Validation of known and novel AKT, S6K and RSK targets by targeted MS
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Cell Culture
SUBMITTER: 
Friedel Drepper
LAB HEAD: Prof. Dr. Bettina Warscheid
PROVIDER: PXD029633 | Pride | 2023-02-22
REPOSITORIES: Pride
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Project description:The signaling network of skeletal muscle cells is controlled by a variety of protein kinases. Although many kinases are known players, their downstream targets are still largely unexplored. To gain further knowledge about the PI3K-AKT-mTOR-S6K and the RAF-MEK-ERK-RSK signaling networks in myotubes, we analyzed changes in protein phosphorylation levels upon pathway activation and direct kinase inhibition on a global scale. Based on the phosphoproteomics data, we further examined target relationships of the basophilic kinases AKT, RSK and S6K, which share the substrate recognition motif RxRxxp[ST].
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Project description:Various protein kinases are regulating the intracellular signaling network of skeletal muscle cells. Despite that many of the involved kinases are known, their downstream targets have remained largely unexplored. To deepen our understanding of the PI3K-AKT-mTOR-S6K and the RAF-MEK-ERK-RSK signaling network in myotubes, we globally analyzed changes in protein phosphorylation levels upon kinase inhibition within these pathways. The phosphoproteomics data were used to define potential targets of the kinases AKT, RSK and S6K, which share the substrate recognition motif RxRxxp[ST].
2023-02-22 | PXD018667 | Pride
Project description:Cytokinesis is the final stage in cell division. Although integrins can regulate cytokinesis, the mechanisms involved are not fully understood. In this study, we demonstrate that integrin-regulated ERK (extracellular signal-related kinase) and RSK (p90 ribosomal S6 kinase) signaling promotes successful cytokinesis. Inhibiting the activation of ERK and RSK in CHO cells by a mutation in the integrin ?1 cytoplasmic tail or with pharmacological inhibitors results in the accumulation of cells with midbodies and the formation of binucleated cells. Activation of ERK and RSK signaling by the expression of constitutively active RAF1 suppresses the mutant phenotype in a RSK-dependent manner. Constitutively active RSK2 also restores cytokinesis inhibited by the mutant integrin. Importantly, the regulatory role of the RSK pathway is not specific to CHO cells. MCF-10A human mammary epithelial cells and HPNE human pancreatic ductal epithelial cells exhibit a similar dependence on RSK for successful cytokinesis. In addition, depriving mitotic MCF10A cells of integrin-mediated adhesion by incubating them in suspension suppressed ERK and RSK activation and resulted in a failure of cytokinesis. Furthermore, inhibition of RSK or integrins within the 3D context of a developing salivary gland organ explant also leads to an accumulation of epithelial cells with midbodies, suggesting a similar defect in cytokinesis. Interestingly, neither ERK nor RSK regulates cytokinesis in human fibroblasts, suggesting cell-type specificity. Taken together, our results identify the integrin-RSK signaling axis as an important regulator of cytokinesis in epithelial cells. We propose that the proper interaction of cells with their microenvironment through integrins contributes to the maintenance of genomic stability by promoting the successful completion of cytokinesis.
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Project description:During ischemia/reperfusion (I/R), ribosomal S6 kinase (RSK) activates Na(+)/H(+) exchanger 1 (NHE1) by phosphorylating NHE1 at serine 703 (pS703-NHE1), which promotes cardiomyocyte death and injury. Pharmacologic inhibition of NHE1 effectively protects animal hearts from I/R. However, clinical trials using NHE1 inhibitors failed to show benefit in patients with acute myocardial infarction (MI). One possible explanation is those inhibitors block both agonist-stimulated activity (increasing I/R injury) and basal NHE1 activity (necessary for cell survival). We previously showed that dominant-negative RSK (DN-RSK) selectively blocked agonist-stimulated NHE1 activity. Therefore, we hypothesized that a novel RSK inhibitor (BIX02565) would blunt agonist-stimulated NHE1 and protect hearts from I/R.Serum/angiotensin II-stimulated pS703-NHE1 was significantly decreased by BIX02565 in cultured cells. Intracellular pH recovery assay showed that BIX02565 selectively inhibited serum-stimulated NHE1 activity. Ischemia/reperfusion decreased left ventricular-developed pressure (LVDP; inhibited) to 8.7% of the basal level in non-transgenic littermate control (NLC) mouse hearts, which was significantly improved (44.6%) by BIX02565. Similar protection was observed in vehicle-treated, cardiac-specific DN-RSK-Tg mice (43%). No additional protective effect was seen in BIX02565-treated DN-RSK-Tg hearts. BIX02565 also improved LVDP in cardiac-specific wild-type (WT)-RSK-Tg mouse hearts (7.4%-40.9%, P < .01). Finally, Western Blotting results confirmed DN-RSK and BIX02565 significantly decreased I/R-induced pS703-NHE1.The RSK plays a crucial role in I/R-induced activation of NHE1 and cardiac injury. The RSK inhibition may provide an alternative target for patients with MI.
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Project description:Activation of the mitogen-activated protein kinase (MAPK) cascade in mammalian cell lines positively regulates the G2/M transition. The molecular mechanism underlying this biological phenomenon remains poorly understood. Ribosomal S6 kinase (RSK) is a key downstream element of the MAPK cascade. Our previous studies established roles of RSK2 in Cdc25C activation during progesterone-induced meiotic maturation of Xenopus oocytes. In this study we demonstrate that both recombinant RSK and endogenous RSK in Xenopus egg extracts phosphorylate all three isoforms of human Cdc25 at a conserved motif near the catalytic domain. In human HEK293 and PC-3mm2 cell lines, RSK preferentially phosphorylates Cdc25A and Cdc25B in mitotic cells. Phosphorylation of the RSK sites in these Cdc25 isoforms increases their M-phase-inducing activities. Inhibition of RSK-mediated phosphorylation of Cdc25 inhibits G2/M transition. Moreover, RSK is likely to be more active in mitotic cells than in interphase cells, as evidenced by the phosphorylation status of T359/S363 in RSK. Together, these findings indicate that RSK promotes G2/M transition in mammalian cells through activating phosphorylation of Cdc25A and Cdc25B.
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Project description:p90(rsk) is a distal member of the mitogen-activated protein kinase signaling pathway. It has been cloned from a variety of species including Xenopus laevis, mouse, chicken, rat, and human. The clone p90(rsk-mo-1), isolated by others from a mouse library, contains a unique 33-nucleotide deletion not found in the p90(rsk) clones from any other species that have been examined. When p90(rsk-mo-1) was expressed in Cos-7 cells that were subsequently stimulated with epidermal growth factor, the immunoprecipitated p90(rsk-mo-1) protein showed no measurable kinase activity toward the ribosomal protein S6 peptide. By comparison, expression of rat p90(rsk-1) resulted in significant kinase activity. Deletion of the 33-nucleotide region missing in the p90(rsk-mo-1) clone from the p90(rsk-rat-1) cDNA abolished kinase activity in the resulting protein. When these 33 nucleotides were introduced into the p90(rsk-mo-1) cDNA, the expressed protein showed significant kinase activity. Reverse transcription-PCR and direct sequencing of mRNA isolated from several mouse tissues indicated the presence of the full-length form of p90(rsk-1) in the mouse and showed no conclusive evidence for a deletion-containing form. This study indicates the presence of a full-length p90(rsk-1) mRNA in mouse tissues that is homologous to that identified in other species and suggests that the deletion in p90(rsk-mo-1) may be a cloning artifact. The findings provide additional support for the conclusion that the first catalytic domain of p90(rsk) is responsible for its enzymatic activity toward ribosomal protein S6.
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Project description:Rationale: Cartilage stem/progenitor cells (CSPC) are a promising cellular source to promote endogenous cartilage regeneration in osteoarthritis (OA). Our previous work indicates that ribosomal s6 kinase 3 (RSK-3) is a target of 4-aminobiphenyl, a chemical enhancing CSPC-mediated cartilage repair in OA. However, the primary function and mechanism of RSK-3 in CSPC-mediated cartilage pathobiology remain undefined. Methods: We systematically assessed the association of RSK-3 with OA in three mouse strains with varying susceptibility to OA (MRL/MpJ>CBA>STR/Ort), and also RSK-3-/- mice. Bioinformatic analysis was used to identify the possible mechanism of RSK-3 affecting CSPC, which was further verified in OA mice and CSPC with varying RSK-3 expression induced by chemicals or gene modification. Results: We demonstrated that the level of RSK-3 in cartilage was positively correlated with cartilage repair capacities in three mouse strains (MRL/MpJ>CBA>STR/Ort). Enhanced RSK-3 expression by 4-aminobiphenyl markedly attenuated cartilage injury in OA mice and inhibition or deficiency of RSK-3 expression, on the other hand, significantly aggravated cartilage damage. Transcriptional profiling of CSPC from mice suggested the potential role of RSK-3 in modulating cell proliferation. It was further shown that the in vivo and in vitro manipulation of the RSK-3 expression indeed affected the CSPC proliferation. Mechanistically, ribosomal protein S6 (rpS6) was activated by RSK-3 to accelerate CSPC growth. Conclusion: RSK-3 is identified as a key regulator to enhance cartilage repair, at least partly by regulating the functionality of the cartilage-resident stem/progenitor cells.
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Project description:The Ser/Thr protein kinase, RSK, is important in the etiology of tumor progression including invasion and motility. The natural product kaempferol-3-O-(3?,4?-di-O-acetyl-?-l-rhamnopyranoside), called SL0101, is a highly specific RSK inhibitor. Acylation of the rhamnose moiety is necessary for high affinity binding and selectivity. However, the acetyl groups can be cleaved by esterases, which accounts for the poor in vitro biological stability of SL0101. To address this problem a series of analogs containing acetyl group replacements were synthesized and their in vitro stability evaluated. Monosubstituted carbamate analogs of SL0101 showed improved in vitro biological stability while maintaining specificity for RSK. These results should facilitate the development of RSK inhibitors derived from SL0101 as anticancer agents.
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