Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: RPS6KA2's role in ovarian cancer initiation, progression, prognosis, cell proliferation, and apoptosis. ( A ) Comparison of RPS6KA2 expression levels between ovarian tumor tissues (n = 426) and normal tissues (n = 88) using the GEPIA database; ( B ) immunohistochemical staining showing RPS6KA2 expression in ovarian tumor tissues (n = 3) and adjacent normal tissues (n = 3); ( C ) expression of RPS6KA2 expression across early (passages 5–20), intermediate (passages 60–80), and late (passages 120–180) passage mouse ovarian surface epithelial cells (MOSE); ( D ) t-SNE plot depicting single-cell clustering, with distinct colors representing different cell populations; ( E ) t-SNE map illustrating the spatial distribution of RPS6KA2 expression levels across individual cells, where color intensity reflects expression magnitude; ( F ) bar graph summarizing RPS6KA2 expression abundance across different cell types; ( G ) survival analysis based on the Kaplan-Meier Plotter database, evaluating the relationship of RPS6KA2 expression with progression-free survival (PFS) and overall survival (OS). Correlation analysis between RPS6KA2 mRNA levels and tumor proliferation ( H ) or apoptosis ( I ) pathway scores, with the x -axis representing RPS6KA2 expression distribution and the y -axis indicating pathway activity scores. * p < 0.05; NS, not significant ( p > 0.05)

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Comparison, Expressing, Immunohistochemical staining, Staining, Activity Assay

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: The association between RPS6KA2 and cisplatin resistance, along with the identification of its downstream target genes. ( A ) RPS6KA2 mRNA expression levels were compared between cisplatin-resistant (A2780CP and COC1/DDP) and cisplatin-sensitive cell lines (A2780 and COC1); ( B ) RPS6KA2 mRNA expression was evaluated in ovarian cancer tissues from cisplatin-resistant (n = 9) and cisplatin-sensitive (n = 9) patients; ( C ) Wilcoxon rank-sum test was used to assess the correlation between cisplatin IC 50 values and RPS6KA2 expression levels. The x -axis represents different sample groups, and the y -axis shows the distribution of IC 50 scores; ( D ) a CCK-8 assay was performed to evaluate cell viability following treatment with increasing concentrations of cisplatin, after either silencing RPS6KA2 in A2780 cells or overexpressing it in A2780CP cells; ( E ) protein–protein interaction networks involving RPS6KA2 were analyzed using the STRING database; ( F ) the correlation between PI3K-AKT-mTOR pathway activity scores and RPS6KA2 expression levels was examined. The x -axis displays the distribution of RPS6KA2 expression, and the y -axis reflects the distribution of pathway activity score; ( G ) among 188 ovarian cancer patients, stratification into low (n = 92) and high (n = 96) RPS6KA2 expression groups was conducted. Each dot represents an individual gene, with colors indicating whether predefined filtering criteria were met; ( H ) a heatmap was constructed to visualize the expression patterns of differentially expressed genes, with sample groups ordered from the outermost to the innermost layer; ( I ) KEGG pathway enrichment analysis was conducted, where color intensity indicates the level of statistical significance and circle size corresponds to the number of enriched genes (larger circles indicate more enriched genes). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Expressing, CCK-8 Assay, Activity Assay, Construct

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: Involvement of RPS6KA2 in the regulation of autophagy and cisplatin sensitivity in ovarian cancer cells. ( A ) qRT-PCR analysis was conducted to measure RPS6KA2 and autophagy-related proteins in ovarian cancer cell lines following transfection with either an RPS6KA2 overexpression plasmid or sh-RPS6KA2; ( B ) autophagic flux in A2780CP cells was evaluated using mRFP-GFP-LC3 fluorescence assay after transfection with RPS6KA2 plasmid or sh-RPS6KA2. Green signals correspond to autophagosomes, red signals indicate autolysosomes, and yellow signals reflect the early stage of autophagosome formation; ( C ) high-resolution transmission electron microscopy (×10,000 magnification) was utilized to observe ultrastructural changes associated with autophagosome formation in A2780CP cells post-transfection; ( D ) A2780 cells were categorized into five experimental groups: NC, Cisplatin (5 μg/mL), sh-RPS6KA2 + Cisplatin, sh-RPS6KA2 + TSC1 + Cisplatin, and sh-RPS6KA2 + TSC2 + Cisplatin. Cell viability was determined by CCK-8 assay (OD 450 values). ( E ) A2780CP cells were allocated into five groups: NC, Cisplatin (20 μg/mL), RPS6KA2 + Cisplatin, RPS6KA2 + sh-TSC1 + Cisplatin, and RPS6KA2 + sh-TSC2 + Cisplatin. Changes in OD 450 values were measured using the CCK-8 assay. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant ( p > 0.05)

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Quantitative RT-PCR, Transfection, Over Expression, Plasmid Preparation, Fluorescence, Transmission Assay, Electron Microscopy, CCK-8 Assay

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: Involvement of RPS6KA2 in the regulation of ferroptosis in ovarian cancer cells. ( A ) Heatmaps display the expression patterns of ferroptosis-related genes in tumor and normal tissues. The x -axis corresponds to various ferroptosis-related genes, while the y -axis reflects their relative expression levels; ( B ) Western blotting was used to examine alterations in the expression of ferroptosis-related protein expression in A2780CP cells following transfection with either RPS6KA2 overexpression plasmid or sh-RPS6KA2; ( C ) levels of intracellular Fe 2+ levels were detected by fluorescence staining (blue fluorescence marks nuclei; red fluorescence indicates Fe 2+ presence); ( D ) total iron content was quantified in A2780CP cells under different treatment conditions; ( E ) cellular GSH levels were measured as an indicator of antioxidant capacity; ( F ) changes in mitochondrial membrane potential were assessed using fluorescent probes. Green fluorescence indicates high mitochondrial membrane potential (viable cells), while red fluorescence signifies low membrane potential (apoptotic cells); Intracellular ROS accumulation was evaluated using fluorescent probes. With blue fluorescence labeling nuclei, and green fluorescence also reflecting ROS accumulation. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; NS, not significant ( p > 0.05)

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation, Fluorescence, Staining, Membrane, Labeling

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: miR-512-3p enhances cisplatin resistance in ovarian cancer through direct targeting of RPS6KA2. ( A ) A comprehensive bioinformatics analysis was performed to identify upstream miRNAs that regulate RPS6KA2, using four public databases: TargetScan, miRDB, miRWalk, and StarBase databases; ( B ) expression levels of miR-512-3p were analyzed in cisplatin-resistant cell lines (A2780CP and COC1/DDP) vs cisplatin-sensitive counterparts (A2780 and COC1), as well as in clinical samples from cisplatin-resistant (n = 3) and cisplatin-sensitive (n = 3) ovarian cancer tissues; ( C ) cell viability following cisplatin exposure at different concentrations was evaluated using the CCK-8 assay after silencing miR-512-3p in A2780CP cells or overexpressing it in A2780 cells; ( D ) qRT-PCR was employed to determine alterations in RPS6KA2 expression upon transfection with either miR-512-3p mimics or inhibitor; ( E ) relative luciferase activity was measured in A2780 cells co-transfected with miR-512-3p mimics or inhibitor in combination with wild-type (wt) or mutant (mut) RPS6KA2 plasmids; ( F ) A2780 cells were grouped into four conditions: negative control (NC), cisplatin (5 μg/mL), miR-512-3p mimics + cisplatin, and miR-512-3p mimics + sh-RPS6KA2 + cisplatin. Similarly, A2780CP cells were divided into four groups: NC, cisplatin (20 μg/mL), miR-512-3p inhibitor + cisplatin, and miR-512-3p inhibitor + RPS6KA2 + cisplatin. Cell viability was assessed using the CCK-8 assay; ( G ) Apoptosis was examined by TUNEL staining in five experimental groups for each cell line. In A2780 cells: NC, cisplatin (5 μg/mL), sh-RPS6KA2 + cisplatin, miR-512-3p mimics + cisplatin, and miR-512-3p mimics + RPS6KA2 + cisplatin. In A2780CP cells: NC, cisplatin (20 μg/mL), RPS6KA2 + cisplatin, miR-512-3p inhibitor + cisplatin, and miR-512-3p inhibitor + sh-RPS6KA2 + cisplatin. Nuclei were stained blue, and apoptotic cells exhibited green fluorescence. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant ( p > 0.05)

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Expressing, CCK-8 Assay, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Mutagenesis, Negative Control, TUNEL Assay, Staining, Fluorescence

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: The miR-512-3p/RPS6KA2 axis modulates the autophagy signaling pathway. ( A ) A2780 cells were distributed into five experimental groups: negative control (NC), Cisplatin treatment (Cis), sh-RPS6KA2 + Cis, miR-512-3p mimics + Cis, and miR-512-3p mimics + Cis + RPS6KA2; ( B ) similarly, A2780CP cells were also separated into five groups: NC, cisplatin (Cis), RPS6KA2 + Cis, miR-512-3p inhibitor + Cis, and miR-512-3p inhibitor + Cis + sh-RPS6KA2. Cell immunofluorescence staining was performed to evaluate MTOR and RHEB expression levels. Nuclei were labeled in blue, MTOR signal appears in green, and RHEB is shown in red. ** p < 0.01; *** p < 0.001

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Negative Control, Immunofluorescence, Staining, Expressing, Labeling

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: The miR-512-3p/RPS6KA2 axis modulates the autophagy signaling pathway and influences cisplatin resistance in ovarian cancer. ( A ) Subcutaneous xenograft mouse models were allocated into five groups: normal control (NC), Cisplatin treatment (Cis), sh-RPS6KA2 + Cis, miR-512-3p mimics + Cis, and miR-512-3p mimics + Cis + sh-RPS6KA2. Tumor volumes were monitored and compared among groups. Immunohistochemistry analysis was performed to detect the expression levels of ATG5, ATG7, BECN1, and SQSTM1; ( B ) In a parallel experiment, subcutaneous tumor-bearing mice were divided into five groups: NC, Cis, RPS6KA2 + Cis, miR-512-3p inhibitor + Cis, and miR-512-3p inhibitor + Cis + RPS6KA2. Tumor sizes were recorded and compared. Immunohistochemistry was conducted to evaluate the expression of ATG5, ATG7, BECN1, and SQSTM1. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Control, Immunohistochemistry, Expressing

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: Targeting RPS6KA2 improves cisplatin sensitivity in ovarian cancer. A subcutaneous xenograft mouse model was established and animals were randomly assigned to 11 treatment groups receiving various interventions, including an RPS6KA2-targeted drug (Honokiol), a ferroptosis inducer (Erastin), and an autophagy inhibitor (3-MA). Tumor volumes were monitored over time and compared among the groups. Immunohistochemical analysis was also performed to evaluate alterations in the expression of RPS6KA2 and autophagy-related proteins. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: Oncology Research

Article Title: miR-512-3p/RPS6KA2 Axis Regulates Cisplatin Resistance in Ovarian Cancer via Autophagy and Ferroptosis

doi: 10.32604/or.2025.070542

Figure Lengend Snippet: Impact of RPS6KA2 targeting on MTOR expression and apoptosis in ovarian cancer cells. Tumor-bearing mice were randomly distributed into 11 groups and administered various agents, including an RPS6KA2-targeted drug (Honokiol), a ferroptosis inducer (Erastin), and an autophagy inhibitor (3-MA). Immunofluorescence staining was performed to examine changes in MTOR expression across groups; ( A ) nuclei are labeled in blue, and MTOR signal is shown in green. ( B ) TUNEL analysis was conducted to assess apoptotic levels among groups; blue fluorescence represents the nucleus, and green fluorescence marks apoptotic cells. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant ( p > 0.05)

Article Snippet: Following fixation in formaldehyde, immunohistochemical (IHC) analysis was performed to evaluate protein expression levels using primary antibodies against RPS6KA2 (14446-1-AP, 1:100 dilution; Proteintech, Chicago, USA), ATG5 (66744-1-Ig, 1:200 dilution; Proteintech, Chicago, USA), ATG7 (67341-1-Ig, 1:1000 dilution; Proteintech, Chicago, USA), BECN1 (66665-1-Ig, 1:500 dilution; Proteintech, Chicago, USA), SQSTM1 (66184-1-Ig, 1:3000 dilution; Proteintech, Chicago, USA).

Techniques: Expressing, Immunofluorescence, Staining, Labeling, TUNEL Assay, Fluorescence

Journal: Journal of Investigative Dermatology

Article Title: Bcl-2 Reduced and Fas Activated by the Inhibition of Stem Cell Factor/KIT Signaling in Murine Melanocyte Precursors

doi: 10.1111/j.0022-202x.2004.23540.x

Figure Lengend Snippet: Figure 5 Western blot analysis of p-ERK (extracellular signal-related kinase) and p-RSK (ribosomal S6 kinase) family proteins in neural crest cell (NCC)me- lb4 cells incubated in anti-KIT antibody medium. ERK activation was detected using an antibody against phosphorylated ERK, p44 and p42, designed ERK1 and ERK2. NCCmelb4 cells were incubated for 24, 48, or 72 h in the presence or absence of anti-KIT antibody (ACK2) (100 mg per mL). p-ERK1 and p-ERK2 protein expression completely disap- peared by 72 h (a, b). p-RSK1 (Thr 359/Ser 363)-R reacts with Thr-359 and Ser-363 phosphorylated RSK family proteins of mouse origin. Anti- KIT antibody (ACK2) induced a significant decrease in the p-RSK family proteins between ACK2-treated and control sample (c, d). (Lane 1, 24 h; lane 2, 48 h; lane 3, 72 h; lane 4, no ACK2 treatment.)

Article Snippet: After electrophoresis, proteins were transferred onto a polyvinylidene difluoride membrane (Immobilon-p, Millipore, Bedford, Massachusetts) and incubated with ERK, p-ERK, RSK1, p-RSK1 (Thr 359/Ser 363)-R, Bcl-2, Bax, Fas, Fas-L, and caspase8 p20 subunit (1 mg per mL, Santa Cruz Biotechnology, Santa Cruz, California, each) overnight at room temperature (RT).

Techniques: Western Blot, Incubation, Activation Assay, Expressing, Control

Journal: Nature Communications

Article Title: Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases

doi: 10.1038/s41467-018-06787-w

Figure Lengend Snippet: DMF inhibits RSK2. a DMF with Michael acceptor reactive carbons marked with red asterisks. b Schematic representation of RSK2 CTKD and position of cysteine residues. c Inhibition of RSK2 CTKD by DMF (squares) and MMF (triangles). Error bars indicate the standard deviation of mean values ( n = 3, all data points included). d Covalent modification of RSK2 CTKD by DMF. Purified murine RSK2 (5 nmol) was incubated with 14 C-labelled DMF (25 nmol). DMF binding is reduced by preincubation of RSK2 with 10-fold molar excess of iodoacetamide or GSH, respectively. The + and – signs represent inclusion or absence of the respective components

Article Snippet: Proteins run on the same gel, treated by vehicle or DMF, were blotted to a nitrocellulose membrane and tested with monoclonal antibody for RSK2 (Santa Cruz Biotechnology sc-9986, 1:1000) and antibody for GST (Cell Signaling #2624S, 1:2000) to identify the full-length protein of about 116 kDa.

Techniques: Inhibition, Standard Deviation, Modification, Purification, Incubation, Binding Assay

Journal: Nature Communications

Article Title: Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases

doi: 10.1038/s41467-018-06787-w

Figure Lengend Snippet: Degree of in vitro modification of cysteines in RSK2 CTKD by DMF on purified RSK2 CTKD (DMF1 mM) and in HEK293 cells (DMF 140 µM) estimated by mass spectrometry ( n = 1)

Article Snippet: Proteins run on the same gel, treated by vehicle or DMF, were blotted to a nitrocellulose membrane and tested with monoclonal antibody for RSK2 (Santa Cruz Biotechnology sc-9986, 1:1000) and antibody for GST (Cell Signaling #2624S, 1:2000) to identify the full-length protein of about 116 kDa.

Techniques: In Vitro, Modification, Purification, Mass Spectrometry, Activation Assay

Journal: Nature Communications

Article Title: Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases

doi: 10.1038/s41467-018-06787-w

Figure Lengend Snippet: Crystallographic data collection and refinement statistics

Article Snippet: Proteins run on the same gel, treated by vehicle or DMF, were blotted to a nitrocellulose membrane and tested with monoclonal antibody for RSK2 (Santa Cruz Biotechnology sc-9986, 1:1000) and antibody for GST (Cell Signaling #2624S, 1:2000) to identify the full-length protein of about 116 kDa.

Techniques:

Journal: Nature Communications

Article Title: Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases

doi: 10.1038/s41467-018-06787-w

Figure Lengend Snippet: DMF binds to C436 and C599 in RSK2 crystals. a Cartoon representation of RSK2 with DMF covalently bound at C599 (DMF1) and C436 (DMF2). b Covalent binding of DMF (orange) to C599 and C436 of RSK2 (light grey). Final 2F o –F c electron density map depicted in blue represent a contour level of 1.0σ and a bias-reduced simulated annealing F o –F c difference maps is contoured in green at 3.0 σ, both on the C436 and C599 dimethyl binding site, respectively. c DMF binding pocket. The regulatory αL-helix, the activation loop, nitrogens and oxygens are coloured in brown, light green, blue, and red, respectively. d Activation of RSK2 (light grey) leads to the phosphorylation of a threonine residue in the activation loop (light green). The movement of the activation loop has been determined by X-ray crystallography for several kinases and is shown in light blue for the related kinase p70S6K1 (PDB ID 3A62). The hinge region is undergoing large structural rearrangements during activation and covalent binding of DMF (in spheres) to C599 could abolish this by steric hindrance. A second DMF modification site, C436 was observed, but it was less well defined

Article Snippet: Proteins run on the same gel, treated by vehicle or DMF, were blotted to a nitrocellulose membrane and tested with monoclonal antibody for RSK2 (Santa Cruz Biotechnology sc-9986, 1:1000) and antibody for GST (Cell Signaling #2624S, 1:2000) to identify the full-length protein of about 116 kDa.

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Residue, Modification

Journal: Nature Communications

Article Title: Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases

doi: 10.1038/s41467-018-06787-w

Figure Lengend Snippet: Time course of DMF inhibition. In vitro determination of apparent IC 50 values of DMF on RSK2 CTKD activity following incubation with DMF at different time points (1 h: black, 24 h: dark grey, and 48 h light grey) prior to ERK2 activation ( n = 3, all data points indicated by circles)

Article Snippet: Proteins run on the same gel, treated by vehicle or DMF, were blotted to a nitrocellulose membrane and tested with monoclonal antibody for RSK2 (Santa Cruz Biotechnology sc-9986, 1:1000) and antibody for GST (Cell Signaling #2624S, 1:2000) to identify the full-length protein of about 116 kDa.

Techniques: Inhibition, In Vitro, Activity Assay, Incubation, Activation Assay

Journal: Nature Communications

Article Title: Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases

doi: 10.1038/s41467-018-06787-w

Figure Lengend Snippet: Mutational studies of DMF inhibition in cells. Mutational analysis of HEK293 cells transfected with RSK2, MSK1, or cysteine mutants hereof. HEK293 cells were supplemented with DMF (140 μM) and stimulated with epidermal growth factor (EGF, 1 ng ml –1 ). a , b Representative western blots from RSK2 and MSK1 mutational analysis respectively. c , d RSK2 CTKD and MSK1 CTKD activity determined as the autophosphorylation of S386 and S376, respectively, relative to WT. The activity of EGF-induced wild-type was set to 100% ( n = 5 and n = 4, respectively; all data points indicated by black, filled circles)

Article Snippet: Proteins run on the same gel, treated by vehicle or DMF, were blotted to a nitrocellulose membrane and tested with monoclonal antibody for RSK2 (Santa Cruz Biotechnology sc-9986, 1:1000) and antibody for GST (Cell Signaling #2624S, 1:2000) to identify the full-length protein of about 116 kDa.

Techniques: Inhibition, Transfection, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: (A) Activation of the tandem kinase RSK is a multi-step process. Activation of RSK is initiated by ERK docking, which is followed by the phosphorylation of the C-terminal kinase domain (CTKD) ( Alexa et al , 2015 ). The active CTKD will phosphorylate a linker site between the kinase domains, which will create a docking motif for PDK1 ( Frödin et al , 2002 ). In the end, PDK1 will activate the N-terminal kinase domain (NTKD) ( Frödin et al , 2000 ). Usually, only the NTKD is considered as an effector kinase and the CTKD is only associated with a self-regulatory role and one of these activated kinases will phosphorylate its C-terminal PBM. While RSK is an effector of the mitogenic ERK pathway, we have limited information about its intracellular role. (B) Each RSK isoform contains a functional, class 1 PBM. RSK1 contains 3, mutually exclusive autophosphorylation sites (at the -1,-2,-3 positions) and the other isoforms contain only two (at the -2,-3 positions), but only the -3 site (Ser732 in RSK1) is considered as a major feedback site ( Hornbeck et al , 2015 ). The structural panel shows the RSK1 binding to the second PDZ domain of MAGI1.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Activation Assay, Functional Assay, Binding Assay

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: (A) PDZome binding profiles of unphosphorylated and phosphorylated RSK1 PBMs. All profiles use biotin as a control. A red line indicates the cutoff for a significant PDZ-PBM interaction (BI>0.2). PDZ domains in the upper and lower plots are ranked on the basis of their BIs for the indicated peptide. In the middle plot, PDZ domains are ranked on the basis of their BIs for the unphosphorylated peptide, while the plotted BI values are those obtained for the phosphopeptide. Note the considerable reshuffling of binding targets induced by phosphorylation. (B) Domain architecture of the identified interaction partners. The PDZ domains are colored according to the measured BI values. Potential RSK phosphorylation sites are highlighted in the schematic maps with sticks. Dark and light green sticks represent ideal and non ideal RSK phosphorylation sites, respectively. Phosphorylation sites were extracted from the phosphosite ( Hornbeck et al , 2015 ) (an N > 2 filter was applied on the database).

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: The overlay of processed electropherograms between the biotin control and the peptide experiment is shown for the most significant interaction partners of RSK1. The average BI value is highlighted in each panel. The more depleted the PDZ peak in the peptide experiment, the stronger the binding of the PDZ domain to the peptide.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: (A) Comparing orthogonal binding data obtained by holdup assay, SPR and competitive fluorescence polarization (FP). The correlation of binding intensities (BI) obtained by holdup assays to affinity constants deduced from SPR or competitive FP was fitted using a Monte Carlo approach. Despite independent fitting procedures, a similar correlation was observed in both cases. The fitting procedure delivers a value for the peptide concentration in the holdup assay. By combining the peptide concentration obtained in that way, with the concentrations of free and peptide bound PDZ domain (both delivered by the holdup assay); one can then estimate from the holdup data the dissociation constant of all human PDZ domains that interacted detectably with the RSK1 peptides. (B) Phosphorylation induces a complex rearrangement in the RSK1 PDZ interactome. Instead of two definite classes (ON or OFF switching), a continuum (ON or OFF dimming) was measured in the phospho-induced K d differences. Dark gray columns show the experimentally determined K d differences from the competitive FP measurements. The lower panel shows the strongest affinity (minimal K d ) observed with either the native or phosphorylated RSK1 peptide in the same order as in the upper panel. Significant interaction partners can be found in all regions of the observed continuum.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Binding Assay, Fluorescence, Concentration Assay

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: Binding experiments were performed between the RSK1 and the PDZ domains of the strongest interaction partners of each peptides (of ARHGEF12 and SYNJ2BP) at 37°C. The calorimetric measurements confirmed the differential binding upon phosphorylation.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: The four channel of the CM5 chip was split into a negative control, a HPV16E6 internal control (green), an unphosphorylated (black) and a phosphorylated (red) surface and the significant RSK1 interaction PDZ domains (fused to MBP) were injected into the surface. Only steady state analysis was performed due to biphasic sensograms.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Negative Control, Injection

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: Fluorescence polarization measurements were carried out to measure the binding of multiple PDZ domains. Direct binding was measurement with a fluorescein labeled 7 residue long RSK1 peptide, while competitive measurements were measured with a 40 residue long native or monophosphorylated peptide (colored black and red, respectively).

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Fluorescence, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: (A) Monitoring steady-state luminescence, with the interaction sensors between RSK1 and full length PDZ proteins. Full-length proteins fused to two complementary fragments of nanoluc luciferase were co-expressed in serum starved HEK293T cells. The resulting luminescence signal was measured as indicated in material and methods. The luminescence signal obtained for the pair of wild-type constructs is used as reference (relative luminescence). The L714E RSK1 mutant is known to eliminate the interaction between RSK1 and ERKs ( Alexa et al , 2015 ). The ΔC1 RSK1 mutant does not contain the last C-terminal residue of RSK1 and therefore does not contain a functional PBM. The luminescence signal is systematically disrupted by the ΔC1 mutation, indicating that this signal efficiently reports the PBM-mediated binding of RSK1 to its PDZ-containing targets. The L714E mutation disrupts the signal in cases where the interaction partner can significantly interact with the phosphorylated form of RSK1. (n=6) Asterisks indicate statistical significance (*** P<0.001) calculated by two-tailed Student’s t-test between the luminescence signals of mutant and WT RSK1 constructs. (B) The steady-state validated RSK1 based luminescence interaction sensors (with ERK2 and several proteins containing RSK1-binding PDZ domains) were co-expressed in serum-starved HEK293T cells. The luminescence signal in absence and in presence of EGF (20 ng/ml) was monitored for sixty minutes following EGF addition. The measured luminescence signal was normalized to the initial luminescence and to the spontaneous substrate (furimazine) decay based on the unstimulated cells. The dark and grey curves show the luminescence signals of the WT and the L714E mutant, respectively. EGF stimulation provokes a time-modulated decrease of the luminescence signal for co-expressed constructs of RSK1 and ERK2 as observed in our previous work ( Gógl et al , 2018 ). Note that EGF simulation diversely modulates (increase, decrease or no significant change) the luminescence signal for each PDZ-containing protein in a similar timescale of the RSK-ERK dissociation. Remarkably, this EGF-induced luminescence signal modulation in the cellular assay follows the same trend as the phosphorylation-induced modulation (off and on dimmer) of the in vitro binding affinity of RSK1 to individual PDZ domains.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Luciferase, Construct, Mutagenesis, Functional Assay, Binding Assay, Two Tailed Test, In Vitro

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: (A) Left panel: a graphical representation of the intersections of RSK substrate lists from four different HTP phosphoproteomic studies: (i) Galan et al. ( Galan et al , 2014 ), (ii) Moritz et al. ( Moritz et al , 2011 ), (iii) Avey et al. ( Avey et al , 2015 ), (iv) [RK]xx[ST] subset of the ERK compendium ( Ünal et al , 2017 ). Middle panel: the intersection of the four lists contains several previously characterized RSK substrates (underlined), suggesting that other proteins found in this core intersecting ensemble may also represent high-confidence RSK substrates. Right panel:the RSK compendium and the direct ERK compendium significantly overlaps. This indicates that a set of substrates can be phosphorylated on both ERK ([ST]P) and RSK ([RK]xx[ST]) consensus sites. (B) Same representation as in (A), focusing on RSK substrates with PDZ domains. Only a few PDZ domain containing substrates are present in the whole dataset. Moreover, only ARHGEF12 was found in the core intersecting ensemble, and only a handful of PDZ interaction partners were found in more than one HTP study. Uncharacterized PDZ partners could be direct partners of other RSK isoforms, PDZ-independent substrates or false positives. (C) Many RSK1 PDZ interaction partners contain an ERK phosphorylation site. Additionally, a few substrates, such as ARHGEF12, can be phosphorylated by both kinases. (D) The IntAct database was used to estimate the enrichment of ERK and RSK substrates among the interaction partners of the RSK1 PDZ-dependent interaction partners. On the vulcano plot, each dot represents the enrichment of kinase substrates among the interaction partners of a PDZ scaffold. We have identified a high number of potential indirect RSK and ERK substrates among these interaction partners, which are indicated with colors in the upper right corner. P values indicate statistical significance compared to a random pool of intracellular proteins, calculated by Chi-square test. Fold enrichment indicates the increased proportions of substrates compared to the same random pool.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques:

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: (A) We filtered the ERK compendium to [ST]P (phosphorylated directly by ERK), [RK]×[ST] (phosphorylated indirectly by RSK) and other (phosphorylated indirectly by an unknown kinase) phosphosites. We have found that only 60% of the phosphorylation sites were potential direct ERK substrates and 20% of the identified phosphorylation events were potential RSK substrates. (B) Most substrate proteins were phosphorylated on multiple sites by a single kinase, but there is a significant overlap between the different phosphorylation motifs indicating that a fraction of the substrates can be phosphorylated by more than one kinase. We considered these putative RSK substrates as an individual set of potential substrates in further analyses. (C) Ternary complex formation between RSK, ERK and a PDZ domain. The MBP-PDZ domain was used as a prey and RSK peptides, ERK2 or ERK2 bound RSK peptides were used as baits in a pull-down experiment. Proteins were detected on an SDS-PAGE gel stained with Coomassie protein dye. The panel shows the results of a representative MBP pull down assay from two independent experiments. We were able to detect an enhanced interaction between ARHGEF12 and ERK2 in the presence of unphosphorylated RSK1 peptide, confirming a ternary complex formation. Moreover, the phosphorylated RSK peptide was unable to induce such effect, confirming the OFF dimmer effect of the complex formation in this particular interaction.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: SDS Page, Staining, Pull Down Assay

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: (A) Dissociation rates were measured by the direct fluorescence polarization changes in a stopped-flow setup for a set of RSK1-PDZ interactions. Partners with OFF dimmer behavior showed a slower binding kinetics while the ON dimmers preferred faster binding rates. (B) Substrate phosphorylation was in silico estimated using their measured dissociation rate. Asterisks denote that the measured dissociation rates were already corrected to unbiased binding of unlabeled peptide.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Fluorescence, Binding Assay, In Silico

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: (A) A complexed fluorescent RSK1 peptide was mixed with high amount of unlabeled peptide. The change in the fluorescence polarization was monitored during the dissociation phase. (B) Measured off-rates of the labeled peptides. (C) Substrate phosphorylation was in silico estimated using their measured dissociation rate.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Fluorescence, Labeling, In Silico

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: (A) This simplified mathematical model was used to simulate MAPK pathway activation. (B) Network based simulation shows that only a small fraction of activated RSK1 has an unphosphorylated PBM (even in the presence of high amount of PDZ domain). (C) Interaction partners with negative feedbacks show a dissociation upon stimulation. While the dissociation profile is off-rate dependent, the substrate phosphorylation rate is not. The system shows an optimal substrate phosphorylation at a low dissociation rate. (D) In contrast to the OFF dimmers, substrates with a positive feedback show an association profile. Increasing their dissociation kinetics increases their substrate phosphorylation rate. Note that the dynamical profiles of the interactions are very similar to the results of our cell based measurements, but we do not have any periodicity in this isotropic system. (E) A set of RSK substrates were in silico phosphorylated using an artificially slow or fast dissociation rate. Partners, which showed an OFF dimmer behavior preferred a slower binding kinetics while the ON dimmers preferred faster kinetics.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Activation Assay, In Silico, Binding Assay

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: (A) Inhibition of the MAPK pathway alters levels of GTP bound RhoA. To visualize this effect, we overexressed WT ARHGEF12 in HEK293T cells, which resulted in a significant increment in the basal active RhoA levels. While MEK inhibition decreased, RSK inhibition increased the intracellular active RhoA level. (n=4) (B) Mimicking the RSK1 phosphorylation site on ARHGEF12 (S1288E) or introducing a RhoA binding incompetent mutant (W769D) altered the RhoA signalization. Phosphomimicking decreased the signal by 20% and W769D mutation by 50%. (n=4) The schematic model of ARHGEF12/LARG activation is highlighted on the right side, including GAP and GEF activities. (C) RSK1/2 knockout HEK293 cell line was used to measure the role of the PBM of RSK1 in a more native environment. Deletion of the RSK1 PBM does not affect the localization of active RSK1. (D) The presence of intact RSK1 increases the basal RhoA activity but this effect is decreased without a functional PBM. (n=4) (E) Transfected and serum starved cells were stimulated with serum (20%, 5 min). Without intact RSK1 (in the mock transfected knockout cell or in the presence of the PBM-lacking RSK1 construct) only minor increment was observed in the RhoA activity. The presence of intact, wt RSK1 enabled a proper response in RhoA activation upon stimulation. (n=4) Asterisks indicate statistical significance (** P<0.005, * P<0.01, NS P>0.01) calculated by two-tailed Student’s t-test.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Inhibition, Binding Assay, Mutagenesis, Activation Assay, Knock-Out, Activity Assay, Functional Assay, Transfection, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Rewiring of RSK-PDZ interactions by linear motif phosphorylation

doi: 10.1101/419721

Figure Lengend Snippet: (A) The close paralogs, ARHGEF12 and ARHGEF11, display different binding affinities with the unphosphorylated RSK1 peptide. Despite no alteration can be observed in their PBM binding groove, a handful of differences can be found in surface exposed loops. Different residues are colored in red and similar residues are colored in purple. Identical residues are colored in yellow. (B) A few differences can be observed in the PBM binding groove in ON dimmer PDZ domains. Sequence logos were generated from every human PDZ sequences or from identified dimmer subsets of the RSK1 peptide. Important differences are underlined in the ON dimmer sequence logo and their sidechains are showed with sticks in the structure of the OFF dimmer MAGI1 indicated with the preferred residues. In contrast, no preferences can be found for OFF dimmers.

Article Snippet: Unphosphorylated, phosphorylated, fluorescein labeled or unlableled RSK1 729–735 peptides were all chemically synthesized on an automated PSE Peptide Synthesizer (Protein Technologies, Tucson, AZ, USA) with Fmoc strategy.

Techniques: Binding Assay, Sequencing, Generated

Journal: Journal of Biological Chemistry

Article Title: p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

doi: 10.1074/jbc.m109.083642

Figure Lengend Snippet: FIGURE 1. RSK1, via its N-terminal kinase domain, interacts with PKARI. A, shown is a schematic of rat RSK1 and HA-tagged RSK1 constructs used; numbers represent the amino acids in rat RSK1. B–E, shown is pulldown of HA-tagged RSK1 or its polypeptides with cAMP-agarose. HEK293T cells were transfected to express HA-tagged RSK1 or its polypeptides. After depriving of serum overnight, cells were lysed and incu- bated with cAMP-agarose to pull down PKARI in the presence and absence of 50 mM cAMP as indicated. The proteins in the complex were monitored using anti-HA, anti-PKARI, or anti-RSK1 antibodies. IB, immunoblot. B, PKARI binds to RSK1 fragments containing the N-terminal part of RSK1. C, HA-RSK1-(1–317) competes with endogenousRSK1forbindingtoPKARI.ThepanelontherightshowsquantificationofbandintensitiesofRSK1 as a ratio of band intensities of PKARI (mean S.E.) from three experiments. *, p 0.05 as compared with the control.D,theNTKofRSK1bindstoPKARI.E,substitutionofSer-221ofRSK1withanegativelychargedresidue abrogates the interaction between RSK1 and PKARI. Representatives of three similar experiments are shown for all panels. WCL, whole cell lysates.

Article Snippet: The supernatants (500 g of protein except for immunoprecipitation with anti-PKARI antibody, where 1mg protein was used) were incubated for 2 h at 4 °Cwith 0.4 g of anti-RSK1 antibody or 2 g of anti-PKAc antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of PKARI antibody (BD Biosciences) together with 15 l of protein G-conjugated agarose beads.

Techniques: Construct, Transfection, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

doi: 10.1074/jbc.m109.083642

Figure Lengend Snippet: FIGURE 2. RSK1 binds to the pseudosubstrate region of PKARI. A, shown is a schematic of bovine PKARI; numbers represent the amino acids in bovine PKARI. B, the truncated PKARI (deletion of amino acids 1–91, PKARI91) retains the ability to interact with RSK1. Pure PKARI or PKARI91 (10 pmol each) was incubated with glutathione-Sepharose prebound with GST or GST-RSK1-(1–317) (5 g). The amounts of PKARI or PKARI91 in the pulldown complex were detected with anti-PKARI antibody. GST or GST-RSK1-(1–317) was stained with Coomassie Blue. IB, immunoblot. C, substitution of Arg-93/94 or Arg-95/96 on PKARI to Ala selectively abrogates the interactions of PKARI with RSK1 and PKAc. HEK293T cells were transfected with plasmids expressing C-terminal fusion of Wt-PKARI, PKARI (R93A/R94A), or PKARI (R95A/R96A) with enhanced yellow fluorescent protein (wt-PKARI-EYFP, PKARI (R93A/R94A)-EYFP, and PKARI (R95A/R96A)- EYFP, respectively). Cell lysates were immunoprecipitated (IP) with anti-RSK1 or PKAc antibody. The immune complex was probed with anti-EYFP, PKARI, RSK1, or PKAc antibodies. WCL, whole cell lysate. D, the peptide corresponding to the PKARI pseudosubstrate region (Wt-PS) competes for the interaction of PKARI with RSK1. GST-RSK1-(1–317) (5 g) prebound to glutathione-Sepharose was incubated with PKARI peptides, Wt-PS, or Mut-PS at the concentrations indicated at 4 °C for 15 min before being mixed with PKARI (10 pmol). GST-RSK1-(1–317) was stained with Coomassie Blue. The panel on the right shows the quantification of relative intensities of PKARI bands as a ratio of GST-RSK1 bands from two identical experiments. Wt-PS, peptide with wild-type PKARI pseudosubstrate sequence KGRRRRGAI). Mut-PS, peptide with mutated PKARI pseudosub- strate sequence (KGAARRGAI).

Article Snippet: The supernatants (500 g of protein except for immunoprecipitation with anti-PKARI antibody, where 1mg protein was used) were incubated for 2 h at 4 °Cwith 0.4 g of anti-RSK1 antibody or 2 g of anti-PKAc antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of PKARI antibody (BD Biosciences) together with 15 l of protein G-conjugated agarose beads.

Techniques: Incubation, Staining, Western Blot, Transfection, Expressing, Immunoprecipitation, Sequencing

Journal: Journal of Biological Chemistry

Article Title: p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

doi: 10.1074/jbc.m109.083642

Figure Lengend Snippet: FIGURE3.AffinitiesofPKAcandRSK1forPKARI.PKARIwasimmobilized on GLC sensor chips of the Bio-Rad Proteon XPR36 instrument, and binding isotherms were monitored by infusing the different concentrations of PKAc (A), GST-full-length RSK1 (GST-FL-RSK1) (B), or GST-RSK1-(1–317) (C) for the 600-s periods (denoted by vertical lines) as described under “Experimental Procedures.” The amount of immobilized PKARI corresponded to 5000 res- onance units. Controls with GST alone (C) were also performed. Representa- tives of six similar independent experiments are shown. KD values presented are the mean S.E. from six different experiments.

Article Snippet: The supernatants (500 g of protein except for immunoprecipitation with anti-PKARI antibody, where 1mg protein was used) were incubated for 2 h at 4 °Cwith 0.4 g of anti-RSK1 antibody or 2 g of anti-PKAc antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of PKARI antibody (BD Biosciences) together with 15 l of protein G-conjugated agarose beads.

Techniques: Binding Assay

Journal: Journal of Biological Chemistry

Article Title: p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

doi: 10.1074/jbc.m109.083642

Figure Lengend Snippet: FIGURE 4. Competition between PKAc and RSK1 for association with PKARI regulates PKA activity. A, PKAc competes with RSK1-(1–317) for binding to PKARI. PKARI (10 pmol) was preincubated with different indicated amounts of PKAc to form the holoenzyme before mixing with glutathione resin pre- bound to GST-RSK1-(1–317) (5 g) for the pulldown assay. GST-RSK1-(1–317) was stained with Coomassie Blue. The panel on the right is quantification of the ratio of the band intensities of PKARI relative to GST-RSK1 from three similar experiments. *, p 0.05; ** p 0.01, Student’s unpaired t test analysis. B, GST full-length RSK1 (GST-FL-RSK1) competes with PKAc for PKARI and decreases the formation of the PKA holoenzyme. PKAc (2 nM final concentration) and increasing indicated concentrations of GST-FL-RSK1 (or GST 100 nM) were added to PKARI (3 nM). After incubation of the mixture for 1 h, formation of the PKA holoenzyme was monitored by measuring PKAc activity as described under “Experimental Procedures.” C, increasing expression of HA-RSK1 (S221A) decreases the association of endogenous PKAc with PKARI. HEK293T cells were transfected with the indicated different amounts of plasmid expressing HA-RSK1 (S221A). Cell lysates were immunoprecipitated (IP) with anti-PKARI antibody. The right hand panel shows the quantification of band intensities of PKAc or HA-RSK1 as a ratio of the band intensities of PKARI from 3 similar experiments. *p 0.05; **, p 0.01. D–E, overexpression of HA-RSK1 (S221A) activates PKA. Experiments were the same as in panel C, except that the cell lysates were probed with anti-phospho-PKA substrate (D) or anti- phospho-BAD Ser-155 antibodies (E). In D, a representative of three similar experi- ments is shown. IB, immunoblot. In E, the right-hand panel shows quantification of band intensities of phospho-BAD-Ser-155 and Erk1/2 from three experiments; *, p 0.05. F, silencing of RSK1 increases the interactions between endogenous PKAc and PKARI. B82L cells were transfected with RSK1-specific siRNA #1 (20 nM) or #2 (40 nM) for 56 h and then serum-starved overnight. The cell lysates were immunoprecipi- tated with anti-PKARI antibody. The right-hand panel shows quantification of band intensities of PKAc as a ratio of PKARI band intensities from three experiments. *, p 0.05, as compared with control. G, silenc- ing of RSK1 decreases phosphorylation of BAD on Ser-155. RSK1 was silenced as in F. Quantified band intensities of phospho-BAD-Ser-155 and Erk1/2 from three experiments are shown on the right. *, p 0.05; **, p 0.01, as compared with control siRNA (Con). WCL, whole cell lysate.

Article Snippet: The supernatants (500 g of protein except for immunoprecipitation with anti-PKARI antibody, where 1mg protein was used) were incubated for 2 h at 4 °Cwith 0.4 g of anti-RSK1 antibody or 2 g of anti-PKAc antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of PKARI antibody (BD Biosciences) together with 15 l of protein G-conjugated agarose beads.

Techniques: Activity Assay, Binding Assay, Staining, Concentration Assay, Incubation, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Over Expression, Western Blot, Control, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

doi: 10.1074/jbc.m109.083642

Figure Lengend Snippet: FIGURE 5. PKARI/RSK1 interactions regulate RSK1 activation and apoptosis. A, peptide (Wt-PS) corre- sponding to the pseudosubstrate region of PKARI disrupts the interaction of PKARI and RSK1 and increases RSK1 phosphorylation in unstimulated cells. B82L cells were serum-starved overnight and then were treated with 2 M each of Wt-PS or Mut-PS (for sequences, see the legend to Fig. 2) for 10 min. RSK1 from the cell lysates was immunoprecipitated (IP) with anti-RSK1 antibody. The proteins in the immune complex were detected with Western analysis. The quantitative data using band intensities of the indicated proteins from three exper- iments are shown in the panels on the right. B, Wt-PS, but not Mut-PS, disrupts the association of RSK1 with PKARI, increases the binding of endogenous PKAc to PKARI, and decreases phosphorylation of BAD on Ser-155. Cell lysates were prepared as in A, and PKARI was immunoprecipitated with anti-PKARI antibody. The proteins in the immune complexes or whole cell lysates (WCL) were subjected to Western analysis. Quan- tification of data using band intensities of the indicated proteins (n 3 experiments) are shown in the panels on the right. C, Wt-PS, but not Mut-PS, increases basal RSK1 activity and decreases apoptosis. After overnight deprivation of serum, B82L cells were treated with PKARI peptides Wt-PS or Mut-PS (2 M each) for 10 min followed by stimulation with 50 nM EGF for 10 min. Cell lysates were examined for phosphorylation of Ser-380 on RSK1 and Ser-112 on BAD. The panels on the right represent quantification of band intensities of the indi- cated proteins from three experiments. For the apoptosis assay, after treatments with or without EGF for 10 min, cells were treated with TNF- (20 ng/ml) plus cycloheximide (25 g/ml) (TNF/CHX) for 1 h, and DNA fragmentation was monitored. Data are the mean S.E. of A405 per g of protein (n 3). D, silencing of RSK1 abrogates the ability of Wt-PS to inhibit apoptosis. Procedures were as in C, except that cells were transfected with RSK1 siRNAs for 56 h before experimentation. The inset shows silencing of RSK1 by the two siRNAs. Data are the mean S.E. of A405 per g of protein (n 3).

Article Snippet: The supernatants (500 g of protein except for immunoprecipitation with anti-PKARI antibody, where 1mg protein was used) were incubated for 2 h at 4 °Cwith 0.4 g of anti-RSK1 antibody or 2 g of anti-PKAc antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of PKARI antibody (BD Biosciences) together with 15 l of protein G-conjugated agarose beads.

Techniques: Activation Assay, Phospho-proteomics, Immunoprecipitation, Western Blot, Binding Assay, Activity Assay, Apoptosis Assay, Transfection