Phospho-RPS6KB1 (S434) Antibody

What is RPS6KB1 and what functional significance does phosphorylation at S434 have?

RPS6KB1 (also known as p70 S6 kinase or S6K1) belongs to the ribosomal S6 kinase family of serine/threonine kinases. This 70 kDa protein contains two non-identical kinase catalytic domains that phosphorylate multiple residues of the S6 ribosomal protein . The kinase activity directly increases protein synthesis and cell proliferation, making it a critical regulator of cellular growth and metabolism .

Phosphorylation at S434 occurs within a C-terminal regulatory domain and represents one of several key phosphorylation sites that collectively regulate kinase activation. This site is part of a proline-directed motif (IRSPR) recognized by specific kinases including JNK1 . Phosphorylation at S434 differs functionally from the mTOR-mediated phosphorylation at T389, which occurs in the linker region between catalytic domains.

Table 1: Key phosphorylation sites of RPS6KB1 and their functions

How does Phospho-RPS6KB1 (S434) Antibody compare to antibodies targeting other phosphorylation sites?

Phospho-RPS6KB1 (S434) antibody specifically recognizes RPS6KB1 when phosphorylated at the serine 434 residue and does not recognize non-phosphorylated peptides . This specificity provides important advantages for studying this particular activation state of RPS6KB1.

While antibodies targeting T389 phosphorylation detect the primary mTORC1-dependent activation site, the S434 antibody detects phosphorylation events that may occur independently or downstream of mTOR signaling. In experimental contexts, T389 phosphorylation often serves as an indicator of canonical mTOR pathway activation, whereas S434 phosphorylation may reflect inputs from alternative signaling pathways, particularly JNK-mediated stress responses .

Research findings demonstrated that in Alzheimer's disease, regression analyses revealed a significant dependence of total tau and PHF-tau on p70 S6 kinase phosphorylated at T421/S424 rather than at T389 , highlighting the importance of monitoring different phosphorylation sites to understand disease mechanisms.

What are the optimal conditions for Western blot detection using Phospho-RPS6KB1 (S434) Antibody?

For Western blot applications, the following protocol parameters have been validated:

• Sample preparation:

  • Lyse cells in buffer containing phosphatase inhibitors (1 mM sodium orthovanadate, 20 mM glycerophosphate)

  • Use fresh samples when possible, or store at -80°C with minimal freeze-thaw cycles

• Antibody dilution range: 0.1-1 μg/mL (typically 1:500-1:2000)

• Expected band sizes:

  • Primary band at approximately 70 kDa

  • Secondary band at approximately 85 kDa (p85 S6K isoform)

• Recommended controls:

  • Positive control: Lysates from cells treated with growth factors (IGF-1, 100 ng/mL for 20 minutes)

  • Negative control: Lysates from cells treated with rapamycin (20 ng/mL for 1 hour)

Research data shows that in MCF-7 cells treated with IGF-1, phosphorylation at T421/S424 generates distinct bands at 70 kDa and 85 kDa when probed with phospho-specific antibodies . Similar phosphorylation patterns at S434 would be expected following growth factor stimulation.

How should samples be prepared for immunohistochemistry with Phospho-RPS6KB1 (S434) Antibody?

For optimal immunohistochemical detection, follow these validated procedures:

• Tissue fixation and processing:

  • Fix tissues in 10% neutral buffered formalin

  • Paraffin-embed and section at 4-6 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Pressure cooker method (125°C, 30 seconds) yields superior results for phospho-epitopes

• Antibody dilution range: 1:100-1:300

• Detection system:

  • Use polymer-based secondary detection systems for enhanced sensitivity

  • DAB (3,3'-diaminobenzidine) as chromogen with hematoxylin counterstain

• Controls:

  • Include serial sections with primary antibody omitted

  • Include blocking peptide competition (pre-incubation of antibody with immunizing peptide)

Research findings from analysis of human breast carcinoma tissue showed specific nuclear and cytoplasmic staining with phospho-S6K antibodies at 1:50 dilution, with signal abolished by blocking peptide pre-incubation .

What are the recommended procedures for validating antibody specificity in experimental systems?

Multiple approaches should be employed to confirm antibody specificity:

• Peptide competition assay:

  • Pre-incubate antibody with excess phosphorylated peptide (sequence: IRSPR with phosphorylated S434)

  • Include non-phosphorylated peptide control

  • Expected result: Signal abolishment with phospho-peptide but not with non-phospho-peptide

• Phosphatase treatment:

  • Treat duplicate samples with lambda phosphatase (400 U/μL, 30 minutes at 30°C)

  • Expected result: Loss of signal in phosphatase-treated samples

• Kinase assay with recombinant protein:

  • Perform in vitro kinase assays with purified GST-S6K protein

  • Reaction conditions: 37°C for 30 minutes in kinase buffer (20 mM HEPES, pH 7.6, 20 mM MgCl₂, 20 mM glycerophosphate, 1 mM DTT, 10 μM ATP)

  • Compare signal with untreated recombinant protein

• Site-directed mutagenesis:

  • Generate S434A mutant (serine to alanine) by point mutation

  • Express in cellular systems along with wild-type control

  • Expected result: Loss of signal in S434A mutant samples

Research demonstrates that mutation analysis effectively validates phosphorylation sites, as shown in studies where S/T to A mutations blocked JNK1-mediated phosphorylation of S6K .

How is RPS6KB1 phosphorylation at S434 regulated in response to cellular stress?

RPS6KB1 phosphorylation at S434 appears to be regulated by stress-activated protein kinases, particularly JNK1. Research has identified several key regulatory mechanisms:

• JNK1-mediated phosphorylation:

  • JNK1 directly phosphorylates S434 in response to cellular stress signals

  • Non-radioactive kinase assays demonstrated JNK1-specific phosphorylation of S6K at S424, with minimal phosphorylation by IKK2 or p38

• Relationship to protein stability:

  • Phosphorylation at S434 affects RPS6KB1 protein stability

  • In the absence of IKK2 activation, JNK1-mediated phosphorylation makes S6K protein unstable

• Cross-talk with the mTOR pathway:

  • While mTOR primarily regulates T389 phosphorylation, cellular stress can modulate S434 phosphorylation independent of mTOR

  • Rapamycin treatment can indirectly affect S434 phosphorylation through feedback mechanisms

Research findings with SH-SY5Y neuroblastoma cells showed that zinc treatment (100 μmol/L) induced rapid phosphorylation of p70 S6 kinase at T421/S424 within 5 minutes, with maximum activation at 30 minutes . Similar stress-induced phosphorylation patterns may occur at S434.

What is the role of RPS6KB1 phosphorylation in neurodegenerative diseases?

Research has established significant correlations between RPS6KB1 phosphorylation and neurodegenerative pathology, particularly in Alzheimer's disease:

• Altered phosphorylation in Alzheimer's brain tissue:

  • Phosphorylated p70 S6 kinase (both T389 and T421/S424) significantly increased in AD brain compared to control cases

  • Levels of phosphorylated S6K showed strong correlation with total tau and PHF-tau levels

• Relationship to tau pathology:

  • Regression analyses revealed significant dependence of total tau and PHF-tau on p70 S6 kinase phosphorylated at T421/S424

  • Zinc-induced S6K phosphorylation in neuronal models resulted in increased tau expression and phosphorylation

• Mechanistic pathway:

  • Activated p70 S6K appears to mediate upregulation of tau translation

  • Rapamycin pretreatment attenuated zinc-induced increases in phosphorylated p70 S6K and tau levels

Table 2: S6K and tau protein levels in AD vs. control brain tissue (derived from ELISA data)

How does phosphorylation at S434 interact with other post-translational modifications of RPS6KB1?

The functional regulation of RPS6KB1 involves a complex interplay between multiple phosphorylation sites and other post-translational modifications:

• Sequential phosphorylation model:

  • Initial phosphorylation at the C-terminal regulatory domain (including S434) relieves autoinhibition

  • This allows subsequent phosphorylation at T389 by mTORC1, resulting in full activation

  • Research shows JNK1 primarily targets the C-terminal region containing S434 and other S/T-P sites

• Site-specific effects:

  • T389 phosphorylation (mTOR target) was not affected by JNK1, IKK2, or p38 kinases

  • S424 showed strong phosphorylation by JNK1 and minimal phosphorylation by IKK2 or p38

  • S434 phosphorylation likely follows similar kinase specificity patterns

• Functional outcomes of multi-site phosphorylation:

  • Phosphorylation at different sites creates distinct functional states of RPS6KB1

  • In neuronal models, T421/S424 phosphorylation correlated more strongly with tau pathology than T389 phosphorylation

  • Mutation studies where "five serine/threonine residues in the c-terminal were all replaced by alanine" blocked S6K phosphorylation by JNK1

Research with primary cultured cortical neurons demonstrated that zinc treatment (200 μmol/L, 30 minutes) significantly increased levels of phosphorylated p70 S6K at T421/S424, which was attenuated by rapamycin pretreatment , suggesting complex cross-talk between different phosphorylation pathways.

What are common issues when detecting phosphorylated RPS6KB1 and how can they be resolved?

Researchers frequently encounter several challenges when working with phospho-specific antibodies against RPS6KB1:

• Low signal strength:

  • Cause: Rapid dephosphorylation during sample preparation

  • Solution: Use fresh phosphatase inhibitor cocktail (1 mM sodium orthovanadate, 20 mM glycerophosphate) in all buffers

  • Solution: Process samples rapidly at 4°C and snap-freeze aliquots

• High background:

  • Cause: Non-specific antibody binding

  • Solution: Optimize blocking conditions (5% BSA is preferred over milk for phospho-epitopes)

  • Solution: Titrate antibody concentration (start with 1:500 for Western blot)

• Inconsistent band pattern:

  • Cause: Multiple isoforms or partial degradation

  • Solution: Clarify lysates by high-speed centrifugation (14,000×g, 15 minutes)

  • Solution: Use freshly prepared samples and avoid freeze-thaw cycles

• Weak phosphorylation signal:

  • Cause: Inefficient activation of signaling pathways

  • Solution: Include positive controls (cells treated with 100 ng/mL IGF-1 for 20 minutes)

  • Solution: Ensure cells are in appropriate growth conditions before stimulation

How should researchers interpret differences between phosphorylation patterns at S434 versus other sites?

Interpreting differential phosphorylation patterns requires careful consideration of several factors:

• Kinase specificity:

  • T389 phosphorylation primarily reflects mTORC1 activity

  • S434 phosphorylation may indicate JNK pathway activation

  • Discrepancies between sites suggest differential activation of upstream kinases

• Temporal dynamics:

  • Research shows T421/S424 phosphorylation occurred rapidly (5 minutes) after zinc treatment

  • Maximum phosphorylation was observed at 30 minutes, followed by a decrease

  • Compare these patterns with S434 phosphorylation kinetics to assess temporal relationships

• Functional implications:

  • Phosphorylation at C-terminal sites (including S434) primarily relieves autoinhibition

  • T389 phosphorylation directly correlates with kinase activity

  • Mutations in C-terminal sites (S to A) blocked JNK1-mediated phosphorylation

• Pathway-specific responses:

  • Rapamycin sensitivity indicates mTOR dependence

  • Research showed rapamycin (20 ng/mL, 1 hour pretreatment) inhibited zinc-induced phosphorylation of p70 S6K

  • Similar experiments can determine if S434 phosphorylation is rapamycin-sensitive

What strategies can be employed to investigate the functional significance of S434 phosphorylation?

To elucidate the specific role of S434 phosphorylation, several advanced experimental approaches are recommended:

• Site-directed mutagenesis:

  • Generate phospho-mimetic (S434D/E) and phospho-deficient (S434A) mutants

  • Express these constructs in cellular models

  • Compare functional outcomes (protein synthesis rates, cell growth, substrate phosphorylation)

  • Research demonstrates mutation approach effectively blocks phosphorylation at specific sites

• Phosphorylation-specific interactome analysis:

  • Perform co-immunoprecipitation with Phospho-RPS6KB1 (S434) Antibody

  • Identify binding partners specific to the phosphorylated state using mass spectrometry

  • Recommended conditions: 2-5 μg/mL antibody for immunoprecipitation

• Kinase inhibitor profiling:

  • Treat cells with panel of kinase inhibitors (JNK inhibitor SP600125, p38 inhibitor SB203580, mTOR inhibitor rapamycin)

  • Monitor effects on S434 phosphorylation compared to other sites

  • Research showed rapamycin pretreatment attenuated zinc-induced p70 S6K phosphorylation

• Correlation with functional readouts:

  • Monitor ribosomal protein S6 phosphorylation

  • Assess protein synthesis rates using puromycin incorporation

  • Measure cell growth parameters

  • Research found significant correlation between p70 S6K phosphorylation and tau levels in AD brain

How is RPS6KB1 phosphorylation at S434 implicated in cancer biology?

Emerging research suggests important roles for RPS6KB1 phosphorylation in cancer development and progression:

• Altered expression in cancer:

  • "Amplification of the region of DNA encoding this gene and overexpression of this kinase are seen in some breast cancer cell lines"

  • The phosphorylation status at S434 may serve as a biomarker for cancer progression

• Growth factor signaling:

  • IGF-1 (100 ng/mL, 20 minutes) significantly increases p70 S6K phosphorylation in MCF-7 breast cancer cells

  • PDGF induces similar phosphorylation patterns in NIH-3T3 cells

  • These growth factors likely affect S434 phosphorylation via JNK activation

• Integration with stress response pathways:

  • JNK1-mediated phosphorylation of S434 may integrate stress signals with growth regulation

  • This could represent a vulnerability in cancer cells that can be therapeutically targeted

What novel techniques are advancing the study of RPS6KB1 phosphorylation dynamics?

Recent technological advances are enhancing our ability to study phosphorylation dynamics:

• Simple Western™ technology:

  • Capillary-based immunoassay system shows improved quantitation of phospho-proteins

  • Research demonstrated detection of phospho-p70 S6K in MCF-7 cells at 66 kDa following IGF-1 treatment

• Phospho-specific mass spectrometry:

  • Enrichment of phosphopeptides prior to MS/MS analysis

  • Allows site-specific quantitation of phosphorylation stoichiometry

  • Can detect multiple phosphorylation sites simultaneously

• Genetically encoded biosensors:

  • FRET-based sensors for real-time monitoring of RPS6KB1 phosphorylation

  • Enables single-cell analysis of phosphorylation dynamics

  • Permits spatial resolution of phosphorylation events