Phospho-MDM2 (S186/S188) Antibody

What is the biological significance of MDM2 phosphorylation at S186/S188?

MDM2 phosphorylation at S186/S188 plays a crucial role in regulating p53 activity and stability. Akt-mediated phosphorylation of MDM2 at these sites increases its interaction with p300, facilitating MDM2-mediated ubiquitination and subsequent degradation of p53 . Additionally, this phosphorylation blocks MDM2's binding to p19ARF, further enhancing p53 degradation . The S186/S188 phosphorylation sites are located near the Nuclear Localization Signal (NLS) of MDM2 (residues 179-185), and phosphorylation at these sites promotes MDM2 nuclear translocation, enhancing its ability to inactivate p53 in response to growth factor signaling .

How does MDM2 regulation of p53 contribute to cancer development?

MDM2 functions as a negative regulator of p53 through multiple mechanisms. It binds and inhibits the transactivation function of p53, and as an E3 ubiquitin ligase, targets p53 for proteasomal degradation . Overexpression or amplification of MDM2 can result in excessive inactivation of p53, diminishing its tumor suppressor function and potentially promoting tumor formation . MDM2 is overexpressed in various human malignancies, including soft tissue carcinomas and breast cancer . The protein also affects cell cycle, apoptosis, and tumorigenesis through interactions with other proteins, including retinoblastoma 1 and ribosomal protein L5 .

What are the key structural and functional domains of MDM2 relevant to its phosphorylation?

MDM2 contains both Nuclear Localization Signal (NLS) (residues 179-185, RQRKRHK) and Nuclear Export Signal (NES) (residues 197-199, LSFDESLAL) sequences that facilitate its nuclear-cytoplasmic shuttling . The S186/S188 phosphorylation sites are strategically positioned near the NLS domain. MDM2 phosphorylation sites generally cluster in regions that are structurally disordered, which is typical for many phosphorylation sites . The protein contains multiple functional domains, including an N-terminal p53-binding domain, a central acidic domain, a zinc finger domain, and a C-terminal RING finger domain with E3 ubiquitin ligase activity.

How does phosphorylation at different sites on MDM2 create a regulatory network?

The MDM2 protein contains multiple phosphorylation sites that form a complex regulatory network. In addition to S186/S188, MDM2 can be phosphorylated at S166 by Akt1, which works in concert with S186/S188 phosphorylation to enhance nuclear localization and p53 degradation . After DNA damage, MDM2 can be phosphorylated at S395 by ATM, at S407 by ATR, and at Y394 by c-Abl . Recent mass spectrometric analysis has revealed additional phosphorylation sites (S386, T419, S425, and S429) that are induced by DNA damage .

These phosphorylation sites demonstrate significant functional redundancy in regulating p53 degradation, with phosphomimetic substitution of a single site capable of strongly inhibiting p53 degradation . The combination of phosphorylation events creates a sophisticated regulatory system that can respond to various cellular signals and stresses.

What are the methodological challenges in studying phosphorylation-specific MDM2 functions?

Studying phosphorylation-specific MDM2 functions presents several challenges. One significant issue involves the antibodies used for detection. Some commonly used antibodies, such as SMP14 and 2A10, have epitopes that can be masked by phosphorylation . For example, the 2A10 epitope contains S395 and can be masked by ATM-mediated phosphorylation, while SMP14 reactivity is also blocked by phosphorylation of an unknown site in its epitope .

This can lead to misinterpretation of results, as decreased antibody reactivity after treatments like irradiation may be incorrectly interpreted as decreased MDM2 levels rather than epitope masking. Researchers must carefully select appropriate antibodies and validate their findings using multiple detection methods to avoid such artifacts.

How do MDM2 phosphorylation events integrate with other post-translational modifications?

MDM2 undergoes various post-translational modifications beyond phosphorylation, including ubiquitination and deubiquitination. Phosphorylation on S166 by SGK1 activates ubiquitination of p53/TP53 . Autoubiquitination of MDM2 leads to its proteasomal degradation, resulting in p53/TP53 activation, which may be regulated by SFN . MDM2 can also be ubiquitinated by TRIM13 .

The deubiquitination landscape adds another layer of complexity, as MDM2 can be deubiquitinated by USP2, leading to MDM2 accumulation and increased deubiquitination and degradation of p53/TP53 . Alternatively, deubiquitination by USP7 leads to MDM2 stabilization . These various modifications create a sophisticated regulatory network that fine-tunes MDM2 function in response to different cellular contexts and signals.

What are the optimal application conditions for Phospho-MDM2 (S186/S188) antibody in different experimental techniques?

The antibody is typically formulated as a liquid in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide . For long-term storage, it should be kept at -20°C, with aliquoting recommended to avoid repeated freeze-thaw cycles .

How can researchers validate the specificity of Phospho-MDM2 (S186/S188) antibody?

To validate antibody specificity, researchers should consider several approaches:

• Phosphatase treatment: Treating samples with lambda phosphatase should eliminate the signal from a phospho-specific antibody.

• Blocking peptide experiments: Using the phosphorylated peptide immunogen to block antibody binding. As noted in the search results: "Blocking peptides are peptides that bind specifically to the target antibody and block antibody binding. These peptides usually contain the epitope recognized by the antibody. Antibodies bound to the blocking peptide no longer bind to the epitope on the target protein."

• Kinase activation/inhibition: Treating cells with Akt pathway activators (e.g., growth factors) or inhibitors to modulate the phosphorylation status of MDM2 at S186/S188.

• Mutational analysis: Using cells expressing MDM2 with S186A/S188A mutations as a negative control.

• Multiple detection methods: Confirming results using different techniques (WB, IF, ELISA) to ensure consistent findings.

What experimental controls are essential when working with Phospho-MDM2 (S186/S188) antibody?

When designing experiments with Phospho-MDM2 (S186/S188) antibody, the following controls are essential:

• Positive control: Samples known to have high levels of S186/S188 phosphorylation, such as cells treated with growth factors that activate Akt. Search results suggest SGC7901 and Panc1 cell lysates as positive controls for Western blot .

• Negative control: Samples with low or no phosphorylation at these sites, such as serum-starved cells or cells treated with Akt inhibitors.

• Loading control: An antibody against total MDM2 to normalize for total protein expression, allowing accurate quantification of phosphorylation levels.

• Secondary antibody control: Samples treated with secondary antibody only to identify any non-specific binding.

• Phosphatase-treated samples: Treatment with lambda phosphatase to remove phosphorylation and confirm antibody specificity.

What are common causes of false negative or weak signals when detecting Phospho-MDM2 (S186/S188)?

Several factors can contribute to false negative or weak signals:

• Rapid dephosphorylation: MDM2 phosphorylation is dynamic and can be rapidly lost during sample preparation. Always include phosphatase inhibitors in lysis buffers.

• Low abundance: Phosphorylated forms of proteins often represent a small fraction of the total protein. Consider using enrichment techniques such as immunoprecipitation.

• Epitope masking: Other post-translational modifications or protein-protein interactions may mask the phospho-epitope.

• Cell type variations: Different cell types may have different basal levels of MDM2 phosphorylation. Cell lines with known MDM2 overexpression (like certain cancer cells) may provide better detection.

• Antibody degradation: Repeated freeze-thaw cycles can diminish antibody activity. Aliquot antibodies upon receipt to minimize this issue.

How does MDM2 phosphorylation status change during the DNA damage response?

During DNA damage response, the phosphorylation pattern of MDM2 undergoes significant changes. DNA damage activates kinases like ATM, which phosphorylates MDM2 at several sites (S386, T419, S425, and S429) . These phosphorylation events inhibit MDM2-mediated degradation of p53, allowing p53 to accumulate and initiate cell cycle arrest or apoptosis.

Interestingly, while many studies reported transient down-regulation of MDM2 levels after DNA damage, recent findings suggest caution in interpreting such data. The commonly used antibodies 2A10 and SMP14 have epitopes that can be masked by phosphorylation, making it appear that MDM2 levels decrease when in fact the protein may still be present but not detectable by these antibodies . Studies using other antibodies have shown that MDM2 levels do not undergo significant decreases after irradiation .

What considerations are important when comparing results across different Phospho-MDM2 (S186/S188) antibody sources?

When comparing results from different antibody sources, researchers should consider:

• Immunogen differences: Although all Phospho-MDM2 (S186/S188) antibodies target the same phosphorylation sites, the exact immunogen sequence and length may vary. For example, some antibodies are raised against synthetic phosphopeptides corresponding to residues surrounding S186/S188 , while others specify the immunogen as being within amino acids 151-200 of human MDM2 .

• Host species and clonality: Most Phospho-MDM2 (S186/S188) antibodies in the search results are rabbit polyclonal antibodies, but variations in rabbit immunization protocols and purification methods can lead to batch-to-batch variability.

• Validation methods: Different manufacturers may use different validation methods, affecting the reliability of specificity claims.

• Formulation differences: Variations in buffer composition, preservatives, and stabilizers may affect antibody performance in specific applications.

• Recommended dilutions: Optimal dilutions vary between manufacturers, reflecting differences in antibody concentration and affinity.