Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet: Characterization of the roles of the four major mitotic kinases in MEE-induced supershifts of Cdc25C (A) Summary of the major Cdk1, MAPK, Plx1 and RSK2 phosphorylation sites previously identified in Cdc25C. (B and C) Phosphorylation of myc-Cdc25C with the four major mitotic kinases individually (B) or collectively (C) in parallel with equivalent strengths of MEE for indicated hr, followed by myc-tag immunoblotting. (D) Phosphorylation of myc-Cdc25C with MEE or p9-Cdk1 for indicated hr, followed by myc-tag immunoblotting. (E) Phosphorylation of myc-Cdc25C with MEE in the presence of drug vehicle DMSO, the 4 specific kinase inhibitors combined (RS + VX + SL + BI) or a pan kinase inhibitor (ST) for indicated min, followed by myc-tag immunoblotting. (F) Phosphorylation of myc-Cdc25C with control IgG-absorbed or 4 kinase-depleted MEE for indicated min, followed by myc-tag immunoblotting. The ≈ symbols indicate equivalent timepoint matching.

Article Snippet: Anti-RSK2 antibodies for multiple species were purchased from Santa Cruz Biotechnology (sc-9986).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet:

Article Snippet: Anti-RSK2 antibodies for multiple species were purchased from Santa Cruz Biotechnology (sc-9986).

Techniques: Western Blot, Immunoprecipitation, Sequencing, Modification, Mass Spectrometry, Recombinant, Protease Inhibitor, Mutagenesis, Purification, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: Endogenous LARG phosphorylation at S1288 and RhoA activation in GBM tissues . A , the phospho-LARG S1288 specific antibody was tested in U87MG cells transfected with Myc-LARG-WT, Myc-LARG-S1288 A, or Myc-EV. After 24 h of starvation, cells were treated with EGF for 10 min in the presence or absence of BI-D1870. B , the phospho-LARG S1288 antibody demonstrates specificity to the phosphorylated form of the protein. U87MG cells were transfected with Myc-LARG-WT of Myc-EV. After 24 h of starvation, cells were treated with EGF for 10 min. Protein was harvested and lysates were treated with λ phosphatase prior to immunoblotting. C , RSK2 phosphorylates LARG at S1288 in PDX-derived GBM39 cells. Cells maintained in complete medium with 10% FBS were treated with BI-D1870 for 4 h. Subsequently cells were starved in 0.1% FBS medium for 24 h prior to 10 min EGF treatment alone or after prior 4 h BID treatment. Immunoblotting was used to determine total expression and phosphorylation of target proteins. The results are representative of three independent experiments. D , levels of activated RhoA-GTP positively correlate with levels of phosphorylated LARG S1288 in human GBM tumor lysates. Endogenous total LARG, pLARG-S1288 and RhoA expression in seven human GBM tumor lysates were analyzed by immunoblotting. Activated RhoA-GTP was isolated by immunoprecipitation and detected with a RhoA antibody. Signals for each target protein expression were normalized to corresponding sample β-tubulin expression. A linear regression analysis was performed to assess the relationship between Activated RhoA expression and pLARG S1288 expression in all seven tumors.

Article Snippet: RhoA (sc-418) mouse, α tubulin (sc-8035) mouse, YB1 (sc-101198) mouse, RSK2 (sc-9986) mouse, LARG H-3 (sc-166318) mouse, GST (sc-374171) mouse monoclonal antibodies, and RSK2 inhibitor BI-D1870 were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Activation Assay, Transfection, Western Blot, Derivative Assay, Expressing, Isolation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: Model of LARG activation of RhoA in response to RSK2 phosphorylation . Schematic of the proposed signaling mechanism in which EGF stimulation activates RSK2, which leads to the phosphorylation of LARG at serine 1288. Phosphorylation of LARG at serine 1288 then promotes plasma membrane localization of LARG and binding to RhoA and subsequent RhoA activation.

Article Snippet: RhoA (sc-418) mouse, α tubulin (sc-8035) mouse, YB1 (sc-101198) mouse, RSK2 (sc-9986) mouse, LARG H-3 (sc-166318) mouse, GST (sc-374171) mouse monoclonal antibodies, and RSK2 inhibitor BI-D1870 were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Clinical Proteomics, Membrane, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: EGFR activation leads to phosphorylation of LARG at S1288 through RSK . A , schematic representation of of the LARG protein showing the functional domains: PDZ, RH, DH, PH, and CC. These domains coordinate LARG activation, RhoA interaction and localization. B , posttranslational modifications determined by Mass Spec are shown as curated by PhosphoSitePlus ( www.phosphosite.com ). The height of the lines relates to the number of reports in which that modification was identified. C , Western blot analysis of LARG phosphorylation from U87 MG cells transfected with Myc-LARG-WT, Myc-LARG-S1288 A, or Myc-EV for 48h. Cells were starved for 24h, then treated with EGF (100 ng/ml) for 10 min. Cell lysates were immunoprecipitated with a Myc antibody, and LARG phosphorylation was detected using a phospho-AKT substrate antibody. LARG phosphorylation was quantified relative to Myc tag expression ( right ). D , starved cells were subjected to RSK inhibitors B-ID1780 or PMD-026 for 24h or 4h before treatment with 100 ng/ml EGF for 10 min. LARG, RSK and YB1 phosphorylation levels were quantified as fold changes ( right ). Results are representative of three independent experiments, each of which was done in triplicate and presented as the mean ± standard deviation. ∗ p ˂ 0.05.

Article Snippet: RhoA (sc-418) mouse, α tubulin (sc-8035) mouse, YB1 (sc-101198) mouse, RSK2 (sc-9986) mouse, LARG H-3 (sc-166318) mouse, GST (sc-374171) mouse monoclonal antibodies, and RSK2 inhibitor BI-D1870 were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Functional Assay, Mass Spectrometry, Modification, Western Blot, Transfection, Immunoprecipitation, Expressing, Standard Deviation

Journal: Biochemical and biophysical research communications

Article Title: Novel wiring of the AKT-RSK2 signaling pathway plays an essential role in cancer cell proliferation via a G 1 /S cell cycle transition.

doi: 10.1016/j.bbrc.2022.12.048

Figure Lengend Snippet: Fig. 1. RSK2 is a key kinase preferentially enhancing cell proliferation in malignant cancer cells. (A) Comparison of magnolin and epimagnolin on the colony growth of ovarian cancer cells in soft agar (B) RSK2 knockdown effects on the cell proliferation of ovarian cancer cells by MTS assay. Top panels, RSK2 knockdown was confirmed by Western blotting. Bottom graphs, Cell proliferation of sh-mock and -RSK2 stable cells were measured by MTS assay. (C) RSK2 knockdown effects on the colony growth of ovarian cancer cells by soft agar assay using ovarian cancer cells. (D) RSK2 knockdown effects on the colony growth of lung cancer cells by soft agar assay using lung cancer cells. Photographs, (CeD) Graphs, Colony numbers grown in soft agar were taken randomly and scored automatically using an ECLIPSE Ti inverted microscope and the NIS-Elements AR (V. 4.0) computer software program (NIKON Instruments Korea, Gangnam, Seoul, Korea). (AeD) Error bars, ±SEM. Statistical significance *, p < 0.001.

Article Snippet: The antibodies for the MAPK signaling pathways, including phospho-ERK1/2 (Cat#: 9106), ERK1/2 (Cat#: 9102), phospho-RSK T359/S363 (Cat#: 9344), total-RSKs (Cat#: SC-9986), and phospho-RSK2 S227 (Cat#: 3556) were acquired from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Comparison, Knockdown, MTS Assay, Western Blot, Soft Agar Assay, Inverted Microscopy, Software

Journal: Biochemical and biophysical research communications

Article Title: Novel wiring of the AKT-RSK2 signaling pathway plays an essential role in cancer cell proliferation via a G 1 /S cell cycle transition.

doi: 10.1016/j.bbrc.2022.12.048

Figure Lengend Snippet: Fig. 2. RSK2 is a novel substrate of AKTs. (A) inhibition of PI3K/AKT signaling suppresses RSK2 phosphorylation. Phospho-protein profile of AKTs and RSK2 were evaluated to compare the effects of Wortmannin on the EGF-induced RSK2 phosphorylation in TOV-112D and SKOV3 cells. The proteins were visualized by Western blotting using specific antibodies as indicated. NTKD, N-terminal kinase domain; CTKD, C-terminal kinase domain. (B) AKTs interacted with RSK2. Table, Similarity of AKT1 target amino acid sequence in p90 RSK family including RSK 1-4 and MSK 1-2. AKT target phosphorylation motif, RxRxxS/T, are boxed. Bottom panels, HEK293T cell lysates transiently expressed each of constitutive active AKTs and His-RSK2 were used to confirm the interaction of AKTs and RSK2 by IP/WB using specific antibodies as indicated. (C) Upper panels, Construction maps for RSK2 deletion mutants. RSK2 proteins were fused with the GAL4 tag at the N-terminal end of RSK2 using a pBIND mammalian two-hybrid vector. RSK2-wt, full length of RSK2; RSK2-D1, aa 1-68 deleted RSK2; RSK2-D2, aa 1-323 deleted RSK2; RSK2-D3, aa 68-323 deleted RSK2; RSK2-D4, aa 422-675 deleted RSK2. Bottom panels, Mammalian two-hybrid assay. RSK2 domains interacting with AKT1 were analyzed by the mammalian two-hybrid assay. The interaction strength was indicated by the luciferase activity. Equal transfection efficiency was equalized by compensation with Renilla luciferase activity. Error bars, ±SEM. Statistical significance *, p < 0.01. (D) In vitro kinase assay. Commercially active AKT1 was combined with partial purified RSK2 from E. coli with [g-32p]-ATP and conducted kinase reaction at 30 C. The phosphorylated RSK2 is visualized by autoradiography. The input of Akt and RSK2 were visualized by Western blotting using the specific antibodies as indicated. (AeB) b-actin was used to verify the equal protein loading.

Article Snippet: The antibodies for the MAPK signaling pathways, including phospho-ERK1/2 (Cat#: 9106), ERK1/2 (Cat#: 9102), phospho-RSK T359/S363 (Cat#: 9344), total-RSKs (Cat#: SC-9986), and phospho-RSK2 S227 (Cat#: 3556) were acquired from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Sequencing, Plasmid Preparation, Two Hybrid Assay, Luciferase, Activity Assay, Transfection, In Vitro, Kinase Assay, Autoradiography

Journal: Biochemical and biophysical research communications

Article Title: Novel wiring of the AKT-RSK2 signaling pathway plays an essential role in cancer cell proliferation via a G 1 /S cell cycle transition.

doi: 10.1016/j.bbrc.2022.12.048

Figure Lengend Snippet: Fig. 3. AKT1 phosphorylates RSK2 at Thr115. (A) Conserved RxRxxS/T motif at NTKD. The RxRxxS/T motif located at the NTKD was aligned with that of different species, as indicated. (B) the NTKD of RSK2 contains an AKT-mediated phosphorylation target. Upper panels, Construction maps for GST-RSK2 fusion expression vectors in E. coli. Bottom panels, An in vitro kinase assay was performed using active AKT1 and each of the RSK2 proteins partially purified from E. coli. The phosphorylated RSK2 and the input AKT1and RSK2 were visualized by Western blotting using the specific antibodies as indicated. (C) RSK2 Thr115 is a phosphorylation target of AKT1. Upper panels, Construction map of RSK2-T115A point mutant expression vector in E. coli. Bottom panels. In vitro kinase assay to confirm that T115 of RSK2 is a target amino acid for AKT1. RSK2-wt or -T115A was combined with commercially active AKT1 and [g-32p]-ATP and conducted kinase reaction at 30 C. The phosphorylated RSK2 is visualized by autoradiography by exposure to x-ray film. (D) RSK2-T115A abolished AKT1-mediated phosphorylation. Upper panels, Construction maps of RSK2 point mutant using the GST-RSK2-FL and RSK2-D4 construct. Bottom panels, Each of the RSK2 proteins partially purified from E. coli were combined with commercially active AKT1 and 100 mM of cold ATP and conducted kinase reaction 30 C for 30 min. The phosphorylated RSK2 AND input AKT and RSK2 were visualized by Western blotting using the specific specific antibodies as indicated.

Article Snippet: The antibodies for the MAPK signaling pathways, including phospho-ERK1/2 (Cat#: 9106), ERK1/2 (Cat#: 9102), phospho-RSK T359/S363 (Cat#: 9344), total-RSKs (Cat#: SC-9986), and phospho-RSK2 S227 (Cat#: 3556) were acquired from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Expressing, In Vitro, Kinase Assay, Western Blot, Mutagenesis, Plasmid Preparation, Autoradiography, Construct

Journal: Biochemical and biophysical research communications

Article Title: Novel wiring of the AKT-RSK2 signaling pathway plays an essential role in cancer cell proliferation via a G 1 /S cell cycle transition.

doi: 10.1016/j.bbrc.2022.12.048

Figure Lengend Snippet: Fig. 4. AKT-mediated RSK2 phosphorylation plays an important role in G1/S cell cycle transition by growth factor stimulation. (A) In vitro cascade kinase reaction. Upper panels, The primary kinase reaction was conducted by a combination of active AKT1 and GST-RSK2-wt or -T115A as described. Bottom panels, Each of the primary reaction mixture (8 ml) in the upper panels was combined with GST-NFAT3-261-360 for a secondary in vitro kinase reaction. The phosphorylated RSK2 (upper panels) and GST-NFAT3-261-360 protein (bottom panels) were visualized by Western blotting using RxxS/T specific antibody. (B) Left panels, Conformation of RSK2 and RSK2-T115A expression in RSK2/ MEFs. RKS2/ MEFs stably expressing mock, RSK2-wt or RSK2-T115A were used to confirm the RSK2 expression by Western blotting using RSK2-specific antibodies. b-actin was used to verify the equal protein loading. Graphs, RSK2-T115A attenuates cell proliferation. The cells in (A) (1 103 cells/well) were seeded into 96-well plates and used to compare the effects of RSK2-wt- and -T115D-mediated cell proliferation by MTS assay. (C-D) AKT1-mediated RSK2 phosphorylation plays an important role in the G1/S cell cycle transition induced by growth factors, such as EGF (C) and IGF-1 (D). The cells (2.5 103 cells/cm2) in (A) were seeded in 60-mm dishes and cultured overnight. The cells were then starved overnight, and stimulated with EGF (C) and IGF-1 (D) for 12 h. The cells were harvested and analyzed to evaluate the cell cycle changed by propidium iodide staining. The cell cycle population (percentage) was graphed. (BeD) Error bars, ±SEM. Statistical significance *, p < 0.05; **, p,0.01.

Article Snippet: The antibodies for the MAPK signaling pathways, including phospho-ERK1/2 (Cat#: 9106), ERK1/2 (Cat#: 9102), phospho-RSK T359/S363 (Cat#: 9344), total-RSKs (Cat#: SC-9986), and phospho-RSK2 S227 (Cat#: 3556) were acquired from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, In Vitro, Western Blot, Expressing, Stable Transfection, MTS Assay, Cell Culture, Staining

Journal: Cells

Article Title: Targeting the p90RSK/MDM2/p53 Pathway Is Effective in Blocking Tumors with Oncogenic Up-Regulation of the MAPK Pathway Such as Melanoma and Lung Cancer

doi: 10.3390/cells13181546

Figure Lengend Snippet: p90RSK is capable of controlling DNA synthesis and apoptosis by regulating p53 levels. ( A ) A549 and A375 cells were incubated with 4 μM BI-D1870 for the indicated times (h). The immunoblot was checked using anti-p21, anti-Bcl2, anti-Bax and anti-p53 antibodies; the anti-tubulin antibody was used for normalization. ( B ) BrdU assay in A549 and A375 cells incubated or not with 4 μM BI-D1870 for 72 h. We used Hoechst solution to stain the nuclei blue, and anti-BrdU antibody was used to highlight apoptotic cells (green stain). In each sample, we counted at least 500 cells. The percentage of BrdU-positive cells was obtained from the ratio of BrdU-labeled nuclei to total nuclei. The histogram shows the average of the counts performed in different fields of BrdU-stained cells (more than 100 cells were counted in each field). The experiment was conducted in triplicate. The standard deviation of the acquired measurements was determined (left panel). The images in the figure represent photographs obtained with a fluorescence microscope at 10× magnification. A representative area for each sample is shown in the figure (right panel). ( C ) TUNEL assay in A549 and A375 cells treated or not with 4 μM BI-D1870 for 72 h. Hoechst solution was used to stain the nuclei blue, and apoptotic cells were stained using the TUNEL assay (red staining). The percentage of apoptotic cells was evaluated from the ratio of red-stained nuclei to blue-stained nuclei. The histogram shows the average of number of apoptotic cells counted in several fields (more than 500 cells were counted in total) and the standard deviation of the values obtained (left panel) in three different experiments. The images in the figure represent photographs obtained with a fluorescence microscope at 10× magnification. One representative field for is sample is displayed (right panel).

Article Snippet: Anti-RSK2 (sc-9986), anti-p21 (sc-397), anti-RSK1 (sc-231), anti-MDM2 (SMP-14, sc-965) and anti-p53 (sc-126) antibodies were obtained from SantaCruz Biotechnology (Dallas, TX, USA).

Techniques: DNA Synthesis, Incubation, Western Blot, BrdU Staining, Staining, Labeling, Standard Deviation, Fluorescence, Microscopy, TUNEL Assay

Journal: Cells

Article Title: Targeting the p90RSK/MDM2/p53 Pathway Is Effective in Blocking Tumors with Oncogenic Up-Regulation of the MAPK Pathway Such as Melanoma and Lung Cancer

doi: 10.3390/cells13181546

Figure Lengend Snippet: Final model: the inhibition of p90RSK induces the suppression of proliferation and survival. In tumor cells characterized by constitutive oncogenic activation of the ERK pathway, p90RSK inhibition destabilizes MDM2, inducing its proteasome degradation. The MDM2 reduction leads to an increase in p53 that, in turn, induces p21 transcription and reduces Bcl-2 transcription. The PI3K/AKT pathway is able to contribute to this mechanism in tumor cells where it is overactive.

Article Snippet: Anti-RSK2 (sc-9986), anti-p21 (sc-397), anti-RSK1 (sc-231), anti-MDM2 (SMP-14, sc-965) and anti-p53 (sc-126) antibodies were obtained from SantaCruz Biotechnology (Dallas, TX, USA).

Techniques: Inhibition, Activation Assay

Journal: Molecular Cancer

Article Title: miR-634 restores drug sensitivity in resistant ovarian cancer cells by targeting the Ras-MAPK pathway

doi: 10.1186/s12943-015-0464-4

Figure Lengend Snippet: MiR-634 regulates genes involved in cell cycle regulation and the Ras-MAPK pathway. Ovarian cancer cell lines (A2780, A2780 DDP) and patient derived tumor cells (culture #3) were transiently transfected with scrambled control (-) or miR-634 mimic (+). 48 h after transfection protein lysates were analyzed for the expression of putative miR-634 target genes by Western blotting (A, B) or RT-PCR (C). a Transfection with miR-634 reduces Cyclin D1 protein levels in the A2780 cell lines and in primary ovarian cancer cell cultures. β-actin is used as a loading control. b MiR-634 overexpression reduces protein levels of GRB2, ERK2, RSK1 and RSK2, components of the Ras-MAPK pathway in the A2780 and A2780 DDP cell lines and primary ovarian cancer cell cultures. β-actin is used as a loading control. c MiR-634 overexpression lowers mRNA levels of CCND1, GRB2, ERK2, RSK1 and RSK2 in the A2780 DDP cell lines 48 h after transfection. Expression was normalized to HPRT and GAPDH expression. Depicted are average values ± SD ( n = 2). The mRNA level in cells transfected with scrambled mimic is set at 100 %. d MiR-634 binds directly to elements of the 3’UTR of CCND1, GRB2, ERK2 and RSK2. A2780 DDP cells were transfected with a scrambled mimic or a miR-634 mimic. After 8 h, the same cells were transfected with a Renilla luciferase reporter construct (psiCHECK™-2) containing a region of 500 bp surrounding the predicted target sites (Additional file : Figure S7), or a luciferase construct in which the miR-634 binding sites were mutated. Since RSK2 contains both a canonical and a non-canonical miR-634 binding site, one construct was generated that contained a mutation in the canonical binding site (RSK2_2 mut 1) and another construct that contained mutations in both the canonical and non-canonical site (RSK2_2 mut 2). The Renilla luciferase activity was measured and normalized using the Firefly luciferase activity. For each construct, the relative luciferase activity of cells transfected with the scrambled mimic was set at 1. Depicted are the average values ± SD ( n = 3). * p < 0.05, ** p < 0.01

Article Snippet: Specific proteins were detected with antibodies against mouse GRB2 (BD Pharmingen, 610111), mouse RSK2 (Santa Cruz, Heerhugowaard, The Netherlands, SC-9986), rabbit ERK1/2 (Cell signaling, Leiden, The Netherlands, #9102), rabbit RSK1 (Santa Cruz, SC-231), rabbit CCND1 (Thermoscientific, Etten-Leur, The Netherlands, RM-9104-S) and mouse anti-β-actin (Sigma, A5441).

Techniques: Derivative Assay, Transfection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Over Expression, Luciferase, Construct, Binding Assay, Generated, Mutagenesis, Activity Assay