RPS6KB1 Human
Description
RPS6KB1 produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 533 amino acids (1-525a.a.) and having a molecular mass of 60.2kDa. (Molecular size on SDS-PAGE will appear at approximately 70-100kDa).
RPS6KB1 is expressed with a 8 amino acid His tag at C-Terminus and purified by proprietary chromatographic techniques.
Product Specs
Ribosomal Protein S6 Kinase Beta-1 Isoform A, also known as RPS6KB1, is a serine/threonine kinase. This protein plays a role in the PI3 kinase pathway, specifically interacting with PIP3 and phosphoinositide-dependent kinase-1. RPS6KB1 is involved in protein synthesis by phosphorylating ribosomal protein S6. Historically, phosphorylation of p70S6K at threonine 389 has been linked to mTOR activation and autophagy inhibition. However, recent research suggests that p70S6K activity may actually promote autophagy.
Produced using Sf9 insect cells, our RPS6KB1 is a single, glycosylated polypeptide chain that contains 533 amino acids (specifically amino acids 1 through 525). This protein has a molecular weight of 60.2 kDa. Note: On SDS-PAGE, the molecular size will appear between 70-100 kDa. This RPS6KB1 protein features an 8 amino acid His tag located at the C-terminus. Purification is achieved through proprietary chromatographic techniques.
This 0.25mg/ml solution of RPS6KB1 protein is prepared with the following components: 50mM Tris-HCl (pH 7.5), 30% glycerol, 0.5M NaCl, 2mM DTT, and 0.1mM PMSF.
For long-term storage, adding a carrier protein such as 0.1% HSA or BSA is advised.
Repeated freezing and thawing should be avoided.
Analysis by SDS-PAGE confirms that the purity of this product exceeds 85%.
Ribosomal protein S6 kinase beta-1 isoform a, p70 S6KA, p70(S6K)-alpha, p70-alpha, p70-S6K, PS6K, S6K, S6K-beta-1, S6K1, STK14A, 70 kDa ribosomal protein S6 kinase 1, p70-S6K 1, Ribosomal protein S6 kinase I, Serine/threonine-protein kinase 14A, p70 ribosomal S6 kinase alpha, p70 S6 kinase alpha, p70 S6K-alpha, p70 S6KA.
Sf9, Baculovirus cells.
MRRRRRRDGF YPAPDFRDRE AEDMAGVFDI DLDQPEDAGS EDELEEGGQL NESMDHGGVG PYELGMEHCE KFEISETSVN RGPEKIRPEC FELLRVLGKG GYGKVFQVRK VTGANTGKIF AMKVLKKAMI VRNAKDTAHT KAERNILEEV KHPFIVDLIY AFQTGGKLYL ILEYLSGGEL
FMQLEREGIF MEDTACFYLA EISMALGHLH QKGIIYRDLK PENIMLNHQG HVKLTDFGLC KESIHDGTVT HTFCGTIEYM APEILMRSGH NRAVDWWSLG ALMYDMLTGA PPFTGENRKK TIDKILKCKL NLPPYLTQEA RDLLKKLLKR NAASRLGAGP GDAGEVQAHP FFRHINWEEL
LARKVEPPFK PLLQSEEDVS QFDSKFTRQT PVDSPDDSTL SESANQVFLG FTYVAPSVLE SVKEKFSFEP KIRSPRRFIG SPRTPVSPVK FSPGDFWGRG ASASTANPQT PVEYPMETSG IEQMDVTMSG EASAPLPIRQ PNSGPYKKQA FPMISKRPEH LRMNLLEHHH HHH.
Q&A
What are the primary molecular functions of RPS6KB1 in human cells?
Methodological Framework:
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Kinase Activity Profiling: Use phosphorylation-specific antibodies (e.g., anti-p-RPS6KB1 Thr389) in western blotting to quantify activation status under nutrient-rich vs. deprived conditions .
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Substrate Identification: Combine siRNA-mediated RPS6KB1 knockdown with phosphoproteomic screens to map downstream targets like ribosomal protein S6 (RPS6), EIF4B, and BAD .
Key Findings:
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RPS6KB1 phosphorylates RPS6 to regulate ribosome biogenesis and protein synthesis .
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Inactive RPS6KB1 associates with the EIF3 complex under nutrient depletion, dissociating upon mTORC1 activation .
How can researchers reliably detect RPS6KB1 expression and activity in tissue samples?
Methodological Framework:
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Immunohistochemistry (IHC): Use validated antibodies against total RPS6KB1 (Cell Signaling Technology #9202) and phosphorylated RPS6KB1 (Thr389, #9205) on formalin-fixed paraffin-embedded (FFPE) tissues. Include antigen retrieval with citrate buffer (pH 6.0) .
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Quantitative Scoring: Apply the H-score system (0–300) incorporating staining intensity (0–3) and percentage of positive cells .
Data Table: Association of p-RPS6KB1 with NSCLC Clinicopathology
| Clinicopathologic Feature | p-RPS6KB1+ (%) | p-value |
|---|---|---|
| Lymph Node Metastasis | 78.9 | <0.001 |
| High TNM Stage (III–IV) | 82.4 | 0.003 |
| 5-Year Survival Rate | 32.1 vs. 68.9* | 0.008 |
| *Compared to p-RPS6KB1- group |
How do conflicting reports on RPS6KB1’s role in cancer progression arise, and how can they be reconciled?
Data Contradiction Analysis:
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Prostate Cancer: miR-195 downregulation elevates RPS6KB1, promoting metastasis via MMP-9/VEGF upregulation .
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NSCLC: Total RPS6KB1 overexpression lacks prognostic value, whereas hyperphosphorylation (p-RPS6KB1) correlates with poor survival .
Resolution Strategies:
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Context-Specific Activation: Assess phosphorylation status alongside total protein levels using multiplex assays (e.g., Luminex xMAP).
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Pathway Crosstalk: Profile upstream regulators (e.g., mTORC1 activity) and parallel pathways (ERK1/2) in patient-derived xenografts .
What experimental models best capture the functional consequences of RPS6KB1 variants in cardiomyopathy?
Methodological Framework:
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In Vitro Models: Transfect HEK293 cells with mutant RPS6KB1 (e.g., p.G47W, p.Q49K) and quantify ERK1/2 phosphorylation via ELISA .
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In Vivo Validation: Generate cardiomyocyte-specific RPS6KB1 knock-in mice using CRISPR-Cas9 to monitor hypertrophy via echocardiography .
Key Findings:
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RPS6KB1 p.G47W increases S6K1 activity by 2.8-fold and ERK1/2 phosphorylation by 1.9-fold compared to wild type .
How does RPS6KB1 phosphorylation influence therapeutic resistance in mTOR inhibitor-treated cancers?
Experimental Design:
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Longitudinal Cohorts: Treat NSCLC PDX models with mTOR inhibitors (e.g., rapamycin) and monitor p-RPS6KB1 rebound using serial IHC .
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Combinatorial Targeting: Co-administer LY2584702 (p-RPS6KB1 inhibitor) with cisplatin to assess synergy in apoptosis assays (Annexin V/PI flow cytometry) .
Data Table: LY2584702 Effects on NSCLC Cell Lines
| Cell Line | Proliferation (IC50, nM) | G0-G1 Arrest (%) | Apoptosis Rate (%) |
|---|---|---|---|
| A549 | 12.3 ± 1.2 | 58.4 ± 3.1 | 24.7 ± 2.5 |
| SK-MES-1 | 18.9 ± 2.1 | 42.6 ± 2.8 | 18.3 ± 1.9 |
Why do RPS6KB1 knockdown studies yield variable phenotypic outcomes across cancer types?
Troubleshooting Guide:
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Off-Target Effects: Validate siRNA efficiency (>80% knockdown) via qRT-PCR (PrimeTime Hs01055559_m1 assay) and rescue experiments with siRNA-resistant RPS6KB1 .
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Cell Line Heterogeneity: Use low-passage patient-derived organoids instead of immortalized lines to preserve native signaling contexts .
How can researchers differentiate RPS6KB1-driven signaling from parallel mTORC1 effectors?
Advanced Methodologies:
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Kinase-Dead Mutants: Introduce T389A mutations to disrupt RPS6KB1 activity without affecting mTORC1 complex formation .
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Spatial Proteomics: Apply multiplexed ion beam imaging (MIBI) to quantify subcellular RPS6KB1 localization relative to RPTOR/MTOR complexes .
What role does RPS6KB1 play in non-canonical tissues like brown adipose tissue (BAT)?
Preliminary Data:
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ODT imaging reveals RPS6KB1 modulates BAT differentiation under high-fructose conditions, increasing lipid droplet volume by 37% compared to glucose-only media .
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Unresolved Question: Does RPS6KB1 coordinate BAT thermogenesis via UCP1 phosphorylation?
Are RPS6KB1 variants associated with non-cardiac metabolic syndromes?
Research Gap Analysis:
Product Science Overview
Ribosomal Protein S6 Kinase B1 (RPS6KB1), also known as p70S6K, is a serine/threonine kinase that plays a crucial role in the regulation of cell growth, cell cycle progression, and protein synthesis. It is a member of the AGC (protein kinase A, G, and C) family of protein kinases and is regulated by the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway .
RPS6KB1 was first identified in the late 1980s in the laboratory of George Thomas. It was detected in extracts of quiescent 3T3 cells that were stimulated to proliferate with epidermal growth factor or serum . The kinase is named for its ability to phosphorylate the ribosomal protein S6, which is a component of the 40S ribosomal subunit .
RPS6KB1 is activated by the PI3K/mTOR signaling pathway, which is a central regulator of cell survival, proliferation, and metabolism . Upon activation, RPS6KB1 phosphorylates the S6 protein of the ribosomal subunit 40S, leading to the selective translation of a unique family of mRNAs that contain an oligopyrimidine tract at the 5’ transcriptional site (5′TOP). These mRNAs encode components of the translational apparatus, including ribosomal proteins and elongation factors .
Due to its role in protein synthesis and cell growth, RPS6KB1 is involved in various human diseases, including cancer, diabetes, and obesity . It is considered a potential therapeutic target for drug development, particularly in the context of cancer treatment. Inhibitors of the mTOR pathway, which regulate RPS6KB1, are being explored for their anticancer and immunosuppressant effects .
Recent studies have suggested that plasma levels of RPS6KB1 can serve as a reliable biomarker for monitoring patient response to mTOR inhibitors . This has significant implications for the development of strategies to aid in the diagnosis, prognosis, and treatment of various pathological conditions.
