Activation Of Ribosomal S6 Kinase Research Articles

Activation of ribosomal p70 S6 kinase by selective serotonin (5-HT)<sub>2B</sub> receptor agonist BW723C86 is mediated by epidermal growth factor/transforming growth factor-α receptor tyrosine kinase in primary cultures of adult rat hepatocytes.

Activation of ribosomal p70 S6 kinase by selective serotonin (5-HT)<sub>2B</sub> receptor agonist BW723C86 is mediated by epidermal growth factor/transforming growth factor-α receptor tyrosine kinase in primary cultures of adult rat hepatocytes.

Aberrant expression of RSK1 characterizes high-grade gliomas with immune infiltration.

The p90 ribosomal S6 kinase (RSK) family, a downstream target of Ras/extracellular signal‐regulated kinase signaling, can mediate cross‐talk with the mammalian target of rapamycin complex 1 pathway. As RSK connects two oncogenic pathways in gliomas, we investigated the protein levels of the RSK isoforms RSK1–4 in nontumoral brain (NB) and grade I‐IV gliomas. When compared to NB or low‐grade gliomas (LGG), a group of glioblastomas (GBMs) that excluded long‐survivor cases expressed higher levels of RSK1 (RSK1hi). No difference was observed in RSK2 median‐expression levels among NB and gliomas; however, high levels of RSK2 in GBM (RSK2hi) were associated with worse survival. RSK4 expression was not detected in any brain tissues, whereas RSK3 expression was very low, with GBM demonstrating the lowest RSK3 protein levels. RSK1hi and, to a lesser extent, RSK2hi GBMs showed higher levels of phosphorylated RSK, which reveals RSK activation. Transcriptome analysis indicated that most RSK1hi GBMs belonged to the mesenchymal subtype, and RSK1 expression strongly correlated with gene expression signature of immune infiltrates, in particular of activated natural killer cells and M2 macrophages. In an independent cohort, we confirmed that RSK1hi GBMs exclude long survivors, and RSK1 expression was associated with high protein levels of the mesenchymal subtype marker lysosomal protein transmembrane 5, as well as with high expression of CD68, which indicated the presence of infiltrating immune cells. An RSK1 signature was obtained based on differentially expressed mRNAs and validated in public glioma datasets. Enrichment of RSK1 signature followed glioma progression, recapitulating RSK1 protein expression, and was associated with worse survival not only in GBM but also in LGG. In conclusion, both RSK1 and RSK2 associate with glioma malignity, but displaying isoform‐specific peculiarities. The progression‐dependent expression and association with immune infiltration suggest RSK1 as a potential progression marker and therapeutic target for gliomas.

Abstract C093: Substituted pteridinones as p90 ribosomal S6 protein kinase 2 (RSK2) inhibitors and their evaluation in acute myeloid leukemia

Abstract The activity of p90 ribosomal S6 kinase 2 (RSK2) has emerged as an attractive target for cancer therapy due to its role in the regulation of diverse cellular processes, such as cell transformation and proliferation and the maintenance of cancer stem cells (CSCs). Several pan-RSK inhibitors have been identified with BI-D1870 and the simple LJH685 and LJI308 analogs being the most selective, potent, and frequently used small molecules to identify the physiological substrates and functional roles for RSK in cells. However, these RSK inhibitors have poor PK profiles, display high clearance and a short plasma half-life and this has prevented their transition to clinical evaluation. Therefore, we designed and synthesized a series of pteridinones and pyrimidines to evaluate the structural features of BI-D1870 that are required for RSK2 inhibition, with the overall aim of developing a RSK inhibitor with improved drug-like properties. We have identified inhibitors of recombinant RSK2 activity, evaluated their target engagement in cells, and measured their effect on cell viability and cytotoxicity in the molm-13 acute myeloid leukemia (AML) cell line. The results of our initial structure-activity studies indicate that halogen substitutions on the 4-hydroxyanilino ring can greatly influence RSK inhibitory potency, elimination of C7 chirality does not appear to effect RSK inhibitory potency, removal of the N5-methyl maintains RSK inhibitory activity, and the N7-alkyl substitution is required for potent RSK inhibition. We have identified compounds that potently inhibit RSK2 activity in cells using the nanoBRET assay, and coupled with the results from our cellular viability and toxicity studies our data supports that RSK2 inhibition is independent from acute cytotoxicity in molm-13 cells. This finding was surprising as the RSKs phosphorylate a number of substrates involved in many diverse cellular processes critical for cell survival. Our studies have provided important structural information for the further development of RSK inhibitors and potential insights into structural modifications that may limit metabolism and incorporate isoform selectivity. Further studies are required to determine the impact of RSK inhibition in cancer cells as synergistic combination therapies may need to be identified. Alternatively, RSK inhibitors may be more therapeutically effective against specific cancer cell populations, such as in the elimination of CSCs. Citation Format: Kimberly Casalvieri, Christopher Matheson, Philip Reigan, Donald Backos. Substituted pteridinones as p90 ribosomal S6 protein kinase 2 (RSK2) inhibitors and their evaluation in acute myeloid leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C093. doi:10.1158/1535-7163.TARG-19-C093

LKB1 Deficiency Renders NSCLC Cells Sensitive to ERK Inhibitors.

Serine/threonine kinase 11 (LKB1/STK11) is one of the most mutated genes in NSCLC accounting for approximately one-third of cases and its activity is impaired in approximately half of KRAS-mutated NSCLC. At present, these patients cannot benefit from any specific therapy. Through CRISPR/Cas9 technology, we systematically deleted LKB1 in both wild-type (WT) and KRAS-mutated human NSCLC cells. By using these isogenic systems together with genetically engineered mouse models we investigated the cell response to ERK inhibitors both invitro and invivo. In all the systems used here, the loss of LKB1 creates vulnerability and renders these cells particularly sensitive to ERK inhibitors both invitro and invivo. The same cells expressing a WT LKB1 poorly respond to these drugs. At the molecular level, in the absence of LKB1, ERK inhibitors induced a marked inhibition of p90 ribosomal S6 kinase activation, which in turn abolished S6 protein activation, promoting the cytotoxic effect. This work shows that ERK inhibitors are effective in LKB1 and LKB1/KRAS-mutated tumors, thus offering a therapeutic strategy for this prognostically unfavorable subgroup of patients. Because ERK inhibitors are already in clinical development, our findings could be easily translatable to the clinic. Importantly, the lack of effect in cells expressing WT LKB1, predicts that treatment of LKB1-mutated tumors with ERK inhibitors should have a favorable toxicity profile.

In situ study of RSK2 kinase activity in a single living cell by combining single molecule spectroscopy with activity-based probes.

Protein phosphorylation is a very important regulatory mechanism in a majority of biological processes, and the determination of protein kinase activity plays a key role in the pathological study and drug development of kinase-related diseases. However, it is very challenging to in situ study endogenous protein kinase activity in a single living cell due to the shortage of in vivo efficient methods. Here, we propose a new strategy for direct determination of protein kinase activity in a single living cell by combining single molecule fluorescence correlation spectroscopy (FCS) with activity-based probes (ABPs). Ribosomal S6 kinase-2 (RSK2) was used as a model, and the ABPs were synthesized on the basis of RSK2 inhibitor FMK to specially label active RSK2 in living cells. Conventional FCS and MEMFCS (maximum entropy method) single molecule techniques were used to in situ determine RSK2 activity in living cells based on the difference in molecular weight between free probes and probe-RSK2 complexes. Furthermore, wild-type and mutated RSK2 were fused with enhanced green fluorescent protein (EGFP) using lentivirus infection, and fluorescence cross-correlation spectroscopy (FCCS) was used to verify the selective binding of ABPs to RSK2-EGFP fusion protein in living cells. Finally, FCS with ABPs was applied for in situ monitoring of the activation of endogenous RSK2 in the stimulation of serum, epidermal growth factor, kinase inhibitors and ultraviolet irradiation; we observed that endogenous RSK2 showed different behaviors in the cytoplasm and the nucleus in some stimulation. Our results document that FCS with ABPs is a very promising method for studying endogenous protein kinases in living cells.

Fisetin targets YB-1/RSK axis independent of its effect on ERK signaling: insights from in vitro and in vivo melanoma models

The anti-proliferative activity of dietary flavonoid fisetin has been validated in various cancer models. Establishing its precise mechanism of action has proved somewhat challenging given the multiplicity of its targets. We demonstrated that YB-1 promotes epithelial-to-mesenchymal transition and its inhibition suppressed tumor cell proliferation and invasion. The p90 ribosomal S6 kinase (RSK), an important ERK effector, activates YB-1 to drive melanoma growth. We found that fisetin treatment of monolayer/3-D melanoma cultures resulted in YB-1 dephosphorylation and reduced transcript levels. In parallel, fisetin suppressed mesenchymal markers and matrix-metalloproteinases in melanoma cells. Data from cell-free/cell-based systems indicated that fisetin inhibited RSK activity through binding to the kinase. Affinity studies for RSK isoforms evaluated stronger interaction for RSK2 than RSK1. Competition assays performed to monitor binding responses revealed that YB-1 and RSK2 do not compete, rather binding of fisetin to RSK2 promotes its binding to YB-1. Fisetin suppressed YB-1/RSK signaling independent of its effect on ERK, and reduced MDR1 levels. Comparable efficacy of fisetin and vemurafenib for inhibiting melanoma growth was noted albeit through divergent modulation of ERK. Our studies provide insight into additional modes of regulation through which fisetin interferes with melanoma growth underscoring its potential therapeutic efficacy in disease progression.

Stress-Induced Phosphorylation of Nuclear YB-1 Depends on Nuclear Trafficking of p90 Ribosomal S6 Kinase.

Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced DNA double-strand breaks (DSBs) in the nucleus. Thus far, the YB-1 nuclear translocation pattern after cell exposure to various cellular stressors is not clear. In the present study, we investigated the pattern of YB-1 phosphorylation and its possible translocation to the nucleus in KRASwt cells after exposure to IR, EGF treatment, and conditional expression of mutated KRAS(G12V). IR, EGF, and conditional KRAS(G12V) expression induced YB-1 phosphorylation in both the cytoplasmic and nuclear fractions of KRASwt cells. None of the stimuli induced YB-1 nuclear translocation, while p90 ribosomal s6 kinase (RSK) translocation was enhanced in KRASwt cells after any of the stimuli. EGF-induced RSK translocation to the nucleus and nuclear YB-1 phosphorylation were completely blocked by the EGF receptor kinase inhibitor erlotinib. Likewise, RSK inhibition blocked RSK nuclear translocation and nuclear YB-1 phosphorylation after irradiation and KRAS(G12V) overexpression. In summary, acute stimulation of YB-1 phosphorylation does not lead to YB-1 translocation from the cytoplasm to the nucleus. Rather, irradiation, EGF treatment, or KRAS(G12V) overexpression induces RSK activation, leading to its translocation to the nucleus, where it activates already-existing nuclear YB-1. Our novel finding illuminates the signaling pathways involved in nuclear YB-1 phosphorylation and provides a rationale for designing appropriate targeting strategies to block YB-1 in oncology as well as in radiation oncology.

Actin Dysfunction Induces Cell Cycle Delay at G2/M with Sustained ERK and RSK Activation in IMR-90 Normal Human Fibroblasts.

The actin cytoskeleton plays a key role in the entry of mitosis as well as in cytokinesis. In a previous study, we showed that actin disruption delays mitotic entry at G2/M by sustained activation of extracellular signal-related kinase 1/2 (ERK1/2) in primary cells but not in transformed cancer cell lines. Here, we examined the mechanism of cell cycle delay at G2/M by actin dysfunction in IMR-90 normal human fibroblasts. We observed that de-polymerization of actin with cytochalasin D (CD) constitutively activated ribosomal S6 kinase (RSK) and induced inhibitory phosphorylation of Cdc2 (Tyr 15) in IMR-90 cells. In the presence of an actin defect in IMR-90 cells, activating phosphorylation of Wee1 kinase (Ser 642) and inhibitory phosphorylation of Cdc25C (Ser 216) was also maintained. However, when kinase-dead RSK (DN-RSK) was over-expressed, we observed sustained activation of ERK1/2, but no delay in the G2/M transition, demonstrating that RSK functions downstream of ERK in cell cycle delay by actin dysfunction. In DN-RSK overexpressing IMR-90 cells treated with CD, phosphorylation of Cdc25C (Ser 216) was blocked and phosphorylation of Cdc2 (Tyr 15) was decreased, but the phosphorylation of Wee1 (Ser 642) was maintained, demonstrating that RSK directly controls phosphorylation of Cdc25C (Ser 216), but not the activity of Wee1. These results strongly suggest that actin dysfunction in primary cells activates ERK1/2 to inhibit Cdc2, delaying the cell cycle at G2/M by activating downstream RSK, which phosphorylates and blocks Cdc25C, and by directly activating Wee1.

Abstract B72: Signaling and mRNA translation regulation in RSK-deficient LN-18 glioblastoma cell line

Abstract The deregulation of p90 ribosomal S6 kinases (RSKs) seems to be responsible for different oncogenic outcomes in several types of tumors. The effects on mRNA translation regulation via RSKs in glioblastomas (GBMs) were not described so far; thus it is essential to understand the molecular pathways regulated by RSKs that control protein synthesis in GBM. Recent data from our group (unpublished) indicate that different GBM cell lines express distinct levels of the RSK1 isoform, while RSK2 levels are much less variable, and RSK3 and RSK4 are not detected. LN-18 cells show higher levels of RSK1 when compared to A172 or U-118 MG; thus CRISPR/Cas9 system technology was used in this cell line to generate RSK-deficient cells. Briefly, two different sets of small guide RNAs directed to both RSK1 and RSK2 were constructed. LN-18 cells were transfected to express Cas9 along with either one of the constructs targeting RSK1 and/or RSK2. Selection of individual cell clones was followed by Western blotting detection of RSK1 and RSK2 isoforms to identify RSK-deficient cells. We were able to efficiently generate clones with no detection of RSK1 (RSK1-/-), RSK2 (RSK2-/-) with both sets of sgRNAs and cells that had both isoforms knocked out (RSK1-/-/RSK2-/-) only with one of the sgRNA sets. Considering that mTORC1 signaling pathway is the main mediator of RSKs-dependent translation activation, the crosstalk between these pathways and its effects on translation levels were studied in RSKs-deficient cells. Therefore, cells were serum starved for 48 h and stimulated with phorbol-12-myristate-13-acetate (PMA) to activate the Ras-ERK1/2-RSK pathway. Signaling pathways involved in RSK and mTORC1 translation activation were assessed. Interestingly, RSK-deficient cells show lower levels of phosphorylation of Tuberous Sclerosis Complex 2 (TSC-2) at 1798 serine residue, confirming the importance of RSK phosphorylation on this residue and its relevance for evaluation of active RSK levels on cells. In order to describe the influence of RSK1 in the translation initiation rate of GBM cells, polysome profiling was assayed in LN-18, A172, and U-118 MG cells, containing high, low, or nondetected levels of RSK1, respectively. Treatment with PMA for 1, 2, or 4 hours affected translation in GBM cells in a different manner, with the cell lines that display lower levels of RSK1 being less sensitive to the treatment, when compared to LN-18 cells. Polysome profiling was also performed with CRISPR/Cas9-generated cells. RSK1-/- and RSK2-/- cells showed reduced translation initiation levels in basal conditions; however, translation initiation was stimulated after PMA treatment, probably because the remaining isoform is enough to elicit this response. Surprisingly, translation levels were comparable between control cells and RSK1-/-/RSK2-/- cells, suggesting the presence of a compensatory mechanism during the generation of double knockout cells. Of note, this same response could be observed in cells that display alterations in the elongation phase, as it can occur by the lack of phosphorylation of eEF2 kinase in the absence of RSKs. In conclusion, the generation of RSKs-deficient GBM-cell line by the CRISPR/Cas9 system is a useful model for studying and characterizing RSK effects on signaling and translation in GBM cells. Supported by FAPESP. Citation Format: Danielle Nascimento, Martín Roffé, Vilma Regina Martins. Signaling and mRNA translation regulation in RSK-deficient LN-18 glioblastoma cell line [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B72.

Molecular Targeting of ERKs/RSK2 Signaling in Cancers.

Accumulating studies have provided concrete evidence that p90 ribosomal S6 kinase 2 (RSK2) is a key signaling molecule involved in cell proliferation, transformation, and cancer development. RSK2 is known to be an etiological gene of Coffin-Lowry Syndrome (CLS). Recently, signaling analysis and molecular biological approaches have provided concrete evidence that RSK2 plays an essential role in human cancers. Here, we will extensively discuss signaling pathways regulating RSK2 activity, the role of RSK2 in human cancer development, inhibitors suppressing RSK2 activity, and why RSK2 is an important target to develop drugs for human cancers.

Male hormones activate EphA2 to facilitate Kaposi's sarcoma-associated herpesvirus infection: Implications for gender disparity in Kaposi's sarcoma.

There is increasing consensus that males are more vulnerable than females to infection by several pathogens. However, the underlying mechanism needs further investigation. Here, it was showed that knockdown of androgen receptor (AR) expression or pre-treatment with 5α-dihydrotestosterone, the AR agonist, led to a considerably dysregulated Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. In endothelial cells, membrane-localized AR promoted the endocytosis and nuclear trafficking of KSHV. The AR interacted with ephrin receptor A2 (EphA2) and increased its phosphorylation at residue Ser897, which was specifically upregulated upon KSHV infection. This phosphorylation resulted from the AR-mediated recruitment of Src, which resulted in the activation of p90 ribosomal S6 kinase 1 (RSK1), which directly phosphorylates EphA2 at Ser897. Finally, the EphA2-mediated entry of KSHV was abolished in a Ser897Asn EphA2 mutant. Taken together, membrane-localized AR was identified as a KSHV entry factor that cooperatively activates Src/RSK1/EphA2 signaling, which subsequently promotes KSHV infection of both endothelial and epithelial cells.

Defining the role of the RSK isoforms in cancer.

The 90kDa ribosomal S6 kinase (RSK) family is a group of Ser/Thr protein kinases (RSK1-4) that function downstream of the Ras/mitogen-activated protein kinase (MAPK) signalling pathway. RSK regulates many substrates involved in cell survival, growth, and proliferation, and as such, deregulated RSK activity has been associated with multiple cancer types. RSK expression and activity are dysregulated in several malignancies, including breast, prostate, and lung cancer, and available evidence suggests that RSK may be a promising cancer therapeutic target. Current limitations include the lack of RSK inhibitors with suitable pharmacokinetics and selectivity toward particular isoforms. This review briefly describes the current knowledge on RSK activation and function, with a particular emphasis on RSK-dependent mechanisms associated with tumorigenesis and pharmacological inhibition.

Human melanoma cells resistant to MAPK inhibitors can be effectively targeted by inhibition of the p90 ribosomal S6 kinase.

The clinical availability of small molecule inhibitors specifically targeting mutated BRAF marked a significant breakthrough in melanoma therapy. Despite a dramatic anti-tumour activity and improved patient survival, rapidly emerging resistance, however, greatly limits the clinical benefit. The majority of the already described resistance mechanisms involve a reactivation of the MAPK signalling pathway. The p90 ribosomal S6 kinase (RSK), a downstream effector of the MAPK signalling cascade, has been reported to enhance survival of melanoma cells in response to chemotherapy. Here, we can show that RSK activity is significantly increased in human melanoma cells with acquired resistance to the BRAFV600E/K inhibitor vemurafenib. Interestingly, inhibition of RSK signalling markedly impairs the viability of vemurafenib resistant melanoma cells and is effective both in two-dimensional and in three-dimensional culture systems, especially in a chronic, long-term application. The effect of RSK inhibition can be partly replicated by downregulation of the well-known RSK target, Y-box binding protein 1 (YB-1). Intriguingly, RSK inhibition also retains its efficacy in melanoma cells with combined resistance to vemurafenib and the MEK inhibitor trametinib. These data suggest that active RSK signalling might be an attractive novel therapeutic target in melanoma with acquired resistance to MAPK pathway inhibitors.

Electroacupuncture relieves depression-like symptoms in rats exposed to chronic unpredictable mild stress by activating ERK signaling pathway

Electroacupuncture (EA) has been shown to alleviate the symptoms associated with major depressive disorder; however, the underlying mechanisms remain unclear. While the mainstay treatment for depression are pharmacological agents that modulate serotonergic and/or noradrenergic activity of the brain, recent data suggest that, neurotrophins may play a larger role in the pathogenesis of depression and may offer better therapeutic potential in alleviating symptoms associated with depression. One downstream target of neurotrophins is the extracellular signal-regulated kinase (ERK)/Mitogen-activated protein kinase (MAPK) cascade, a major mediator of cellular stress often associated with clinical depression. In this study, we assessed whether the efficacy of EA is due to regulation of these novel pathways using an animal model of depression induced by chronic unpredictable mild stress (CUMS). We found that EA stimulation at specific locations, Baihui (GV20), and Yintang (GV29) ameliorated the behavioral responses of CUMS, which included reduced locomotion, decreased sucrose intake and weight loss. Furthermore, EA increased the activation of ERK and ribosomal s6 kinase (RSK) levels under stress. Both the behavioral and biochemical responses to EA were attenuated with administration of ERK inhibitor, suggesting that EA improves depression-like symptoms in stressed rats, in part, by activation of ERK signaling.

SiRNA Targeting mTOR Effectively Prevents the Proliferation and Migration of Human Lens Epithelial Cells.

Posterior capsule opacification (PCO) is the most common complication that causes visual decrease after extracapsular cataract surgery. The primary cause of PCO formation is the proliferation of the residual lens epithelial cells (LECs). The mammalian target of rapamycin (mTOR) plays an important role in the growth and migration of LECs. In the current study, we used small interfering RNA (siRNA) to specifically attenuate mTOR in human lens epithelial B3 cells (HLE B3). We aimed to examine the effect of mTOR-siRNA on the proliferation, migration and epithelial-to-mesenchymal transition (EMT) of HLE B3 cells and explore the underlying mechanisms. The mTOR-siRNA was transfected into HLE B3 cells using lipofectamine 2000. The mRNA and protein levels of mTOR were examined to confirm the efficiency of mTOR-siRNA. The levels of mRNA and protein as well as the activity of mTOR down-stream effectors p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB, AKT) were examined using real-time PCR or Western blot, respectively. The cell proliferation was determined using cell counting kit (CCK) 8 and cell growth curve assay. The cell migration was examined using Transwell system and Scratch assay. MTOR-siRNA effectively eliminated mTOR mRNA and protein. The proliferation and migration were significantly suppressed by mTOR-siRNA transfection. mTOR-siRNA reduced the mRNA of p70S6K and AKT in a time-dependent manner. Furthermore, the phosphorylation of p70S6K and AKT was decreased by mTOR-siRNA. MTOR-siRNA also eliminated the formation of mTORC1 and mTORC2 protein complex and blocked the transforming growth factor (TGF)-β-induced EMT. Our results suggested that mTOR-siRNA could effectively inhibit the proliferation, migration and EMT of HLE B3 cells through the inhibition of p70S6K and AKT. These results indicated that mTOR-siRNA might be an effective agent inhibiting HLE cells growth and EMT following cataract surgery and provide an alternative therapy for preventing PCO.

Calmodulin Inhibition Rescues DBA Models with Ribosomal Protein Deficiency through Reduction of RSK Signaling

Ribosomal protein (RP) mutations are found in many diseases, including Diamond Blackfan anemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancer risk are major complications. RP mutations are thought to cause p53 activation through accumulation of free RPs that bind and sequester MDM2, the negative regulator of p53. We previously characterized a zebrafish mutant in rps29, a ribosomal gene found mutated in DBA patients. Rps29-/- embryos have hematopoietic and endothelial defects, including decreased cmyb and flk1 expression and defects in hemoglobinization. Consistent with other animal models of RP dysfunction, p53 knockdown in rps29-/- embryos rescued these defects. To uncover novel compounds that correct the phenotypes of DBA, we performed a chemical screen in rps29-/- embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued hemoglobin (Hb) levels in the mutant embryo. To confirm that CaM inhibitors could rescue mammalian models of DBA, we tested them in human and murine models. Treating cord blood-derived CD34+ cells deficient in RPS19, the CaM inhibitor trifluoperazine (TFP) relieved the erythroid differentiation block. Injection of TFP in a DBA murine model significantly increased red blood cell number and Hb levels and reduced p53 activity in the bone marrow. Of note, the effect of TFP was specific to RP deficiency and had no effect on erythroid differentiation or p53 activity in WT cells or mice. This prompted us to hypothesize that TFP is selectively blocking signaling in the RP deficient state, possibly through inhibition of CaM-dependent kinases. In vitro kinase profiling of over 100 kinases revealed that TFP and other positive hits from our screen inhibited the activity of p70 ribosomal S6 kinase (p70S6K) and multiple members of p90 ribosomal S6 kinase (RSK) family. RSKs are highly conserved serine/threonine kinases that regulate cell growth, migration, and survival. They can activate mTOR and directly phosphorylate RPS6. Strikingly, RSK protein levels are elevated in lysates from rps29-/- embryos and treatment with RSK or p70S6K inhibitors increased Hb in rps29-/- embryos in vivo, mimicking CaM inhibitors. RSK phosphorylation is also increased in human RPS19-deficient peripheral blood-derived CD34+ cells compared to WT cells. Treatment with TFP reduced RSK phosphorylation and decreased signaling downstream of RSK, including p70S6K, but only in the presence of RP deficiency. Similarly, TFP reduced p53 and phosphorylation of p53 at S392, but only in the presence of RP deficiency. An in vitro kinase assay determined that p70S6K directly phosphorylated p53 at S392 but not other commonly modified residues, S15 and S20. Mass spectrometry analysis of posttranslational modifications of p53 isolated from TFP treated cells revealed a significant reduction of peptides that contain phosphorylated S392. We hypothesized that TFP inhibited phosphorylation of S392. To test this hypothesis, phosphomimetic mutants were transfected into Saos2 cells and p53 transcriptional activity was evaluated using p21mRNA levels. TFP treatment of cells containing WT p53 or a negative control transactivation domain mutant, p53-S15D, resulted in a 4-fold reduction in p21 mRNA levels, while cells containing p53-S392D had no reduction in p21 mRNA in response to TFP. In conclusion, we have shown that RP deficiency increases RSK phosphorylation and CaM inhibitors decrease signaling downstream of RSK, which leads to a reduction of p53 activity and rescues the phenotypes of multiple in vitro and in vivo models of DBA. Our data strongly suggests that CaM inhibitors may be effective therapies for DBA patients, and a clinical trial is being planned with TFP for 2017. DisclosuresZon:Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Bacterial Expression, Purification and In Vitro Phosphorylation of Full-Length Ribosomal S6 Kinase 2 (RSK2).

Ribosomal S6 kinases (RSK) play important roles in cell signaling through the mitogen-activated protein kinase (MAPK) pathway. Each of the four RSK isoforms (RSK1-4) is a single polypeptide chain containing two kinase domains connected by a linker sequence with regulatory phosphorylation sites. Here, we demonstrate that full-length RSK2—which is implicated in several types of cancer, and which is linked to the genetic Coffin-Lowry syndrome—can be overexpressed with high yields in Escherichia coli as a fusion with maltose binding protein (MBP), and can be purified to homogeneity after proteolytic removal of MBP by affinity and size-exclusion chromatography. The purified protein can be fully activated in vitro by phosphorylation with protein kinases ERK2 and PDK1. Compared to full-length RSK2 purified from insect host cells, the bacterially expressed and phosphorylated murine RSK2 shows the same levels of catalytic activity after phosphorylation, and sensitivity to inhibition by RSK-specific inhibitor SL0101. Interestingly, we detect low levels of phosphorylation in the nascent RSK2 on Ser386, owing to autocatalysis by the C-terminal domain, independent of ERK. This observation has implications for in vivo signaling, as it suggests that full activation of RSK2 by PDK1 alone is possible, circumventing at least in some cases the requirement for ERK.

Chronic High Dose Alcohol Induces Osteopenia via Activation of mTOR Signaling in Bone Marrow Mesenchymal Stem Cells.

Chronic consumption of excessive alcohol results in reduced bone mass, impaired bone structure, and increased risk of bone fracture. However, the mechanisms underlying alcohol-induced osteoporosis are not fully understood. Here, we show that high dose chronic alcohol consumption reduces osteogenic differentiation and enhances adipogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs), leading to osteopenia in a mouse model. Mechanistically, impaired osteo/adipogenic lineage differentiation of BMMSCs is due to activation of a phosphatidylinositide 3-kinase/AKT/mammalian target of rapamycin (mTOR) signaling cascade, resulting in downregulation of runt-related transcription factor 2 and upregulation of peroxisome proliferator-activated receptor gamma via activation of p70 ribosomal protein S6 kinase. Blockage of the mTOR pathway by rapamycin treatment ameliorates alcohol-induced osteopenia by rescuing impaired osteo/adipogenic lineage differentiation of BMMSCs. In this study, we identify a previously unknown mechanism by which alcohol impairs BMMSC lineage differentiation and reveal a potential rapamycin-based drug therapy for alcohol-induced osteoporosis. Stem Cells 2016;34:2157-2168.

Abstract 563: Oxidized LDL Promotes Endothelial NF-kappaB Activation and Inflammation Through Focal Adhesion Kinase-dependent RSK Signaling

Oxidized LDL (oxLDL) accumulates early in atherosclerosis and promotes endothelial NF-kappaB activation, proinflammatory gene expression, and monocyte adhesion. Like other atherogenic factors, oxLDL-induced proinflammatory responses requires integrin-dependent focal adhesion kinase (FAK) signaling; however, the mechanism by which FAK mediates oxLDL-dependent NF-kappaB signaling has yet to be revealed. We now show that oxLDL induces NF-kappaB activation and VCAM-1 expression through FAK-dependent IkappaB kinase beta (IKKbeta) activation. We further identify FAK-dependent activation of p90 ribosomal S6-kinase (RSK) as a critical mediator of oxLDL-dependent IKKbeta/NF-kappaB signaling, as inhibiting RSK blocks oxLDL-induced IKKbeta/NF-kappaB activation, VCAM-1 expression, and monocyte adhesion. Lastly, transgenic mice containing a kinase-dead mutation in FAK specifically in the endothelial cells show reduced RSK activity, decreased VCAM-1 expression, and reduced macrophage accumulation in regions of early atherosclerosis. Taken together, our data elucidates a novel mechanism whereby oxLDL-induced endothelial FAK signaling drives an ERK/RSK pathway to activate IKKbeta/NF-kappaB signaling and proinflammatory gene expression.

Activation of mTORC1 is essential for β-adrenergic stimulation of adipose browning.

A classic metabolic concept posits that insulin promotes energy storage and adipose expansion, while catecholamines stimulate release of adipose energy stores by hydrolysis of triglycerides through β-adrenergic receptor (βARs) and protein kinase A (PKA) signaling. Here, we have shown that a key hub in the insulin signaling pathway, activation of p70 ribosomal S6 kinase (S6K1) through mTORC1, is also triggered by PKA activation in both mouse and human adipocytes. Mice with mTORC1 impairment, either through adipocyte-specific deletion of Raptor or pharmacologic rapamycin treatment, were refractory to the well-known βAR-dependent increase of uncoupling protein UCP1 expression and expansion of beige/brite adipocytes (so-called browning) in white adipose tissue (WAT). Mechanistically, PKA directly phosphorylated mTOR and RAPTOR on unique serine residues, an effect that was independent of insulin/AKT signaling. Abrogation of the PKA site within RAPTOR disrupted βAR/mTORC1 activation of S6K1 without affecting mTORC1 activation by insulin. Conversely, a phosphomimetic RAPTOR augmented S6K1 activity. Together, these studies reveal a signaling pathway from βARs and PKA through mTORC1 that is required for adipose browning by catecholamines and provides potential therapeutic strategies to enhance energy expenditure and combat metabolic disease.