Phospho-RPS6 (S235) Antibody

What is RPS6 and why is S235 phosphorylation significant?

RPS6 is a critical component of the 40S ribosomal subunit that plays an important role in controlling cell growth and proliferation through the selective translation of particular classes of mRNA . Phosphorylation at S235 is one of five phosphorylation sites (S235, S236, S240, S244, S247) identified on the carboxy-terminal domain of RPS6 . This specific phosphorylation is widely used as a marker for neuronal activity and as a readout of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway activation . Unlike other phosphorylation sites, S235 can be targeted by multiple kinases, making it a sensitive indicator of various signaling cascades.

How does phosphorylation occur at the S235 site?

Phosphorylation of RPS6 occurs in an ordered manner, beginning with Ser-236 and followed sequentially by phosphorylation of Ser-235, Ser-240, Ser-244, and Ser-247 . The S235 site can be phosphorylated by multiple kinases including:

• p70/p85 S6 kinase 1 (S6K1), which can catalyze phosphorylation at all five sites

• p90 Ribosomal S6 Kinases (RSK1-4), which specifically target S235 and S236

• Protein Kinase A (PKA), which directly phosphorylates S235/S236

• Protein Kinase C (PKC)

• Protein Kinase G (PKG)

• Death-Associated Protein Kinase (DAPK)

The dephosphorylation of all sites, including S235, is primarily carried out by Protein Phosphatase-1 (PP-1) .

Which signaling pathways regulate RPS6 phosphorylation at S235?

Multiple signaling pathways converge on RPS6 phosphorylation at S235:

• mTORC1 pathway: Activates S6K1/2, a major regulator of RPS6 phosphorylation at all sites including S235

• RAS/ERK pathway: Regulates S235 phosphorylation through activation of RSK1 and RSK2

• cAMP/PKA pathway: Contributes to S235 phosphorylation as demonstrated by increased phosphorylation following forskolin treatment or cAMP analog administration

• PKA/DARPP-32/PP-1 pathway: Particularly important in the striatum, where PKA phosphorylates DARPP-32, which inhibits PP-1, thereby enhancing RPS6 phosphorylation

These pathways can work independently or synergistically, creating complex regulatory patterns in different cellular contexts and tissue types.

What are best practices for antibody selection and validation?

When selecting a phospho-RPS6 (S235) antibody, consider the following best practices:

• Specificity validation: Choose antibodies validated with phospho-mutants (such as S235A) or competing phospho-peptides

• Species reactivity: Verify the antibody works in your species of interest. Many antibodies recognize human, mouse, and rat phospho-RPS6

• Application-specific validation: Ensure the antibody is validated for your specific application (Western blot, immunohistochemistry, flow cytometry)

• Dual recognition: Many commercial antibodies recognize both phospho-S235 and phospho-S236 (dual phospho-specific)

• Vendor comparison: Review validation data from multiple vendors, including customer reviews and published citations

• Lot consistency: Request information about lot-to-lot consistency when purchasing

Validation should include positive controls (tissues/cells with known high phospho-RPS6 levels) and negative controls (phosphatase-treated samples or tissues from phospho-mutant mice) .

How should I optimize Western blot conditions for phospho-RPS6 (S235) detection?

Optimizing Western blot conditions for phospho-RPS6 (S235) detection requires careful attention to several key factors:

• Sample preparation:

  • Use phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers

  • Maintain samples at 4°C during processing to prevent dephosphorylation

  • Use NP-40 or similar mild detergent buffer for protein extraction

• Protein loading:

  • Load 10-20 μg of total protein for most tissue samples

  • Mouse skeletal muscle and bone marrow samples show good signal with 10 μg of protein

• Gel separation:

  • Use 10-12% acrylamide gels or 4-12% gradient gels for optimal resolution

• Blocking:

  • Use 5% BSA in TBS-T rather than milk, as milk contains phospho-proteins that interfere with antibody binding

  • Block for 1 hour at room temperature

• Antibody incubation:

  • Typical dilutions range from 1:400 to 1:1000 for primary antibody

  • Incubate overnight at 4°C for optimal results

• Detection:

  • Use chemiluminescence for high sensitivity

  • Consider fluorescent secondary antibodies for precise quantification and multiplexing

• Controls:

  • Always run total RPS6 detection in parallel for normalization

  • Include positive controls from cells treated with growth factors or forskolin

What controls are essential when studying phospho-RPS6 (S235)?

When studying phospho-RPS6 (S235), the following controls are essential:

• Total protein controls:

  • Always detect total RPS6 for normalization to account for changes in protein expression

  • Use housekeeping proteins (β-actin, GAPDH) as loading controls

• Phosphorylation site controls:

  • Compare phosphorylation at S235/236 with S240/244 to distinguish pathway-specific effects

  • Consider examining all five phosphorylation sites when possible

• Pharmacological controls:

  • Rapamycin treatment to inhibit mTORC1/S6K pathway

  • MEK inhibitors (U0126, PD98059) to block ERK/RSK pathway

  • PKA inhibitors (H89) to block cAMP/PKA pathway

• Genetic controls:

  • When available, use tissues from RPS6 phospho-mutant mice (rpS6P−/−) as negative controls

  • S6K1/2 knockout models to determine kinase-specific effects

• Dephosphorylation controls:

  • Lambda phosphatase-treated samples as negative controls

  • PP-1 inhibitor treatment to enhance phosphorylation signal

• Stimulation controls:

  • Positive controls with known stimuli: forskolin (PKA), phorbol esters (PKC), growth factors (mTORC1)

How can I distinguish between different kinase contributions to S235 phosphorylation?

Distinguishing between different kinase contributions requires a combinatorial approach:

• Selective inhibitor strategy:

  • Apply specific kinase inhibitors sequentially and in combination:

    • Rapamycin or Torin (mTORC1/S6K inhibition)

    • U0126 or PD98059 (MEK/ERK/RSK inhibition)

    • H89 (PKA inhibition)

    • Bisindolylmaleimide (PKC inhibition)

• Phosphorylation site analysis:

  • Compare phosphorylation patterns across sites:

    • S6K1/2 phosphorylates all five sites

    • RSK, PKA, PKC target only S235/S236

  • Ratio analysis of pS235/236 to pS240/244 can indicate relative pathway contributions

• Genetic approaches:

  • Use of knockout or knockdown models:

    • S6K1/S6K2 double knockout mice

    • RSK-deficient models

    • PKA catalytic subunit knockdowns

• Temporal dynamics:

  • Analyze phosphorylation kinetics following stimulus:

    • Fast S235/236 phosphorylation (minutes) often indicates RSK or PKA activity

    • Slower responses (tens of minutes) may reflect mTORC1/S6K activation

• Pathway-specific stimuli:

  • Apply selective pathway activators:

    • Forskolin or cAMP analogs (PKA activation)

    • Phorbol esters (PKC activation)

    • Growth factors (mTORC1 activation)

What is the relationship between RPS6 phosphorylation and neuronal activity?

RPS6 phosphorylation is widely used as a marker for neuronal activity in neuroscience research :

• Synaptic plasticity correlation:

  • Enhanced RPS6 phosphorylation occurs during:

    • Long-term potentiation (LTP) following high-frequency stimulation

    • mGluR-dependent long-term depression (LTD)

    • Forskolin-induced synaptic strengthening

• Activity-dependent signaling pathways:

  • Neuronal activity triggers multiple cascades converging on RPS6:

    • Glutamate receptor activation → ERK/RSK → pS235/236

    • BDNF/TrkB → mTORC1/S6K → phosphorylation at all sites

    • Dopamine/D1R → PKA/DARPP-32/PP-1 → enhanced phosphorylation

• Cell-type specificity:

  • Phosphorylation patterns vary across neuronal populations:

    • S240/244 phosphorylation estimates activity in striatal cholinergic interneurons

    • D1 receptor-expressing neurons show distinct regulation from D2-expressing neurons

• Pharmacological responses:

  • Various neuroactive compounds alter RPS6 phosphorylation:

    • D-amphetamine enhances pS235/236 in D1R-containing MSNs

    • Haloperidol increases phosphorylation through DARPP-32/PP-1 signaling

• Functional significance:

  • RPS6 phosphorylation may regulate:

    • Translation of specific mRNA subsets rather than global translation

    • Mitochondria-related mRNAs in the nucleus accumbens

    • Synaptic function through LTP in specific brain regions

How should I interpret contradictory results between phospho-S235/236 and phospho-S240/244 antibodies?

Contradictory results between phospho-S235/236 and phospho-S240/244 antibodies can provide valuable insights into underlying signaling mechanisms:

• Pathway-specific activation:

  • Different kinases target specific sites:

    • S235/236-only phosphorylation suggests RSK, PKA, or PKC activity

    • All-site phosphorylation indicates S6K1/2 activity

  • Discrepancies may reveal differential pathway activation in your experimental system

• Temporal dynamics consideration:

  • Phosphorylation occurs sequentially:

    • S236→S235→S240→S244→S247

  • Time point selection may capture different stages of this sequence

• Bidirectional regulation analysis:

  • Complex interdependencies exist:

    • Phosphorylation at S240/244 influences S247 phosphorylation

    • Phospho-S247 affects S240/244 phosphorylation

  • This bidirectionality creates complex patterns that may appear contradictory

• Phosphatase regulation evaluation:

  • PP-1 dephosphorylates all sites but its regulation may vary:

    • DARPP-32-mediated inhibition in striatum

    • Other regulatory mechanisms in different tissues

  • Differential phosphatase activity can create site-specific differences

• Technical validation:

  • Verify antibody specificity:

    • Test with phospho-mutants (S235/236A or S240/244/247A)

    • Use competing phospho-peptides

    • Include dephosphorylation controls

What is the relationship between RPS6 phosphorylation and mRNA translation?

The relationship between RPS6 phosphorylation and mRNA translation is complex and still being elucidated:

• Historical perspective:

  • Initially believed to specifically regulate translation of TOP mRNAs (5' terminal oligopyrimidine tract)

  • TOP mRNAs often encode components of translational machinery

• Biochemical evidence:

  • Phosphorylation of RPS6 enhances its affinity for the m7GpppG cap structure

  • This suggests a role in enhancing mRNA translation initiation

  • Both S235 and S236 phosphorylation are required for optimal cap binding

• Recent findings from phospho-mutant models:

  • rpS6P−/− mice (all five phosphorylation sites mutated) show:

    • Normal global translation rates

    • Impaired translation of specific mRNA subsets

    • Particularly affected are mitochondria-related mRNAs in the nucleus accumbens

• Polysome profiling evidence:

  • Analysis of heavy polysomal fractions (actively translated mRNAs) reveals:

    • 998 differentially expressed mRNAs in nucleus accumbens of rpS6P−/− mice

    • Equal numbers up- and down-regulated (497 down, 501 up)

    • No changes in cytosolic steady-state mRNA levels of these genes

• Functional implications:

  • Rather than global translation control, RPS6 phosphorylation may:

    • Fine-tune translation of specific mRNA subsets

    • Regulate specialized cellular processes

    • Have cell-type and brain-region specific effects

How does the PKA/DARPP-32/PP-1 pathway regulate RPS6 phosphorylation in neurons?

The PKA/DARPP-32/PP-1 pathway plays a crucial role in regulating RPS6 phosphorylation, particularly in the striatum :

• Dual regulatory mechanism:

  • Direct phosphorylation:

    • PKA directly phosphorylates RPS6 at S235/236 sites

    • Demonstrated by increased phosphorylation following forskolin or cAMP analog treatment

  • Phosphatase regulation:

    • PKA phosphorylates DARPP-32 at T34

    • Phospho-DARPP-32 becomes a potent PP-1 inhibitor

    • PP-1 inhibition prevents dephosphorylation of RPS6 at all sites

• Experimental evidence:

  • Pharmacological:

    • D-amphetamine enhances pS235/236-rpS6 selectively in D1R-expressing MSNs

    • This effect requires D1R/Gαolf/PKA-mediated activation of DARPP-32/PP-1 signaling

    • Independent of mTORC1/p70S6K1/2, ERK, and PKC pathways

  • Inhibitor studies:

    • Forskolin-induced RPS6 phosphorylation in striatal slices is reduced by PKA inhibitors

    • PKA stimulation with cAMP analog cBIMPS increases pS235/236-rpS6 in striatal culture

• Brain region specificity:

  • Well-characterized in striatum due to high DARPP-32 expression

  • Similar mechanisms may operate in other brain regions with different PP-1 regulators

• Physiological significance:

  • Links dopaminergic signaling to translational control

  • May contribute to activity-dependent protein synthesis

  • Potential role in adaptation to drugs of abuse and antipsychotics

What is the function of RPS6 phosphorylation in different cellular contexts?

The function of RPS6 phosphorylation varies across cellular contexts, with emerging evidence for tissue-specific roles:

What are the emerging methods for studying RPS6 phosphorylation dynamics?

Emerging methods for studying RPS6 phosphorylation dynamics include:

• Phospho-proteomic approaches:

  • Mass spectrometry-based quantification:

    • Allows simultaneous detection of multiple phosphorylation sites

    • Can identify novel interaction partners

    • Enables unbiased analysis of signaling networks

• Live-cell imaging techniques:

  • Phospho-specific fluorescent reporters:

    • FRET-based sensors for real-time monitoring

    • Enables single-cell resolution analysis

    • Reveals subcellular localization patterns

• High-throughput screening:

  • Kinase/phosphatase library screening:

    • Identifies novel regulators of RPS6 phosphorylation

    • Reveals potential therapeutic targets

    • Enables pathway mapping

• Genetic approaches:

  • Site-specific phospho-mutants:

    • Beyond all-site mutants (rpS6P−/−)

    • Specific S235A or S235D (phospho-mimetic) mutations

    • CRISPR/Cas9-mediated knockin models

• Translatomic analysis:

  • TRAP (Translating Ribosome Affinity Purification):

    • Cell-type-specific analysis of translated mRNAs

    • Correlates with RPS6 phosphorylation state

    • Reveals functional consequences

• Multi-modal approaches:

  • Integrated analysis combining:

    • Phosphorylation state (Western blot, mass spectrometry)

    • Ribosome profiling (translation dynamics)

    • RNAseq (transcriptional changes)

    • Functional assays (cellular phenotypes)

How does RPS6 phosphorylation relate to neurological and psychiatric disorders?

RPS6 phosphorylation has been implicated in various neurological and psychiatric disorders:

• Neurodevelopmental disorders:

  • Autism spectrum disorders:

    • Hyperactivated mTORC1 signaling and increased RPS6 phosphorylation

    • Models of Tuberous Sclerosis and Fragile X syndrome show aberrant regulation

  • Intellectual disability:

    • Mutations in mTOR pathway components affect RPS6 phosphorylation

    • Altered protein synthesis during critical developmental periods

• Neurodegenerative diseases:

  • Alzheimer's disease:

    • Dysregulated mTORC1/S6K signaling

    • Altered RPS6 phosphorylation in affected brain regions

  • Parkinson's disease:

    • Impaired PKA/DARPP-32/PP-1 signaling in striatum

    • Changes in RPS6 phosphorylation state

• Psychiatric disorders:

  • Depression and stress models:

    • Altered RPS6 phosphorylation in response to antidepressants

    • Potential role in stress adaptation mechanisms

  • Schizophrenia:

    • Antipsychotic drugs modulate RPS6 phosphorylation

    • Haloperidol increases phosphorylation through DARPP-32/PP-1 signaling

• Substance use disorders:

  • D-amphetamine exposure:

    • Enhances pS235/236-rpS6 selectively in D1R-containing MSNs

    • Requires D1R/PKA/DARPP-32/PP-1 signaling

  • Potential therapeutic target:

    • Manipulation of RPS6 phosphorylation may affect drug-seeking behaviors

    • May contribute to neuroadaptations underlying addiction