Phospho-RPS6KB1 (S371) Antibody
Product Specs
Target Background
RPS6KB1 (Ribosomal Protein S6 Kinase Beta-1), also known as p70S6 kinase, is a serine/threonine-specific protein kinase that functions downstream of mTOR signaling. It is activated in response to growth factors and nutrients, promoting cell proliferation, growth, and cell cycle progression. Its mechanisms of action include:
- Regulation of protein synthesis: RPS6KB1 phosphorylates key translation factors such as EIF4B, RPS6, and EEF2K, influencing protein synthesis rates.
- Cell survival: It represses the pro-apoptotic function of BAD, contributing to cell survival.
- Translation initiation control: Under nutrient depletion, inactive RPS6KB1 associates with the EIF3 complex. Mitogenic stimulation and subsequent mTORC1-mediated phosphorylation lead to dissociation from EIF3 and activation, influencing EIF2B and EIF4B.
- Regulation of mTORC2: RPS6KB1 participates in a feedback loop, phosphorylating RICTOR to inhibit mTORC2 and AKT1 signaling.
- Mitochondrial function: Phosphorylation of mitochondrial URI1 leads to the dissociation of a URI1-PPP1CC complex, potentially impacting RPS6KB1's anti-apoptotic function via PPP1CC-mediated dephosphorylation of RPS6KB1 at Thr-412.
- Insulin resistance: RPS6KB1 mediates TNF-alpha-induced insulin resistance by phosphorylating IRS1, causing its degradation.
- GSK3B regulation: In cells lacking a functional TSC1-2 complex, RPS6KB1 constitutively phosphorylates and inhibits GSK3B.
- Cytoskeletal rearrangement: RPS6KB1 may bind to neurabin, potentially influencing cytoskeletal dynamics.
- Pyrimidine biosynthesis: It phosphorylates and activates CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a key enzyme in pyrimidine biosynthesis.
- Fatty acid uptake: Following mTORC1 activation, RPS6KB1 phosphorylates EPRS, playing a crucial role in adipocyte fatty acid uptake and interferon-gamma-induced translation inhibition.
The role of RPS6KB1 in various biological processes and disease states is supported by extensive research:
- miR-96, miR-557, and miR-3182 target mTOR and S6K1 in triple-negative breast cancer. (PMID: 29862445)
- p70S6K activation promotes myofibroblast transdifferentiation in pterygium fibroblasts. (PMID: 29270715)
- Akt and p70S6K signaling are highly activated in ER-negative premalignant breast lesions and breast cancer. (PMID: 28877935)
- ADAR1 contributes to gastric cancer through the mTOR/p70S6K/S6 ribosomal protein signaling axis. (PMID: 27863387)
- PICT-1 triggers pro-death autophagy by inhibiting rRNA transcription and the AKT/mTOR/p70S6K pathway in glioblastoma. (PMID: 27729611)
- miR-145 targets p70S6K1, inhibiting its expression and influencing gemcitabine chemoresistance in pancreatic adenocarcinoma. (PMID: 27765914)
- Fenofibrate inhibits PC-3 cell proliferation via apoptosis, associated with inactivation of the mTOR/p70S6K pathway. (PMID: 29305864)
- mTORC1 activation of S6K1 and MYC activity co-regulate rDNA transcription. (PMID: 27385002)
- PYK2 modulates AR function and prostate cancer cell growth via S6K1 activation. (PMID: 27492635)
- Inhibitors of Erk1/2 or S6K1 may prevent BAFF-induced aggressive B-cell malignancies. (PMID: 27235588)
- AIM2 overexpression suppresses the mTOR-S6K1 pathway and inhibits hepatocellular carcinoma cell proliferation. (PMID: 27167192)
- p70S6K and IL-6 are upregulated in high-metastatic head and neck squamous cell carcinoma cells undergoing EMT. (PMID: 27174914)
- S6K plays a critical role in dopaminergic neuronal differentiation in human neural stem cells. (PMID: 26143260)
- Elevated p-Mnk1, p-eIF4E, and p-p70S6K are associated with tumor recurrence and poor prognosis in astrocytomas. (PMID: 27900644)
- ULK1 is involved in RPS6KB1-NCOR1 repression of NR1H/LXR-mediated Scd1 transcription and augments lipotoxicity. (PMID: 27846372)
- RPS6KB1 function is mimicked by the Kaposi's sarcoma-associated herpesvirus ORF36 kinase. (PMID: 27342859)
- p-RPS6KB1 is over-activated in non-small cell lung cancer, potentially serving as a prognostic marker. (PMID: 28792981)
- p54-S6K2 is predominantly nuclear, while p70-S6K1 is predominantly cytosolic. (PMID: 27493124)
- S6K1 regulates mitochondrial morphology and function in HeLa cells. (PMID: 27634387)
- S6K1 promotes self-renewal and leukemia progression through multiple mTOR pathway targets. (PMID: 27294524)
- S6K1 is a potential tumor-specific target for enhancing NSCLC radiosensitivity, possibly via increased PDCD4 expression. (PMID: 28276898)
- Spheroids exhibit lower AKT, mTOR, and S6K signaling activity compared to 2D cultures. (PMID: 27663511)
- S6K1 phosphorylation of H2B mediates EZH2 trimethylation of H3 in adipogenesis, promoting obesity. (PMID: 27151441)
- Golgi growth is modulated by the G1 cell growth checkpoint through S6K1 activity. (PMID: 27325676)
- S6K1 activation in an adjuvant trastuzumab setting may predict patient response to trastuzumab in breast cancer. (PMID: 27993682)
- YAP1 may predict sensitivity to dual AKT/P70S6K inhibitors. (PMID: 28376174)
- RPS6KB1 SNPs are associated with colorectal cancer patient survival. (PMID: 28138309)
- S6K1-mediated PIPKIgamma90 phosphorylation regulates cell migration and invasion by controlling PIPKIgamma90 degradation. (PMID: 27780861)
- Notch3 and pS6 are associated with ovarian epithelial cancer development and prognosis. (PMID: 27445438)
- FXR suppresses human liver cancer cell proliferation by inhibiting the mTOR/S6K signaling pathway. (PMID: 27109477)
- p-p70S6K is involved in ESCC invasion and metastasis; its downregulation improves rapamycin sensitivity. (PMID: 27595116)
- RPS6KB1 SNPs are associated with multiple sclerosis susceptibility. (PMID: 28079472)
- S6K1 Iso-2 overexpression promotes cancer cell growth and inhibits apoptosis in NSCLC. (PMID: 27460085)
- S6K phosphorylation via the PI3K-PD1 pathway is involved in tau and actin pathology in neurodegenerative diseases. (PMID: 26582459)
- Rapamycin's inhibitory effect may be due to increased p14, p15, and p57 expression and decreased mTOR and p70S6K expression in ALL cell lines. (PMID: 26362858)
- The miR-195-RPS6KB1 axis is involved in prostate cancer progression. (PMID: 26080838)
- eIF3 controls cell size independently of S6K1 activity. (PMID: 26172298)
- miR-497 decreases cisplatin resistance in ovarian cancer cells by targeting mTOR/P70S6K1. (PMID: 26238185)
- Increased p70 S6 kinase levels are observed in Progressive Supranuclear Palsy and Corticobasal Degeneration brains. (PMID: 26818518)
- Th17 cell differentiation is positively regulated by p70(S6K1). (PMID: 26514620)
- AT1R silencing inhibits HG-induced EMT in HK-2 cells via inactivation of the mTOR/p70S6K pathway. (PMID: 26626074)
- Blocking both mTOR kinase downstream targets 4E-BP1 and p70 S6 kinase 1 prevents RPE cell migration. (PMID: 26427479)
- Microcystin-LR promotes HL7702 cell proliferation via Akt/S6K1 activation. (PMID: 26506538)
- Oleic acid prevents ER stress, lipoapoptosis, and insulin resistance by inhibiting palmitic acid-induced S6K1 activation. (PMID: 25846498)
- S6K1 may link aging and Alzheimer's disease. (PMID: 26468204)
- Increased S6K1 levels are associated with obesity, insulin resistance, and inflammation. (PMID: 25118997)
- mTORC1 regulates cell adhesion through S6K1 and 4E-BP1, while mTORC2 uses an Akt-independent mechanism. (PMID: 25762619)
- pS6 expression is associated with a high Ki-67 subset in ER+ and HER2- breast cancer. (PMID: 25600244)
- Leucine stimulates mTORC1 signaling and S6K1 phosphorylation. (PMID: 26169935)
- Inactivated Sendai virus induces apoptosis and autophagy via the PI3K/Akt/mTOR/p70S6K pathway in NSCLC cells. (PMID: 26235873)
Q&A
Basic Research Questions
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What is Phospho-RPS6KB1 (S371) and why is it important in cellular signaling?
RPS6KB1 (p70S6K) is a serine/threonine-protein kinase that functions downstream of mTOR signaling in response to growth factors and nutrients. It plays crucial roles in promoting cell proliferation, growth, and cell cycle progression by regulating protein synthesis through phosphorylation of substrates including EIF4B, RPS6, and EEF2K .
The S371 phosphorylation site is particularly important as it is part of the complex regulatory mechanism of RPS6KB1 activity. GSK3 can directly phosphorylate S6K1 at S371 (the turn motif), which is antagonized by phosphatase 2A (PP2A) . This phosphorylation contributes to the stabilization of the kinase domain and is required for full activation of RPS6KB1, working in concert with other phosphorylation sites.
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How does phosphorylation at S371 differ functionally from other RPS6KB1 phosphorylation sites?
RPS6KB1 contains multiple phosphorylation sites that function in concert to regulate its activity:
While Thr389 is considered the primary mTORC1-dependent activation site, S371 phosphorylation appears to be part of a distinct regulatory mechanism that contributes to proper kinase folding and function. Unlike other sites, S371 phosphorylation remains relatively stable under conditions where Thr389 phosphorylation may be dynamic .
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What are the recommended experimental conditions for using Phospho-RPS6KB1 (S371) Antibody in Western blotting?
For optimal Western blot results with Phospho-RPS6KB1 (S371) Antibody:
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Sample preparation: Include phosphatase inhibitors in lysis buffer
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Protein amount: Load 20-40 μg of total protein per lane
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Gel percentage: 8-10% SDS-PAGE for optimal separation
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Transfer conditions: Wet transfer at 30V overnight at 4°C for high molecular weight proteins
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Blocking solution: 5% BSA in TBST (not milk, which contains phosphatases)
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Antibody incubation: Overnight at 4°C in 5% BSA/TBST
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Detection method: ECL with exposure times optimized for signal-to-noise ratio
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Storage: Store antibody at -20°C and avoid freeze-thaw cycles
Always run positive controls such as lysates from insulin or IGF-1 stimulated cells, which activate the mTOR pathway .
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What controls should be included when using Phospho-RPS6KB1 (S371) Antibody?
A robust experimental design should include these controls:
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Positive control: Lysates from cells treated with IGF-1, insulin, or serum after starvation to induce phosphorylation
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Negative control:
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Lysates from cells treated with mTOR inhibitors (rapamycin, Torin)
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Samples treated with lambda phosphatase to remove phosphorylation
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Antibody validation controls:
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Loading controls:
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Total RPS6KB1 antibody on parallel blots
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Housekeeping protein (β-actin, GAPDH)
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Specificity control:
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