P90 ribosomal S6 kinase confers cancer cell survival by mediating checkpoint kinase 1 degradation in response to glucose stress.
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ABSTRACT:
SUBMITTER: Ma Y
PROVIDER: S-EPMC8748233 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Serine/Threonine protein kinase family, p90 ribosomal S6 kinases (RSK) are downstream effectors of extracellular signal regulated kinase 1/2 (ERK1/2) and are activated in response to tyrosine kinase receptor or G-protein coupled receptor signaling. RSK contains two distinct kinase domains, an N-terminal kinase (NTKD) and a C-terminal kinase (CTKD). The sole function of the CTKD is to aid in the activation of the NTKD, which is responsible for substrate phosphorylation. RSK regulates various homeostatic processes including those involved in transcription, translation and ribosome biogenesis, proliferation and survival, cytoskeleton, nutrient sensing, excitation and inflammation. RSK also acts as a major negative regulator of ERK1/2 signaling. RSK is associated with numerous cancers and has been primarily studied in the context of transformation and metastasis. The development of specific RSK inhibitors as cancer therapeutics has lagged behind that of other members of the mitogen-activated protein kinase signaling pathway. Importantly, a pan-RSK inhibitor, PMD-026, is currently in phase I/1b clinical trials for metastatic breast cancer. However, there are four members of the RSK family, which have overlapping and distinct functions that can vary in a tissue specific manner. Thus, a problem for transitioning a RSK inhibitor to the clinic may be the necessity to develop isoform specific inhibitors, which will be challenging as the NTKDs are very similar to each other. CTKD inhibitors have limited use as therapeutics as they are not able to inhibit the activity of the NTKD but could be used in the development of proteolysis-targeting chimeras.
| S-EPMC10758423 | biostudies-literature
Project description:RationaleCardiac myocyte hypertrophy is the main compensatory response to chronic stress on the heart. p90 ribosomal S6 kinase (RSK) family members are effectors for extracellular signal-regulated kinases that induce myocyte growth. Although increased RSK activity has been observed in stressed myocytes, the functions of individual RSK family members have remained poorly defined, despite being potential therapeutic targets for cardiac disease.ObjectiveTo demonstrate that type 3 RSK (RSK3) is required for cardiac myocyte hypertrophy.Methods and resultsRSK3 contains a unique N-terminal domain that is not conserved in other RSK family members. We show that this domain mediates the regulated binding of RSK3 to the muscle A-kinase anchoring protein scaffold, defining a novel kinase anchoring event. Disruption of both RSK3 expression using RNA interference and RSK3 anchoring using a competing muscle A-kinase anchoring protein peptide inhibited the hypertrophy of cultured myocytes. In vivo, RSK3 gene deletion in the mouse attenuated the concentric myocyte hypertrophy induced by pressure overload and catecholamine infusion.ConclusionsTaken together, these data demonstrate that anchored RSK3 transduces signals that modulate pathologic myocyte growth. Targeting of signaling complexes that contain select kinase isoforms should provide an approach for the specific inhibition of cardiac myocyte hypertrophy and for the development of novel strategies for the prevention and treatment of heart failure.
| S-EPMC3537852 | biostudies-literature
Project description:In the canonical model of smooth muscle (SM) contraction, the contractile force is generated by phosphorylation of the myosin regulatory light chain (RLC20) by the myosin light chain kinase (MLCK). Moreover, phosphorylation of the myosin targeting subunit (MYPT1) of the RLC20 phosphatase (MLCP) by the RhoA-dependent ROCK kinase, inhibits the phosphatase activity and consequently inhibits dephosphorylation of RLC20 with concomitant increase in contractile force, at constant intracellular [Ca(2+)]. This pathway is referred to as Ca(2+)-sensitization. There is, however, emerging evidence suggesting that additional Ser/Thr kinases may contribute to the regulatory pathways in SM. Here, we report data implicating the p90 ribosomal S6 kinase (RSK) in SM contractility. During both Ca(2+)- and agonist (U46619) induced SM contraction, RSK inhibition by the highly selective compound BI-D1870 (which has no effect on MLCK or ROCK) resulted in significant suppression of contractile force. Furthermore, phosphorylation levels of RLC20 and MYPT1 were both significantly decreased. Experiments involving the irreversible MLCP inhibitor microcystin-LR, in the absence of Ca(2+), revealed that the decrease in phosphorylation levels of RLC20 upon RSK inhibition are not due solely to the increase in the phosphatase activity, but reflect direct or indirect phosphorylation of RLC20 by RSK. Finally, we show that agonist (U46619) stimulation of SM leads to activation of extracellular signal-regulated kinases ERK1/2 and PDK1, consistent with a canonical activation cascade for RSK. Thus, we demonstrate a novel and important physiological function of the p90 ribosomal S6 kinase, which to date has been typically associated with the regulation of gene expression.
| S-EPMC3596281 | biostudies-literature
Project description:The p90 ribosomal protein kinase 2 (RSK2) is a highly expressed Ser/Thr kinase activated by growth factors and is involved in cancer cell proliferation and tumor promoter-induced cell transformation. RSK2 possesses two non-identical kinase domains, and the structure of its N-terminal domain (NTD), which is responsible for phosphorylation of a variety of substrates, is unknown. The crystal structure of the NTD RSK2 was determined at 1.8 A resolution in complex with AMP-PNP. The N-terminal kinase domain adopted a unique active conformation showing a significant structural diversity of the kinase domain compared to other kinases. The NTD RSK2 possesses a three-stranded betaB-sheet inserted in the N-terminal lobe, resulting in displacement of the alphaC-helix and disruption of the Lys-Glu interaction, classifying the kinase conformation as inactive. The purified protein was phosphorylated at Ser227 in the T-activation loop and exhibited in vitro kinase activity. A key characteristic is the appearance of a new contact between Lys216 (betaB-sheet) and the beta-phosphate of AMP-PNP. Mutation of this lysine to alanine impaired both NTDs in vitro and full length RSK2 ex vivo activity, emphasizing the importance of this interaction. Even though the N-terminal lobe undergoes structural re-arrangement, it possesses an intact hydrophobic groove formed between the alphaC-helix, the beta4-strand, and the betaB-sheet junction, which is occupied by the N-terminal tail. The presence of a unique betaB-sheet insert in the N-lobe suggests a different type of activation mechanism for RSK2.
| S-EPMC2779450 | biostudies-literature
Project description:Previously, we reported that the catalytic subunit of cAMP-dependent protein kinase (PKAc) binds to the active p90 ribosomal S6 kinase 1 (RSK1) (Chaturvedi, D., Poppleton, H. M., Stringfield, T., Barbier, A., and Patel, T. B. (2006) Mol. Cell. Biol. 26, 4586-4600). Herein, by overexpressing hemagglutinin-tagged RSK1 fragments in HeLa cells we have identified the region of RSK1 that is responsible for the interaction with PKAc. PKAc bound to the last 13 amino acids of RSK1, which overlaps the Erk1/2 docking site. This interaction between PKAc and RSK1 required the phosphorylation of Ser-732 in the C terminus of RSK1. Depending upon its phosphorylation status, RSK1 switched interactions between Erk1/2 and PKAc. In addition, a peptide corresponding to the last 13 amino acids of RSK1 with substitution of Ser-732 with Glu (peptide E), but not Ala (peptide A), decreased interactions between endogenous active RSK1 and PKAc. RSK1 attenuated the ability of cAMP to activate PKA in vitro and this modulation was abrogated by peptide E, but not by peptide A. Similarly, in intact cells, cAMP-mediated phosphorylation of Bcl-xL/Bcl-2-associated death promoter on Ser-115, the PKA site, was reduced when RSK1 was activated by epidermal growth factor, and this effect was blocked by peptide E, but not by peptide A. These findings demonstrate that interactions between endogenous RSK1 and PKAc in intact cells regulate the ability of cAMP to activate PKA and identify a novel mechanism by which PKA activity is regulated by the Erk1/2 pathway.
| S-EPMC2785148 | biostudies-other
Project description:Chaperonin containing TCP-1 (CCT) is a large multisubunit complex that mediates protein folding in eukaryotic cells. CCT participates in the folding of newly synthesized polypeptides, including actin, tubulin, and several cell cycle regulators; therefore, CCT plays an important role in cytoskeletal organization and cell division. Here we identify the chaperonin CCT as a novel physiological substrate for p90 ribosomal S6 kinase (RSK) and p70 ribosomal S6 kinase (S6K). RSK phosphorylates the beta subunit of CCT in response to tumor promoters or growth factors that activate the Ras-mitogen-activated protein kinase (MAPK) pathway. CCTbeta Ser-260 was identified as the RSK site by mass spectrometry and confirmed by site-directed mutagenesis. RSK-dependent Ser-260 phosphorylation was sensitive to the MEK inhibitor UO126 and the RSK inhibitor BID-1870. Insulin weakly activates RSK but strongly activates the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway and utilizes S6K to regulate CCTbeta phosphorylation. Thus, the Ras-MAPK and PI3K-mTOR pathways converge on CCTbeta Ser-260 phosphorylation in response to multiple agonists in various mammalian cells. We also show that RNA interference-mediated knockdown of endogenous CCTbeta causes impaired cell proliferation that can be rescued with ectopically expressed murine CCTbeta wild-type or phosphomimetic mutant S260D, but not the phosphorylation-deficient mutant S260A. Although the molecular mechanism of CCTbeta regulation remains unclear, our findings demonstrate a link between oncogene and growth factor signaling and chaperonin CCT-mediated cellular activities.
| S-EPMC2685676 | biostudies-literature
Project description:In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.
| S-EPMC3037642 | biostudies-literature
Project description:RSK2 (p90 ribosomal S6 kinase 2) is activated via the ERK (extracellular-signal-regulated kinase) pathway by phosphorylation on four sites: Ser227 in the activation loop of the N-terminal kinase domain, Ser369 in the linker, Ser386 in the hydrophobic motif and Thr577 in the C-terminal kinase domain of RSK2. In the present study, we demonstrate that RSK2 is associated in vivo with PP2Cdelta (protein phosphatase 2Cdelta). In epidermal growth factorstimulated cells, RSK2 is partially dephosphorylated on all four sites in an Mn2+-dependent manner, leading to reduced protein kinase activity. Furthermore, PP2Cd is phosphorylated by ERK on Thr315 and Thr333 in the catalytic domain. Mutation of Thr315 and Thr333 to alanine in a catalytically inactive mutant PP2Cdelta (H154D) (His154-->Asp) increases the association with RSK2 significantly, whereas mutation to glutamate, mimicking phosphorylation, reduces the binding of RSK2. The domains of interaction are mapped to the N-terminal extension comprising residues 1-71 of PP2Cd and the N-terminal kinase domain of RSK2. The interaction is specific, since PP2Cd associates with RSK1-RSK4, MSK1 (mitogen- and stress-activated kinase 1) and MSK2, but not with p70 S6 kinase or phosphoinositide-dependent kinase 1. We conclude that RSK2 is associated with PP2Cd in vivo and is partially dephosphorylated by it, leading to reduced kinase activity.
| S-EPMC1133798 | biostudies-literature
Project description:The p90 ribosomal S6 kinases (RSKs) contain two distinct catalytic kinase domains, the N-terminal and C-terminal kinase domains (NTKD and CTKD, respectively). The activation of CTKD is regulated by phosphorylation by extracellular signal-regulated kinase (ERK1/2) and an autoinhibitory αL helix. Through a mutational series in vitro of the RSK CTKDs, we found a complex mechanism lifting autoinhibition that led us to design constitutively active RSK CTKDs. These are based on a phosphomimetic mutation and a C-terminal truncation (e.g., RSK2 T577E D694*) where a high activity in absence of ERK phosphorylation is obtained. Using these constructs, we characterize IC50 values of ATP-competitive inhibitors and provide a setup for determining specificity constants (kinact/Ki) of covalent CTKD inhibitors.
| S-EPMC9941302 | biostudies-literature
Project description:All known protein kinases share a bilobal kinase domain with well conserved structural elements. Because of significant structural similarities of nucleotide binding pocket, the development of highly selective kinase inhibitors is a very challenging task. Flavonols, naturally occurring plant metabolites, have long been known to inhibit kinases by mimicking the adenine moiety. Interestingly, recent data show that some flavonol glycosides are more selective, although underlying mechanisms were unknown. Crystallographic data from our laboratory revealed that the N-terminal kinase domain of p90 ribosomal S6 kinase, isoform 2, binds three different flavonol rhamnosides in a highly unusual manner, distinct from other kinase inhibitor interactions. The kinase domain undergoes a reorganization of several structural elements in response to the binding of the inhibitors. Specifically, the main ?-sheet of the N-lobe undergoes a twisting rotation by ~56° around an axis passing through the N- and C-lobes, leading to the restructuring of the canonical ATP-binding pocket into pockets sterically adapted to the inhibitor shape. The flavonol rhamnosides appear to adopt compact, but strained conformations with the rhamnose moiety swept under the B-ring of flavonol, unlike the structure of the free counterparts in solution. These data suggest that the flavonol glycoside scaffold could be used as a template for new inhibitors selective for the RSK family. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
| S-EPMC3683371 | biostudies-literature