Phospho-MEF2C (S396) Antibody

What is MEF2C and why is phosphorylation at S396 significant?

MEF2C (Myocyte Enhancer Factor 2C) is a member of the MADS box transcription factor family that plays crucial roles in multiple biological processes. MEF2C functions as a transcription activator that binds specifically to the MEF2 element present in regulatory regions of many muscle-specific genes . Beyond muscle development, MEF2C controls cardiac morphogenesis, vascular development, neuronal function, and immune cell development .

Phosphorylation at serine 396 (S396) represents a critical post-translational modification that regulates MEF2C's transcriptional activity. This specific phosphorylation site is located within the γ-domain of MEF2C and has been demonstrated to negatively regulate its transcriptional activity . The significance of this phosphorylation lies in its ability to facilitate subsequent sumoylation at nearby lysine 391 (K391), creating a phosphorylation-dependent regulatory mechanism that modulates MEF2C function .

How do I distinguish between total MEF2C and the S396-phosphorylated form?

Distinguishing between total MEF2C and its phosphorylated form requires specific antibodies that recognize either the phosphorylated epitope or the general protein regardless of its phosphorylation state:

• For phosphorylated MEF2C detection: Use a phospho-specific antibody like Phospho-MEF2C (S396) that specifically recognizes MEF2C only when phosphorylated at serine 396 .

• For total MEF2C detection: Use antibodies that bind to regions of MEF2C independent of its phosphorylation status.

When performing Western blot analysis, the calculated molecular weight of MEF2C is approximately 51kDa, but the observed molecular weight is typically around 60kDa due to post-translational modifications . Running parallel samples with both types of antibodies allows for calculating the ratio of phosphorylated to total protein, providing insight into the phosphorylation state under different experimental conditions.

What is the relationship between S396 phosphorylation and other post-translational modifications?

The relationship between S396 phosphorylation and other post-translational modifications, particularly sumoylation, represents a sophisticated regulatory mechanism:

• Phosphorylation-facilitated sumoylation: Phosphorylation of S396 enhances sumoylation of MEF2C at lysine 391 (K391), which is located in close proximity to S396. This represents a prime example of a phosphorylation-dependent sumoylation process .

• Functional consequences: Sumoylation of MEF2C at K391 inhibits its transcriptional activity. The S396A mutation reduces sumoylation of MEF2C in vivo and enhances the transcriptional activity of MEF2C in reporter assays .

• Regulatory mechanism: This suggests a sequential post-translational modification pathway where phosphorylation at S396 serves as a priming event that facilitates sumoylation at K391, which in turn recruits transcriptional repressors to inhibit MEF2C-mediated gene expression .

This interdependence between phosphorylation and sumoylation creates a dynamic regulatory system for fine-tuning MEF2C transcriptional activity in response to various cellular signals.

What are the optimal conditions for Western blot detection of phosphorylated MEF2C?

For optimal Western blot detection of phosphorylated MEF2C at S396, researchers should follow these methodological guidelines:

When troubleshooting Western blot detection, researchers should pay special attention to sample preparation, as phosphorylation marks are easily lost during cell lysis if appropriate inhibitors are not included. Additionally, using serum-starved 3T3 cells has been reported as an effective positive control system for detecting phosphorylated MEF2C .

How can I validate the specificity of Phospho-MEF2C (S396) antibody?

Validating antibody specificity is crucial for ensuring reliable results in phospho-protein research. For Phospho-MEF2C (S396) antibody, consider these validation approaches:

• Phosphopeptide competition assay: Pre-incubation of the antibody with the immunizing phosphopeptide should abolish specific signal in Western blot or immunohistochemistry. This demonstrates the phospho-specificity of the antibody .

• Phosphatase treatment: Treating part of your sample with lambda phosphatase should eliminate signal from a genuine phospho-specific antibody while leaving total protein detection unaffected .

• Genetic models: Using samples from MEF2C S396A mutant models (where the serine is mutated to alanine) should show reduced or absent signal compared to wild-type samples .

• Comparison with mass spectrometry data: For advanced validation, correlation of antibody-based detection with quantitative targeted mass spectrometry can provide orthogonal confirmation of phosphorylation status .

A comprehensive validation should incorporate multiple approaches to ensure the antibody specifically recognizes MEF2C phosphorylated at S396 and not other phosphorylation sites or related proteins.

What applications beyond Western blot are suitable for Phospho-MEF2C (S396) antibody?

While Western blot is the most common application, Phospho-MEF2C (S396) antibodies can be utilized in several other techniques:

• Immunohistochemistry (IHC): Useful for detecting phosphorylated MEF2C in tissue sections, typically at dilutions of 1:50-1:200. This application allows visualization of the subcellular localization of phosphorylated MEF2C, which is primarily nuclear .

• ELISA: Several antibodies are validated for ELISA applications at dilutions around 1:10000, enabling quantitative measurement of phosphorylated MEF2C levels in complex samples .

• Transcription Factor Activity Assays: Specialized kits using phospho-specific antibodies can measure the transcriptional activity of phosphorylated MEF2C, providing functional insights beyond mere presence of the phosphorylated protein .

• Immunofluorescence (IF): Enables subcellular localization studies at recommended dilutions of 1:50-1:200, particularly useful for co-localization studies with potential interacting partners .

Each application requires specific optimization, and researchers should validate the antibody for their particular experimental system before proceeding with large-scale experiments.

How does phosphorylation at S396 regulate MEF2C transcriptional activity?

Phosphorylation at S396 represents a key regulatory mechanism that modulates MEF2C transcriptional activity through several interconnected pathways:

• Facilitation of sumoylation: S396 phosphorylation enhances sumoylation of MEF2C at K391, a nearby lysine residue. This sumoylation has been demonstrated to negatively regulate MEF2C transcriptional activity .

• Recruitment of transcriptional repressors: The phosphorylation-facilitated sumoylation appears to recruit as-yet unidentified transcriptional repressors that inhibit MEF2C-mediated gene activation .

• Relationship with γ-domain function: S396 is located within the γ-domain of MEF2C, a 32-residue domain that contributes to transcriptional repression. The S396A mutation diminishes the transcriptional repression activity of this domain .

• Effect on DNA binding: Unlike some post-translational modifications that affect DNA binding, phosphorylation at S396 does not appear to block MEF2C's DNA-binding activity, suggesting it primarily affects co-activator/co-repressor recruitment .

These mechanisms collectively establish phosphorylation at S396 as a "molecular switch" that can toggle MEF2C between more active and more repressed transcriptional states.

What is known about MEF2C S396 phosphorylation in disease contexts?

MEF2C S396 phosphorylation has been implicated in several disease contexts, though with varying levels of mechanistic understanding:

• Neurodevelopmental disorders: MEF2C phosphorylation has been linked to neurodevelopmental disorders. MEF2C is crucial for normal neuronal development, distribution, and electrical activity in the neocortex, and mutations in MEF2C have been associated with severe mental retardation, stereotypic movements, epilepsy, and cerebral malformation .

• Cardiovascular diseases: Given MEF2C's role in cardiac morphogenesis and myogenesis, altered phosphorylation at S396 may contribute to cardiovascular pathologies, though direct evidence linking S396 phosphorylation to specific cardiac diseases remains limited .

• Cancer: Phosphorylation of MEF2C has been implicated in chemotherapy resistance in acute myeloid leukemia (AML). While much of this research has focused on S222 phosphorylation, S396 phosphorylation has also been detected in AML specimens, though in sub-stoichiometric amounts compared to S222 .

• Immune system dysfunction: MEF2C is required for B-cell survival and proliferation in response to BCR stimulation and for efficient antibody responses to T-cell-dependent antigens. Dysregulation of its phosphorylation status may contribute to immune dysfunction .

The multifaceted roles of MEF2C across different tissues make its phosphorylation status a potentially important biomarker and therapeutic target in various pathological conditions.

What cell signaling pathways regulate MEF2C S396 phosphorylation?

The regulation of MEF2C S396 phosphorylation involves several signaling pathways, though the complete mechanistic picture remains to be fully elucidated:

• Kinase involvement: While the specific kinase(s) responsible for S396 phosphorylation have not been definitively identified in the search results, there are indications of potential links to the AMPK signaling pathway .

• Transcriptional control circuitry: The phosphorylation status of MEF2C appears to be part of a complex transcriptional control circuit, where phosphorylation-dependent recruitment of repressors modulates gene expression patterns .

• Interaction with 14-3-3 proteins: MEF2D, a related MEF2 family member, associates with 14-3-3τ protein, which competes with HDAC4 for binding to MEF2D. Similar regulatory mechanisms may exist for MEF2C, potentially involving phosphorylation-dependent protein-protein interactions .

• Context-dependent regulation: The kinases responsible for S396 phosphorylation may vary depending on cell type and physiological context, contributing to the diverse roles of MEF2C across different tissues .

Further research is needed to definitively identify the kinases and upstream signaling events that regulate MEF2C S396 phosphorylation in different cellular contexts.

How can I develop experimental models to study the functional consequences of MEF2C S396 phosphorylation?

Developing appropriate experimental models is crucial for studying the functional consequences of MEF2C S396 phosphorylation. Several sophisticated approaches can be employed:

• Phosphomimetic and phosphodeficient mutants: Generate MEF2C constructs with S396D (phosphomimetic) or S396A (phosphodeficient) mutations. These can be expressed in cell culture systems or animal models to study the functional consequences of constitutive phosphorylation or dephosphorylation .

• Knock-in mouse models: Generate Mef2c S222A/S222A or Mef2c S222D/S222D knock-in mice to study the physiological consequences of altered MEF2C phosphorylation in vivo. Similar approaches could be used for S396 phosphorylation studies .

• Cellular transformation assays: Use bone marrow GMP cells transduced with oncogenes like MLL-AF9 in combination with MEF2C phosphorylation mutants to study the role of MEF2C phosphorylation in cellular transformation and leukemogenesis .

• Reporter gene assays: Utilize MEF2C-responsive luciferase reporter constructs to measure the transcriptional activity of wild-type versus phosphorylation site mutants, allowing quantitative assessment of how phosphorylation affects gene expression .

• Proteomic interactome analysis: Employ mass spectrometry-based approaches to identify proteins that differentially interact with MEF2C based on its phosphorylation status at S396, potentially revealing the co-repressors recruited by phosphorylated MEF2C .

These experimental models enable comprehensive investigation of MEF2C S396 phosphorylation's role in normal physiology and disease contexts.

How does MEF2C S396 phosphorylation compare to phosphorylation at other sites on MEF2C?

MEF2C contains multiple phosphorylation sites that regulate its function through distinct mechanisms:

• S396 vs. S222 phosphorylation: While S396 phosphorylation facilitates sumoylation and transcriptional repression, S222 phosphorylation has been more directly implicated in chemotherapy resistance in acute myeloid leukemia. High levels of S222 phosphorylation were significantly associated with primary chemotherapy resistance, while S396 phosphorylation was present in sub-stoichiometric amounts in AML specimens .

• Functional consequences: Different phosphorylation sites appear to regulate different aspects of MEF2C function:

  • S396 phosphorylation primarily affects transcriptional activity through sumoylation-dependent mechanisms

  • S222 phosphorylation appears critical for leukemia maintenance in MLL-AF9-transformed cells

• Tissue-specific relevance: The relative importance of different phosphorylation sites may vary depending on the tissue context:

  • S396 has been studied extensively in muscle and neural contexts

  • S222 has been highlighted particularly in hematopoietic malignancies

Understanding the interplay between different phosphorylation sites and how they collectively regulate MEF2C function remains an important area for future research.

What are the challenges in detecting phosphorylated MEF2C in complex biological samples?

Detecting phosphorylated MEF2C in complex biological samples presents several technical challenges that researchers should consider:

• Low abundance: Phosphorylated forms of proteins often represent a small fraction of the total protein pool. For instance, S396 phosphorylation was present in sub-stoichiometric amounts compared to S222 phosphorylation in AML samples .

• Phosphatase activity during sample preparation: Endogenous phosphatases can rapidly dephosphorylate proteins during cell lysis unless appropriate inhibitors are included. This is particularly important for potentially labile phosphorylation sites .

• Antibody cross-reactivity: Ensuring antibody specificity is critical, as cross-reactivity with other phosphorylated epitopes or related MEF2 family members (MEF2A, MEF2B, MEF2D) can complicate interpretation .

• Context-dependent phosphorylation: The phosphorylation status of MEF2C can vary dramatically depending on cell type, stimulation conditions, and disease state, requiring careful experimental design .

• Co-occurring modifications: The presence of other post-translational modifications (such as sumoylation) can affect antibody accessibility to the phosphorylated epitope or alter protein migration patterns in gel electrophoresis .

Advanced techniques such as quantitative targeted mass spectrometry, phospho-specific flow cytometry, or proximity ligation assays may provide complementary approaches to overcome these challenges in detecting phosphorylated MEF2C in complex biological samples.

What are the emerging therapeutic implications of targeting MEF2C S396 phosphorylation?

The potential therapeutic implications of targeting MEF2C S396 phosphorylation are beginning to emerge, particularly in several disease contexts:

• Cancer therapy: Understanding the role of MEF2C phosphorylation in chemotherapy resistance suggests that inhibiting specific phosphorylation events could sensitize cancer cells to conventional therapies. This approach might be particularly relevant in acute myeloid leukemia, where MEF2C phosphorylation has been associated with therapy resistance .

• Neurodevelopmental disorders: Given MEF2C's critical role in neuronal development and function, modulating its phosphorylation status might represent a novel approach for treating neurodevelopmental disorders associated with MEF2C dysfunction .

• Cardiovascular diseases: MEF2C's involvement in cardiac morphogenesis and myogenesis suggests that targeting its phosphorylation could have applications in cardiovascular disorders, though this remains to be fully explored .

• Immunomodulation: MEF2C's role in B-cell development and function indicates that manipulating its phosphorylation could have immunomodulatory effects, potentially applicable to autoimmune disorders or immunodeficiencies .

Developing therapeutic approaches would require identifying and targeting the specific kinases responsible for S396 phosphorylation or the downstream effectors of phosphorylated MEF2C, representing an important area for future research.

How can advanced technologies enhance our understanding of MEF2C phosphorylation dynamics?

Emerging technologies offer new opportunities to deepen our understanding of MEF2C phosphorylation dynamics:

• Single-cell phosphoproteomics: This technique allows examination of phosphorylation heterogeneity within cell populations, potentially revealing subpopulations with distinct MEF2C phosphorylation states that might have different functional properties or disease relevance .

• CRISPR-based phosphorylation site editing: CRISPR-Cas9 genome editing enables precise modification of endogenous phosphorylation sites, allowing study of phosphorylation events in their native genomic context without overexpression artifacts .

• Phospho-specific biosensors: Developing FRET-based biosensors for specific MEF2C phosphorylation events could enable real-time monitoring of phosphorylation dynamics in living cells in response to various stimuli.

• Spatial transcriptomics combined with phosphoproteomics: This approach could reveal how MEF2C phosphorylation affects gene expression patterns with spatial resolution in tissues, providing insights into the local consequences of MEF2C phosphorylation.

• AI-driven modeling of phosphorylation networks: Machine learning approaches could help predict kinase-substrate relationships and model how MEF2C phosphorylation fits within broader signaling networks, generating testable hypotheses about regulation and function.