phosphorylation site

  • Over-representation of Arg/Lys at position -3 relative to the phosphorylation site

  • For substrates with basic residues at -3:

    • Preference for hydrophobic residues at positions -5, -1, and +4

    • Under-representation of Thr at -1

    • Under-representation of basic residues at +2 and +3

    • Under-representation of acidic residues at -2 and -4

• Substrate Prediction Methods:

  • Use position-specific scoring matrices based on known CHEK1 substrates

  • Apply phosphoproteomics data to identify proteins with matching motifs

  • Consider structural elements that may influence accessibility

• Validation Approaches:

  • In vitro kinase assays with recombinant CHEK1 and candidate substrates

  • Analog-sensitive CHEK1 mutants combined with ATP analogs for specific labeling

  • Phospho-specific antibodies to monitor candidate phosphorylation

• Functional Context:

  • Consider cellular processes where CHEK1 is known to function:

    • DNA damage response

    • Replication stress

    • Cell cycle checkpoints

    • Mitotic regulation

This sequence specificity information can be especially valuable when combined with high-throughput phosphoproteomic screens to identify novel CHEK1 substrates .

What experimental approaches can be used to study the functional consequences of CHEK1 Ser280 phosphorylation?

Investigating the functional impact of CHEK1 Ser280 phosphorylation requires multiple complementary approaches:

• Phospho-Mutant Expression:

  • Generate stable cell lines expressing:

    • Wild-type CHEK1 (control)

    • S280A (non-phosphorylatable)

    • S280E (phosphomimetic)

  • Use inducible systems (Tet-On) for controlled expression

  • Combine with siRNA-mediated depletion of endogenous CHEK1

• Cell Cycle Analysis:

  • Flow cytometry to assess cell cycle distribution

  • Live-cell imaging to measure mitotic timing and fidelity

  • Immunofluorescence for mitotic markers (H3S10ph, cyclin B1)

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation to assess interactions with CHEK1-S and other partners

  • Proximity ligation assays for in situ detection of interactions

  • FRET-based approaches for dynamic interaction monitoring

• Kinase Activity Assays:

  • In vitro kinase assays with immunoprecipitated CHEK1

  • Monitor phosphorylation of known CHEK1 substrates (CDC25A/B/C, PLK1)

  • ATP consumption measurements

• Subcellular Localization:

  • Fractionation followed by western blotting

  • Immunofluorescence microscopy

  • Live-cell imaging with fluorescently tagged constructs

• Response to DNA Damage:

  • Treatment with genotoxic agents (camptothecin, cisplatin)

  • Assessment of checkpoint activation

  • DNA repair efficiency measurements

• Manipulation of Upstream Kinases:

  • Inhibition or depletion of PIM2, p90RSK, or AKT

  • Overexpression of constitutively active kinase forms

  • Analysis of resulting effects on CHEK1 function

These approaches have revealed that Ser280 phosphorylation controls nuclear localization and enables CHEK1 to phosphorylate PLK1 during mitosis , highlighting its role in both normal cell cycle progression and stress responses.

How does CHEK1-S splice variant regulation interface with Ser280 phosphorylation?

The interaction between CHEK1-S (a splice variant of CHEK1) and Ser280 phosphorylation represents a sophisticated regulatory mechanism:

• CHEK1-S Structure and Function:

  • N-terminally truncated alternative splice variant of CHEK1

  • Functions as an endogenous repressor of CHEK1

  • Expressed at higher levels in fetal and cancer tissues compared to normal tissues

• Regulatory Mechanism Under Normal Conditions:

  • CHEK1-S interacts with and antagonizes CHEK1

  • This interaction promotes S-to-G2/M phase transition

  • Maintains appropriate cell cycle progression

• Phosphorylation-Mediated Regulation:

  • During DNA damage, ATR phosphorylates CHEK1 at Ser345/Ser317

  • This phosphorylation disrupts the CHEK1-CHEK1-S interaction

  • Disruption releases CHEK1 from CHEK1-S inhibition

• Dependencies and Requirements:

  • The dissociation of CHEK1 from CHEK1-S requires:

    • ATR activity (dominant-negative ATR prevents dissociation)

    • Phosphorylation sites Ser345/Ser317 (S345A/S317A mutant remains bound to CHEK1-S)

• Experimental Evidence:

  • Co-immunoprecipitation shows CHEK1-CHEK1-S interaction under normal conditions

  • DNA damage induced by camptothecin or cisplatin attenuates this interaction

  • Inhibition of ATR prevents the dissociation