Phospho-MTOR (S2481) Recombinant Monoclonal Antibody

What is Phospho-MTOR (S2481) and why is it significant?

Phospho-MTOR (S2481) refers to the mechanistic target of rapamycin (MTOR) protein when phosphorylated at the serine 2481 residue. MTOR is a serine/threonine protein kinase that functions as a central regulator of cellular metabolism, growth, and survival in response to various signals including hormones, growth factors, nutrients, energy levels, and stress . The phosphorylation at S2481 has particular significance as a biomarker for intact mTORC2 (MTOR Complex 2), one of the two functionally distinct signaling complexes formed by MTOR . While MTOR directly or indirectly regulates the phosphorylation of at least 800 proteins, the S2481 phosphorylation site provides researchers with a specific indicator of mTORC2 activity and integrity .

How does MTOR phosphorylation at S2481 differ functionally from other phosphorylation sites?

MTOR exhibits differential phosphorylation patterns depending on which complex it participates in. Research has demonstrated that mTORC1 predominantly contains MTOR phosphorylated at S2448, while mTORC2 predominantly contains MTOR phosphorylated at S2481 . This distinction makes S2481 phosphorylation a valuable marker for specifically monitoring mTORC2 activity. Unlike other phosphorylation sites, S2481 phosphorylation requires intact mTORC2, as demonstrated by experiments showing that depletion of essential mTORC2 components (mSin1 and Rictor) abolishes S2481 phosphorylation . This specificity allows researchers to use S2481 phosphorylation as a more direct marker of intact mTORC2 than downstream targets such as phosphorylation of S473 on Akt .

What experimental applications are Phospho-MTOR (S2481) antibodies suitable for?

Phospho-MTOR (S2481) recombinant monoclonal antibodies have been validated for multiple experimental applications:

These antibodies have been extensively validated across multiple species including human, mouse, rat, and monkey samples, making them versatile tools for comparative studies across model organisms .

What are the optimal conditions for using Phospho-MTOR (S2481) antibody in Western Blotting?

For optimal Western Blotting results with Phospho-MTOR (S2481) antibody, researchers should follow these methodological guidelines:

• Sample preparation: Lyse cells in a buffer containing phosphatase inhibitors to preserve phosphorylation status

• Protein loading: Load 20-50 μg of total protein per lane

• Separation: Use lower percentage (6-8%) SDS-PAGE gels as MTOR is a large protein (~289 kDa)

• Transfer: Employ longer transfer times or specialized protocols for high molecular weight proteins

• Blocking: Use 5% BSA in TBST rather than milk (which contains phosphatases)

• Primary antibody: Dilute antibody 1:1000 in 5% BSA/TBST and incubate overnight at 4°C

• Detection: Use enhanced chemiluminescence systems with appropriate exposure times

Researchers should note that insulin stimulation of cells prior to lysis can enhance detection of phosphorylated MTOR, as this pathway is responsive to insulin signaling .

How can specificity of Phospho-MTOR (S2481) antibody be validated?

Validating the specificity of Phospho-MTOR (S2481) antibody is critical for experimental integrity. Multiple approaches should be employed:

• Peptide competition assay: Pre-incubate the antibody with phospho-S2481 peptide to confirm signal elimination

• Phosphatase treatment controls: Treat half of your sample with lambda phosphatase to demonstrate loss of signal

• Genetic manipulation: Use cells with MTOR knockdown or knockout as negative controls

• mTORC2 component knockdown: As demonstrated in research, knockdown of mTORC2-specific components (mSin1 and Rictor) should reduce S2481 phosphorylation

• Rapamycin treatment: In rapamycin-sensitive cell lines, prolonged treatment should reduce S2481 phosphorylation due to mTORC2 disruption

The observation that mTOR S2481 phosphorylation is abolished in mSin1-null cells provides a powerful validation approach, confirming that this phosphorylation is indeed mTORC2-dependent .

How can Phospho-MTOR (S2481) be used to distinguish between mTORC1 and mTORC2 activation?

Phospho-MTOR (S2481) antibody provides researchers with a powerful tool to distinguish between mTORC1 and mTORC2 activation due to the differential phosphorylation patterns of MTOR in these complexes:

• mTORC1 predominantly contains MTOR phosphorylated on S2448

• mTORC2 predominantly contains MTOR phosphorylated on S2481

This distinction allows for experimental designs that can specifically monitor mTORC2 formation and activity by tracking S2481 phosphorylation. To comprehensively analyze both complexes simultaneously, researchers can:

• Perform immunoprecipitation of complex-specific components (Raptor for mTORC1, Rictor for mTORC2)

• Analyze the precipitates for phosphorylation at both S2448 and S2481

• Compare the relative abundance of each phosphorylation site within the complexes

Research has shown that when Rictor immunoprecipitates (representing mTORC2) are analyzed, the reduction in MTOR bound to Rictor parallels the reduction in S2481 phosphorylation, confirming the specific association of this phosphorylation site with mTORC2 .

What is the relationship between mTORC2 formation and MTOR S2481 phosphorylation?

The relationship between mTORC2 formation and MTOR S2481 phosphorylation is direct and causative. Studies have demonstrated that:

• Intact mTORC2 is necessary for S2481 phosphorylation

• Depletion of mTORC2-specific components (mSin1 and Rictor) abolishes insulin-induced phosphorylation of MTOR on S2481

• When analyzed in mSin1-null cells, S2481 phosphorylation is completely absent

This relationship makes S2481 phosphorylation an excellent biomarker for the presence of intact, functional mTORC2. Researchers investigating mTORC2 assembly dynamics can monitor S2481 phosphorylation as a direct readout of complex formation and integrity, providing advantages over less direct markers such as phosphorylation of downstream targets like Akt on S473 .

How does rapamycin treatment affect MTOR S2481 phosphorylation in different cell types?

Rapamycin's effect on MTOR S2481 phosphorylation varies by cell type, duration of treatment, and inherent sensitivity of mTORC2 to rapamycin in particular cancer cell lines:

In rapamycin-sensitive cell lines, both S2481 phosphorylation and downstream S473 phosphorylation of Akt are reduced upon treatment . This makes S2481 phosphorylation a valuable biomarker for predicting rapamycin sensitivity of mTORC2 in different cancer cell types. Researchers can use S2481 phosphorylation status to determine whether mTORC2 formation is rapamycin-sensitive in their particular experimental system, which has significant implications for cancer therapy research .

Why might I see reduced or absent Phospho-MTOR (S2481) signal in my experiments?

Several factors can contribute to reduced or absent Phospho-MTOR (S2481) signal:

• Technical issues:

  • Inadequate phosphatase inhibition during sample preparation

  • Inefficient transfer of high molecular weight proteins

  • Antibody degradation or incorrect storage

  • Insufficient incubation time with primary antibody

• Biological factors:

  • Disruption of mTORC2 complex formation

  • Decreased expression of mTORC2 components like Rictor or mSin1

  • Cell type-specific differences in mTORC2 activity

  • Treatment effects (e.g., rapamycin in sensitive cell lines)

Research has demonstrated that depletion of mTORC2 components through shRNA, rapamycin treatment, or both leads to reduction in MTOR phosphorylated on S2481 . Notably, partial depletion of mSin1 has a more profound effect on reducing S2481 phosphorylation than partial depletion of Rictor, likely because decreased mSin1 protein levels lead to a concomitant decrease in Rictor protein levels .

How should contradictory results between S2481 phosphorylation and downstream target activation be interpreted?

When faced with contradictory results between MTOR S2481 phosphorylation and downstream target activation (e.g., Akt S473 phosphorylation), researchers should consider:

• Temporal dynamics: mTORC2 phosphorylation may precede or follow downstream target activation depending on the context

• Signal amplification: Small changes in mTORC2 activity (S2481) may result in larger changes in downstream signaling

• Compensatory mechanisms: Alternative pathways may maintain Akt phosphorylation despite reduced mTORC2 activity

• Specificity differences: S2481 phosphorylation is specifically linked to intact mTORC2, whereas Akt S473 can potentially be phosphorylated by other kinases

Research has demonstrated that S2481 phosphorylation is a more direct marker of intact mTORC2 than phosphorylation of S473 of Akt . Therefore, when contradictions arise, the S2481 phosphorylation status should generally be considered the more reliable indicator of mTORC2 integrity and function. This is exemplified by studies showing that in certain cancer cell lines, Akt S473 phosphorylation may appear insensitive to rapamycin while S2481 phosphorylation shows sensitivity, providing a more accurate representation of mTORC2 status .

How can Phospho-MTOR (S2481) antibody be used to study cancer cell sensitivity to rapamycin?

Phospho-MTOR (S2481) antibody offers a sophisticated approach to studying cancer cell sensitivity to rapamycin:

• Biomarker function: S2481 phosphorylation serves as a biomarker for predicting rapamycin-induced mTORC2 suppression in different cancer cell types

• Comparative analysis: Researchers can compare S2481 phosphorylation patterns across cancer cell lines to identify intrinsic differences in mTORC2 sensitivity

• Treatment response monitoring: Changes in S2481 phosphorylation following rapamycin treatment provide direct evidence of mTORC2 disruption, even when downstream markers like Akt S473 phosphorylation remain unchanged

• Combination therapy assessment: S2481 phosphorylation can help determine whether combinatorial approaches effectively target mTORC2 in rapamycin-resistant cells

This approach is particularly valuable given that several cancer cell lines show resistance to prolonged rapamycin treatment when assessed solely by Akt phosphorylation . The use of S2481 phosphorylation as a marker provides a more direct assessment of mTORC2 status and can guide the development of more effective therapeutic strategies targeting the MTOR pathway.

What techniques can be combined with Phospho-MTOR (S2481) detection to comprehensively analyze mTORC2 activity?

For comprehensive analysis of mTORC2 activity, researchers should consider combining Phospho-MTOR (S2481) detection with the following techniques:

• Co-immunoprecipitation studies:

  • Immunoprecipitate Rictor (mTORC2-specific component)

  • Analyze for bound MTOR and S2481 phosphorylation status

  • Quantify complex component stoichiometry

• Genetic manipulation approaches:

  • shRNA or CRISPR targeting of mTORC2 components (mSin1, Rictor)

  • Restoration experiments with wild-type vs. mutant components

  • Analysis of S2481 phosphorylation in response to these manipulations

• Downstream signaling analysis:

  • Parallel assessment of Akt phosphorylation at S473

  • Analysis of additional mTORC2 targets (PKCα, SGK1)

  • Correlation between S2481 phosphorylation and functional outcomes

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize mTORC2 localization

  • FRET-based approaches to monitor complex assembly

  • Live-cell imaging with phospho-specific sensors

Research has demonstrated that the combination of S2481 phosphorylation analysis with genetic manipulation of mTORC2 components provides powerful insights into complex formation and function . For example, studies combining S2481 phosphorylation analysis with shRNA-mediated depletion of Rictor and mSin1 revealed that mSin1 knockdown has a more profound effect on reducing mTORC2 function than Rictor knockdown alone .

What are the latest methodological advances in studying MTOR S2481 phosphorylation dynamics?

Recent methodological advances for studying MTOR S2481 phosphorylation dynamics include:

• Temporal resolution techniques:

  • Real-time monitoring using genetically encoded biosensors

  • Microfluidic systems for precise control of stimulation timing

  • Pulse-chase approaches to track phosphorylation turnover rates

• Spatial resolution approaches:

  • Subcellular fractionation to analyze compartment-specific phosphorylation

  • Proximity labeling methods to identify proteins near phosphorylated MTOR

  • In situ phosphorylation detection with proximity ligation assays

• Quantitative analysis:

  • Absolute quantification using phosphopeptide standards

  • Phosphoproteomic analysis with tandem mass tag labeling

  • Single-cell analysis of phosphorylation heterogeneity

• Computational methods:

  • Mathematical modeling of phosphorylation dynamics

  • Network analysis integrating multiple phosphorylation sites

  • Machine learning approaches to predict functional consequences

These advanced methods build upon fundamental research showing that S2481 phosphorylation requires intact mTORC2 and can serve as a biomarker for mTORC2 integrity . By combining these innovative approaches with established techniques like Western blotting and immunofluorescence, researchers can gain unprecedented insights into the dynamics and regulation of MTOR signaling in both normal physiology and disease states.