The RSK family of kinases: emerging roles in cellular signalling

The 90 kDa ribosomal S6 kinase (RSK) family of proteins is a group of highly conserved Ser/Thr kinases that regulate diverse cellular processes, such as cell growth, cell motility, cell survival and cell proliferation. RSKs are downstream effectors of the Ras-extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling cascade. Significant advances in the field of RSK and ERK/MAPK signalling have occurred in the past few years, including biological insights and the discovery of novel substrates and new RSK regulatory mechanisms. Collectively, these data expand the current models of RSK signalling and highlight potential directions of research in RSK-mediated survival, growth, proliferation and migration.     

Phosphorylation of RSK2 at Tyr529 by FGFR2-p38 enhances human mammary epithelial cells migration

The members of p90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases are downstream effectors of MAPK/ERK pathway that regulate diverse cellular processes including cell growth, proliferation and survival. In carcinogenesis, RSKs are thought to modulate cell motility, invasion and metastasis. Herein, we have studied an involvement of RSKs in FGF2/FGFR2-driven behaviours of mammary epithelial and breast cancer cells. We found that both silencing and inhibiting of FGFR2 attenuated phosphorylation of RSKs, whereas FGFR2 overexpression and/or its stimulation with FGF2 enhanced RSKs activity. Moreover, treatment with ERK, Src and p38 inhibitors revealed that p38 kinase acts as an upstream RSK2 regulator. We demonstrate for the first time that in FGF2/FGFR2 signalling, p38 but not MEK/ERK, indirectly activated RSK2 at Tyr529, which facilitated phosphorylation of its other residues (Thr359/Ser363, Thr573 and Ser380). In contrast to FGF2-triggered signalling, inhibition of p38 in the EGF pathway affected only RSK2-Tyr529, without any impact on the remaining RSK phosphorylation sites. p38-mediated phosphorylation of RSK2-Tyr529 was crucial for the transactivation of residues located at kinase C-terminal domain and linker-region, specifically, in the FGF2/FGFR2 signalling pathway. Furthermore, we show that FGF2 promoted anchorage-independent cell proliferation, formation of focal adhesions and cell migration, which was effectively abolished by treatment with RSKs inhibitor (FMK). These indicate that RSK2 activity is indispensable for FGF2/FGFR2-mediated cellular effects. Our findings identified a new FGF2/FGFR2-p38-RSK2 pathway, which may play a significant role in the pathogenesis and progression of breast cancer and, hence, may present a novel therapeutic target in the treatment of FGFR2-expressing tumours.     

RSK3 encodes a novel pp90rsk isoform with a unique N-terminal sequence: growth factor-stimulated kinase function and nuclear translocation.

A novel pp90rsk Ser/Thr kinase (referred to as RSK3) was cloned from a human cDNA library. The RSK3 cDNA encodes a predicted 733-amino-acid protein with a unique N-terminal region containing a putative nuclear localization signal. RSK3 mRNA was widely expressed (but was predominant in lung and skeletal muscle). By using fluorescence in situ hybridization, the human RSK3 gene was localized to band q27 of chromosome 6. Hemagglutinin epitope-tagged RSK3 was expressed in transiently transfected COS cells. Growth factors, serum, and phorbol ester stimulated autophosphorylation of recombinant RSK3 and its kinase activity toward several protein substrates known to be phosphorylated by RSKs. However, the relative substrate specificity of RSK3 differed from that reported for other isoforms. RSK3 also phosphorylated potential nuclear target proteins including c-Fos and histones. Furthermore, although RSK3 was inactivated by protein phosphatase 2A in vitro, the enzyme was not activated by ERK2/mitogen-activated protein (MAP) kinase. In contrast, the kinase activity of another epitope-tagged RSK isoform (RSK-1) was significantly increased by in vitro incubation with ERK2/MAP kinase. Finally, we used affinity-purified RSK3 antibodies to demonstrate by immunofluorescence that endogenous RSK3 undergoes serum-stimulated nuclear translocation in cultured HeLa cells. These results provide evidence that RSK3 is a third distinct isoform of pp90rsk which translocates to the cell nucleus, phosphorylates potential nuclear targets, and may have a unique upstream activator. RSK3 may therefore subserve a discrete physiologic role(s) that differs from those of the other two known mammalian RSK isoforms.     

Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1

PDK1 (3-phosphoinositide-dependent protein kinase 1) activates a group of protein kinases belonging to the AGC [PKA (protein kinase A)/PKG (protein kinase G)/PKC (protein kinase C)]-kinase family that play important roles in mediating diverse biological processes. Many cancer-driving mutations induce activation of PDK1 targets including Akt, S6K (p70 ribosomal S6 kinase) and SGK (serum- and glucocorticoid-induced protein kinase). In the present paper, we describe the small molecule GSK2334470, which inhibits PDK1 with an IC?? of ~10 nM, but does not suppress the activity of 93 other protein kinases including 13 AGC-kinases most related to PDK1 at 500-fold higher concentrations. Addition of GSK2334470 to HEK (human embryonic kidney)-293, U87 or MEF (mouse embryonic fibroblast) cells ablated T-loop residue phosphorylation and activation of SGK isoforms and S6K1 induced by serum or IGF1 (insulin-like growth factor 1). GSK2334470 also inhibited T-loop phosphorylation and activation of Akt, but was more efficient at inhibiting Akt in response to stimuli such as serum that activated the PI3K (phosphoinositide 3-kinase) pathway weakly. GSK2334470 inhibited activation of an Akt1 mutant lacking the PH domain (pleckstrin homology domain) more potently than full-length Akt1, suggesting that GSK2334470 is more effective at inhibiting PDK1 substrates that are activated in the cytosol rather than at the plasma membrane. Consistent with this, GSK2334470 inhibited Akt activation in knock-in embryonic stem cells expressing a mutant of PDK1 that is unable to interact with phosphoinositides more potently than in wild-type cells. GSK2334470 also suppressed T-loop phosphorylation and activation of RSK2 (p90 ribosomal S6 kinase 2), another PDK1 target activated by the ERK (extracellular-signal-regulated kinase) pathway. However, prolonged treatment of cells with inhibitor was required to observe inhibition of RSK2, indicating that PDK1 substrates possess distinct T-loop dephosphorylation kinetics. Our data define how PDK1 inhibitors affect AGC signalling pathways and suggest that GSK2334470 will be a useful tool for delineating the roles of PDK1 in biological processes.     

Phosphorylation of p90 ribosomal S6 kinase (RSK) regulates extracellular signal-regulated kinase docking and RSK activity

Stimulation of the Ras/extracellular signal-regulated kinase (ERK) pathway can modulate cell growth, proliferation, survival, and motility. The p90 ribosomal S6 kinases (RSKs) comprise a family of serine/threonine kinases that lie at the terminus of the ERK pathway. Efficient RSK activation by ERK requires its interaction through a docking site located near the C terminus of RSK, but the regulation of this interaction remains unknown. In this report we show that RSK1 and ERK1/2 form a complex in quiescent HEK293 cells that transiently dissociates upon mitogen stimulation. Complex dissociation requires phosphorylation of RSK1 serine 749, which is a mitogen-regulated phosphorylation site located near the ERK docking site. Using recombinant RSK1 proteins, we find that serine 749 is phosphorylated by the N-terminal kinase domain of RSK1 in vitro, suggesting that ERK1/2 dissociation is mediated through RSK1 autophosphorylation of this residue. Consistent with this hypothesis, we find that inactivating mutations in the RSK1 kinase domains disrupted the mitogen-regulated dissociation of ERK1/2 in vivo. Analysis of different RSK isoforms revealed that RSK1 and RSK2 readily dissociate from ERK1/2 following mitogen stimulation but that RSK3 remains associated with active ERK1/2. RSK activity assays revealed that RSK3 also remains active longer than RSK1 and RSK2, suggesting that prolonged ERK association increased the duration of RSK3 activation. These results provide new evidence for the regulated nature of ERK docking interactions and reveal important differences among the closely related RSK family members.     

Activation of p90 ribosomal S6 kinase by ORF45 of Kaposi's sarcoma-associated herpesvirus and its role in viral lytic replication

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway is essential for infection by a variety of viruses. The p90 ribosomal S6 kinases (RSKs) are direct substrates of ERK and functional mediators of ERK MAPK signaling, but their roles in viral infection have never been examined. We demonstrate that ORF45 of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with RSK1 and RSK2 and strongly stimulates their kinase activities. The activation of RSK by ORF45 is correlated with ERK activation but does not require MEK. We further demonstrate that RSK1/RSK2 is activated during KSHV primary infection and reactivation from latency; a subset of RSK1/RSK2 is present in the viral replication compartment in the nucleus. Depletion of RSK1/RSK2 by small interfering RNA or the specific inhibitor BI-D1870 suppresses KSHV lytic gene expression and progeny virion production, suggesting an essential role of RSK1/RSK2 in KSHV lytic replication.     

Crystal structures of the N-terminal kinase domain of human RSK1 bound to three different ligands: Implications for the design of RSK1 specific inhibitors

The p90 ribosomal S6 kinases (RSKs) also known as MAPKAP-Ks are serine/threonine protein kinases that are activated by ERK or PDK1 and act as downstream effectors of mitogen-activated protein kinase (MAPK). RSK1, a member of the RSK family, contains two distinct kinase domains in a single polypeptide chain, the regulatory C-terminal kinase domain (CTKD) and the catalytic N-terminal kinase domain (NTKD). Autophosphorylation of the CTKD leads to activation of the NTKD that subsequently phosphorylates downstream substrates. Here we report the crystal structures of the unactivated RSK1 NTKD bound to different ligands at 2.0 A resolution. The activation loop and helix alphaC, key regulatory elements of kinase function, are disordered. The DFG motif of the inactive RSK1 adopts an "active-like" conformation. The beta-PO(4) group in the AMP-PCP complex adopts a unique conformation that may contribute to inactivity of the enzyme. Structures of RSK1 ligand complexes offer insights into the design of novel anticancer agents and into the regulation of the catalytic activity of RSKs.     

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

Summary: The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries.     

Yersinia Virulence Factor YopM Induces Sustained RSK Activation by Interfering with Dephosphorylation

Background

Pathogenic yersiniae inject several effector proteins (Yops) into host cells, which subverts immune functions and enables the bacteria to survive within the host organism. YopM, whose deletion in enteropathogenic yersiniae results in a dramatic loss of virulence, has previously been shown to form a complex with and activate the multifunctional kinases PKN2 and RSK1 in transfected cells.

Methodology/Principal Findings

In a near physiological approach with double-affinity-tagged YopM being translocated into the macrophage cell line J774A.1 via the natural type three secretion system of Yersinia we verified the interaction of YopM with PKN2 and RSK1 and detected association with additional PKN and RSK isoforms. In transfected and infected cells YopM induced sustained phosphorylation of RSK at its activation sites serine-380 and serine-221 even in the absence of signalling from its upstream kinase ERK1/2, suggesting inhibition of dephosphorylation. ATP-depletion and in vitro assays using purified components directly confirmed that YopM shields RSK isoforms from phosphatase activity towards serines 380 and 221.

Conclusions/Significance

Our study suggests that during Yersinia infection YopM induces sustained activation of RSK by blocking dephosphorylation of its activatory phosphorylation sites. This may represent a novel mode of action of a bacterial virulence factor.     

Cloning and characterization of Xenopus Rsk2, the predominant p90 Rsk isozyme in oocytes and eggs

The 90-kDa ribosomal S6 kinases, the p90 Rsks, are a family of intracellular serine/threonine protein kinases distinguished by two distinct kinase domains. Rsks are activated downstream of the ERK1 (p44) and ERK2 (p42) mitogen-activated protein (MAP) kinases in diverse biological contexts, including progression through meiotic and mitotic M phases in Xenopus oocytes and cycling Xenopus egg extracts, and are critical for the M phase functions of Xenopus p42 MAPK. Here we report the cloning and biochemical characterization of Xenopus Rsk2. Xenopus Rsk1 and Rsk2 are specifically recognized by commercially available RSK1 and RSK2 antisera on immunoblots, but both Rsk1 and Rsk2 are immunoprecipitated by RSK1, RSK2, and RSK3 sera. Rsk2 is about 20-fold more abundant than the previously described Xenopus Rsk1 protein; their concentrations are approximately 120 and 5 nm, respectively. Rsk2, like Rsk1, forms a heteromeric complex with p42 MAP kinase. This interaction depends on sequences at the extreme C terminus of Rsk2 and can be disrupted by a synthetic peptide derived from the C-terminal 20 amino acids of Rsk2. Finally, we demonstrate that p42 MAP kinase can activate recombinant Rsk2 in vitro to a specific activity comparable to that found in Rsk2 that has been activated maximally in vivo. These findings underscore the importance of the Rsk2 isozyme in the M phase functions of p42 MAP kinase and provide tools for further examining Rsk2 function.     

Negative feedback via RSK modulates Erk‐dependent progression from naïve pluripotency

Mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) signalling is implicated in initiation of embryonic stem (ES) cell differentiation. The pathway is subject to complex feedback regulation. Here, we examined the ERK‐responsive phosphoproteome in ES cells and identified the negative regulator RSK1 as a prominent target. We used CRISPR/Cas9 to create combinatorial mutations in RSK family genes. Genotypes that included homozygous null mutations in Rps6ka1, encoding RSK1, resulted in elevated ERK phosphorylation. These RSK‐depleted ES cells exhibit altered kinetics of transition into differentiation, with accelerated downregulation of naïve pluripotency factors, precocious expression of transitional epiblast markers and early onset of lineage specification. We further show that chemical inhibition of RSK increases ERK phosphorylation and expedites ES cell transition without compromising multilineage potential. These findings demonstrate that the ERK activation profile influences the dynamics of pluripotency progression and highlight the role of signalling feedback in temporal control of cell state transitions.     

Novel role for p90 ribosomal S6 kinase in the regulation of cardiac myofilament phosphorylation

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.     

Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase

Elongation factor 2 kinase (eEF2k) phosphorylates and inactivates eEF2. Insulin induces dephosphorylation of eEF2 and inactivation of eEF2 kinase, and these effects are blocked by rapamycin, which inhibits the mammalian target of rapamycin, mTOR. However, the signalling mechanisms underlying these effects are unknown. Regulation of eEF2 phosphorylation and eEF2k activity is lost in cells in which phosphoinositide-dependent kinase 1 (PDK1) has been genetically knocked out. This is not due to loss of mTOR function since phosphorylation of another target of mTOR, initiation factor 4E-binding protein 1, is not defective. PDK1 is required for activation of members of the AGC kinase family; we show that two such kinases, p70 S6 kinase (regulated via mTOR) and p90(RSK1) (activated by Erk), phosphorylate eEF2k at a conserved serine and inhibit its activity. In response to insulin-like growth factor 1, which activates p70 S6 kinase but not Erk, regulation of eEF2 is blocked by rapamycin. In contrast, regulation of eEF2 by stimuli that activate Erk is insensitive to rapamycin, but blocked by inhibitors of MEK/Erk signalling, consistent with the involvement of p90(RSK1).     

Ribosomal S6 kinase 1 (RSK1) activation requires signals dependent on and independent of the MAP kinase ERK.

BACKGROUND: The rsk1 gene encodes the 90 kDa ribosomal S6 kinase 1 (RSK1) protein, which contains two kinase domains. RSK1, which is involved in regulating cell survival and proliferation, lies at the end of the signaling cascade mediated by the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinases. ERK activation and subsequent phosphorylation of the RSK1 carboxy-terminal catalytic loop stimulates phosphotransferase activity in the RSK1 amino-terminal kinase domain. When activated, RSK1 phosphorylates both nuclear and cytoplasmic substrates through this amino-terminal catalytic domain. It is thought that stimulation of the ERK/MAP kinase pathway is sufficient for RSK1 activation, but how ERK phosphorylation activates the RSK1 amino-terminal kinase domain is not known. RESULTS: The individual isolated RSK1 kinase domains were found to be under regulatory control. In vitro kinase assays established that ERK phosphorylates RSK1 within the carboxy-terminal kinase domain, and the phosphoinositide-dependent kinase 1 (PDK1) phosphorylates RSK1 within the amino-terminal kinase domain. In transiently transfected HEK 293E cells, PDK1 alone stimulated phosphotransferase activity of an isolated RSK1 amino-terminal kinase domain. Nevertheless, activation of full-length RSK1 in the absence of serum required activation by both PDK1 and ERK. CONCLUSIONS: RSK1 is phosphorylated by PDK1 in the amino-terminal kinase-activation loop, and by ERK in the carboxy-terminal kinase-activation loop. Activation of phosphotransferase activity of full-length RSK1 in vivo requires both PDK1 and ERK. RSK1 activation is therefore regulated by both the mitogen-stimulated ERK/MAP kinase pathway and a PDK1-dependent pathway.     

Increased Membrane/Nuclear Translocation and Phosphorylation of p90 KD Ribosomal S6 Kinase in the Brain of Hypoxic Preconditioned Mice

Our previous studies have demonstrated that hypoxic precondition (HPC) increased membrane translocation of protein kinase C isoforms and decreased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the brain of mice. The goal of this study was to determine the involvement of p90 KD ribosomal S6 kinase (RSK) in cerebral HPC of mice. Using Western-blot analysis, we found that the levels of membrane/nuclear translocation, but not protein expression of RSK increased significantly in the frontal cortex and hippocampus of HPC mice. In addition, we found that the phosphorylation levels of RSK at the Ser227 site (a PDK1 phosphorylation site), but not at the Thr359/Ser363 sites (ERK1/2 phosphorylated sites) increased significantly in the brain of HPC mice. Similar results were confirmed by an immunostaining study of total RSK and phospho-Ser227 RSK. To further define the cellular populations to express phospho-Ser227 RSK, we found that the expression of phospho-Ser227 RSK co-localized with neurogranin, a neuron-specific marker, in cortex and hippocampus of HPC mice by using double-labeled immunofluorescent staining method. These results suggest that increased RSK membrane/nuclear translocation and PDK1 mediated neuron-specific phosphorylation of RSK at Ser227 might be involved in the development of cerebral HPC of mice.     

Spatial organisation of AKAP18 and PDE4 isoforms in renal collecting duct principal cells

A plethora of stimuli including hormones and neurotransmitters mediate a rise of the cellular level of cAMP and thereby activation of protein kinase A (PKA). PKA phosphorylates and thereby modulates the activity of a wide range of cellular targets. It is now appreciated that different stimuli induce the activation of PKA at specific sites where the kinase phosphorylates particular substrates in close proximity. The tethering of PKA to cellular compartments is facilitated by A kinase-anchoring proteins (AKAPs). The incorporation of phosphodiesterases (PDEs) into AKAP-based signalling complexes provides gradients of cAMP that regulate PKA activity locally. An example for a process depending on compartmentalised cAMP/PKA signalling is the arginine-vasopressin (AVP)-mediated water reabsorption in renal collecting duct principal cells. Upon activation through AVP, PKA phosphorylates the water channel aquaporin-2 (AQP-2) located on intracellular vesicles. The phosphorylation triggers the redistribution of AQP2 to the plasma membrane. AKAP-anchored PKA has been shown to be involved in AQP2 shuttling. Here, AKAP18 isoforms and members of the PDE4 family of PDEs are shown to be differentially localised in renal principal cells.     

Subcellular localization and biological actions of activated RSK1 are determined by its interactions with subunits of cyclic AMP-dependent protein kinase

Cyclic AMP (cAMP)-dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1) share several cellular proteins as substrates. However, to date no other similarities between the two kinases or interactions between them have been reported. Here, we describe novel interactions between subunits of PKA and RSK1 that are dependent upon the activation state of RSK1 and determine its subcellular distribution and biological actions. Inactive RSK1 interacts with the type I regulatory subunit (RI) of PKA. Conversely, active RSK1 interacts with the catalytic subunit of PKA (PKAc). Binding of RSK1 to RI decreases the interactions between RI and PKAc, while the binding of active RSK1 to PKAc increases interactions between PKAc and RI and decreases the ability of cAMP to stimulate PKA. The RSK1/PKA subunit interactions ensure the colocalization of RSK1 with A-kinase PKA anchoring proteins (AKAPs). Disruption of the interactions between PKA and AKAPs decreases the nuclear accumulation of active RSK1 and, thus, increases its cytosolic content. This subcellular redistribution of active RSK1 is manifested by increased phosphorylation of its cytosolic substrates tuberous sclerosis complex 2 and BAD by epidermal growth factor along with decreased cellular apoptosis.