Phospho-CARM1 (Ser228) Antibody

Basic Research Questions

• What is CARM1 and why is the phosphorylated form at Ser228 significant in research?

  CARM1 (Coactivator-associated arginine methyltransferase 1, also known as PRMT4) functions as a key regulator of gene expression and plays crucial roles in transcriptional regulation, cell cycle progression, and tumorigenesis . The phosphorylation of CARM1 at serine 228 (Ser228 in human, Ser229 in mouse/rat) represents a critical regulatory mechanism that negatively modulates its methyltransferase activity .

  This phosphorylation serves as a molecular switch, as it has been demonstrated to prevent CARM1 homodimerization, which is essential for its enzymatic function . Researchers investigating transcriptional regulation mechanisms, particularly those involving nuclear receptors like estrogen receptor, will find this phosphorylation site particularly relevant, as it directly impacts CARM1's coactivator functions .

• What experimental applications are supported by commercially available Phospho-CARM1 (Ser228) antibodies?

  Most commercial Phospho-CARM1 (Ser228) antibodies have been validated for Western blot (WB) applications . Some antibodies are also validated for ELISA and immunofluorescence (IF) . When planning experiments, researchers should consider:

  • Western blot is the most widely validated application, typically using dilutions of 1:500-1:1000

  • Positive control samples often include Jurkat cells and EGF-treated A431 cell lysates

  • Most antibodies are rabbit polyclonal antibodies, prepared by immunizing with synthetic phosphopeptides corresponding to the region surrounding Ser228

  For optimal results, researchers should follow manufacturer-recommended protocols for sample preparation, including proper cell lysis conditions and phosphatase inhibitor usage during extraction to preserve the phosphorylation state .

• How can researchers confirm the specificity of Phospho-CARM1 (Ser228) antibodies?

  To validate antibody specificity, implement these methodological approaches:

  • Phosphatase treatment control: Treat half of your sample with λ-phosphatase before Western blotting. The disappearance of the band detected by the phospho-specific antibody confirms specificity for the phosphorylated form .

  • Mutant expression: Compare detection between wild-type CARM1 and S228A mutant (non-phosphorylatable) in transfected cells. The absence of signal in the S228A mutant lane confirms phospho-specificity .

  • Peptide competition assay: Pre-incubate the antibody with the phosphopeptide immunogen to block specific binding sites before probing your samples .

  • Cross-reactivity testing: Validate against other phosphorylated proteins, particularly other PRMT family members, to ensure the antibody doesn't detect related phosphorylation sites .

  Several commercial antibodies have been purified using affinity chromatography with phospho-specific peptides, with non-phospho specific antibodies removed through chromatography using non-phosphopeptides .

Advanced Research Applications

• What molecular mechanisms explain how Ser228 phosphorylation inhibits CARM1 methyltransferase activity?

  The inhibition of CARM1 methyltransferase activity through Ser228 phosphorylation involves several key molecular mechanisms:

  1. Disruption of dimerization: Structural modeling based on the PRMT1 crystal structure suggests that phosphorylation at Ser228 (human)/Ser229 (mouse) interferes with the interaction between Asn-230 and Asp-323, which is critical for CARM1 dimerization . This has been experimentally confirmed through coimmunoprecipitation studies where the phosphomimetic S229E mutant fails to form dimers .

  2. Impaired adenosylmethionine (AdoMet) binding: Phosphorylation at Ser228 reduces CARM1's ability to bind its methyl donor, S-adenosylmethionine (AdoMet) . This can be assessed experimentally through photoaffinity labeling using [³H]AdoMet UV cross-linking assays .

  3. Altered protein conformation: The phosphorylated form of CARM1 exhibits a slower migration pattern on SDS-PAGE, indicating a conformational change that likely affects its enzymatic activity .

  To investigate these mechanisms, researchers typically employ a combination of site-directed mutagenesis (S228A or S228E mutations), protein-protein interaction assays, and enzyme activity measurements using histone substrates .

• How does CARM1 phosphorylation at Ser228 regulate estrogen receptor-dependent gene expression?

  CARM1 phosphorylation at Ser228 regulates estrogen receptor (ER)-dependent gene expression through a multi-faceted mechanism:

  1. Transcriptional impact: The S229E phosphomimetic mutation impairs CARM1's ability to stimulate estrogen-induced ER transcription . This can be measured using reporter gene assays with estrogen-responsive elements.

  2. Target gene modulation: Phosphorylation of CARM1 compromises its ability to upregulate endogenous ER target genes. Studies have demonstrated that unlike wild-type CARM1, the S229E mutant fails to stimulate key ER target genes such as EGFR and EBAG9 . This can be quantified using RT-PCR analysis.

  3. Coactivator synergy disruption: CARM1 normally acts synergistically with GRIP1/TIF2 to activate ER-dependent transcription, but phosphorylation disrupts this coordinated activity .

  Experimentally, researchers can investigate this regulation by:

  • Transfecting wild-type, S228A (non-phosphorylatable), or S228E (phosphomimetic) CARM1 constructs into CARM1-deficient cells

  • Measuring expression of ER target genes after estrogen treatment using qRT-PCR

  • Analyzing ER-dependent promoter activity using luciferase reporter assays

  • Examining coactivator complex formation through coimmunoprecipitation studies

• What is known about the kinases responsible for CARM1 Ser228 phosphorylation and how can they be identified?

  Several kinases have been implicated in CARM1 phosphorylation at different sites, with specific evidence for Ser228:

  Phosphorylation Site	Kinase	Effect	Reference
  S228 (human)/S229 (mouse)	PKC	Prevents CARM1 homodimerization
  S216 (human)/S217 (mouse)	Unknown	Blocks SAM binding, promotes cytoplasmic localization
  S447 (human)/S448 (mouse)	PKA	Facilitates CARM1 binding to ERα
  S595 (human)	p38 MAPK	Prevents nuclear translocation

  To identify and confirm the kinases responsible for Ser228 phosphorylation, researchers can employ:

  1. Kinase inhibitor screening: Treat cells with specific inhibitors (e.g., PKC inhibitors like staurosporine) and assess CARM1 phosphorylation status using phospho-specific antibodies .

  2. In vitro kinase assays: Incubate purified CARM1 with candidate kinases and ATP, then detect phosphorylation using phospho-specific antibodies or mass spectrometry .

  3. Kinase knockdown/knockout: Use siRNA or CRISPR to deplete specific kinases and examine effects on CARM1 phosphorylation .

  4. Mass spectrometry-based phosphoproteomics: Analyze CARM1 phosphorylation sites under various conditions to identify regulated sites and potentially implicate specific kinases based on consensus motifs .

• How can researchers induce and detect CARM1 phosphorylation at Ser228 in cellular models?

  To effectively induce and detect CARM1 phosphorylation at Ser228, researchers can implement the following methodological approaches:

  1. Cell cycle synchronization: CARM1 phosphorylation has been observed in mitotic cells, so researchers can synchronize cells using nocodazole (a microtubule inhibitor) treatment to enrich for mitotic populations .

  2. Growth factor stimulation: Some growth factors affect CARM1 phosphorylation levels. For example, EGF treatment of A431 cells has been used as a positive control for phospho-CARM1 (Ser228) antibodies .

  3. Detection methods:

     • Western blotting: The phosphorylated form of CARM1 appears as a slightly slower migrating band that can be detected using specific phospho-CARM1 (Ser228) antibodies .

     • Phospho-amino acid analysis: For detailed characterization, researchers can culture cells in phosphate-free media supplemented with [³²P] phosphoric acid, immunoprecipitate CARM1, and perform phospho-amino acid analysis .

     • Mass spectrometry: For precise identification of phosphorylation sites, mass spectrometric analysis can be performed on immunoprecipitated CARM1 .

  4. Validation controls:

     • λ-phosphatase treatment: Treating samples with λ-phosphatase before Western blotting should eliminate the phospho-specific signal .

     • Phosphorylation-deficient mutants: Compare with S228A mutant-expressing cells as a negative control .

• What is the relationship between CARM1 phosphorylation and cancer development?

  CARM1 phosphorylation has significant implications for cancer development and progression:

  1. Differential phosphorylation patterns: Studies have shown varying levels of CARM1 phosphorylation across different cancer cell lines. For example, phosphorylated CARM1 is detectable in SK-BR3 breast cancer cells that express high levels of Her2/Neu/ErbB2, while it was initially not detected in MCF7 breast cancer cells .

  2. Impact on estrogen receptor signaling: Since CARM1 phosphorylation negatively regulates ER-dependent gene expression, alterations in CARM1 phosphorylation status could impact estrogen-driven cancers, particularly breast cancer .

  3. Potential as a biomarker: The phosphorylation status of CARM1 could potentially serve as a biomarker for certain cancer types or stages .

  Research methodologies to investigate this relationship include:

  • Comparative analysis of CARM1 phosphorylation across cancer cell lines using phospho-specific antibodies

  • Correlation of CARM1 phosphorylation status with clinical parameters in patient samples

  • Functional studies examining how modulation of CARM1 phosphorylation affects cancer cell proliferation, migration, and response to therapies

  • Investigation of the relationship between oncogenic signaling pathways and CARM1 phosphorylation

• How do other post-translational modifications of CARM1 interact with Ser228 phosphorylation?

  CARM1 undergoes multiple post-translational modifications that may interact with Ser228 phosphorylation:

  Human AA	Mouse AA	Type of PTM	Enzymes	Effect
  S228	S229	Phosphorylation	PKC	Prevents CARM1 homodimerization
  S216	S217	Phosphorylation	Unknown	Blocks SAM binding, promotes cytoplasmic localization
  S447	S448	Phosphorylation	PKA	Facilitates CARM1 binding to ERα
  S595	S595	Phosphorylation	p38 MAPK	Prevents nuclear translocation

  Investigating the interplay between these modifications requires sophisticated methodologies:

  1. Multi-site mutant analysis: Generate CARM1 constructs with combinations of phosphomimetic (S→E) and phospho-deficient (S→A) mutations at different sites to analyze functional interactions.

  2. Sequential immunoprecipitation: Use antibodies against one modification to immunoprecipitate CARM1, then probe for other modifications to determine co-occurrence.

  3. Mass spectrometry approaches: Employ techniques like parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) to simultaneously quantify multiple phosphorylation sites on CARM1.

  4. Temporal dynamics analysis: Study the order and timing of different phosphorylation events during cell cycle progression or in response to stimuli using synchronized cell populations .

• How can researchers distinguish between CARM1 isoforms and their phosphorylation states in experimental systems?

  CARM1 exists in multiple isoforms, and distinguishing between them and their phosphorylation states requires careful experimental design:

  1. Isoform-specific detection:

     • Use antibodies targeting unique regions of specific isoforms

     • Employ RT-PCR with primers spanning unique exon junctions to detect isoform-specific mRNAs

     • Use mass spectrometry to identify isoform-specific peptides

  2. Phosphorylation state analysis:

     • For Western blotting, use isoform-specific antibodies in parallel with phospho-specific antibodies

     • Run samples on Phos-tag gels, which can separate proteins based on their phosphorylation state

     • Use phosphatase treatment controls to confirm phospho-specific bands

  3. Expression systems for controlled studies:

     • Generate expression vectors for specific CARM1 isoforms with tags for detection

     • Introduce phospho-null (S228A) or phosphomimetic (S228E) mutations in different isoform backgrounds

     • Express these constructs in CARM1-deficient cells (like MEF-/- cells) to avoid endogenous CARM1 interference

  4. Functional validation:

     • Compare the methyltransferase activity of different isoforms and their phosphorylation variants using in vitro methylation assays

     • Assess subcellular localization patterns using immunofluorescence

     • Evaluate transcriptional coactivator function using reporter gene assays

• What methods can be used to study the functional impact of CARM1 Ser228 phosphorylation on target gene expression?

  To investigate how CARM1 Ser228 phosphorylation affects target gene expression, researchers can implement these methodological approaches:

  1. CARM1 variant expression systems:

     • Express wild-type CARM1, phospho-null (S228A), or phosphomimetic (S228E) mutants in CARM1-deficient cells

     • Use inducible expression systems to control timing and level of expression

     • Consider viral delivery systems for efficient transduction in hard-to-transfect cells

  2. Gene expression analysis:

     • Quantitative RT-PCR to measure expression of known CARM1 target genes (e.g., EGFR, EBAG9 for ER-regulated genes)

     • RNA-seq for genome-wide transcriptome analysis to identify differentially expressed genes

     • ChIP-seq to examine CARM1 recruitment and histone arginine methylation at target gene promoters

  3. Reporter gene assays:

     • Use luciferase reporters driven by promoters of CARM1 target genes

     • Assess HTLV-1 LTR-Luc activity, which has been shown to be enhanced by CARM1

     • Co-transfect with interacting transcription factors (e.g., estrogen receptor) and evaluate synergistic effects

  4. Single-cell analysis:

     • Examine heterogeneity in response using single-cell RNA-seq

     • Combine with phospho-flow cytometry to correlate CARM1 phosphorylation state with gene expression at the single-cell level

  These approaches should be complemented with appropriate controls, including catalytically inactive CARM1 mutants (e.g., VLD 189-191-AAA) as negative controls for methyltransferase-dependent effects .

Technical Considerations

• What are the best experimental controls when working with Phospho-CARM1 (Ser228) antibodies?

  Implementing appropriate controls is critical for reliable results with phospho-specific antibodies:

  1. Positive controls:

     • Nocodazole-treated cells: Mitotic cells show enhanced CARM1 phosphorylation at Ser228

     • EGF-treated A431 cells: Validated as positive controls for many commercial antibodies

     • Jurkat cells: Identified as positive samples for some Phospho-CARM1 (Ser228) antibodies

  2. Negative controls:

     • Phosphatase treatment: Samples treated with λ-phosphatase should show diminished or absent signal

     • S228A mutant expression: Cells expressing the non-phosphorylatable mutant should show minimal signal

     • CARM1 knockdown/knockout: siRNA against CARM1 or CARM1-deficient cells should show no specific signal

  3. Specificity controls:

     • Peptide competition assay: Pre-incubating the antibody with the phosphopeptide immunogen should abolish specific signal

     • Total CARM1 antibody comparison: Run parallel blots with phospho-specific and total CARM1 antibodies to distinguish changes in phosphorylation from changes in expression

  4. Cross-reactivity assessment:

     • Test against other PRMT family members, particularly those with similar sequences around the phosphorylation site

     • Include samples from multiple species if working across species boundaries, as the equivalent site is Ser229 in mouse/rat CARM1

• How can researchers optimize Western blot protocols for detecting phosphorylated CARM1?

  Optimizing Western blot protocols for phosphorylated CARM1 detection requires attention to several critical factors:

  1. Sample preparation:

     • Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers

     • Prepare samples quickly and maintain cold temperatures throughout to minimize dephosphorylation

     • Consider using specialized lysis buffers designed for phosphoprotein preservation

  2. Protein separation:

     • Use lower percentage (7-8%) SDS-PAGE gels for better resolution of the subtle mobility shift of phosphorylated CARM1

     • Consider Phos-tag acrylamide gels for enhanced separation of phosphorylated and non-phosphorylated forms

     • Ensure complete protein denaturation to avoid artifacts from incomplete SDS binding

  3. Antibody conditions:

     • Optimize primary antibody dilution: Most Phospho-CARM1 (Ser228) antibodies work best at 1:500-1:1000 dilutions

     • Include appropriate blocking agents to minimize background

     • Consider longer incubation times at 4°C to enhance specific binding

  4. Detection optimization:

     • Use enhanced chemiluminescence (ECL) or fluorescent secondary antibodies for sensitive detection

     • Extended exposure times may be necessary, as the phosphorylated form can represent a small fraction of total CARM1

     • Consider signal amplification methods for low-abundance phosphorylated forms

  5. Data analysis:

     • Always normalize phospho-CARM1 signal to total CARM1 levels

     • Use appropriate software for densitometric analysis

     • Include multiple biological replicates for statistical validation

Emerging Research Directions

• What are the implications of CARM1 Ser228 phosphorylation in cellular senescence and aging research?

  CARM1 has emerging roles in cellular senescence and aging processes, with phosphorylation potentially playing a regulatory role:

  1. CARM1 and cellular senescence:

     • Reduced CARM1 expression contributes to senescence of alveolar epithelial cells

     • CARM1 haploinsufficiency is associated with decreased anti-senescence factor SIRT1 and increased senescence markers p16 and β-galactosidase

     • In vitro knockdown of CARM1 in ATII-like cell line LA-4 leads to decreased SIRT1 expression and increased expression of senescence markers p16 and p21

  2. Methodological approaches to study phosphorylation in this context:

     • Compare wild-type CARM1 with phosphomimetic S228E mutant effects on senescence markers

     • Analyze correlation between CARM1 phosphorylation status and cellular senescence across different tissues

     • Investigate age-dependent changes in CARM1 phosphorylation patterns

     • Examine the impact of senescence-inducing stressors on CARM1 phosphorylation

  3. Potential mechanisms:

     • Phosphorylation at Ser-217 serves as a molecular switch for controlling CARM1 enzymatic activity during the cell cycle

     • CARM1 phosphorylation may affect its interaction with anti-senescence factors like SIRT1

     • Altered CARM1 methyltransferase activity due to phosphorylation could influence the expression of genes involved in senescence pathways

  Future investigations should explore whether Ser228 phosphorylation specifically modulates CARM1's role in cellular senescence and aging processes, potentially opening new avenues for interventions targeting age-related diseases.

• How can Phospho-CARM1 (Ser228) antibodies be used in cancer therapeutics research?

  Phospho-CARM1 (Ser228) antibodies offer valuable tools for cancer therapeutics research through several methodological applications:

  1. Biomarker development:

     • Screen cancer tissue microarrays to correlate CARM1 phosphorylation patterns with clinical outcomes

     • Evaluate phospho-CARM1 levels before and after treatment to assess therapeutic response

     • Develop immunohistochemistry protocols using phospho-specific antibodies for potential diagnostic applications

  2. Target validation studies:

     • Monitor changes in CARM1 phosphorylation in response to kinase inhibitors

     • Correlate phosphorylation status with sensitivity to epigenetic therapies

     • Use phospho-CARM1 antibodies in high-content screening to identify compounds that modulate CARM1 phosphorylation

  3. Mechanism-of-action studies:

     • Investigate how existing cancer therapies affect CARM1 phosphorylation

     • Examine whether therapeutic resistance correlates with changes in CARM1 phosphorylation status

     • Study combination therapies targeting both CARM1 activity and its phosphorylation

  4. Therapeutic development strategies:

     • Use knowledge of phosphorylation-dependent CARM1 inactivation to design peptide inhibitors mimicking the phosphorylated region

     • Develop small molecules that stabilize the phosphorylated conformation of CARM1

     • Screen for compounds that enhance CARM1 phosphorylation as a strategy to inhibit its activity in cancers where CARM1 is overactive

  The relevance of this approach is supported by evidence that CARM1 has diverse physiological functions in cancer, including roles in oxidative stress response, cell death, metabolism, tumor development, metastasis, and therapeutic resistance .