Phospho-MSN/RDX/EZR (T558) Antibody

What are ERM proteins and what is their biological significance?

ERM (Ezrin, Radixin, Moesin) proteins function as crucial linkers between the plasma membrane and the actin cytoskeleton. These proteins play essential roles in cell adhesion, membrane ruffling, and microvilli formation . ERM proteins exist in two conformational states: inactive (closed) cytosolic monomers/dimers where there is intramolecular interaction between amino- and carboxy-terminal domains, and active (open) forms where this interaction is disrupted .

The active state allows ERM proteins to bind to membrane proteins and the actin cytoskeleton simultaneously, facilitating their role as membrane-cytoskeleton crosslinkers. This activation-inactivation cycle is primarily regulated through phosphorylation events at conserved threonine residues in their C-terminal domains: Thr567 of ezrin, Thr564 of radixin, and Thr558 of moesin . This phosphorylation is critical for proper cytoskeletal organization and cell morphology maintenance.

Why is phosphorylation of ERM proteins significant in cellular processes?

Phosphorylation at the C-terminal threonine residues (Thr567 of ezrin, Thr564 of radixin, Thr558 of moesin) disrupts the amino- and carboxy-terminal association, effectively activating the proteins by allowing them to bind both membrane proteins and actin filaments . This phosphorylation plays a key regulatory role in controlling ERM protein conformation and function, allowing the proteins to respond dynamically to cellular signaling events .

Research has demonstrated that phosphorylation at Thr567 of ezrin is specifically required for cytoskeletal rearrangements and oncogene-induced transformation . Additionally, experimental evidence using site-directed mutagenesis has shown that phosphorylation status at these threonine residues directly affects cell migration, adhesion, and cytoskeletal organization . For example, cells expressing constitutively activated MSN T558E (phosphomimetic) exhibit increased proliferation, invasion, and anchorage-independent growth compared to wild-type cells, while cells expressing constitutively inactivated MSN T558A show the opposite effect .

What biological information does the Phospho-MSN/RDX/EZR (T558) Antibody specifically detect?

The Phospho-MSN/RDX/EZR (T558) Antibody specifically recognizes any of the three ERM proteins only when they are phosphorylated at their respective threonine residues: Thr567 of ezrin, Thr564 of radixin, and Thr558 of moesin . This antibody does not react with unphosphorylated forms of these proteins, making it a valuable tool for detecting the activated state of ERM proteins .

The antibody is typically raised against a synthetic phosphorylated peptide derived from the sequences surrounding these specific threonine residues in human ERM proteins . The consensus sequence recognized is often "YKTLR" with the threonine being phosphorylated . This specificity allows researchers to monitor the activation status of ERM proteins under various experimental conditions and in different cell types.

What are the recommended protocols for using Phospho-MSN/RDX/EZR (T558) Antibody in Western blot analysis?

For optimal Western blot results with Phospho-MSN/RDX/EZR (T558) Antibody, the following protocol is recommended:

Sample preparation and electrophoresis:

• Extract proteins from cells using an appropriate lysis buffer containing phosphatase inhibitors to preserve phosphorylation status

• Separate proteins by SDS-PAGE using a 4-15% gradient gel

• Transfer proteins to nitrocellulose or PVDF membranes at 30V for 18h or 100V for 1.5h

Immunoblotting procedure:

• Block membrane with 5% BSA in TBST for 1 hour at room temperature

• Incubate with Phospho-MSN/RDX/EZR (T558) Antibody at a dilution of 1:500-1:2000 in blocking solution overnight at 4°C

• Wash membrane thoroughly with TBST (3-5 times for 5 minutes each)

• Incubate with HRP-conjugated secondary antibody for 1 hour at room temperature

• Develop using ECL reagent according to manufacturer's instructions

Expected results:
The phosphorylated ERM proteins typically appear as bands around 80 kDa . In some cell types, you may observe differential expression of the three ERM proteins, leading to multiple bands or bands of varying intensity.

How can researchers validate the specificity of phosphorylation detection in their experiments?

Several approaches can be used to validate the specificity of phosphorylation detection:

• Phosphatase treatment control: Treat half of your sample with lambda phosphatase before immunoblotting. The phosphatase-treated sample should show reduced or absent signal with the phospho-specific antibody, confirming that the detected signal is indeed phosphorylation-dependent .

• Phosphopeptide competition: Pre-incubate the antibody with the phosphorylated peptide that was used as the immunogen. This should block specific binding and eliminate true phospho-specific signals . As demonstrated in validation studies, immunoblotting, immunofluorescence, and immunohistochemistry signals are blocked when the antibody is pre-incubated with the phosphopeptide .

• Phosphorylation site mutants: Express wild-type protein alongside phospho-deficient (e.g., T558A) and phospho-mimetic (e.g., T558E) mutants. The phospho-deficient mutant should not be recognized by the antibody, while the phospho-mimetic mutant may or may not be recognized depending on how well the substitution mimics phosphorylation .

• Physiological stimulation: Treat cells with known activators of ERM phosphorylation, such as ATP (5mM) which has been shown to induce phosphorylation in PC-12 cells . This should increase the phospho-ERM signal if the antibody is specific.

What technical considerations should be addressed when performing immunofluorescence with this antibody?

When performing immunofluorescence with Phospho-MSN/RDX/EZR (T558) Antibody, consider the following technical aspects:

• Fixation method: For optimal preservation of phosphorylation epitopes, use 3.7% formaldehyde in PBS for 10 minutes at 4°C . Avoid methanol fixation as it can cause dephosphorylation of some epitopes.

• Permeabilization: Use 0.2% Triton X-100 in PBS-Tween for 30 minutes at room temperature .

• Blocking: Block with 2% BSA in PBS-Tween for 30 minutes to reduce non-specific binding .

• Antibody dilution: Use the antibody at a dilution of 1:200-1:1000 in blocking solution .

• Co-staining recommendations: To visualize the relationship between phosphorylated ERM proteins and actin filaments, co-stain with fluorescently-labeled phalloidin (e.g., Texas red-conjugated phalloidin) .

• Controls: Include a negative control without primary antibody and a peptide competition control to validate specificity .

• Subcellular localization: Phosphorylated ERM proteins are typically localized to membrane structures, particularly membrane protrusions such as microvilli and membrane ruffles. This characteristic localization pattern can help confirm specificity of the staining.

How can site-directed mutagenesis of threonine phosphorylation sites be used to study ERM protein function?

Site-directed mutagenesis of the threonine phosphorylation sites provides a powerful approach to study ERM protein function. The following methodological approach has been validated in research settings:

• Generate mutant constructs:

  • Phospho-deficient (inactive) mutant: Replace threonine with alanine (T558A for moesin, T564A for radixin, T567A for ezrin)

  • Phospho-mimetic (constitutively active) mutant: Replace threonine with glutamic acid (T558E for moesin, T564E for radixin, T567E for ezrin)

• Expression system:

  • Transfect constructs into appropriate cell lines (e.g., MDA-MB-231 cells for cancer studies)

  • Verify expression by qRT-PCR and Western blot

• Functional assays:

  • Cell proliferation: MTT or BrdU incorporation assays

  • Invasion: Transwell invasion assays

  • Anchorage-independent growth: Soft agar colony formation assays

  • In vivo tumor growth: Xenograft models

Research using this approach has demonstrated that MSN T558E-overexpressing cells show significantly higher proliferation, invasion, and anchorage-independent growth compared to wild-type MSN-expressing cells, while MSN T558A-overexpressing cells show decreased capabilities in these assays . In xenograft models, tumors expressing MSN T558E grew faster and had higher Ki67-positive rates compared to wild-type tumors, while MSN T558A tumors grew slower .

This methodological approach allows researchers to directly assess the role of phosphorylation in various cellular processes and can help establish causative relationships between ERM protein activation and phenotypic outcomes.

What are the known upstream kinases and signaling pathways that regulate ERM protein phosphorylation?

Several kinases and signaling pathways have been implicated in the regulation of ERM protein phosphorylation:

• Protein Kinase C (PKC):

  • PKCζ has been shown to phosphorylate MSN at T558

  • PKC inhibitor (Go 6983) treatment can reverse the increased CREB phosphorylation caused by MSN overexpression

• Rho-associated protein kinase (ROCK):

  • Downstream of the small GTPase Rho

  • Directly phosphorylates ERM proteins at their C-terminal threonine residues

  • Inhibition of ROCK leads to decreased ERM phosphorylation

• Phosphatidylinositol 4,5-bisphosphate (PIP2):

  • Binding of PIP2 to the FERM domain induces conformational changes that expose the C-terminal threonine for phosphorylation

  • PIP2 depletion reduces ERM phosphorylation

The signaling pathway often follows this sequence:

• Extracellular stimuli → Activation of membrane receptors

• Activation of Rho GTPases

• Activation of ROCK or PKC

• Phosphorylation of ERM proteins at C-terminal threonine residues

• Conformational change leading to ERM activation

• Membrane-cytoskeleton linkage and downstream cellular effects

Research using co-immunoprecipitation has confirmed that PKCζ and phosphorylated PKCζ interact with FLAG-tagged MSN in MDA-MB-231 cells, whereas PKA does not . Furthermore, knockdown of MSN or its binding partner NONO significantly reduced nuclear localization of phosphorylated PKCζ, suggesting a role for the MSN-NONO complex in PKCζ signaling .

How does ERM protein phosphorylation relate to pathological conditions such as cancer?

ERM protein phosphorylation has been implicated in various pathological conditions, particularly cancer:

• Triple-negative breast cancer (TNBC):

  • MSN and its phosphorylation at T558 are positively associated with TNBC progression

  • T558 phosphorylation level of MSN positively correlates with total MSN levels in tumor samples

  • The MSN-NONO complex and its activated CREB signaling pathway have been proposed as potential therapeutic targets for TNBC

• Cell migration and metastasis:

  • Phosphorylation of ERM proteins promotes cytoskeletal reorganization required for cell migration

  • Enhanced ERM phosphorylation correlates with increased metastatic potential in various cancer types

  • Constitutively activated MSN (T558E) enhances invasion capabilities in vitro

• Endothelial barrier function:

  • ERM proteins differentially regulate endothelial hyperpermeability

  • Phosphorylation status of ERM proteins affects vascular integrity

  • This has implications for conditions involving vascular leakage, such as inflammation and tumor angiogenesis

Research has shown that inhibiting MSN phosphorylation (through T558A mutation) significantly reduces tumor growth in xenograft models, with decreased Ki67-positive rates compared to tumors expressing wild-type MSN . This suggests that targeting ERM phosphorylation could be a potential therapeutic strategy for cancers where these proteins play a significant role.

What techniques can be combined with Phospho-MSN/RDX/EZR (T558) antibody detection to study ERM protein dynamics?

Several advanced techniques can be combined with Phospho-MSN/RDX/EZR (T558) antibody detection to gain deeper insights into ERM protein dynamics:

• Live-cell imaging with fluorescent protein fusions:

  • Express ERM proteins fused to fluorescent proteins (e.g., GFP-ezrin)

  • Monitor real-time changes in localization

  • Fix cells at different time points and immunostain with the phospho-ERM antibody to correlate localization with phosphorylation status

• FRET-based biosensors:

  • Design biosensors containing ERM proteins that undergo conformational changes upon phosphorylation

  • Use to monitor ERM activation in real-time in living cells

  • Validate findings using fixed-cell immunostaining with Phospho-MSN/RDX/EZR (T558) antibody

• Proximity ligation assay (PLA):

  • Detect interactions between ERM proteins and their binding partners

  • Can be used to detect conformational changes by probing the proximity of N-terminal and C-terminal domains

  • Combine with phospho-specific antibodies to correlate interaction with phosphorylation status

• Mass spectrometry-based phosphoproteomics:

  • Identify all phosphorylation sites on ERM proteins

  • Quantify changes in phosphorylation under different conditions

  • Validate findings using Phospho-MSN/RDX/EZR (T558) antibody in Western blots

• Co-immunoprecipitation and RIP-qRT-PCR:

  • Investigate protein-protein and protein-RNA interactions of phosphorylated ERM proteins

  • Use phospho-ERM antibodies for immunoprecipitation

  • Analyze co-precipitated proteins or RNAs to identify novel interactions

A comprehensive approach combining these techniques has revealed that phosphorylated MSN interacts more strongly with binding partners like NONO, and that this interaction influences downstream signaling through PKCζ and CREB . These integrated methods provide a more complete picture of ERM activation dynamics and their functional consequences.

What are common challenges in detecting phosphorylated ERM proteins and how can they be addressed?

Researchers may encounter several challenges when detecting phosphorylated ERM proteins:

• Low signal intensity:

  • Ensure complete inhibition of phosphatases during sample preparation by using fresh phosphatase inhibitors

  • Optimize antibody concentration (try higher concentrations within the recommended range)

  • Extend primary antibody incubation time to overnight at 4°C

  • Use enhanced chemiluminescence (ECL) detection systems with higher sensitivity

• High background:

  • Increase blocking time or concentration (try 5% BSA instead of 3%)

  • Use more stringent washing conditions (increase number of washes or time)

  • Dilute primary antibody further if the background is specific to the primary antibody

  • Use a different blocking agent (milk vs. BSA) depending on the application

• Multiple bands or unexpected molecular weights:

  • ERM proteins have similar molecular weights (approximately 80 kDa)

  • Verify specificity using phosphopeptide competition

  • Consider that proteolytic degradation may produce lower molecular weight bands

  • Use high-percentage or gradient gels (4-15%) for better separation

• Inconsistent phosphorylation levels:

  • Standardize cell culture conditions and treatment times

  • Use positive controls such as ATP-stimulated PC-12 cells

  • Consider the rapid turnover of phosphorylation – fix or lyse cells quickly

  • Avoid phosphatase activation during sample preparation by keeping samples cold and using phosphatase inhibitors

How can researchers quantitatively analyze phosphorylation levels of ERM proteins in different experimental conditions?

For quantitative analysis of ERM protein phosphorylation:

How does phosphorylation at other sites affect ERM protein function beyond the T558/T564/T567 residues?

While phosphorylation at T558/T564/T567 is the primary regulatory mechanism for ERM proteins, other phosphorylation sites also influence their function:

• Tyrosine phosphorylation of ezrin:

  • Ezrin is phosphorylated at tyrosine residues upon growth factor stimulation

  • Phosphorylation at Tyr353 transmits a survival signal during epithelial differentiation

  • This phosphorylation can affect protein-protein interactions distinct from those regulated by threonine phosphorylation

• Serine phosphorylation:

  • Various serine residues in ERM proteins can be phosphorylated by different kinases

  • These modifications may fine-tune protein function or provide additional regulatory layers

  • Less well-characterized than threonine phosphorylation but potentially important for specific contexts

• Sequential phosphorylation events:

  • In some cases, phosphorylation at one site may prime the protein for modification at another site

  • This creates complex regulatory networks that integrate multiple signaling pathways

  • The temporal sequence of phosphorylation events may determine ultimate functional outcomes

Research methodologies to study these additional phosphorylation events include:

• Mass spectrometry-based phosphopeptide mapping to identify all phosphorylation sites

• Site-directed mutagenesis of multiple phosphorylation sites to assess their combined effects

• Phospho-specific antibodies targeting different phosphorylation sites for comparative analysis

• Kinase inhibitor studies to determine which signaling pathways regulate which phosphorylation events

Understanding the interplay between different phosphorylation events provides a more complete picture of ERM protein regulation and function in various cellular contexts.

What innovative research applications are emerging for Phospho-MSN/RDX/EZR antibodies?

Several innovative applications for Phospho-MSN/RDX/EZR antibodies are emerging in cutting-edge research:

• Single-cell phosphoproteomics:

  • Combining phospho-specific antibodies with mass cytometry (CyTOF) to analyze ERM phosphorylation at the single-cell level

  • Integrating with other phospho-protein markers to map signaling networks

  • Characterizing cellular heterogeneity in phosphorylation states within tissues

• Tissue microarray analysis:

  • High-throughput screening of phospho-ERM levels across multiple tumor samples

  • Correlation with clinical outcomes and other biomarkers

  • Development of phospho-ERM as a potential prognostic marker for certain cancers

• Intravital imaging:

  • Using labeled phospho-specific antibodies or biosensors to monitor ERM activation in living organisms

  • Tracking changes in phosphorylation during developmental processes or disease progression

  • Correlating with cellular behaviors such as migration or differentiation

• Drug discovery applications:

  • Screening for compounds that modulate ERM phosphorylation

  • Development of targeted therapies for conditions with dysregulated ERM function

  • Use as pharmacodynamic biomarkers to assess drug efficacy

• Combination with CRISPR/Cas9 technology:

  • Genome-wide screens to identify regulators of ERM phosphorylation

  • Creation of knock-in models with fluorescent tags on endogenous ERM proteins

  • Generation of cell lines with phospho-deficient or phospho-mimetic mutations at endogenous loci

What are the current research gaps in understanding ERM protein phosphorylation dynamics?

Despite significant advances, several knowledge gaps remain in understanding ERM protein phosphorylation:

• Isoform-specific functions:

  • While the Phospho-MSN/RDX/EZR (T558) antibody detects all three phosphorylated ERM proteins, they may have distinct functions

  • Current methods often cannot distinguish which isoform is responsible for specific cellular effects

  • Development of isoform-specific phospho-antibodies or other approaches is needed to address this limitation

• Temporal dynamics:

  • The kinetics of phosphorylation and dephosphorylation in response to different stimuli remain poorly characterized

  • Real-time monitoring methods are needed to understand these dynamics fully

  • The relationship between phosphorylation duration and biological outcomes requires further investigation

• Spatial regulation:

  • How phosphorylation is regulated in different subcellular domains is not well understood

  • Local activation versus global phosphorylation may lead to different functional outcomes

  • Advanced imaging techniques combined with phospho-specific detection are needed to address this gap

• Integration with other post-translational modifications:

  • Cross-talk between phosphorylation and other modifications (methylation, acetylation, ubiquitination) is largely unexplored

  • Comprehensive analysis of multiple modifications simultaneously is technically challenging

  • New methodologies combining different approaches may help address this limitation

• Tissue-specific regulation:

  • ERM proteins are expressed in multiple tissues but may be regulated differently

  • Tissue-specific interaction partners could modify the effects of phosphorylation

  • Comparative studies across different tissue types are needed to understand context-dependent regulation

Addressing these research gaps will require interdisciplinary approaches combining advanced imaging, biochemical analyses, genetic models, and computational methods. The continued development and refinement of phospho-specific antibodies and related tools will be essential for progress in this field.