Phospho-RPS6KA1 (T359+S363) Recombinant Monoclonal Antibody

What is the functional significance of RPS6KA1 phosphorylation at T359/S363 sites?

Phosphorylation at T359/S363 sites is crucial for the activation and regulation of p90RSK (RPS6KA1), a key signaling protein involved in cell growth, survival, and proliferation pathways . These specific phosphorylation events occur within the activation loop of the N-terminal kinase domain and are necessary for full catalytic activity of RPS6KA1. This phosphorylation is typically mediated by ERK1/2 following growth factor stimulation and represents a critical step in the MAPK signaling cascade . Functionally, this phosphorylation enables RPS6KA1 to subsequently phosphorylate downstream substrates involved in transcriptional regulation, protein synthesis, and cell cycle progression, making it a pivotal regulatory point in cellular signaling networks .

Which experimental applications are most suitable for Phospho-RPS6KA1 (T359/S363) antibodies?

Phospho-RPS6KA1 (T359/S363) antibodies have been validated for several experimental applications with varying optimal dilutions:

When selecting the appropriate application, researchers should consider their specific experimental goals. Western blotting provides quantitative measurement of phosphorylation status across different experimental conditions, while flow cytometry allows for analysis at the single-cell level, which is particularly valuable for heterogeneous cell populations . All applications require proper controls, including λ phosphatase-treated samples to confirm antibody specificity for the phosphorylated form of the protein .

How does RPS6KA1 function within the MAPK signaling cascade?

RPS6KA1 (RSK1) functions as a downstream effector of the MAPK pathway, particularly ERK1/2 signaling . Following growth factor stimulation, the signaling cascade progresses as follows:

• Growth factors bind to receptor tyrosine kinases, activating the RAS-RAF-MEK-ERK pathway

• Activated ERK1/2 phosphorylates RPS6KA1 at multiple sites, including T359/S363, which are critical for activation

• Once activated, RPS6KA1 mediates several cellular processes by phosphorylating various downstream substrates:

  • Transcription factors: CREB1, ETV1/ER81, and NR4A1/NUR77

  • Translation factors: RPS6 and EIF4B, promoting protein synthesis

  • Cell survival regulators: Inhibition of pro-apoptotic proteins BAD and DAPK1

  • Cell cycle regulators: CDK inhibitor CDKN1B

This positions RPS6KA1 as a crucial mediator that translates extracellular signals into specific cellular responses related to growth, differentiation, and survival . Understanding this pathway is essential for interpreting experimental results when working with phospho-specific antibodies targeting this protein.

What positive and negative controls should be included when validating Phospho-RPS6KA1 (T359/S363) antibody specificity?

Proper controls are essential for validating the specificity of phospho-specific antibodies:

Validation should include side-by-side comparison between stimulated and unstimulated conditions to demonstrate the dynamic range of phosphorylation detection. For Western blotting, treatment with λ phosphatase is particularly important as it removes phosphate groups, allowing researchers to confirm that the observed signal is truly phosphorylation-dependent rather than non-specific binding . Based on available data, the expected molecular weight of phosphorylated RPS6KA1 is approximately 83-90 kDa when detected on Western blots .

How should researchers optimize sample preparation to preserve phosphorylation status?

Preservation of phosphorylation status is critical when working with phospho-specific antibodies:

• Rapid sample processing: Harvest and process cells quickly to minimize dephosphorylation by endogenous phosphatases

• Phosphatase inhibitor cocktails: Always include fresh phosphatase inhibitors in lysis buffers:

  • Serine/threonine phosphatase inhibitors (e.g., sodium fluoride, β-glycerophosphate)

  • Tyrosine phosphatase inhibitors (e.g., sodium orthovanadate)

  • Broad-spectrum inhibitors (e.g., sodium pyrophosphate, microcystin-LR)

• Cold temperature maintenance: Keep samples on ice throughout processing

• Optimal lysis buffer composition:

  • RIPA or NP-40 based buffers with phosphatase inhibitors for Western blot

  • For flow cytometry, use paraformaldehyde fixation followed by methanol permeabilization to preserve phospho-epitopes

• Storage considerations: Aliquot lysates and store at -80°C; avoid repeated freeze-thaw cycles

For phosphorylation analysis, timing of sample collection post-stimulation is crucial as T359/S363 phosphorylation typically peaks within 5-15 minutes following growth factor treatment. Sample preparation techniques should be thoroughly documented in research protocols to ensure reproducibility and reliable phosphorylation detection .

What are the optimal stimulation conditions to induce RPS6KA1 T359/S363 phosphorylation?

To effectively induce and detect RPS6KA1 T359/S363 phosphorylation:

Cell line selection is important as expression and phosphorylation patterns of RPS6KA1 vary across tissue types. HeLa cells have been validated as a positive control system for detecting T359/S363 phosphorylation . For experimental design, researchers should establish a time course of stimulation, as phosphorylation events are often transient. When testing new cell systems, researchers should verify RPS6KA1 expression levels before proceeding with phosphorylation studies, as expression levels impact detection sensitivity. Additionally, serum starvation (0.1-0.5% serum for 16-24 hours) prior to stimulation often enhances detection of induced phosphorylation by reducing baseline signaling activity .

How does T359/S363 phosphorylation coordinate with other phosphorylation sites on RPS6KA1?

RPS6KA1 activation involves a complex sequence of phosphorylation events at multiple sites:

The activation of RPS6KA1 follows a specific order: ERK1/2 first phosphorylates T573 in the C-terminal kinase domain, followed by phosphorylation of T359/S363 in the N-terminal kinase domain. The C-terminal kinase domain then phosphorylates S380, creating a docking site for PDK1, which phosphorylates S221, completing the activation process . Researchers investigating RPS6KA1 should consider analyzing multiple phosphorylation sites simultaneously to gain a comprehensive understanding of activation status. The interconnected nature of these phosphorylation events means that disruption at any step (through mutations or inhibitors) can affect the entire activation cascade, highlighting the importance of phospho-specific antibodies that target distinct sites .

What are the technical challenges in distinguishing RPS6KA1 phosphorylation from other RSK family members?

The RSK family consists of four isoforms (RSK1-4) with high sequence homology, presenting challenges for isoform-specific phosphorylation detection:

• Sequence similarity around phosphorylation sites: The T359/S363 region has high conservation among RSK family members, potentially causing cross-reactivity

• Verification strategies:

  • Knockout/knockdown validation: Use siRNA or CRISPR targeting specific RSK isoforms

  • Isoform-specific expression: Overexpress individual RSK family members

  • Mass spectrometry: For definitive identification of phosphorylated peptides

• Antibody validation data: Review manufacturer's cross-reactivity testing data

• Peptide competition assays: Confirm specificity using blocking peptides containing the phosphorylated epitope

When investigating RPS6KA1-specific functions, researchers should employ multiple approaches to confirm isoform specificity. This could include using cell lines with differential expression of RSK family members or verifying results with multiple antibodies targeting different epitopes. For advanced studies, considering the use of phosphatase-resistant phosphomimetic mutants (T359D/S363D) can help distinguish the functional consequences of phosphorylation at these specific sites versus other regulatory sites .

How can researchers integrate phospho-RPS6KA1 (T359/S363) analysis with broader signaling pathway investigations?

For comprehensive signaling pathway analysis:

• Multiplex analysis approaches:

  • Sequential blotting with antibodies against multiple pathway components

  • Phospho-proteomics to identify global phosphorylation changes

  • Kinase activity assays to measure functional outcomes

• Key pathway components to analyze alongside RPS6KA1:

  • Upstream: ERK1/2 phosphorylation status

  • Parallel: AKT, p38 MAPK, and JNK activation

  • Downstream: S6, CREB, and BAD phosphorylation

• Inhibitor studies to dissect pathway connectivity:

  • MEK inhibitors (U0126, PD98059) to block ERK1/2-mediated RPS6KA1 phosphorylation

  • RSK inhibitors (BI-D1870, SL0101) to distinguish RPS6KA1-specific effects

  • mTOR inhibitors (rapamycin) to differentiate RPS6KA1 from S6K1 effects on downstream targets

• Temporal analysis:

  • Time-course experiments to map activation sequence

  • Pulse-chase approaches to determine phosphorylation stability

By examining multiple components simultaneously, researchers can place RPS6KA1 T359/S363 phosphorylation in proper context within the signaling network . This integrated approach also helps identify potential compensatory mechanisms or feedback loops that might impact experimental outcomes when targeting RPS6KA1 specifically.

What are common sources of false positive or negative results when detecting phospho-RPS6KA1?

When troubleshooting phosphorylation-specific detection, researchers should systematically evaluate each step of their protocol. For Western blotting, transferring phosphorylated proteins efficiently may require optimization of transfer conditions. For flow cytometry applications, fixation and permeabilization protocols significantly impact epitope preservation and accessibility. Additionally, certain cell types may exhibit different baseline phosphorylation levels or activation kinetics, necessitating protocol adjustments .

How can researchers accurately quantify changes in RPS6KA1 T359/S363 phosphorylation?

For accurate quantification of phosphorylation changes:

• Normalization approaches:

  • Ratio of phospho-RPS6KA1 to total RPS6KA1 (preferred method)

  • Normalization to housekeeping proteins (GAPDH, β-actin) when comparing total lysate loading

  • Use of loading controls specific to subcellular fractions if performing fractionation

• Quantification methods:

  • Densitometry for Western blots with linear range validation

  • Mean fluorescence intensity (MFI) measurements for flow cytometry

  • ELISA-based quantification for high-throughput analysis

• Statistical considerations:

  • Perform at least three independent biological replicates

  • Use appropriate statistical tests for the experimental design

  • Consider the dynamic range of the assay and avoid saturation

• Technical validation:

  • Include a stimulation dose-response to demonstrate proportional signal changes

  • Use both positive (stimulated) and negative (unstimulated or phosphatase-treated) samples as references

These approaches help ensure that observed changes in phosphorylation are biologically meaningful rather than technical artifacts. When reporting quantitative changes, researchers should clearly describe their normalization approach and include both representative images and quantitative data with statistical analysis .

What are the limitations of using phospho-RPS6KA1 antibodies in different experimental systems?

Understanding the limitations of phospho-specific antibodies across experimental systems is crucial:

• Species cross-reactivity considerations:

  • Most antibodies are validated against human and mouse RPS6KA1

  • Sequence verification is necessary for other species

  • Test species-specific positive controls before proceeding with experiments

• Cell/tissue-specific challenges:

  • Primary tissues may require optimized extraction protocols to preserve phosphorylation

  • High phosphatase activity in certain tissues may necessitate stronger inhibitor cocktails

  • Autofluorescence in certain tissues may interfere with immunofluorescence/flow cytometry applications

• Fixed vs. fresh samples:

  • Formalin fixation can mask phospho-epitopes or cause epitope degradation over time

  • Antigen retrieval methods may need optimization for immunohistochemistry

  • Phosphorylation status may not be preserved in long-term stored samples

• Application-specific limitations:

  • Western blot: Limited spatial information within cell populations

  • Flow cytometry: Requires single-cell suspensions, limiting tissue architecture information

  • Immunohistochemistry: More qualitative than quantitative for phospho-epitopes

  • ELISA: May have cross-reactivity issues unless highly validated

Researchers should validate antibodies in their specific experimental system rather than relying solely on manufacturer data. Preliminary experiments should include appropriate controls to establish sensitivity and specificity boundaries for the chosen application .

How does RPS6KA1 T359/S363 phosphorylation status correlate with cellular phenotypes in cancer research?

RPS6KA1 T359/S363 phosphorylation has significant implications in cancer biology:

• Cancer-related phenotypes associated with RPS6KA1 hyperactivation:

  • Enhanced cell proliferation through regulation of cell cycle proteins

  • Increased cell survival via inhibition of pro-apoptotic BAD and DAPK1

  • Altered protein synthesis through regulation of translation machinery

  • Contributions to therapy resistance mechanisms

• Correlation with clinical parameters:

  • Increased phosphorylation often correlates with more aggressive disease in certain cancers

  • May serve as a biomarker for MAPK pathway activation

  • Potential predictive value for response to targeted therapies

• Therapeutic implications:

  • RSK inhibitors under investigation as potential cancer therapeutics

  • Combination strategies targeting both upstream (MEK/ERK) and downstream (RSK) components

  • Potential synthetic lethality approaches with other pathway inhibitors

Researchers investigating cancer-related signaling should consider analyzing RPS6KA1 T359/S363 phosphorylation alongside other markers of MAPK pathway activation. The dynamic relationship between RPS6KA1 activation and cellular phenotypes makes phospho-specific antibodies valuable tools for monitoring pathway activity in response to therapeutic interventions .

What insights can phospho-RPS6KA1 analysis provide about cross-talk between MAPK and other signaling pathways?

Phospho-RPS6KA1 analysis reveals important pathway interconnections:

• MAPK and mTOR pathway cross-talk:

  • RPS6KA1 phosphorylates TSC2 at Ser-1798, inhibiting its ability to suppress mTOR signaling

  • RPS6KA1 mediates phosphorylation of RPTOR, regulating mTORC1 activity

  • These interactions create feedback loops between growth factor and nutrient-sensing pathways

• MAPK and PI3K/AKT pathway integration:

  • RPS6KA1 and AKT share several downstream substrates (e.g., BAD, GSK3β)

  • Differential phosphorylation of shared substrates can reveal pathway dominance

  • Analysis of substrate phosphorylation patterns can identify compensatory mechanisms

• Cell cycle regulation cross-talk:

  • RPS6KA1 phosphorylates the CDK inhibitor CDKN1B, affecting cell cycle progression

  • Integrated analysis of cell cycle regulators and RPS6KA1 phosphorylation can reveal mechanism of growth control

• Experimental approaches to study cross-talk:

  • Combinatorial inhibitor treatments targeting multiple pathways

  • Temporal analysis of phosphorylation events across pathways

  • Genetic manipulation of pathway components with phospho-RPS6KA1 readouts

This multi-pathway perspective is crucial for understanding cellular responses to targeted therapies and developing more effective combination strategies. Researchers should design experiments that simultaneously monitor key nodes in interconnected pathways to fully understand the network-level consequences of specific interventions .

How can phospho-RPS6KA1 (T359/S363) analysis contribute to drug discovery and targeted therapy development?

Phospho-RPS6KA1 analysis offers several applications in drug development:

• Target engagement biomarker:

  • Direct measure of MAPK pathway inhibition for ERK-targeted therapies

  • Early pharmacodynamic marker in drug development

  • Dose-finding tool in preclinical and clinical studies

• Resistance mechanism identification:

  • Persistent RPS6KA1 phosphorylation despite upstream inhibition suggests bypass mechanisms

  • Temporal changes in phosphorylation patterns may reveal adaptive resistance

  • Combination therapy rationale development based on phosphorylation profiles

• Patient stratification strategies:

  • Baseline phosphorylation status may predict response to pathway-targeted therapies

  • Dynamic changes upon treatment could serve as early response indicators

  • Integration with other biomarkers for refined patient selection

• Novel therapeutic approaches:

  • Identification of synthetic lethal interactions based on phosphorylation status

  • Development of degraders or inhibitors specifically targeting the phosphorylated form

  • Combination strategies targeting both the phosphorylation event and downstream effectors

Pharmaceutical researchers can leverage phospho-specific antibodies as tools throughout the drug development process, from target validation to clinical trial biomarker analysis. The specificity of phosphorylation-state detection provides valuable insights into both on-target activity and potential off-target effects of candidate compounds targeting this signaling pathway .