Phospho-RUNX1 (S276) Antibody

What is the biological significance of RUNX1 phosphorylation at S276 in hematopoiesis?

RUNX1 phosphorylation at S276 is part of a complex post-translational modification system that regulates RUNX1 activity in hematopoiesis. Research has shown that phosphorylation at S276, along with other sites (S293, T300, S303, and S462), is crucial for RUNX1's biological functions. While phospho-deficient mutations at S276 and S293 alone (RUNX1-2A) retain hematopoietic activity, additional mutations at T300 and S303 (RUNX1-4A) impair T-cell differentiation while still supporting early hematopoiesis. When S462 is also mutated (RUNX1-5A), hematopoietic activity is completely lost .

How can researchers detect and quantify RUNX1 phosphorylated at S276 in experimental samples?

Researchers can detect and quantify phosphorylated RUNX1 at S276 using multiple complementary approaches. Western blotting (WB) is commonly employed using specific anti-phospho-RUNX1 (S276) antibodies, with recommended dilutions of 1:500-1:2000 . This technique allows for semi-quantitative assessment of phosphorylation levels across different experimental conditions.

Immunohistochemistry (IHC) at dilutions of 1:100-1:300 enables visualization of phosphorylated RUNX1 in tissue sections, revealing spatial distribution patterns that might be physiologically relevant . For example, researchers have observed highly variable p-RUNX1 immunoreactivity among matrix-attached cells, indicating heterogeneous phosphorylation states within a seemingly uniform population .

ELISA techniques (recommended at 1:5000 dilution) provide more quantitative measurements when analyzing multiple samples simultaneously . For all applications, proper controls are essential, including phosphorylation-deficient mutants (S276A) and samples treated with phosphatase inhibitors to preserve phosphorylation status during sample preparation.

What factors influence RUNX1 phosphorylation at S276, and how can these be experimentally manipulated?

RUNX1 phosphorylation at S276 is primarily regulated by cyclin-dependent kinases (CDKs), particularly CDK1, CDK2, and CDK6 . This phosphorylation subsequently renders RUNX1 vulnerable to degradation mediated by the CDC20-containing anaphase promoting complex (APC) and, to a lesser extent, the SCF-SKP2 complex during different cell cycle phases .

Researchers can experimentally manipulate RUNX1 phosphorylation through several approaches:

• CDK inhibitors: Terameprocol (targeting CDK1), roscovitine (targeting CDK1/CDK2), and R547 (targeting CDK1/CDK2) have been shown to reduce levels of detectable RUNX1 protein phosphorylated at S276 . Roscovitine treatment strongly reduced p-RUNX1 levels in cellular assays, while ERK1/2 inhibition by U0126 had minimal effects, suggesting specificity of CDK-mediated phosphorylation .

• Proteasome inhibitors: Bortezomib, carfilzomib, ixazomib, and oprozomib can indirectly affect phosphorylation levels by preventing degradation of phosphorylated RUNX1 .

• Genetic approaches: Site-directed mutagenesis to create phosphomimetic (S to D/E) or phospho-deficient (S to A) mutations provides powerful tools to study the functional consequences of phosphorylation at specific sites .

These experimental manipulations enable researchers to investigate the relationship between RUNX1 phosphorylation, protein stability, and downstream biological functions in hematopoiesis and T-cell differentiation.

How does S276 phosphorylation interact with other phosphorylation sites to regulate RUNX1 activity in different hematopoietic contexts?

The interaction between S276 phosphorylation and other RUNX1 phosphorylation sites represents a sophisticated regulatory network that varies across hematopoietic contexts. Research reveals a hierarchical importance of these sites, where combinatorial phosphorylation determines functional outcomes. The RUNX1-2A mutant (S276A/S293A) maintains hematopoietic activity comparable to wild-type RUNX1, indicating some redundancy or compensatory mechanisms .

Significantly, phosphomimetic mutations (RUNX1-4D/E and RUNX1-5D/E) restore normal RUNX1 function, confirming that the phosphorylation state, rather than the specific amino acids, dictates activity . This creates a model where different hematopoietic lineages require distinct phosphorylation patterns, with S276 contributing to this code in context-dependent ways.

For researchers investigating these interactions, mutational analyses combined with lineage-specific functional assays are necessary to fully dissect the phosphorylation requirements across different hematopoietic populations.

What are the methodological considerations when using phospho-RUNX1 (S276) antibodies to investigate heterogeneous cellular populations?

When investigating heterogeneous cellular populations with phospho-RUNX1 (S276) antibodies, researchers must address several critical methodological challenges:

• Phosphorylation state preservation: Phosphorylation is highly labile, requiring rapid sample processing with phosphatase inhibitors to prevent artificial dephosphorylation. This is particularly important when comparing populations with potentially different phosphatase activities .

• Detecting cellular heterogeneity: Studies have revealed highly variable p-RUNX1 immunoreactivity among seemingly uniform cell populations. For example, matrix-attached cells showed heterogeneous p-RUNX1 staining despite homogeneous total RUNX1 expression . This requires:

  • Single-cell resolution techniques (immunofluorescence, flow cytometry)

  • Co-staining with lineage markers to correlate phosphorylation with cell identity

  • Quantitative image analysis methods to objectively measure staining intensity variations

• Accounting for cell cycle effects: As RUNX1 phosphorylation is mediated by cell cycle-dependent kinases, cell cycle phase heterogeneity within populations can create phosphorylation variations independent of cell identity . Cell cycle synchronization or co-staining with cell cycle markers becomes essential for accurate interpretation.

• Antibody validation: Cross-reactivity with other phosphoproteins is a potential concern. Researchers should validate specificity using:

  • Phospho-deficient mutants (S276A) as negative controls

  • Phosphatase treatment of samples as additional controls

  • Multiple antibody clones targeting the same epitope when possible

These considerations are particularly important when investigating rare hematopoietic progenitors or heterogeneous primary samples, where subtle variations in phosphorylation status may correlate with functional differences.

How can phospho-RUNX1 (S276) antibodies be used to investigate the relationship between RUNX1 phosphorylation and protein degradation pathways?

Phospho-RUNX1 (S276) antibodies provide powerful tools for investigating the intricate relationship between RUNX1 phosphorylation and protein degradation pathways. RUNX1 stability is tightly regulated by phosphorylation-dependent degradation mechanisms involving the ubiquitin-proteasome system .

To effectively study this relationship, researchers can employ several experimental strategies:

• Temporal correlation studies: Combining phospho-specific antibody detection with total RUNX1 protein level analysis after treatment with CDK inhibitors (terameprocol, roscovitine) can reveal the kinetics of phosphorylation-induced degradation. Reduced p-RUNX1(S276) levels following terameprocol treatment have been observed, confirming this relationship .

• Proteasome inhibition experiments: Treatment with proteasome inhibitors (bortezomib, carfilzomib) while monitoring p-RUNX1(S276) levels can determine if phosphorylated forms preferentially accumulate, indicating selective degradation of the phosphorylated protein .

• Cell cycle phase analysis: Since RUNX1 degradation is mediated by different complexes during specific cell cycle phases (CDC20-APC during G2/M, SCF-SKP2 during S phase), combining phospho-S276 detection with cell cycle markers can identify phase-specific degradation patterns .

• Ubiquitination assays: Co-immunoprecipitation experiments using phospho-RUNX1(S276) antibodies followed by ubiquitin detection can directly demonstrate the link between S276 phosphorylation and subsequent ubiquitination.

These approaches can address fundamental questions about how S276 phosphorylation, compared to other phosphorylation sites, contributes to RUNX1 protein turnover and consequent biological functions in hematopoiesis and T-cell differentiation.

What experimental approaches can resolve contradictory findings regarding the functional significance of S276 phosphorylation across different experimental systems?

Research findings regarding RUNX1 S276 phosphorylation sometimes appear contradictory across experimental systems. For example, while RUNX1-2A (S276A/S293A) retains function in some contexts, other studies suggest critical roles for these sites . Resolving these discrepancies requires sophisticated experimental approaches:

• Context-dependent analysis: Design experiments that directly compare S276 phosphorylation across:

  • Primary cells versus cell lines

  • Different hematopoietic lineages

  • Various developmental stages

  • Normal versus malignant states

  This approach can identify conditional requirements for S276 phosphorylation that explain seemingly contradictory results.

• Combinatorial mutation analysis: Generate comprehensive mutation sets that systematically vary phosphorylation sites individually and in combinations, testing each in standardized functional assays. This can identify compensatory mechanisms and synergistic interactions between phosphorylation sites .

• Quantitative phosphoproteomics: Mass spectrometry-based phosphoproteomics can quantify the stoichiometry of phosphorylation at multiple sites simultaneously, revealing whether contradictory findings stem from variations in phosphorylation efficiency across systems.

• Developmental timing studies: Since RUNX1 functions change during hematopoietic development, temporal analysis of S276 phosphorylation effects at defined developmental stages can reconcile contradictory findings from studies at different timepoints. Studies indicate that proliferation effects become apparent at day 14 of morphogenesis but not earlier (day 10) .

• Compensatory signaling analysis: Investigate potential compensatory pathways, such as FOXO signaling, which can mask or amplify S276 phosphorylation effects depending on cellular context. The interaction between RUNX1 and FOXO transcription factors suggests potential mechanisms for context-dependent requirements .

These approaches collectively create a framework for systematically resolving contradictions and building a more nuanced understanding of S276 phosphorylation significance.