Identification of RSK substrates using an analog-sensitive kinase approach.

The p90 ribosomal S6 kinases (RSK) family of serine/threonine kinases comprises four isoforms (RSK1-4) that lie downstream of the ERK1/2 mitogen-activated protein kinase pathway. RSKs are implicated in fine tuning of cellular processes such as translation, transcription, proliferation, and motility. Previous work showed that pathogens such as Cardioviruses could hijack any of the four RSK isoforms to inhibit PKR activation or to disrupt cellular nucleocytoplasmic trafficking. In contrast, some reports suggest nonredundant functions for distinct RSK isoforms, whereas Coffin-Lowry syndrome has only been associated with mutations in the gene encoding RSK2. In this work, we used the analog-sensitive kinase strategy to ask whether the cellular substrates of distinct RSK isoforms differ. We compared the substrates of two of the most distant RSK isoforms: RSK1 and RSK4. We identified a series of potential substrates for both RSKs in cells and validated RanBP3, PDCD4, IRS2, and ZC3H11A as substrates of both RSK1 and RSK4, and SORBS2 as an RSK1 substrate. In addition, using mutagenesis and inhibitors, we confirmed analog-sensitive kinase data showing that endogenous RSKs phosphorylate TRIM33 at S1119. Our data thus identify a series of potential RSK substrates and suggest that the substrates of RSK1 and RSK4 largely overlap and that the specificity of the various RSK isoforms likely depends on their cell- or tissue-specific expression pattern.

The ribosomal S6 kinase 2 (RSK2)-SPRED2 complex regulates the phosphorylation of RSK substrates and MAPK signaling.

Sprouty-related EVH-1 domain-containing (SPRED) proteins are a family of proteins that negatively regulate the RAS-Mitogen-Activated Protein Kinase (MAPK) pathway, which is involved in the regulation of the mitogenic response and cell proliferation. However, the mechanism by which these proteins affect RAS-MAPK signaling has not been elucidated. Patients with mutations in SPRED give rise to unique disease phenotypes; thus, we hypothesized that distinct interactions across SPRED proteins may account for alternative nodes of regulation. To characterize the SPRED interactome and evaluate how members of the SPRED family function through unique binding partners, we performed affinity purification mass spectrometry. We identified 90-kDa ribosomal S6 kinase 2 (RSK2) as a specific interactor of SPRED2 but not SPRED1 or SPRED3. We identified that the N-terminal kinase domain of RSK2 mediates the interaction between amino acids 123 to 201 of SPRED2. Using X-ray crystallography, we determined the structure of the SPRED2-RSK2 complex and identified the SPRED2 motif, F145A, as critical for interaction. We found that the formation of this interaction is regulated by MAPK signaling events. We also find that this interaction between SPRED2 and RSK2 has functional consequences, whereby the knockdown of SPRED2 resulted in increased phosphorylation of RSK substrates, YB1 and CREB. Furthermore, SPRED2 knockdown hindered phospho-RSK membrane and nuclear subcellular localization. We report that disruption of the SPRED2-RSK complex has effects on RAS-MAPK signaling dynamics. Our analysis reveals that members of the SPRED family have unique protein binding partners and describes the molecular and functional determinants of SPRED2-RSK2 complex dynamics.

RSK inhibitors as potential anticancer agents: Discovery, optimization, and challenges.

Ribosomal S6 kinase (RSK) family is a group of serine/threonine kinases, including four isoforms (RSK1/2/3/4). As a downstream effector of the Ras-mitogen-activated protein kinase (Ras-MAPK) pathway, RSK participates in many physiological activities such as cell growth, proliferation, and migration, and is intimately involved in tumor occurrence and development. As a result, it is recognized as a potential target for anti-cancer and anti-resistance therapies. There have been several RSK inhibitors discovered or designed in recent decades, but only two have entered clinical trials. Low specificity, low selectivity, and poor pharmacokinetic properties in vivo limit their clinical translation. Published studies performed structure optimization by increasing interaction with RSK, avoiding hydrolysis of pharmacophores, eliminating chirality, adapting to binding site shape, and becoming prodrugs. Besides enhancing efficacy, the focus of further design will move towards selectivity since there are functional differences among RSK isoforms. This review summarized the types of cancers associated with RSK, along with the structural characteristics and optimization process of the reported RSK inhibitors. Furthermore, we addressed the importance of RSK inhibitors' selectivity and discussed future drug development directions. This review is expected to shed light on the emergence of RSK inhibitors with high potency, specificity, and selectivity.

Comparison of Conformational Analyses of Naturally Occurring Flavonoid-O-Glycosides with Unnatural Flavonoid-CF2-Glycosides Using Molecular Modeling.

Many glycosylated natural products display biological activity and are deglycosylated by the metabolic processes of the body. Although unnatural CF2-glycosides have been proposed as nonhydrolyzable analogues, CF2-derivatives of natural products are exceedingly challenging to synthesize and few examples exist. These difluorinated molecules may have unique conformational behavior as a consequence of changing the glycosidic linkage. In this study, we performed conformational searches using MacroModel followed by molecular dynamics simulations to investigate the conformational behavior of the glycosidic bonds in flavonoid-O-glycosides and in corresponding CF2-glycosylated derivatives. Compared to their O-glycosylated analogues, flavonoid-3-CF2-glycosides and flavonoid-5-CF2-glycosides showed conformational bias, whereas flavonoid-7-CF2-glycosides showed more flexibility. Flavonoid-5-CF2-glycosides were the least flexible compared to all others. Our results show that the site of the glycosylation and the substitution pattern on the flavonoid determine the conformational properties of these molecules. These two factors influence the steric destabilization and/or stereoelectronic stabilization which govern the conformational behavior of the flavonoid glycosides. Moreover, a docking study of quercitrin and its CF2-analogue into murine ribosomal kinase RSK2 demonstrated the potential for flavonoid-CF2-glycosides to retain a similar binding pose as the parent O-glycoside. These findings will assist in designing stable flavonoid-CF2-glycosides for carbohydrate research.

A non-catalytic herpesviral protein reconfigures ERK-RSK signaling by targeting kinase docking systems in the host.

The Kaposi's sarcoma associated herpesvirus protein ORF45 binds the extracellular signal-regulated kinase (ERK) and the p90 Ribosomal S6 kinase (RSK). ORF45 was shown to be a kinase activator in cells but a kinase inhibitor in vitro, and its effects on the ERK-RSK complex are unknown. Here, we demonstrate that ORF45 binds ERK and RSK using optimized linear binding motifs. The crystal structure of the ORF45-ERK2 complex shows how kinase docking motifs recognize the activated form of ERK. The crystal structure of the ORF45-RSK2 complex reveals an AGC kinase docking system, for which we provide evidence that it is functional in the host. We find that ORF45 manipulates ERK-RSK signaling by favoring the formation of a complex, in which activated kinases are better protected from phosphatases and docking motif-independent RSK substrate phosphorylation is selectively up-regulated. As such, our data suggest that ORF45 interferes with the natural design of kinase docking systems in the host.

Discovery, Optimization, and Structure-Activity Relationship Study of Novel and Potent RSK4 Inhibitors as Promising Agents for the Treatment of Esophageal Squamous Cell Carcinoma.

Ribosomal S6 protein kinase 4 (RSK4) was identified to be a promising target for the treatment of esophageal squamous cell carcinoma (ESCC) in our previous research, whose current treatments are primarily chemotherapy and radiotherapy due to the lack of targeted therapy. However, few potent and specific RSK4 inhibitors are reported. In this study, a series of 1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-ones derivatives were designed and synthesized as novel and potent RSK4 inhibitors. Compound 14f was identified with potent RSK4 inhibitory activity both in vitro and in vivo. 14f significantly inhibited the proliferation and invasion of ESCC cells in vitro with IC50 values of 0.57 and 0.98 µM, respectively. It dose dependently inhibited the phosphorylation of RSK4 downstream substrates while exerting little effect on the substrates of RSK1-3 in ESCC cells. The markedly suppressed tumor growth and no observed toxicity to main organs in the ESCC xenograft mouse model suggested 14f to be a promising RSK4-targeting agent for ESCC treatment.

Involvement of RSK phosphorylation in PTTH-stimulated ecdysone secretion in prothoracic glands of the silkworm, Bombyx mori.

It is well known that phosphorylation of extracellular signal-regulated kinase (ERK) is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we further investigated the downstream signalling pathways. Our results showed that PTTH stimulated p90 ribosomal S6 kinase (RSK) phosphorylation at Thr573 in Bombyx mori PGs both in vitro and in vivo. The in vitro PTTH stimulation was stage- and dose-dependent. The absence of Ca2+ reduced PTTH-stimulated RSK phosphorylation. Stimulation of RSK phosphorylation was also observed after treatment with either A23187 or thapsigargin. A phospholipase C (PLC) inhibitor, U73122, blocked PTTH-stimulated RSK phosphorylation. These results indicate the involvement of Ca2+ and PLC. Treatment with diphenylene iodonium (DPI), a mitochondrial oxidative phosphorylation inhibitor, blocked PTTH-regulated RSK phosphorylation, indicating its redox regulation. A mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, but not a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, decreased PTTH-stimulated RSK phosphorylation, indicating that ERK is an upstream signalling. A protein kinase C (PKC) inhibitor, chelerythrine C, inhibited PTTH-stimulated RSK phosphorylation, and a PKC activator, phorbol 12-myristate acetate (PMA) stimulated RSK phosphorylation, indicating the involvement of PKC. BI-D1870, a specific RSK inhibitor, partly prevented PTTH-stimulated RSK phosphorylation and significantly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated RSK phosphorylation is involved in ecdysteroidogenesis. Taken together, these data indicate that PTTH activates RSK phosphorylation which plays important roles in PTTH-stimulated ecdysteroidogenesis.

MAP Kinase-Mediated Activation of RSK1 and MK2 Substrate Kinases.

Mitogen-activated protein kinases (MAPKs) control essential eukaryotic signaling pathways. While much has been learned about MAPK activation, much less is known about substrate recruitment and specificity. MAPK substrates may be other kinases that are crucial to promote a further diversification of the signaling outcomes. Here, we used a variety of molecular and cellular tools to investigate the recruitment of two substrate kinases, RSK1 and MK2, to three MAPKs (ERK2, p38α, and ERK5). Unexpectedly, we identified that kinase heterodimers form structurally and functionally distinct complexes depending on the activation state of the MAPK. These may be incompatible with downstream signaling, but naturally they may also form structures that are compatible with the phosphorylation of the downstream kinase at the activation loop, or alternatively at other allosteric sites. Furthermore, we show that small-molecule inhibitors may affect the quaternary arrangement of kinase heterodimers and thus influence downstream signaling in a specific manner.

Dual Specificity PDZ- and 14-3-3-Binding Motifs: A Structural and Interactomics Study.

Protein-protein interaction motifs are often alterable by post-translational modifications. For example, 19% of predicted human PDZ domain-binding motifs (PBMs) have been experimentally proven to be phosphorylated, and up to 82% are theoretically phosphorylatable. Phosphorylation of PBMs may drastically rewire their interactomes, by altering their affinities for PDZ domains and 14-3-3 proteins. The effect of phosphorylation is often analyzed by performing "phosphomimetic" mutations. Here, we focused on the PBMs of HPV16-E6 viral oncoprotein and human RSK1 kinase. We measured the binding affinities of native, phosphorylated, and phosphomimetic variants of both PBMs toward the 266 human PDZ domains. We co-crystallized all the motif variants with a selected PDZ domain to characterize the structural consequence of the different modifications. Finally, we elucidated the structural basis of PBM capture by 14-3-3 proteins. This study provides novel atomic and interactomic insights into phosphorylatable dual specificity motifs and the differential effects of phosphorylation and phosphomimetic approaches.

Activation of RSK by phosphomimetic substitution in the activation loop is prevented by structural constraints.

The activation of the majority of AGC kinases is regulated by two phosphorylation events on two conserved serine/threonine residues located on the activation loop and on the hydrophobic motif, respectively. In AGC kinase family, phosphomimetic substitutions with aspartate or glutamate, leading to constitutive activation, have frequently occurred at the hydrophobic motif site. On the contrary, phosphomimetic substitutions in the activation loop are absent across the evolution of AGC kinases. This observation is explained by the failure of aspartate and glutamate to mimic phosphorylatable serine/threonine in this regulatory site. By detailed 3D structural simulations of RSK2 and further biochemical evaluation in cells, we show that the phosphomimetic residue on the activation loop fails to form a critical salt bridge with R114, necessary to reorient the αC-helix and to activate the protein. By a phylogenetic analysis, we point at a possible coevolution of a phosphorylatable activation loop and the presence of a conserved positively charged amino acid on the αC-helix. In sum, our analysis leads to the unfeasibility of phosphomimetic substitution in the activation loop of RSK and, at the same time, highlights the peculiar structural role of activation loop phosphorylation.

Isobavachalcone exerts anti­proliferative and pro­apoptotic effects on human liver cancer cells by targeting the ERKs/RSK2 signaling pathway.

Aberrant activation of the extracellular signal­regulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathway is frequently determined in various human tumor types, including liver cancer, and has been considered as a promising target for cancer chemoprevention and therapy. In the present study, using computer­aided virtual screening and molecular docking, isobavachalcone (IBC), a natural chalcone compound, was identified to be an ATP­competitive inhibitor targeting ERK1/2 and RSK2. Cell Counting Kit­8, EdU incorporation and colony formation assays were used to detect the effects of IBC on cell viability and proliferation, and the results demonstrated that IBC effectively inhibited the proliferation of liver cancer HepG2 and Hep3B cells, whereas it had no notable cytotoxic effect on immortal liver L02 cells. Flow cytometric analysis and western blotting further revealed that IBC caused significant levels of apoptosis on liver cancer cells via the caspase­dependent mitochondria pathway. The computer prediction was confirmed with pull­down and in vitro kinase assays, in which IBC directly bound with ERK1/2 and RSK2, and dose­dependently blocked RSK2 kinase activity in liver cancer cells. Treatment of HepG2 or Hep3B cells with IBC significantly attenuated epidermal growth factor­induced phosphorylation of RSK2 and resulted in the reduced activation of its downstream substrates including cAMP response element­binding protein, activating transcription factor 1, histone H3 and activating protein­1. Enforced RSK2 expression in L02 cells could increase the effect of IBC on suppressing cell growth. Conversely, knockdown of RSK2 reduced the inhibitory effect of IBC on HepG2 cell proliferation. Overall, the present data indicated that ERKs/RSK2 signaling serves a pivotal role in IBC­induced suppression of liver cancer cells and that IBC may be a potential therapeutic candidate for human cancer with elevated ERKs/RSK2 activity.

Rewiring of RSK-PDZ Interactome by Linear Motif Phosphorylation.

Phosphorylation of short linear peptide motifs is a widespread process for the dynamic regulation of protein-protein interactions. However, the global impact of phosphorylation events on the protein-protein interactome is rarely addressed. The disordered C-terminal tail of ribosomal S6 kinase 1 (RSK1) binds to PDZ domain-containing scaffold proteins, and it harbors a phosphorylatable PDZ-binding motif (PBM) responsive to epidermal growth factor stimulation. Here, we examined binding of two versions of the RSK1 PBM, either phosphorylated or unphosphorylated at position -3, to almost all (95%) of the 266 PDZ domains of the human proteome. PBM phosphorylation dramatically altered the PDZ domain-binding landscape of RSK1, by strengthening or weakening numerous interactions to various degrees. The RSK-PDZome interactome analyzed in this study reveals how linear motif-based phospho-switches convey stimulus-dependent changes in the context of related network components.

Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases.

Dimethyl fumarate (DMF) has been applied for decades in the treatment of psoriasis and now also multiple sclerosis. However, the mechanism of action has remained obscure and involves high dose over long time of this small, reactive compound implicating many potential targets. Based on a 1.9 Å resolution crystal structure of the C-terminal kinase domain of the mouse p90 Ribosomal S6 Kinase 2 (RSK2) inhibited by DMF we describe a central binding site in RSKs and the closely related Mitogen and Stress-activated Kinases (MSKs). DMF reacts covalently as a Michael acceptor to a conserved cysteine residue in the αF-helix of RSK/MSKs. Binding of DMF prevents the activation loop of the kinase from engaging substrate, and stabilizes an auto-inhibitory αL-helix, thus pointing to an effective, allosteric mechanism of kinase inhibition. The biochemical and cell biological characteristics of DMF inhibition of RSK/MSKs are consistent with the clinical protocols of DMF treatment.

Identification of p90 Ribosomal S6 Kinase 2 as a Novel Host Protein in HBx Augmenting HBV Replication by iTRAQ-Based Quantitative Comparative Proteomics.

PURPOSE: The aim of this study was to screen for novel host proteins that play a role in HBx augmenting Hepatitis B virus (HBV) replication. EXPERIMENTAL DESIGN: Three HepG2 cell lines stably harboring different functional domains of HBx (HBx, HBx-Cm6, and HBx-Cm16) were cultured. ITRAQ technology integrated with LC-MS/MS analysis was applied to identify the proteome differences among these three cell lines. RESULTS: In brief, a total of 70 different proteins were identified among HepG2-HBx, HepG2-HBx-Cm6, and HepG2-HBx-Cm16 by double repetition. Several differentially expressed proteins, including p90 ribosomal S6 kinase 2 (RSK2), were further validated. RSK2 was expressed at higher levels in HepG2-HBx and HepG2-HBx-Cm6 compared with HepG2-HBx-Cm16. Furthermore, levels of HBV replication intermediates were decreased after silencing RSK2 in HepG2.2.15. An HBx-minus HBV mutant genome led to decreased levels of HBV replication intermediates and these decreases were restored to levels similar to wild-type HBV by transient ectopic expression of HBx. After silencing RSK2 expression, the levels of HBV replication intermediates synthesized from the HBx-minus HBV mutant genome were not restored to levels that were observed with wild-type HBV by transient HBx expression. CONCLUSION AND CLINICAL RELEVANCE: Based on iTRAQ quantitative comparative proteomics, RSK2 was identified as a novel host protein that plays a role in HBx augmenting HBV replication.

Dynamic control of RSK complexes by phosphoswitch-based regulation.

Assembly and disassembly of protein-protein complexes needs to be dynamically controlled and phosphoswitches based on linear motifs are crucial in this process. Extracellular signal-regulated kinase 2 (ERK2) recognizes a linear-binding motif at the C-terminal tail (CTT) of ribosomal S6 kinase 1 (RSK1), leading to phosphorylation and subsequent activation of RSK1. The CTT also contains a classical PDZ domain-binding motif which binds RSK substrates (e.g. MAGI-1). We show that autophosphorylation of the disordered CTT promotes the formation of an intramolecular charge clamp, which efficiently masks critical residues and indirectly hinders ERK binding. Thus, RSK1 CTT operates as an autoregulated phosphoswitch: its phosphorylation at specific sites affects its protein-binding capacity and its conformational dynamics. These biochemical feedbacks, which form the structural basis for the rapid dissociation of ERK2-RSK1 and RSK1-PDZ substrate complexes under sustained epidermal growth factor (EGF) stimulation, were structurally characterized and validated in living cells. Overall, conformational changes induced by phosphorylation in disordered regions of protein kinases, coupled to allosteric events occurring in the kinase domain cores, may provide mechanisms that contribute to the emergence of complex signaling activities. In addition, we show that phosphoswitches based on linear motifs can be functionally classified as ON and OFF protein-protein interaction switches or dimmers, depending on the specific positioning of phosphorylation target sites in relation to functional linear-binding motifs. Moreover, interaction of phosphorylated residues with positively charged residues in disordered regions is likely to be a common mechanism of phosphoregulation. DATABASE: Structural data are available in the PDB database under the accession numbers 5N7D, 5N7F and 5N7G. NMR spectral assignation data are available in the BMRB database under the accession numbers 27213 and 27214.

The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1.

The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.

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.

[Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1].

Objective To investigate the regulatory effect of post-translation modification of ribosomal protein S6 kinase 1 (S6K1) on the polyploidization of megakaryocytes. Methods SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and H-89, a cAMP-dependent protein kinase (PKA) inhibitor, were used to treat CMK cells separately or in combination. With propidium iodide (PI) to dye DNA in the treated cells, the relative DNA content was detected by flow cytometry, and then the DNA polyploidy was analyzed. The change of expression and phosphorylation of ribosomal protein S6 kinase 1 (S6K1), an important mammalian target of rapamycin (mTOR) downstream target molecule, was analyzed by Western blotting. Molecular docking study and kinase activity assay were performed to analyze the combination of H-89 with S6K1 and the effect of H-89 on the activity of S6K1 kinase. Results SP600125 induced CMK cell polyploidization in a time-dependent and dose-dependent manner. At the same time, it increased the phosphorylation of S6K1 at Thr421/Ser424 and decreased the phosphorylation of S6K1 at Thr389. H-89 not only blocked polyploidization, but also decreased the phosphorylation of S6K1 at Thr421/Ser424 and increased the phosphorylation of S6K1 at Thr389. Molecular docking and kinase activity assay showed that H-89 occupied the ATP binding sites of S6K1 and inhibited its activity. Noticeably, both H-89 and SP600125 inhibited the activity of PKA. Moreover, the two drugs further inhibited the activity of PKA when used together. Therefore, these data indicated that H-89 blocked the SP600125-induced polyploidization of CMK cells mainly by changing S6K1 phosphorylation state, rather than its inhibitory effect on PKA. Conclusion H-89 can block the polyploidization of SP600125-induced CMK cells by regulating S6K1 phosphorylation state.

Functional Basis and Biophysical Approaches to Characterize the C-Terminal Domain of Human-Ribosomal S6 Kinases-3.

Ribosomal S6 kinases (RSKs) are the major functional components in mitogen-activated protein kinase (MAPK) pathway, and these are activated by upstream Extracellular signal-regulated kinase. Upon activation, RSKs activate a number of substrate molecules involved in transcription, translation and cell-cycle regulation. But how cellular binding partners are engaged in the MAPK pathways and regulate the molecular mechanisms have not been explored. Considering the importance of protein-protein interactions in cell signalling and folding pattern of native protein, functional C-terminal kinase domain of RSK3 has been characterized using in vitro, in silico and biophysical approaches. RSKs discharge different functions by binding to downstream kinase partners. Hence, depending upon cellular binding partners, RSKs translocate between cytoplasm and nucleus. In our study, it has been observed that the refolded C-terminal Kinase domain (CTKD) of RSK 3 has a compact domain structure which is predominantly α-helical in nature by burying the tryptophans deep into the core, which was confirmed by CD, Fluorescence spectroscopy and limited proteolysis assay. Our study also revealed that RSK 3 CTKD was found to be a homotrimer from DLS experiments. A model was also built for RSK 3 CTKD and was further validated using PROCHECK and ProSA webservers.

p90 ribosomal S6 kinase: a potential therapeutic target in lung cancer.

A global survey of cancer has shown that lung cancer is the most common cause of the new cancer cases and cancer deaths in men worldwide. The mortality from lung cancer is more than the combined mortality from breast, prostate and colorectal cancers. The two major histological types of lung cancer are non-small cell lung cancer (NSCLC) accounting for about 85 % of cases and small cell lung cancer accounting for 15 % of cases. NSCLC, the more prevalent form of lung cancer, is often diagnosed at an advanced stage and has a very poor prognosis. Many factors have been shown to contribute to the development of lung cancer in humans including tobacco smoking, exposure to environmental carcinogens (asbestos, or radon) and genetic factors. Despite the advances in treatment, lung cancer remains one of the leading causes of cancer death worldwide. Interestingly, the overall 5 year survival from lung cancer has not changed appreciably in the past 25 years. For this reason, novel and more effective treatments and strategies for NSCLC are critically needed. p90 ribosomal S6 kinase (RSK), a serine threonine kinase that lies downstream of the Ras-MAPK (mitogen activated protein kinase) cascade, has been demonstrated to be involved in the regulation of cell proliferation in various malignancies through indirect (e.g., modulation of transcription factors) or direct effects on the cell-cycle machinery. Increased expression of RSK has been demonstrated in various cancers, including lung cancer. This review focuses on the role of RSK in lung cancer and its potential therapeutic application.