Phospho-MYC (T58+S62) Recombinant Monoclonal Antibody

What is MYC and why is phosphorylation at T58 and S62 significant?

MYC is a proto-oncogene that encodes a nuclear phosphoprotein playing crucial roles in cell cycle progression, apoptosis, and cellular transformation. It forms a heterodimer with transcription factor MAX to bind E-box DNA consensus sequences and regulate transcription of specific target genes . Phosphorylation at T58 and S62 residues is critical for regulating MYC protein stability and function. Specifically, S62 phosphorylation primarily by ERK stabilizes MYC, while subsequent T58 phosphorylation by GSK3β initiates a cascade leading to MYC degradation . These phosphorylation events thus serve as a molecular switch controlling MYC's cellular activities and abundance, with dysregulation of this process being implicated in various cancers.

How do T58 and S62 phosphorylation events regulate MYC protein stability?

MYC protein stability is regulated through a sequential phosphorylation mechanism. In response to growth signals, MYC is first phosphorylated at S62 by proline-directed kinases including ERK or CDKs, which transiently increases MYC stability. Subsequently, phosphorylation at T58 is mediated by GSK3β or BRD4, which initiates the dephosphorylation of S62 by Protein Phosphatase 2A (PP2A) . This dephosphorylation is facilitated by the peptidyl prolyl isomerase PIN1. Following these events, the E3 ligase complex SCF-FBW7 ubiquitinates MYC, targeting it for proteasomal degradation . This phosphorylation-dependent degradation pathway is a critical regulatory mechanism that prevents excessive MYC activity in normal cells.

What are the different MYC isoforms and how do they affect antibody selection?

MYC has multiple isoforms, with the 439 amino acid isoform (P01106-1, UniProt) being the canonical form. Importantly, the S62 phosphorylation site in this canonical isoform corresponds to S77 in isoform 2 (P01106-2) . When selecting antibodies, researchers must consider which isoform they're studying and ensure the antibody recognizes the appropriate phosphorylation site. For example, an antibody specific to phospho-S62 in the canonical isoform may not effectively detect the equivalent modification in isoform 2 unless it's designed to recognize the conserved motif around this phosphorylation site regardless of the exact residue number.

What are the key differences between phospho-specific antibodies targeting T58, S62, or both sites?

Phospho-specific antibodies can target T58 alone, S62 alone, or both sites simultaneously, with each offering distinct research advantages:

What are the validated applications for Phospho-MYC (T58+S62) antibodies?

Phospho-MYC (T58+S62) antibodies have been validated for several experimental applications:

It's recommended to titrate the antibody in each testing system to obtain optimal results, as sample type can significantly affect performance .

What are the critical controls needed when using Phospho-MYC (T58+S62) antibodies?

When designing experiments with phospho-MYC antibodies, several controls are essential:

• Positive control: Cells treated with MG132 (proteasome inhibitor) to accumulate phosphorylated MYC. HEK-293T cells treated with MG132 have been validated for this purpose .

• Negative controls:

  • Phosphatase treatment of lysates to remove phosphorylation

  • siRNA or CRISPR knockout of MYC to confirm antibody specificity

  • Blocking peptide competition assay using the phosphorylated peptide used as immunogen

• Activation control: Serum-starved cells stimulated with growth factors to induce ERK activation and subsequent S62 phosphorylation

• Inhibitor controls: Using MEK inhibitors (to block ERK activity and reduce S62 phosphorylation) or GSK3β inhibitors (to reduce T58 phosphorylation)

These controls help validate antibody specificity and ensure experimental rigor.

How should researchers optimize Western blot protocols for detecting phospho-MYC?

Optimizing Western blot protocols for phospho-MYC detection requires several specific considerations:

• Sample preparation:

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation status

  • Process samples quickly and keep them cold to minimize dephosphorylation

  • Consider using proteasome inhibitors (e.g., MG132) in cell treatment to enhance detection of the otherwise rapidly degraded phospho-MYC forms

• Gel electrophoresis:

  • Use fresh samples when possible, as freeze-thaw cycles can affect phosphorylation

  • Adjust polyacrylamide percentage to optimize separation (typically 8-10% gels)

• Transfer and detection:

  • Use PVDF membranes for better protein retention

  • Block with BSA rather than milk, as milk contains phosphatases

  • Dilute antibody appropriately (typically 1:500-1:2000 for Western blotting)

  • Consider using enhanced chemiluminescence detection systems for better sensitivity

• Data interpretation:

  • The expected molecular weight of phospho-MYC is approximately 51-55 kDa

  • Validate results with total MYC antibody in parallel samples

How can researchers address conflicting results between phospho-MYC antibody detection and functional assays?

When facing discrepancies between phospho-MYC antibody detection and functional outcomes, consider the following strategies:

• Phosphorylation kinetics assessment: MYC phosphorylation is dynamic, so perform time-course experiments to capture transient phosphorylation events that might be missed in single timepoint analyses.

• Antibody validation: Confirm antibody specificity using phosphatase treatments, mutant MYC constructs (T58A and/or S62A), and phospho-mimetic mutants (T58D/E and/or S62D/E).

• Pathway interrogation: Analyze upstream kinases (ERK for S62, GSK3β for T58) and downstream effectors simultaneously to validate the signaling pathway's integrity.

• Alternative detection methods: Complement antibody-based detection with mass spectrometry to quantify phosphorylation stoichiometry at specific sites.

• Subcellular fractionation: MYC functions primarily in the nucleus, so separate nuclear and cytoplasmic fractions to determine if the detected phospho-MYC is in the appropriate cellular compartment.

Integrating these approaches provides a more comprehensive understanding of phospho-MYC biology beyond simple detection.

What are common pitfalls in interpreting Phospho-MYC antibody results and how can they be avoided?

Several pitfalls can complicate phospho-MYC antibody result interpretation:

• Cross-reactivity: Some phospho-MYC antibodies may cross-react with related proteins or with non-phosphorylated MYC. Solution: Always validate antibody specificity using appropriate controls and consider using multiple antibodies targeting different epitopes.

• Isoform complexity: The S62 site in canonical MYC (P01106-1) corresponds to S77 in isoform 2 (P01106-2) . Solution: Clearly identify which MYC isoform is being studied and select antibodies accordingly.

• Rapid phosphorylation turnover: The dynamic nature of MYC phosphorylation can lead to false negatives. Solution: Use proteasome inhibitors or phosphatase inhibitors to stabilize the phosphorylated form during sample preparation.

• Context-dependent phosphorylation: MYC phosphorylation patterns vary across cell types and conditions. Solution: Always include appropriate positive controls specific to your experimental system.

• Signal intensity misinterpretation: Changes in total MYC levels can be misinterpreted as changes in phosphorylation. Solution: Always normalize phospho-MYC signals to total MYC levels.

How can Phospho-MYC antibodies be integrated with other techniques to study MYC-dependent alternative splicing?

Recent research has revealed MYC's role in regulating alternative splicing, which can be studied using integrated approaches:

• Combined RNA-seq and phospho-MYC ChIP-seq: This approach can identify direct splicing targets of phospho-MYC. Pathway-guided analysis has linked MYC to exon changes in various genes, suggesting a broader role in post-transcriptional regulation .

• PAIRADISE statistical model application: When studying MYC-dependent alternative splicing, the PAIRADISE model can be used for conducting paired tests between MYC +/- conditions, with filtering criteria including ≥10 splice junction reads per event and |deltaPSI| > 0.05 .

• Correlation analysis protocol:

  • Calculate pathway activity scores across samples

  • Identify alternatively spliced exons using rMATS-turbo

  • Compute Pearson correlation coefficients between pathway scores and exon inclusion

  • Apply empirical p-value cutoffs (e.g., p < 2 × 10^-4) through permutation testing

• Validation experiments:

  • Use phospho-MYC antibodies to immunoprecipitate and identify bound RNA targets

  • Perform splicing reporter assays with wild-type and phospho-mutant MYC variants

  • Compare results from cells expressing phospho-mimetic MYC mutants versus phospho-deficient mutants

These integrated approaches can reveal how different phosphorylation states of MYC might differentially regulate alternative splicing events.

What are the emerging techniques for studying the dynamic interplay between T58 and S62 phosphorylation?

Several cutting-edge techniques are advancing our understanding of the dynamic relationship between T58 and S62 phosphorylation:

• Live-cell biosensors: FRET-based biosensors that can detect changes in MYC phosphorylation states in real-time within living cells.

• Sequential immunoprecipitation: Using antibodies against different phosphorylation states in sequence to isolate MYC populations with specific combinations of modifications.

• Phospho-proteomics time course analysis: Mass spectrometry-based approaches to quantify the temporal dynamics of T58 and S62 phosphorylation following stimulation.

• Proximity ligation assays (PLA): To visualize and quantify interactions between phospho-MYC and its regulatory partners like PIN1, PP2A, and FBW7 in situ.

• Single-molecule imaging: Techniques to track individual MYC molecules and their phosphorylation-dependent degradation in real-time.

• Computational modeling: Developing mathematical models of the MYC phosphorylation/dephosphorylation cycle to predict how perturbations affect MYC stability.

These emerging techniques provide higher resolution understanding of the temporal and spatial aspects of MYC phosphorylation dynamics.

How do alterations in MYC T58/S62 phosphorylation contribute to lymphomagenesis?

Research using transgenic mouse models has revealed critical insights into how dysregulated phosphorylation at T58 and S62 impacts lymphoma development:

• Differential effects of phosphorylation site mutations:

  • MYC T58A mice (which cannot be phosphorylated at T58) developed clonal T-cell lymphomas with high penetrance

  • MYC S62A mice (which cannot be phosphorylated at S62) developed clonal T-cell lymphomas at a much lower penetrance

• Interaction with endogenous MYC:

  • Loss of endogenous MYC accelerated lymphomagenesis in MYC T58A mice

  • In contrast, loss of endogenous MYC reduced lymphoma penetrance in MYC S62A mice and increased the appearance of non-transgene driven B-cell lymphomas with splenomegaly

• Mechanistic implications:

  • T58 phosphorylation normally promotes MYC degradation; T58A mutation prevents this, leading to MYC stabilization and oncogenic potential

  • S62 phosphorylation normally stabilizes MYC; S62A mutation reduces MYC activity, potentially explaining the lower lymphoma penetrance

These findings highlight the importance of regulated phosphorylation at T58 and S62 for normal T-cell development and tumor suppression, suggesting that disruption of this regulatory mechanism is a key contributor to lymphomagenesis .

What research approaches can be used to target the MYC phosphorylation pathway therapeutically?

Targeting the MYC phosphorylation pathway presents promising therapeutic opportunities:

• Kinase modulation approaches:

  • Inhibiting kinases that phosphorylate S62 (ERK, CDKs) could destabilize MYC in cancers where it's overactive

  • Inhibiting GSK3β to prevent T58 phosphorylation might stabilize MYC in contexts where increased MYC activity is desirable

  • BRD4 inhibitors may affect T58 phosphorylation, offering another regulatory point

• Phosphatase targeting:

  • Modulating PP2A activity could affect the dephosphorylation of S62 following T58 phosphorylation

  • Small molecules that activate PP2A could potentially increase MYC degradation in cancer contexts

• Isomerase interference:

  • PIN1 inhibitors could block the conformational change required for PP2A-mediated S62 dephosphorylation

  • This might maintain the T58/S62 dual-phosphorylated state, altering MYC's degradation dynamics

• E3 ligase complex modulation:

  • Approaches to enhance SCF-FBW7 recognition of phosphorylated MYC could accelerate its degradation

  • Proteolysis-targeting chimeras (PROTACs) could be designed to recognize phospho-MYC and target it for degradation

• Research validation methodology:

  • Phospho-specific antibodies are crucial tools for validating the efficacy of these approaches

  • Monitoring changes in T58/S62 phosphorylation ratios serves as a biomarker for treatment efficacy

  • Cell-based assays measuring MYC half-life can assess the functional impact of these therapeutic strategies

These research directions represent promising avenues for developing novel therapeutics targeting MYC, a historically "undruggable" oncogene.