Phospho-MUC1 (Y1243) Antibody

What is MUC1 and what is its normal physiological role?

MUC1 (CD227) is a transmembrane glycoprotein belonging to the mucin family. It is a type-I transmembrane heterodimer composed of two non-covalently bound subunits. The larger α subunit is located extracellularly and consists of the N-terminal region, Variable Number of Tandem Repeats (VNTR) region, and C-terminal region. The smaller β subunit consists of extracellular, transmembrane, and cytoplasmic regions .

In normal physiology, MUC1 serves several important functions:

• The alpha subunit has cell adhesive properties and can act both as an adhesion and anti-adhesion protein

• Provides a protective layer on epithelial cells against bacterial and enzyme attack

• The beta subunit contains a C-terminal domain involved in cell signaling through phosphorylations and protein-protein interactions

• Modulates signaling in ERK, SRC, and NF-kappa-B pathways

• In activated T-cells, influences directly or indirectly the Ras/MAPK pathway

MUC1 is normally expressed at the apical surface of epithelial cells and during fetal development is expressed at low levels in the colonic epithelium from 13 weeks of gestation .

Why is phosphorylation of MUC1 important in cellular signaling?

Phosphorylation of MUC1 plays a critical role in regulating its cellular functions, particularly in signaling pathways:

• Phosphorylation on tyrosines in the C-terminal increases the nuclear location of MUC1 and beta-catenin

• Phosphorylation by PKC delta induces binding of MUC1 to beta-catenin/CTNNB1, decreasing the formation of the beta-catenin/E-cadherin complex

• Src-mediated phosphorylation inhibits interaction with GSK3B

• Src- and EGFR-mediated phosphorylation on Tyr-1229 increases binding to beta-catenin/CTNNB1

• GSK3B-mediated phosphorylation on Ser-1227 decreases this interaction but restores the formation of the beta-cadherin/E-cadherin complex

• On T-cell receptor activation, MUC1 is phosphorylated by LCK

• PDGFR-mediated phosphorylation increases nuclear colocalization of MUC1CT and CTNNB1

These phosphorylation events act as molecular switches that regulate MUC1's interactions with various signaling molecules and transcription factors, thereby influencing multiple cellular processes including proliferation, adhesion, and survival.

What is Phospho-MUC1 (Y1243) Antibody and how is it generated?

Phospho-MUC1 (Y1243) Antibody is a polyclonal antibody that specifically recognizes MUC1 when it is phosphorylated at tyrosine residue 1243. According to the search results:

• It is typically raised in rabbits using a synthetic peptide derived from human MUC1 around the phosphorylation site of Y1243

• The antibody is affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogen

• It specifically binds to the phosphorylated form of MUC1 at Y1243, allowing researchers to detect this specific post-translational modification

The typical form of this antibody is supplied as a liquid in PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide . Blocking peptides are also available for confirming antibody specificity in experimental workflows .

What are the validated applications for Phospho-MUC1 (Y1243) Antibody?

Based on the search results, Phospho-MUC1 (Y1243) Antibody has been validated for several research applications:

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): Typically used at dilutions of 1:100-1:300

• Enzyme-Linked Immunosorbent Assay (ELISA): Typically used at dilutions of 1:20000

The antibody has demonstrated reactivity with both human and mouse samples, making it valuable for comparative studies across species .

How should researchers optimize IHC protocols with this antibody?

For optimal IHC results with Phospho-MUC1 (Y1243) Antibody, researchers should consider:

• Sample Preparation:

  • Use freshly fixed tissues (10% neutral buffered formalin recommended)

  • Ensure thorough deparaffinization and rehydration

  • Include phosphatase inhibitors in all buffers to prevent loss of phosphorylation

• Antibody Dilution and Incubation:

  • Start with the recommended dilution range of 1:100-1:300 for IHC-P

  • Perform a titration experiment to determine optimal concentration for your specific tissue

  • Include positive controls (tissues known to express phosphorylated MUC1)

• Specificity Controls:

  • Use the available blocking peptide to confirm specificity

  • Include a negative control omitting primary antibody

  • Consider using tissues from MUC1 knockout models if available

Following these guidelines will help ensure specific detection of phosphorylated MUC1 at Y1243 in tissue samples.

How is MUC1 dysregulated in cancer and what is its significance?

MUC1 shows significant alterations in cancer that make it an important research target:

• MUC1 is overexpressed and aberrantly glycosylated in most human epithelial cancers

• The aberrantly glycosylated MUC1 expressed on malignant cells (tumor-associated MUC1 or tMUC1) exposes epitopes that are usually inaccessible

• In cancer cells, MUC1 displays hypoglycosylation of the core glycans, like sialylation of Tn and T antigens leading to premature chain termination

• MUC1 expression has been shown to be up to 10 times higher in many human carcinomas than in normal tissues, which provides resistance to chemotherapy

MUC1 contributes to cancer progression through multiple mechanisms:

• Inhibits cell death and promotes metastasis

• Induces signaling through the EGFR family and enhances ERK activation and cell proliferation

• Interacts with β-catenin, stabilizes it and co-activates Wnt signaling

• Dampens drug-induced apoptosis by interacting with p53 and FOXO3a

• Reduces pro-apoptotic signaling via HSP90, PI3K/Akt and Caspase-8 pathways

• Confers drug resistance in pancreatic ductal adenocarcinoma cells

These properties have led the National Cancer Institute to rank tMUC1 as the second most targetable antigen out of 75 for developing cancer vaccines .

How does phosphorylation at Y1243 compare with other MUC1 phosphorylation sites?

While the search results don't provide direct comparisons specific to Y1243, several other phosphorylation sites on MUC1 have been characterized:

Researchers interested in Y1243-specific signaling would need to:

• Conduct phosphorylation-specific functional studies

• Perform site-directed mutagenesis (e.g., Y1243F) to compare functional consequences

• Investigate if there's hierarchical phosphorylation or cross-talk between these sites

This comparative approach would help elucidate the unique contributions of Y1243 phosphorylation to MUC1 function.

What is the clinical significance of MUC1 in patient samples?

MUC1 has established clinical significance as a biomarker:

• MUC1/CA 15-3 is used as a serological clinical marker of breast cancer to monitor response to treatment and disease recurrence

• Decreased levels over time may indicate a positive response to treatment

• Increased levels may indicate disease progression

• At an early stage of disease, only 21% of patients exhibit high MUC1/CA 15-3 levels, which limits its usefulness as a screening test

Studies have also found:

Additionally, autoantibodies to MUC1 glycopeptides (MUC1-STn and MUC1-Core3) showed 44.6% sensitivity at 95% specificity for detecting colorectal cancer .

How does aberrant glycosylation in cancer affect MUC1 phosphorylation detection?

This is a critical consideration when studying MUC1 in cancer contexts:

MUC1 glycosylation is extensive and tissue-dependent:

• O-glycosylation is initiated by adding N-acetyl-galactosamine (GalNAc) to the VNTR region rich in threonine and serine residues

• A large family of up to 20 distinct polypeptide GalNAc transferases form the initial O-linked GalNAcα-Ser/Thr structure (Tn antigen)

• In cancer cells, MUC1 displays hypoglycosylation with sialylation of Tn and T antigens via sialyltransferase enzymes that lead to premature chain termination

Nevertheless, researchers should be aware that:

• Changes in extracellular domain glycosylation might indirectly affect cytoplasmic domain conformation

• Different cancer types may exhibit distinct glycosylation profiles that could influence detection consistency

• Validation across multiple cancer types with different glycosylation patterns is advisable

How is MUC1 being targeted for cancer immunotherapy?

The search results highlight several approaches for targeting MUC1 in cancer immunotherapy:

• Antibody-Based Approaches:

  • Humanized antibodies like HMFG1 (AS1402, huHMFG1) that recognize the PDTR epitope within the VNTR region

  • Novel bispecific antibodies such as Muc1-Bi-1 that link anti-Muc1-VHH and anti-CD16-VHH to recruit Natural Killer (NK) cells

  • Humanized forms like Muc1-Bi-2 that can suppress tumor growth in the presence of human peripheral blood mononuclear cells (PBMC)

• Cancer Vaccines:

  • MUC1 was ranked as the second most promising target by the National Cancer Institute Translational Research Working Group

  • Vaccines targeting aberrantly glycosylated MUC1 epitopes

• Adoptive Cell Therapies:

  • Antibodies against tMUC1 are used to design human T cells targeting MUC1, called Chimeric Antigen Receptor T-cells (CAR T cells)

• Antibody-Drug Conjugates (ADC):

  • Mentioned as one of the approaches being evaluated for MUC1-targeted therapy

These diverse approaches capitalize on MUC1's properties as a tumor-associated antigen that is adequately immunogenic to trigger strong antitumor effects .

What are the major technical challenges when studying phosphorylated MUC1?

Researchers working with phosphorylated MUC1 face several technical challenges:

• Phosphorylation Lability:

  • Phosphorylations are easily lost during sample collection and processing

  • Endogenous phosphatases can rapidly dephosphorylate the target

• MUC1 Glycosylation Complexity:

  • Extensive and heterogeneous glycosylation patterns between patients and tissues

  • Aberrant glycosylation in cancer adds another layer of variability

  • Potential interference with antibody access to epitopes

• Sample Processing Effects:

  • Variations in fixation time and conditions between clinical samples

  • Differences in tissue preservation methods affecting phospho-epitope integrity

• Detection Sensitivity:

  • Low abundance of specific phosphorylation among total MUC1 protein

  • Signal-to-noise challenges in complex tissue environments

Recommended Solutions:

• Incorporate phosphatase inhibitors in all buffers during sample processing

• Use standardized protocols for tissue handling and fixation

• Validate antibody performance across different cancer types with varied glycosylation profiles

• Consider using blocking peptides (as mentioned in result #11) to confirm specificity

• Combine multiple detection methods (IHC, ELISA, western blotting) for validation

• Include appropriate controls in each experiment

Addressing these challenges is essential for obtaining reliable and reproducible results when studying MUC1 phosphorylation in research and clinical settings.