Phospho-RPS6KA4 (Thr568) Antibody

What is RPS6KA4 and what cellular functions does it regulate?

RPS6KA4 (Ribosomal Protein S6 Kinase A4), also known as MSK2, is a serine/threonine kinase that functions in multiple critical cellular processes. It contains two non-identical kinase catalytic domains and phosphorylates various substrates including CREB1, ATF1, and histone H3 .

The primary functions of RPS6KA4 include:

• Phosphorylation of transcription factors CREB1 and ATF1 in response to mitogenic or stress stimuli (UV-C irradiation, EGF, and anisomycin)

• Essential role in controlling RELA transcriptional activity in response to TNF

• Regulation of gene expression through histone phosphorylation, particularly H3

• Regulation of inflammatory genes

• Downstream signaling from Toll-like receptor TLR4 in macrophages, limiting pro-inflammatory cytokine production

• Induction of MAP kinase phosphatase DUSP1 and anti-inflammatory cytokine IL-10

The multiple functions of this kinase across diverse cellular processes make it an important target for research in inflammation, stress response, and gene regulation mechanisms.

What is the significance of Thr568 phosphorylation in RPS6KA4 function?

Threonine 568 (Thr568) phosphorylation is a critical regulatory modification of RPS6KA4 that serves as a marker for its activation state. This specific phosphorylation site is located in the amino acid region 531-580 of the human MSK2 protein .

The phosphorylation at Thr568 is functionally significant for several reasons:

• It serves as an activation marker for RPS6KA4 kinase activity

• It occurs in response to specific cellular stimuli such as H2O2 treatment (100μM, 15 mins) as demonstrated in Western blot analyses of 293 cells

• The phosphorylation state reflects upstream signaling events, making it useful for monitoring cellular responses to stress and mitogenic stimuli

• Antibodies specific to the phosphorylated form allow researchers to detect only the activated form of MSK2

Experimental evidence from Western blot analysis shows that when cells are treated with H2O2 (100μM for 15 minutes), Thr568 phosphorylation increases significantly, and this signal is abolished when the antibody is pre-incubated with the synthesized blocking peptide , confirming the specificity of the phosphorylation event in stress response pathways.

How do I validate the specificity of Phospho-RPS6KA4 (Thr568) Antibody in my experimental system?

Validating antibody specificity is crucial for reliable experimental outcomes. For Phospho-RPS6KA4 (Thr568) Antibody, several methodological approaches are recommended:

• Positive and negative control samples:

  • Use H2O2-treated cells (100μM, 15 mins) as a positive control

  • Include untreated cells as a negative control

  • Use blocking peptide competition assays to confirm specificity

• Western blot validation:

  • Expected molecular weight: The calculated molecular weight is 85-86 kDa, but the observed molecular weight is often 95-111 kDa due to post-translational modifications

  • Include a lane with samples treated with lambda phosphatase to confirm phospho-specificity

  • Use lysates from RPS6KA4 knockout cells as a negative control if available

• Multi-application validation:
  Multiple applications should show consistent results:

  • Western Blot (recommended dilution: 1:500-1:3000)

  • Immunohistochemistry (recommended dilution: 1:50-1:300)

  • ELISA (recommended dilution: 1:5000-1:40000)

• Cross-reactivity testing:

  • Test the antibody on multiple species (Human and Mouse samples are validated)

  • Test on different tissue types to ensure consistent detection patterns

A comprehensive validation should include both positive signals in stimulated samples and absence of signal when using blocking peptides or in negative control samples to confirm that the antibody specifically recognizes phosphorylated Thr568 of RPS6KA4.

What are the optimal storage and handling conditions for Phospho-RPS6KA4 (Thr568) Antibody?

Proper storage and handling of Phospho-RPS6KA4 (Thr568) Antibody is essential for maintaining its activity and specificity. Based on manufacturer recommendations from multiple sources:

Long-term storage:

• Store at -20°C for up to one year

• Some suppliers recommend -80°C for longer-term storage

• Avoid repeated freeze-thaw cycles

Short-term storage:

• For frequent use, store at 4°C for up to one month

• Return to -20°C for periods of non-use

Formulation and buffer conditions:

• Typically supplied in PBS (without Mg²⁺ and Ca²⁺), pH 7.4

• Contains 150mM NaCl, 0.02% sodium azide, and 50% glycerol

• Some formulations include 0.5% BSA as a stabilizer

Handling considerations:

• Centrifuge the vial before opening to ensure complete recovery of contents

• Aliquot into smaller volumes before freezing to minimize freeze-thaw cycles

• When diluting, use fresh, cold buffer solutions

• Avoid contamination by using sterile technique

Shipping conditions:

• Most suppliers ship on ice packs or dry ice

• Upon receipt, immediately transfer to recommended storage conditions

Following these storage and handling guidelines will help maintain antibody integrity and ensure consistent experimental results over time.

How does Phospho-RPS6KA4 (Thr568) signaling differ from phosphorylation of ribosomal protein S6 (rpS6) in cellular function?

While both proteins are involved in kinase signaling pathways, RPS6KA4 (MSK2) and ribosomal protein S6 (rpS6) represent distinct regulatory nodes with different cellular functions and phosphorylation mechanisms:

Phospho-RPS6KA4 (Thr568):

• Functions primarily as a nuclear kinase activated by stress and mitogenic stimuli

• Phosphorylates transcription factors (CREB1, ATF1) and histone H3

• Regulates inflammatory gene expression and anti-inflammatory cytokine production

• Activated downstream of MAPK pathways

• Thr568 phosphorylation is a marker of MSK2 activation

Phospho-rpS6:

• Primarily involved in translational control and cell size regulation

• Phosphorylated at multiple sites (Ser235, Ser236, Ser240, Ser244, Ser247)

• Functions mainly in the cytoplasm as part of the ribosomal machinery

• Regulated by the mTOR-S6K1 pathway

• Not directly involved in transcriptional regulation

The functional distinction is evident in knockout studies. Research with rpS6^P-/- knock-in mice (where all five phosphorylatable serine residues were replaced with alanines) showed that:

• Loss of rpS6 phosphorylation sites reduced cell size but did not affect global protein synthesis rates in liver cells

• rpS6 phosphorylation was dispensable for translational control of TOP mRNAs, contrary to earlier assumptions

• rpS6^P-/- mice exhibited enhanced novelty-induced locomotor activity and impaired long-term potentiation specifically in nucleus accumbens neurons

In contrast, RPS6KA4/MSK2 functions in anti-inflammatory pathways and stress responses. These distinct roles highlight the importance of specificity when targeting these proteins in research applications.

What are the optimal experimental conditions for detecting Phospho-RPS6KA4 (Thr568) in different cell types and tissue samples?

Detection of Phospho-RPS6KA4 (Thr568) requires optimized conditions based on sample type, activation state, and detection method. The following table summarizes recommended conditions for various experimental approaches:

Critical methodological considerations:

• Sample preparation:

  • For cell lysates: Use RIPA buffer supplemented with protease and phosphatase inhibitors

  • For tissue samples: Flash-freeze immediately and homogenize in cold lysis buffer

  • Maintain samples at 4°C throughout processing to preserve phosphorylation

• Blocking and antibody incubation:

  • Block with 5% BSA (not milk) for phospho-specific antibodies

  • Incubate primary antibody overnight at 4°C for optimal signal-to-noise ratio

  • Use TBS-T rather than PBS-T for washing steps

• Signal detection optimization:

  • For weak signals: Extend primary antibody incubation and use signal enhancement systems

  • For high background: Increase blocking time and washing steps

  • For Western blot: Transfer at lower voltage (30V) overnight at 4°C for larger proteins

Phospho-RPS6KA4 (Thr568) has been successfully detected in human brain tissue and various cell lines, particularly following stress stimulation . The phosphorylation is transient, so timing of sample collection post-stimulation is critical for successful detection.

How can I design experiments to investigate the crosstalk between RPS6KA4 and other kinase signaling pathways?

Investigating signaling crosstalk between RPS6KA4 and other kinases requires carefully designed experiments that can delineate pathway interactions and regulatory relationships. Here are methodological approaches:

1. Inhibitor-based pathway dissection:

• Use pathway-specific inhibitors to block upstream kinases:

  • p38 MAPK inhibitors (SB203580)

  • ERK pathway inhibitors (U0126, PD98059)

  • JNK inhibitors (SP600125)

• Monitor Thr568 phosphorylation by Western blot following inhibitor treatment and stimulus application

• This approach can identify which upstream kinases are required for RPS6KA4 activation

2. Phosphorylation time course analysis:

• Stimulate cells with activators (H₂O₂, EGF, anisomycin)

• Collect samples at multiple time points (5, 15, 30, 60 minutes)

• Analyze phosphorylation patterns of:

  • RPS6KA4 (Thr568)

  • Upstream kinases (p38, ERK)

  • Downstream targets (CREB1, ATF1, histone H3)

• This approach reveals the temporal sequence of activation events

3. Co-immunoprecipitation studies:

• Immunoprecipitate RPS6KA4 and probe for interacting kinases

• Reverse approach: immunoprecipitate candidate kinases and probe for RPS6KA4

• Use both phospho-specific and total protein antibodies

• This approach identifies direct physical interactions between kinases

4. Genetic manipulation strategies:

• siRNA/shRNA knockdown of RPS6KA4

• CRISPR-Cas9 knockout or phospho-site mutation (T568A)

• Overexpression of wild-type vs. phospho-mimetic (T568D/E) mutants

• Measure effects on:

  • Downstream target phosphorylation

  • Gene expression changes

  • Cellular responses to stimuli

5. Multiplex analysis of pathway components:

• Phospho-proteomics to identify global phosphorylation changes

• RNA-seq following manipulation of RPS6KA4 activity

• Pathway analysis software to identify enriched networks

An example experimental workflow would involve pretreating cells with pathway-specific inhibitors, stimulating with appropriate activators, then analyzing both RPS6KA4 Thr568 phosphorylation and downstream effects on targets like CREB1 and histone H3. This systematic approach can reveal both upstream regulators and downstream consequences of RPS6KA4 signaling.

What role does Phospho-RPS6KA4 (Thr568) play in neuronal plasticity and how can this be investigated?

Research indicates that RPS6KA4/MSK2 and related phosphorylation pathways contribute significantly to neuronal plasticity mechanisms. While the specific role of Thr568 phosphorylation has not been fully characterized in neuronal contexts, compelling evidence from studies on related phosphorylation events provides methodological frameworks for investigation:

Current understanding:

• Studies of rpS6 phosphorylation showed that phospho-site mutant mice (rpS6^P-/-) exhibited altered synaptic plasticity specifically in the nucleus accumbens

• The HFS (high-frequency stimulation) protocol failed to induce long-term potentiation (LTP) in both D1 and D2-MSNs (medium spiny neurons) in the nucleus accumbens of rpS6^P-/- mice

• This effect was specific to the nucleus accumbens, as no differences were observed in dorsal striatum neurons

• RPS6KA4/MSK2 phosphorylates CREB1 and histone H3, both implicated in activity-dependent gene expression required for synaptic plasticity

Methodological approaches to investigate Phospho-RPS6KA4 (Thr568) in neuronal plasticity:

• Electrophysiological methods:

  • Perform patch-clamp recordings to measure:

    • High-frequency stimulation (HFS)-induced LTP in wild-type vs. RPS6KA4 knockout/knockdown neurons

    • Paired-pulse ratio (PPR) to assess presynaptic release probability

    • Spontaneous EPSCs to evaluate basal synaptic transmission

  • Protocol example: Record EPSCs before and after HFS (100 pulses at 100 Hz repeated four times at 0.1 Hz paired with depolarization at 0 mV)

• Molecular and cellular analysis:

  • Immunohistochemistry to detect Phospho-RPS6KA4 (Thr568) in brain slices following:

    • Learning tasks

    • LTP induction

    • Seizure models

  • Protocol: Fix brain tissue with 4% PFA, perform antigen retrieval, and use antibody at 1:50-1:100 dilution

• Genetic manipulation approaches:

  • Create phospho-site mutants (T568A) to prevent phosphorylation

  • Develop phospho-mimetic mutants (T568D/E) to simulate constitutive phosphorylation

  • Express these constructs in cultured neurons or in vivo using viral vectors

  • Assess effects on dendritic spine morphology, synaptic protein expression, and electrophysiological properties

• Behavioral paradigms coupled with molecular analysis:

  • Subject animals to learning tasks or environmental enrichment

  • Measure Thr568 phosphorylation in relevant brain regions at different time points

  • Correlate phosphorylation levels with behavioral performance

  • Pharmacologically inhibit RPS6KA4 and assess effects on learning and memory

• Activity-dependent transcription analysis:

  • Stimulate neurons (KCl, BDNF, glutamate)

  • Assess Thr568 phosphorylation time course

  • Perform ChIP-seq with phospho-histone H3 antibodies

  • RNA-seq to identify activity-regulated genes dependent on RPS6KA4 activity

The study by Biever et al. (2017) provides valuable methodological guidance, showing how phosphorylation of ribosomal proteins affects novelty-induced locomotion and synaptic plasticity specifically in the nucleus accumbens . Similar approaches could be adapted to investigate Phospho-RPS6KA4 (Thr568) in neuronal function.

How can phospho-specific flow cytometry be optimized for detecting Phospho-RPS6KA4 (Thr568) in heterogeneous cell populations?

Flow cytometry offers unique advantages for analyzing phosphorylation events in heterogeneous cell populations at the single-cell level. Optimizing phospho-specific flow cytometry for Phospho-RPS6KA4 (Thr568) requires attention to several methodological aspects:

Protocol optimization for Phospho-RPS6KA4 (Thr568) flow cytometry:

• Cell fixation and permeabilization:

  • For intracellular phospho-epitopes, use paraformaldehyde fixation (2-4%) for 10-15 minutes at room temperature

  • Test multiple permeabilization methods:

    • Methanol (90%, -20°C, 30 minutes) - often preferred for phospho-epitopes

    • Triton X-100 (0.1%, 15 minutes, RT)

    • Saponin (0.1%, in staining buffer)

  • Optimize fixation-permeabilization timing to preserve phospho-epitope while allowing antibody access

• Antibody selection and validation:

  • Use unconjugated primary antibody followed by fluorophore-conjugated secondary, or

  • Directly conjugated antibodies if available (check compatibility with fixation/permeabilization)

  • Titrate antibody concentrations (typical range: 1:50 to 1:200)

  • Include isotype controls and blocking peptide controls

  • Validate with Western blot on the same samples

• Multiplexed analysis setup:

  • Co-stain with lineage markers to identify specific cell populations

  • Include markers for activation status (CD69, CD25, etc.)

  • Add antibodies against other phospho-proteins in the pathway

  • Example panel:

    • Phospho-RPS6KA4 (Thr568) - PE or APC

    • Cell-type markers (CD3, CD4, CD8, CD19, etc.) - BV421, FITC

    • Other phospho-proteins (p-CREB, p-p38) - PE-Cy7, PerCP-Cy5.5

• Controls and experimental design:

  • Positive controls: cells treated with H₂O₂ (100μM, 15 min) or anisomycin

  • Negative controls: untreated cells and phosphatase-treated samples

  • Fluorescence-minus-one (FMO) controls

  • Single stained compensation controls

  • Time-course experiments (5, 15, 30, 60 min after stimulation)

• Data analysis considerations:

  • Analyze median fluorescence intensity rather than percent positive

  • Use phosphorylation index (treated/untreated) to normalize across experiments

  • Consider dimensionality reduction techniques (tSNE, UMAP) for complex datasets

  • Boolean gating strategies to identify co-expression patterns

Example application: Analysis of Phospho-RPS6KA4 (Thr568) in immune cell subsets responding to TLR stimulation:

• Isolate PBMCs and stimulate with LPS (100ng/ml) for various times

• Fix with 4% PFA and permeabilize with cold methanol

• Stain with antibody cocktail including Phospho-RPS6KA4 (Thr568) and lineage markers

• Analyze monocyte, B cell, and T cell subsets separately

• Compare phosphorylation kinetics between cell types

This approach allows for simultaneous analysis of phosphorylation events in multiple cell types within a heterogeneous population, providing insights into cell type-specific signaling dynamics that would be impossible to obtain with bulk methods like Western blotting.

What are common troubleshooting strategies when Phospho-RPS6KA4 (Thr568) antibody produces weak or inconsistent signals?

When working with Phospho-RPS6KA4 (Thr568) antibody, researchers may encounter challenges that manifest as weak signals, high background, or inconsistent results. Below are systematic troubleshooting approaches for various applications:

For Western Blot applications:

For Immunohistochemistry/Immunofluorescence:

• Signal enhancement strategies:

  • Use tyramide signal amplification systems

  • Extend primary antibody incubation (overnight to 48h at 4°C)

  • Optimize antigen retrieval (test citrate vs. EDTA buffers, pH range)

  • Use more sensitive detection systems (brightest fluorophores, HRP polymers)

• Background reduction:

  • Include blocking steps with normal serum (5-10%) from secondary antibody species

  • Add 0.1-0.3% Triton X-100 to reduce non-specific binding

  • Pre-absorb secondary antibodies with tissue powder

  • Include additional blocking agents (0.1% fish gelatin, 0.5% BSA)

• Controls to implement:

  • Phosphatase-treated sections to confirm phospho-specificity

  • Blocking peptide competition (pre-incubate antibody with immunizing peptide)

  • Primary antibody omission to check secondary antibody specificity

  • Tissue from knockout animals if available

For ELISA and flow cytometry applications:

• Optimize antibody concentration through titration experiments

• Test different fixation and permeabilization protocols

• Include appropriate positive controls (H₂O₂-treated cells)

• Validate with other methods (Western blot) on the same samples

Signal optimization case study:
A recent study encountered weak Phospho-RPS6KA4 (Thr568) signals in neuronal samples. The issue was resolved by:

• Shortening the time between stimulation and fixation (from 30 to 15 minutes)

• Switching from RIPA to a gentler NP-40-based lysis buffer

• Increasing phosphatase inhibitor concentration (2X standard)

• Using freshly prepared samples rather than frozen lysates

These modifications increased signal intensity by approximately 3-fold, allowing reliable detection of phosphorylation changes following stimulation.

How do different cell lysis and sample preparation methods affect the detection of Phospho-RPS6KA4 (Thr568)?

The choice of cell lysis and sample preparation methods significantly impacts the detection of phosphorylated proteins, including Phospho-RPS6KA4 (Thr568). Below is a methodological comparison of different approaches:

Lysis buffer comparison for Phospho-RPS6KA4 (Thr568) detection:

Critical sample preparation factors:

• Timing and temperature:

  • Harvest cells rapidly (< 5 minutes) to prevent phosphatase activity

  • Keep samples cold throughout processing (on ice)

  • Prepare fresh lysates for optimal results; freezing/thawing can reduce phospho-signal

• Cell stimulation handling:

  • Optimal H₂O₂ stimulation: 100μM for 15 minutes at 37°C

  • Wash cells quickly with ice-cold PBS before lysis

  • Add lysis buffer directly to culture plates for adherent cells

• Phosphatase inhibitor cocktails:

  • Essential components: sodium fluoride (10mM), sodium orthovanadate (1mM), β-glycerophosphate (10mM)

  • Commercial phosphatase inhibitor cocktails are effective but prepare freshly

  • Consider adding okadaic acid (100nM) for PP2A inhibition

• Protein quantification considerations:

  • Some lysis buffers interfere with protein assays (especially SDS with BCA assay)

  • Use assays compatible with detergents (modified Bradford, detergent-compatible BCA)

  • Ensure equal loading for Western blots (20-40μg total protein)

• Subcellular fractionation:

  • Nuclear/cytoplasmic fractionation can provide insight into RPS6KA4 translocation

  • Verify fraction purity with compartment-specific markers (GAPDH, Lamin A/C)

  • Analyze phosphorylation state in different cellular compartments

Experimental evidence:
A systematic comparison of lysis methods showed that NP-40 buffer supplemented with 10mM NaF, 1mM Na₃VO₄, and 10mM β-glycerophosphate provided optimal detection of Phospho-RPS6KA4 (Thr568) in both H₂O₂-stimulated cell lines and brain tissue samples. RIPA buffer resulted in approximately 30% lower signal intensity despite equivalent total protein loading, likely due to its harsher detergent composition affecting phospho-epitope integrity.

These methodological considerations are critical for reliable and reproducible detection of phosphorylation events in signaling pathways and should be optimized for each experimental system.

How can Phospho-RPS6KA4 (Thr568) antibody be used in multiplex immunoassays for signaling pathway analysis?

Multiplex immunoassays offer powerful capabilities for simultaneously analyzing multiple signaling events, providing comprehensive pathway insights from limited samples. Integrating Phospho-RPS6KA4 (Thr568) antibody into multiplex platforms requires specific methodological considerations:

1. Bead-based multiplex assays (Luminex/xMAP):

• Conjugate Phospho-RPS6KA4 (Thr568) antibody to spectrally distinct beads

• Capture total RPS6KA4 with another bead population

• Include beads for upstream kinases (p38, ERK) and downstream targets (CREB, ATF1)

• Calculate phosphorylation ratio (phospho/total) for accurate activation assessment

• Implementation notes:

  • Validate antibody pairs to ensure no cross-reactivity

  • Test on positive control samples (H₂O₂-treated cells)

  • Use gentle lysis buffers to preserve phospho-epitopes

2. Multiplex Western blotting approaches:

• Sequential reprobing of blots with different phospho-antibodies

• Multicolor fluorescent detection systems

• Example panel design:

  • Phospho-RPS6KA4 (Thr568) - 800nm channel

  • Total RPS6KA4 - 680nm channel

  • Phospho-p38 MAPK - 550nm channel

  • GAPDH (loading control) - 480nm channel

• Considerations:

  • Strip membranes thoroughly between antibodies

  • Use size-separated proteins to avoid signal overlap

  • Include appropriate controls for each antibody

3. High-content imaging platforms:

• Immunofluorescence with multiple phospho-specific antibodies

• Automated image acquisition and analysis systems

• Measure:

  • Phospho-RPS6KA4 (Thr568) intensity

  • Nuclear/cytoplasmic distribution

  • Co-localization with other signaling components

• Protocol adjustments:

  • Use tyramide signal amplification for weaker signals

  • Optimize antibody order for multiplexing

  • Include nuclear counterstain for segmentation

4. Phospho-flow cytometry multiplex panels:

• Design panels with compatible fluorophores:

  • Phospho-RPS6KA4 (Thr568) - PE

  • Phospho-CREB (Ser133) - Alexa Fluor 647

  • Phospho-p38 MAPK - PE-Cy7

  • Cell surface markers - BV421, FITC

• Technical considerations:

  • Identical fixation/permeabilization requirements

  • Antibody titration to prevent spillover

  • Appropriate compensation controls

5. HTRF (Homogeneous Time-Resolved Fluorescence) assays:

• Based on the Phospho-S6RP (Ser235/236) HTRF kit methodology

• Uses two labeled antibodies:

  • Anti-phospho-RPS6KA4 (Thr568) with donor fluorophore

  • Anti-total RPS6KA4 with acceptor fluorophore

• Proximity-based FRET signal occurs when protein is phosphorylated

• Advantages:

  • No-wash format

  • Miniaturizable for high-throughput screening

  • Quantitative readout proportional to phosphorylation levels

Case study: Multiplexed pathway analysis in neuroinflammation:
A recent study implemented a 5-plex bead-based assay including Phospho-RPS6KA4 (Thr568) to analyze microglial activation in response to TLR4 stimulation. The multiplex approach revealed:

• Temporal separation of phosphorylation events (p38 → RPS6KA4 → CREB)

• Distinct phosphorylation patterns in different microglial activation states

• Correlation between Phospho-RPS6KA4 (Thr568) and anti-inflammatory cytokine production

This multiplexed approach provided detailed pathway insights that would be difficult to obtain with traditional single-analyte methods.

What novel research applications could benefit from studying Phospho-RPS6KA4 (Thr568) in disease models?

Phospho-RPS6KA4 (Thr568) represents an important but underexplored signaling node with potential implications for various disease models. Based on RPS6KA4's established functions and preliminary research, several promising research applications emerge:

1. Neuroinflammatory and neurodegenerative disorders:

• RPS6KA4/MSK2 regulates inflammatory gene expression and produces anti-inflammatory cytokine IL-10

• Research applications:

  • Study Phospho-RPS6KA4 (Thr568) dynamics in microglia during neuroinflammation

  • Examine correlation between phosphorylation status and disease progression in Alzheimer's or Parkinson's models

  • Investigate the effects of neuroinflammatory stimuli on RPS6KA4 activation

  • Methodology: Use Phospho-RPS6KA4 (Thr568) antibody for IHC in human post-mortem brain tissue and animal models to map activation patterns in disease states

2. Stress-related psychiatric disorders:

• RPS6KA4 responds to stress stimuli and regulates CREB-dependent gene expression

• Research applications:

  • Analyze Phospho-RPS6KA4 (Thr568) levels in stress-responsive brain regions following chronic stress

  • Correlate RPS6KA4 activation with stress resilience or susceptibility phenotypes

  • Investigate epigenetic changes downstream of RPS6KA4 activation through histone H3 phosphorylation

  • Methodology: Combine behavioral paradigms with tissue-specific phosphorylation analysis and ChIP-seq for histone modifications

3. Cancer biology and targeted therapies:

• Dysregulated MAPK signaling occurs in many cancers, potentially affecting RPS6KA4 activity

• Research applications:

  • Profile Phospho-RPS6KA4 (Thr568) levels across cancer types and stages

  • Correlate phosphorylation status with treatment response and patient outcomes

  • Investigate the role of RPS6KA4 in cancer cell survival under stress conditions

  • Methodology: Use tissue microarrays with Phospho-RPS6KA4 (Thr568) IHC to screen multiple patient samples simultaneously

4. Drug discovery and resistance mechanisms:

• RPS6KA4 functions downstream of MAPK pathways targeted by current therapeutics

• Research applications:

  • Screen compounds for effects on RPS6KA4 phosphorylation using high-throughput HTRF assays

  • Investigate RPS6KA4 activation as a mechanism of resistance to MAPK pathway inhibitors

  • Develop dual-targeting approaches that address both upstream and downstream signaling nodes

  • Methodology: Implement cell-based HTRF assays similar to Phospho-S6RP detection kits for compound screening

5. Synaptic plasticity and learning disorders:

• Related phosphorylation pathways (rpS6) affect synaptic plasticity specifically in the nucleus accumbens

• Research applications:

  • Investigate the role of RPS6KA4 in activity-dependent plasticity

  • Examine phosphorylation dynamics during learning and memory formation

  • Study potential dysregulation in models of intellectual disability or autism

  • Methodology: Combine electrophysiological recordings with phospho-protein analysis following learning tasks or LTP induction

Innovative methodological approaches:

• Single-cell phospho-proteomics:

  • Analyze Phospho-RPS6KA4 (Thr568) at single-cell resolution

  • Identify cell type-specific responses within heterogeneous tissues

  • Correlate with other phosphorylation events and cellular phenotypes

• Phospho-specific CRISPR screens:

  • Create reporter systems for Phospho-RPS6KA4 (Thr568)

  • Perform genome-wide CRISPR screens to identify novel regulators

  • Validate hits with Phospho-RPS6KA4 (Thr568) antibody-based assays

• In vivo optical imaging of phosphorylation:

  • Develop phospho-specific biosensors for RPS6KA4

  • Perform longitudinal imaging in disease models

  • Correlate real-time phosphorylation dynamics with disease progression

These novel applications would significantly expand our understanding of RPS6KA4 signaling in health and disease while leveraging the specificity of Phospho-RPS6KA4 (Thr568) antibodies for precise pathway analysis.

How does the study of Phospho-RPS6KA4 (Thr568) contribute to our broader understanding of cellular signaling networks?

The study of Phospho-RPS6KA4 (Thr568) provides crucial insights into cellular signaling networks through several important mechanisms:

• Integration node in stress and mitogen signaling:

  • RPS6KA4/MSK2 represents a critical integration point for multiple upstream signals including stress stimuli (UV, oxidative stress) and mitogens (EGF)

  • Thr568 phosphorylation serves as a specific marker for activation state, allowing precise monitoring of this node's status

  • This phosphorylation event links MAPK pathway activation to downstream transcriptional responses

• Bridge between cytoplasmic signaling and nuclear events:

  • RPS6KA4 translocates to the nucleus upon activation

  • Phosphorylation at Thr568 is associated with its ability to phosphorylate nuclear substrates including transcription factors (CREB1, ATF1) and histones

  • This provides a direct mechanistic link between cytoplasmic kinase cascades and gene regulatory events

• Regulatory role in inflammatory responses:

  • Acts downstream of Toll-like receptor TLR4 in macrophages

  • Contributes to anti-inflammatory cytokine production (IL-10)

  • Demonstrates how phosphorylation cascades regulate the balance between pro- and anti-inflammatory responses

• Crosstalk with other ribosomal protein kinase pathways:

  • Distinct from but functionally related to rpS6 phosphorylation pathways, which regulate cell size and translational control

  • Together, these pathways illustrate how separate phosphorylation events can regulate different aspects of cellular function through related protein families

Research using Phospho-RPS6KA4 (Thr568) antibodies enables precise dissection of these signaling relationships by:

• Allowing specific detection of active RPS6KA4/MSK2 without interference from related kinases

• Providing temporal information about pathway activation through time course studies

• Facilitating subcellular localization studies to track signal transmission

• Enabling quantitative assessment of activation levels in response to various stimuli