Phospho-RPS6KA1 (T359/S363) Antibody

What is the significance of RPS6KA1 phosphorylation at T359/S363 sites in cell signaling?

The phosphorylation of RPS6KA1 (p90RSK) at threonine 359 and serine 363 represents a critical activation event in MAPK signaling pathways. These specific phosphorylation sites are crucial for the activation and regulation of p90RSK, a key signaling protein involved in cell growth, survival, and proliferation . RPS6KA1 contains two nonidentical kinase catalytic domains and phosphorylates various substrates, including members of the mitogen-activated kinase (MAPK) signaling pathway . The phosphorylation at T359/S363 is typically mediated by upstream kinases like MAPK1/ERK2 and MAPK3/ERK1 , creating an essential step in the signal transduction cascade that controls diverse cellular functions.

What are the primary research applications for Phospho-RPS6KA1 (T359/S363) antibodies?

Phospho-RPS6KA1 (T359/S363) antibodies are primarily utilized in:

• Western Blot (WB): For detecting and quantifying phosphorylated RPS6KA1 levels in cell or tissue lysates, with recommended dilutions typically ranging from 1:500 to 1:6000

• ELISA: For quantitative measurement of phosphorylated protein levels in solution

• Flow Cytometry (Intracellular): For analyzing phosphorylation status at single-cell resolution

• Cell Signaling Studies: For investigating MAPK pathway activation in response to various stimuli

• Cancer Research: For studying dysregulated signaling in malignancies, particularly in leukemia and solid tumors

• Drug Response Mechanisms: For elucidating resistance mechanisms to therapeutics like venetoclax/azacitidine in acute myeloid leukemia

How does the structure of RPS6KA1 relate to its function and phosphorylation state?

RPS6KA1 consists of several functional domains with distinct roles:

• N-terminal Domain (NTD): Contains the TOR signaling (TOS) motif for regulatory interactions

• Kinase Domain (KD): Responsible for catalytic activity and substrate phosphorylation

• Linker Region: Contains critical sites for catalytic activity, including the signature mTORC1 phospho-site, Thr 389

• C-terminal Domain (CTD): Contains a cluster of Ser/Thr-Pro motif phospho-sites that contribute to kinase activity and substrate specificity

The phosphorylation at T359/S363 occurs in this regulatory framework and enhances kinase activation. Research has demonstrated that the CTD specifically influences substrate selection - deletion of the CTD from S6K1 decreases phosphorylation of certain substrates like EPRS but not others like RPS6 , indicating that phosphorylation in this region creates a "phospho-code" that directs substrate specificity.

What are the optimal sample preparation methods for detecting phospho-RPS6KA1 (T359/S363) in Western blot applications?

For optimal detection of phospho-RPS6KA1 (T359/S363) in Western blot:

Sample Preparation Protocol:

• Cell Lysis: Use a phosphatase inhibitor-containing buffer (e.g., PBS with 0.02% sodium azide, 50% glycerol, pH 7.3) to preserve phosphorylation states

• Protein Loading: Load 25μg of protein per lane for standard detection

• Blocking Conditions: Use 3% BSA rather than milk-based blockers which contain phosphatases that may dephosphorylate targets

• Antibody Dilution: Optimize within the range of 1:500-1:2000 for polyclonal antibodies or 1:5000-1:50000 for recombinant antibodies

• Secondary Antibody: Use HRP-conjugated anti-rabbit IgG at 1:10000 dilution

• Positive Controls: Include lysates from A431 cells (or similarly validated cell lines) as a positive control

• Validation Control: Include λ phosphatase-treated samples as negative controls to confirm phospho-specificity

This methodology has been validated across multiple research groups and provides reliable detection of the phosphorylated protein at its expected molecular weight of 83-90 kDa.

How can researchers verify the specificity of phospho-RPS6KA1 (T359/S363) antibody signals?

To verify antibody specificity and rule out false positives:

Validation Strategy:

• Phosphatase Treatment: Treat duplicate samples with lambda phosphatase to confirm signal loss

• Stimulation Experiments: Compare samples from unstimulated cells with those treated with known activators (e.g., PDGF, EGF, insulin) to demonstrate inducible phosphorylation

• Peptide Competition: Pre-incubate antibody with phosphorylated and non-phosphorylated peptides corresponding to the T359/S363 region

• Knockdown/Knockout Controls: Use RPS6KA1 siRNA knockdown or CRISPR/Cas9 knockout cells

• Correlation Testing: Confirm consistent results with alternative antibodies targeting different phospho-sites on RPS6KA1 that are co-regulated

• Cross-reactivity Assessment: Test antibody against closely related family members (RSK2-4) to ensure specificity

A comprehensive validation approach employing multiple strategies provides the highest confidence in experimental results.

What experimental considerations are important when studying RPS6KA1 phosphorylation in different cell types?

When investigating RPS6KA1 phosphorylation across different cellular contexts:

Critical Considerations:

• Basal Phosphorylation Levels: Different cell types exhibit varying basal phosphorylation of RPS6KA1; determine baseline levels before stimulation experiments

• Stimulus Duration and Concentration: Optimize treatment conditions as phosphorylation kinetics vary by cell type:

  • A431 cells respond rapidly to EGF stimulation

  • Monocytic cells show strong responses to IFN-γ

  • Adipocytes exhibit insulin-dependent phosphorylation patterns

• Pathway Cross-talk: Account for cell-specific signaling networks:

  • In monocytes, IFN-γ activates both mTORC1 and Cdk5 pathways affecting RPS6KA1

  • In AML cells, RPS6KA1 participates in venetoclax/azacitidine resistance mechanisms

• Tissue-specific Isoforms: Check for expression of alternative splice variants

• Sample Timing: Capture both rapid (minutes) and sustained (hours) phosphorylation events

Comparative Response Table:

How should researchers interpret changes in RPS6KA1 T359/S363 phosphorylation in relation to other phosphorylation sites?

Interpreting RPS6KA1 phosphorylation requires understanding the sequential and hierarchical nature of its activation:

Phosphorylation Interpretation Framework:

• Activation Sequence: The full activation of RPS6KA1 involves sequential phosphorylation events:

  • Initial phosphorylation at Thr359/Ser363 by ERK1/2

  • Subsequent phosphorylation at Ser380 (autophosphorylation)

  • Additional phosphorylation at Thr229 by PDK1

• Hierarchical Relationships:

  • T359/S363 phosphorylation is necessary but not always sufficient for full activation

  • Thr389 phosphorylation by mTORC1 represents a canonical activation marker

  • Ser424/Ser429 phosphorylation by Cdk5 directs substrate specificity

• Substrate-Specific Implications:

  • Changes in T359/S363 phosphorylation without corresponding changes in Ser424/Ser429 may affect only canonical targets like RPS6, not EPRS

  • Multisite phosphorylation creates a "phospho-code" determining target selection

When analyzing phosphorylation data, always consider ratios between different phosphorylation sites rather than absolute values of any single modification to understand the complete activation state.

What are common troubleshooting strategies for weak or inconsistent phospho-RPS6KA1 (T359/S363) detection?

When experiencing detection challenges:

Troubleshooting Decision Tree:

• Weak or No Signal:

  • Verify treatment conditions triggered pathway activation (use positive control like phospho-ERK)

  • Check phosphatase inhibitor freshness and concentration in all buffers

  • Increase antibody concentration or extend incubation time

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Try membrane stripping and reprobing with total RPS6KA1 antibody to confirm protein presence

• High Background:

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

  • Perform additional membrane washes with 0.1% Tween-20

  • Dilute primary antibody further

  • Test alternative secondary antibodies

• Multiple Bands:

  • Verify running conditions to ensure optimal protein separation

  • Check for degradation by adding additional protease inhibitors

  • Purify antibody by pre-absorption with cell lysates from non-stimulated cells

  • Consider if closely-migrating bands represent different RPS6KA1 isoforms

• Inconsistent Results Across Experiments:

  • Standardize lysate preparation timing (phosphorylation states decay)

  • Maintain consistent storage conditions (avoid freeze-thaw cycles)

  • Use internal loading controls for each experiment

  • Create a standard positive control lysate for calibration across experiments

How do cell culture conditions impact RPS6KA1 phosphorylation status and experimental reproducibility?

Cell culture variables significantly affect phosphorylation states:

Critical Culture Parameters:

• Serum Conditions:

  • Serum-containing media can activate MAPK pathways and increase basal phosphorylation

  • Standardize serum starvation (12-16h) before stimulation experiments

  • Document lot-to-lot serum variation effects

• Confluence Effects:

  • High confluence (>80%) can alter contact inhibition and reduce kinase responsiveness

  • Maintain consistent seeding densities across experiments (e.g., 60-70% confluence)

• Passage Number:

  • High passage cells (>20) may show altered signaling responses

  • Document passage number in methods and maintain consistent ranges

• Media Composition:

  • Glucose levels affect mTOR signaling upstream of RPS6KA1

  • Amino acid concentrations influence basal activation

  • Standardize fresh media addition timing prior to stimulation

• Temperature and pH Shifts:

  • Even brief exposure to room temperature can alter phosphorylation

  • Maintain constant 37°C conditions during harvesting

  • Process samples rapidly to prevent phosphatase activation

Pre-Experiment Standardization Protocol:

• Seed cells at consistent density in multi-well plates

• Allow 24-48h growth in complete media

• Synchronize by serum starvation (0.1-0.5% serum) for 12-16h

• Add fresh starvation media 1-2h before stimulation

• Prepare ice-cold lysis buffers with fresh inhibitors

• Process all experimental conditions within a tight time window

How does multisite phosphorylation of RPS6KA1 create a "phospho-code" that determines substrate specificity?

The concept of a RPS6KA1 phospho-code represents an advanced regulatory mechanism:

Phospho-Code Mechanism:
Research by Arif et al. (2019) revealed that multisite phosphorylation of RPS6KA1 creates a sophisticated substrate selection system . This phospho-code operates through:

• Combinatorial Phosphorylation Requirements:

  • Canonical phosphorylation at Thr389 by mTORC1 is necessary but not sufficient for all substrate interactions

  • Additional phosphorylation at Ser424/Ser429 by Cdk5 redirects kinase activity toward specific substrates

• Structural Conformational Changes:

  • Phosphorylation at specific CTD sites induces conformational switches that expose or conceal binding interfaces

  • These conformational changes create high-affinity binding sites for specific substrates only when particular phosphorylation combinations are present

• Substrate-Specific Outcomes:

  • Standard mTORC1-activated RPS6KA1 efficiently phosphorylates RPS6

  • Multiply-phosphorylated RPS6KA1 (mTORC1 + Cdk5 sites) efficiently phosphorylates EPRS and other targets like coenzyme A synthase, lipocalin 2, and cortactin

• Integration of Multiple Signaling Inputs:

  • This system allows cells to integrate signals from different pathways (mTORC1, MAPK, Cdk5)

  • The resulting phospho-pattern determines which downstream substrates become activated

This represents a sophisticated regulatory mechanism beyond simple on/off kinase activation, creating specific functional outputs from the same kinase depending on cellular context.

What roles does RPS6KA1 phosphorylation play in cancer development and therapeutic resistance?

RPS6KA1 phosphorylation has emerged as a critical mediator in cancer biology:

Cancer-Related Functions:

• Therapeutic Resistance Mechanisms:

  • In acute myeloid leukemia (AML), RPS6KA1 was identified as one of the most significantly depleted sgRNA-genes in venetoclax/azacitidine-treated cells, suggesting its role in resistance

  • Pharmacological inhibition of RPS6KA1 with BI-D1870 increased sensitivity to venetoclax/azacitidine in AML cells

  • RPS6KA1 inhibition efficiently targeted monocytic blast subclones, a potential source of relapse

• Signaling Pathway Integration:

  • RPS6KA1 acts as a node connecting MAPK, mTOR, and Cdk5 pathways, all implicated in oncogenic signaling

  • Its phosphorylation status reflects the activation state of these pathways and can serve as a biomarker of pathway inhibitor efficacy

• Cell Growth and Survival Regulation:

  • Phosphorylated RPS6KA1 mediates signals controlling cell growth, survival, and differentiation

  • In some contexts, it contributes to dysregulated proliferation and resistance to apoptosis

• Translational Control:

  • Through the regulation of translational machinery, RPS6KA1 can modulate the expression of specific mRNAs involved in cancer progression

  • In IFNλ signaling, activated RPS6KA1 dissociates from 4E-BP1, enabling cap-dependent translation and upregulation of p21(WAF1/CIP1), suggesting context-dependent tumor-suppressive functions

These findings position RPS6KA1 as both a biomarker and therapeutic target in cancer, particularly in overcoming resistance to established therapies.

How can phospho-proteomic approaches be integrated with traditional immunoblotting to provide comprehensive analysis of RPS6KA1 signaling networks?

Modern research requires integrating multiple analytical approaches:

Integrative Analytical Strategy:

• Orthogonal Validation Workflow:

  • Begin with hypothesis-driven immunoblotting for key phosphorylation sites (T359/S363, T389, S424/S429)

  • Follow with unbiased phospho-proteomics to discover novel phosphorylation events and substrates

  • Confirm findings with targeted approaches (immunoprecipitation, in vitro kinase assays)

• Phospho-Proteomic Methods for RPS6KA1 Research:

  • Titanium dioxide (TiO₂) enrichment for phosphopeptide isolation

  • IMAC (Immobilized Metal Affinity Chromatography) for multi-phosphorylated peptides

  • Label-free quantification (LFQ) for relative abundance measurements

  • Parallel Reaction Monitoring (PRM) for targeted validation of specific phosphosites

• Data Integration Framework:

  • Link phosphorylation status to kinase activity using kinase-substrate enrichment analysis

  • Apply gene set enrichment analysis (GSEA) to connect phosphorylation patterns to biological pathways

  • Develop computational models of phosphorylation dynamics within signaling networks

• Application to Biological Questions:

  • Identify all RPS6KA1 substrates phosphorylated under specific stimulus conditions

  • Map temporal dynamics of phosphorylation cascades

  • Discover feedback and feedforward loops involving RPS6KA1

  • Determine how inhibitors affect the complete phospho-proteome beyond intended targets

Example Integrated Analysis Pipeline:

• Treat cells with stimulus of interest ± RPS6KA1 inhibitors

• Split samples for parallel analysis:

  • Western blotting with phospho-specific antibodies

  • Phosphopeptide enrichment and MS/MS analysis

• Identify phosphoproteins with altered abundance

• Validate key targets with phospho-specific antibodies

• Perform in vitro kinase assays to confirm direct substrates

• Analyze pathway enrichment to place findings in biological context

This integrated approach provides a comprehensive map of RPS6KA1 signaling that cannot be achieved through any single methodology.

What are emerging techniques for studying dynamic phosphorylation events at RPS6KA1-T359/S363 in living cells?

The field continues to advance with new methodologies:

Emerging Technologies:

• Phospho-Specific Biosensors:

  • FRET-based sensors can detect conformational changes upon RPS6KA1 phosphorylation

  • Genetically encoded fluorescent reporters allow real-time visualization of phosphorylation events

• Optogenetic Control Systems:

  • Light-activatable kinases and phosphatases enable precise temporal control of RPS6KA1 phosphorylation

  • Combining with live-cell imaging reveals dynamic regulation patterns

• Proximity Labeling Techniques:

  • BioID or TurboID fusions to RPS6KA1 can identify transient interaction partners dependent on phosphorylation state

  • APEX2-based approaches provide high temporal resolution of proximal proteins

• Single-Cell Phospho-Profiling:

  • Mass cytometry (CyTOF) with phospho-specific antibodies enables multiparameter analysis at single-cell resolution

  • Single-cell phospho-proteomics reveals cell-to-cell variability in signaling responses

These technologies will help resolve outstanding questions regarding the dynamics, heterogeneity, and context-dependence of RPS6KA1 phosphorylation events.

How will understanding RPS6KA1 phosphorylation contribute to precision medicine approaches in cancer therapy?

The clinical translation of RPS6KA1 research holds significant promise:

Precision Medicine Applications:

• Biomarker Development:

  • Phospho-RPS6KA1 status could serve as a predictive biomarker for response to targeted therapies

  • In AML, RPS6KA1 inhibition restored sensitivity to venetoclax/azacitidine in resistant cells

• Combination Therapy Design:

  • Rational combinations targeting both mTORC1 and RPS6KA1 may overcome resistance mechanisms

  • Understanding the phospho-code could help design treatments that inhibit specific substrate interactions

• Patient Stratification Strategies:

  • Tumors with hyperactivated RPS6KA1 might be classified into distinct molecular subtypes

  • Phosphorylation patterns could indicate which signaling pathways are driving individual tumors

• Novel Target Identification:

  • Downstream substrates of specifically phosphorylated RPS6KA1 represent potential drug targets

  • The phospho-code concept suggests targeting specific kinase-substrate interfaces rather than catalytic activity