Phospho-RUNX1 (Ser303) Antibody

What is RUNX1 and why is phosphorylation at Ser303 significant?

RUNX1 (also known as AML1) is a critical transcription factor that regulates hematopoiesis, angiogenesis, muscle function, and neurogenesis. It plays a fundamental role in the formation of definitive hematopoietic stem cells and their subsequent lineage maturation . Phosphorylation at Ser303 is particularly significant because:

• It is one of the key serine residues (along with Ser-48 and Ser-424) phosphorylated by cyclin-dependent kinases (CDKs)

• This specific phosphorylation increases RUNX1's transcriptional activation capacity

• Phosphorylation at Ser303 alters RUNX1's protein interactions, particularly reducing its binding to histone deacetylases (HDAC1 and HDAC3)

• This modification affects cell cycle progression, as RUNX1 levels increase as hematopoietic cells progress from G1 to S and from S to G2/M phases

The precise regulation of Ser303 phosphorylation is crucial, as mutations in RUNX1 can contribute to leukemic transformation .

Which kinases phosphorylate RUNX1 at Ser303?

Research has identified several cyclin-dependent kinases that phosphorylate RUNX1 at Ser303:

• Cdk1/cyclin B can phosphorylate Ser303 in vitro

• Cdk2/cyclin A has demonstrated capability to phosphorylate this site

• Cdk6/cyclin D3 has also been identified as a kinase for Ser303

• CDK9 has recently been identified as a novel kinase for RUNX1, linking the fundamental transcriptional machinery with activation of this cell type-specific transcription factor

Phosphorylation can be experimentally blocked using CDK inhibitors such as roscovitine, which inhibits the activity of Cdk1, Cdk2, and Cdk5 .

What experimental models are suitable for studying RUNX1 Ser303 phosphorylation?

Several experimental models have proven effective for studying RUNX1 Ser303 phosphorylation:

• 293T cells transfected with RUNX1 expression constructs provide a reliable system for studying phosphorylation mechanisms

• GST-RUNX1 fusion proteins containing amino acids 267-315 can be used to specifically examine phosphorylation at Ser303 and surrounding sites

• Human erythroleukemia (HEL) cell lines expressing wild-type or mutant RUNX1 variants serve as models for studying differential gene regulation

• Primary human megakaryocytic-erythroid progenitors (MEPs) represent a physiologically relevant model for investigating the role of phosphorylated RUNX1 in lineage specification

• Lineage-negative murine marrow progenitors can be used to assess the functional consequences of RUNX1 phosphorylation states

How can I validate the specificity of a Phospho-RUNX1 (Ser303) Antibody?

To ensure the specificity of a Phospho-RUNX1 (Ser303) Antibody, implement these validation approaches:

• Phosphatase treatment control: Treat one sample with lambda phosphatase prior to antibody probing. The signal should be abolished in the treated sample if the antibody is specific to the phosphorylated form .

• Mutation analysis: Use RUNX1 constructs with serine-to-alanine (S303A) mutations that cannot be phosphorylated as negative controls, and serine-to-aspartic acid (S303D) mutations as phosphomimetic positive controls .

• Kinase inhibitor treatment: Treat cells with roscovitine or other CDK inhibitors that block Ser303 phosphorylation. This should reduce antibody binding if it's specific for the phosphorylated form .

• Peptide competition assay: Pre-incubate the antibody with a phosphorylated peptide containing the Ser303 site. This should block antibody binding in subsequent applications if it's specific.

• Cross-validation: Compare results with alternative methods of detecting phosphorylation, such as mass spectrometry or 32P-labeling experiments .

What are optimal conditions for detecting RUNX1 Ser303 phosphorylation in different experimental contexts?

Optimal detection conditions vary based on experimental context:

• Western blotting:

  • Use fresh lysates with phosphatase inhibitors

  • Sample buffer should contain SDS and reducing agents

  • Include appropriate phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Run gel at lower voltage to improve resolution around 50-60 kDa (RUNX1 molecular weight)

• Immunoprecipitation:

  • Use mild lysis conditions to preserve protein-protein interactions

  • Pre-clear lysates to reduce background

  • For co-immunoprecipitation studies with HDACs, avoid harsh detergents that might disrupt interactions

• Cell cycle considerations:

  • Phosphorylation levels vary throughout cell cycle, with increased levels as cells progress from G1 to S and from S to G2/M

  • Synchronize cells for more consistent results

  • Consider that RUNX1 degradation occurs during G2/M phase due to Ser303 phosphorylation

• In vitro kinase assays:

  • Use GST-RUNX1 purified from bacterial extracts as substrate

  • Include ATP, MgCl2, and appropriate kinase buffer

  • Purified Cdk1/cyclin B serves as an effective kinase

How does phosphorylation at Ser303 affect RUNX1's protein interactions?

Phosphorylation at Ser303 significantly alters RUNX1's protein interaction network:

• HDAC interactions: Phosphorylation at Ser303 reduces binding to HDAC1 and HDAC3 by approximately 1.5-fold and 1.6-fold, respectively . This reduction in HDAC interaction contributes to increased transcriptional activation.

• Co-activator interactions: Interestingly, phosphorylation at Ser303 does not affect RUNX1's interaction with the p300 co-activator, suggesting selective modulation of protein partners .

• Degradation mechanisms: Ser303 phosphorylation affects RUNX1 stability by marking it for ubiquitin-mediated degradation during G2/M phase . Specifically, this phosphorylation facilitates interaction with Cdc20, a substrate-targeting subunit of the anaphase-promoting complex (APC) .

• Combined effects: Mutation studies have shown that the combined phosphorylation of Ser48, Ser303, and Ser424 has more pronounced effects on protein interactions than modification of individual sites alone .

What controls should be included when studying RUNX1 Ser303 phosphorylation?

A robust experimental design should include these controls:

• Phosphorylation state controls:

  • Phosphomimetic mutants (S303D) to mimic constitutive phosphorylation

  • Non-phosphorylatable mutants (S303A) to prevent phosphorylation

  • Triple mutants (S48A/S303A/S424A or S48D/S303D/S424D) when studying coordinated phosphorylation events

• Kinase activity controls:

  • CDK inhibitors (roscovitine) to block phosphorylation

  • Phosphatase treatment to remove phosphate groups

  • Kinase-dead mutants as negative controls

• Functional readouts:

  • Transcriptional activity assays using RUNX1-responsive promoters

  • Proliferation assays with lineage-negative murine marrow progenitors

  • Gene expression analysis of RUNX1 target genes

• Cell cycle controls:

  • Synchronized cell populations to control for cell cycle variation in phosphorylation

  • Cell cycle markers to correlate phosphorylation with specific phases

What are effective approaches to modulate RUNX1 Ser303 phosphorylation experimentally?

To manipulate RUNX1 Ser303 phosphorylation levels:

• Pharmacological approaches:

  • Roscovitine treatment inhibits CDK activity and reduces Ser303 phosphorylation

  • Specific CDK9 inhibitors can reduce RUNX1 phosphorylation in megakaryocytic-erythroid progenitors

  • Cell cycle synchronization agents can influence phosphorylation status indirectly

• Genetic approaches:

  • Expression of phosphomimetic (S303D) or non-phosphorylatable (S303A) RUNX1 mutants

  • siRNA knockdown of specific CDKs to reduce phosphorylation

  • Expression of constitutively active CDK mutants to increase phosphorylation

• Contextual manipulation:

  • Serum starvation followed by growth factor stimulation can activate CDKs and induce phosphorylation

  • Differentiation induction in hematopoietic progenitors alters RUNX1 phosphorylation states

  • Cell density manipulation affects cell cycle progression and associated phosphorylation events

How does Ser303 phosphorylation affect RUNX1's biological function?

Ser303 phosphorylation influences multiple aspects of RUNX1 biology:

• Transcriptional activity: Phosphorylation increases RUNX1's trans-activation potential, enhancing its ability to regulate target genes .

• Cell proliferation: RUNX1 with phosphomimetic mutations at Ser48, Ser303, and Ser424 (RUNX1-tripleD) stimulates proliferation of lineage-negative murine marrow progenitors more potently than non-phosphorylatable mutants (RUNX1-tripleA) .

• Protein stability: Phosphorylation at Ser303 leads to ubiquitin-mediated degradation during G2/M phase of the cell cycle, regulating RUNX1 protein levels throughout cell division .

• Lineage specification: In primary human megakaryocytic-erythroid progenitors, serine phosphorylation of RUNX1 (including at Ser303) promotes megakaryocytic rather than erythroid fate specification .

• Gene expression patterns: Differential phosphorylation states of RUNX1 regulate distinct gene sets, with phosphomimetic RUNX1 variants (like RUNX1-4D) showing enhanced regulatory capacity compared to non-phosphorylatable variants (RUNX1-4A) .

What methodological approaches are recommended for quantifying RUNX1 Ser303 phosphorylation?

For accurate quantification of RUNX1 Ser303 phosphorylation:

• Western blot analysis:

  • Use both phospho-specific and total RUNX1 antibodies

  • Calculate the ratio of phosphorylated to total RUNX1

  • Include loading controls and phosphorylation standards

  • Use digital imaging software for densitometric analysis

• Mass spectrometry:

  • Employ phosphopeptide enrichment strategies

  • Use stable isotope labeling for relative quantification

  • Monitor multiple phosphorylation sites simultaneously

  • Consider targeted approaches for improved sensitivity

• Functional readouts:

  • Measure transcriptional activity using reporter assays

  • Assess HDAC binding as an inverse correlate of phosphorylation

  • Quantify cell proliferation rates in response to phosphorylation status

• Visualization techniques:

  • Immunofluorescence microscopy with phospho-specific antibodies

  • Flow cytometry for cell-by-cell quantification

  • Live-cell imaging with phosphorylation-sensitive probes

How do research findings on RUNX1 Ser303 phosphorylation relate to hematological disorders?

RUNX1 Ser303 phosphorylation has significant implications for hematological disorders:

• Leukemic transformation: RUNX1 mutations contribute to leukemic transformation, and altered phosphorylation may play a role in this process .

• Megakaryocytic/erythroid lineage balance: Phosphorylation states influence the specification of hematopoietic progenitors towards megakaryocytic or erythroid lineages, potentially contributing to disorders characterized by imbalanced blood cell production .

• Cell cycle regulation: Since RUNX1 phosphorylation affects cell cycle progression and proliferation of hematopoietic progenitors , dysregulation may contribute to proliferative disorders.

• Therapeutic targeting: Understanding the regulation of RUNX1 phosphorylation provides potential therapeutic targets. For example, CDK9 inhibition leads to decreased RUNX1 phosphorylation and increased erythroid commitment , suggesting applications for conditions requiring enhanced erythropoiesis.

• Diagnostic potential: Detection of phosphorylated RUNX1 states could serve as biomarkers for disease progression or treatment response in hematological malignancies.

What are the latest advances in understanding the kinase networks regulating RUNX1 Ser303 phosphorylation?

Recent research has expanded our understanding of the kinase networks involved in RUNX1 phosphorylation:

• Novel kinase identification: CDK9 has been newly identified as a kinase for RUNX1, linking transcriptional machinery with activation of this cell type-specific transcription factor .

• Context-specific regulation: Different kinases may preferentially phosphorylate RUNX1 in different cellular contexts, with CDK9 playing a prominent role in megakaryocytic-erythroid progenitors .

• Coordinated phosphorylation: Research suggests that phosphorylation at multiple sites (Ser48, Ser303, Ser424) occurs in a coordinated manner, potentially through sequential action of different CDKs at different cell cycle stages .

• Phosphatase interactions: While much focus has been on kinases, emerging work is beginning to investigate the phosphatases that remove phosphate groups from RUNX1, adding another layer of regulatory control.