Kinase Rsk Research Articles

Erratum: Corrigendum: The MAPK-activated kinase Rsk controls an acute Toll-like receptor signaling response in dendritic cells and is activated through two distinct pathways

Nat. Immunol. 8, 1227–1235 (2007); published online 30 September 2007; corrected after print 16 November 2007 In the version of this article initially published, the Author Contributions section was incorrect. The correct author contributions are as follows: R.Z and C.W designed the study, interpreted the data and wrote the manuscript; R.

A "Rsk"y Shortcut in Dendritic Cells

Dendritic cells have a key role in protecting organisms from infections. These cells detect antigens derived from invading bacteria or viruses and present them to T cells. Zaru et al. explored the roles of protein kinases activated in response to mitogen-activated protein kinases (MAPKs) and not only demonstrate the importance of various MAPK-activated protein kinases (MKs)--the MKs known as MK2 and MK3 and the p90 ribosomal S6 kinases (Rsks)--but also reveal a previously unrecognized regulatory network for control of downstream kinases in a MAPK system. The authors monitored Toll-like receptor-activated endocytosis by detecting uptake of fluorescently labeled dextran and observed that dendritic cells lacking both MK2 and MK3 had a reduced response. They also observed that pharmacological inhibition of Rsk family members with two structurally distinct inhibitors inhibited Toll-like receptor-induced endocytosis. The surprise was that inhibition of ERK1 and ERK2, the MAPKs known to activate Rsks, could not block receptor-induced Rsk activation in this system. Inhibition of ERKs only blocked Rsk activation in cells lacking MK2 and MK3, kinases that are activated by the MAPK p38 rather then ERKs. Rsks have two kinase domains, and the C-terminal kinase domain of Rsk proteins is phosphorylated by ERKs and autophosphorylates a serine residue located between the two kinase domains [thus recruiting yet another kinase (PDK1) to complete the activation]. Zaru et al . realized, however, that this critical serine residue in Rsk fits the consensus for recognition by MK2 and MK3 and showed that MK2 could phosphorylate Rsk2 at this site in vitro. Thus, the p38 MAPK provides an alternative mechanism for activating Rsks that bypasses the mechanism used for their activation by the ERK MAPKs, a mechanism that appears to be restricted to dendritic cells. The authors and Karin (in associated commentary) speculate on ways in which this modified regulatory scheme might be advantageous in these particular cells. R. Zaru, N. Ronkina, M. Gaestel, J. S. C. Arthur, C. Watts, The MAPK-activated kinase Rsk controls an acute Toll-like receptor signaling response in dendritic cells and is activated through two distinct pathways. Nat. Immunol. 8 , 1227-1235 (2007). [PubMed] M. Karin, Rsk Tolls the bell for endocytosis in DCs. Nat. Immunol. 8 , 1197-1199 (2007). [PubMed]

The MAPK-activated kinase Rsk controls an acute Toll-like receptor signaling response in dendritic cells and is activated through two distinct pathways

Most dendritic cell (DC) responses to Toll-like receptor (TLR) ligands depend on the activation of mitogen-activated protein kinases (MAPKs), but the contributions of the many MAPK-activated kinases (MKs) that act 'downstream' of the MAPKs Erk and p38 are not known. Here we sought to determine which MKs are required for acute TLR-driven, MAPK-dependent DC endocytic responses. Two specific and structurally different inhibitors of the MK Rsk suppressed TLR-induced endocytosis, thus defining in DCs a specific requirement for MKs in TLR responses. In addition, we identify in DCs a previously unknown configuration of the MAPK system whereby Rsk is activated not only by Erk but also by p38 through the intermediates MK2 and MK3. Thus, in DCs, p38 contributes to the activation of all known MK families.

Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation

The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.

Involvement of ERKs, RSK2 and PKR in UVA-induced signal transduction toward phosphorylation of eIF2α (Ser51)

Double-stranded RNA-dependent protein kinase R (PKR) has been implicated in anti-viral (antitumor) and apoptotic responses. PKR is activated by extracellular stresses and phosphorylates the alpha subunit of protein synthesis initiation factor eIF2, thereby inhibiting protein synthesis and impeding virus multiplication. Phosphorylation of eIF2alpha in mammalian cells has been shown to be increased after ultraviolet (UV) stress and to be required for UV-induced repression of protein translation. UVA is an important etiological factor in skin carcinogenesis and we observed that UVA induced phosphorylation of PKR (Thr(451)) and eIF2alpha (Ser(51)) in mouse skin epidermal JB6 Cl41 cells. The induction was suppressed by the MEK1 inhibitor, PD 98059. UVA stimulation of PKR and eIF2alpha phosphorylation was also inhibited by a dominant-negative mutant (DNM) of ERK2- or RSK2-deficient cells (RSK2(-)). An inhibitor of p38, SB 202190 or a DNM of p38alpha kinase (DNM-p38alpha) suppressed UVA-induced phosphorylation of eIF2alpha (Ser(51)) but had no effect on phosphorylation of PKR (Thr(451)). Our data indicated that phosphorylation of PKR at Thr(451) is mediated through ERK2 and RSK2, but not through p38 kinase, and is involved in the regulation of Ser(51) phosphorylation of eIF2alpha in UVA-irradiated JB6 cells. In vitro and in vivo kinase assays indicated that phosphorylation of eIF2alpha at Ser(51) occurred indirectly through ERK2, RSK2 or p38 kinase in the cellular response to UVA. These data may lead to the use of these signaling molecules as targets to develop more effective chemopreventive agents with fewer side effects to control UV-induced skin cancer.

A RSK kinase inhibitor reporting its selectivity in vivo

The presence of 491 very similar protein kinases in humans has proven to be a serious obstacle to generating specific kinase inhibitors and assessing their selectivity. Now, rational drug design has led to the generation of an irreversible kinase inhibitor with built-in features for demonstrating specificity in cells.

The cAMP response element binding protein (CREB) as an integrative HUB selector in metazoans: Clues from the hydra model system

In eukaryotic cells, a multiplicity of extra-cellular signals can activate a unique signal transduction system that at the nuclear level will turn on a variety of target genes, eliciting thus diverse responses adapted to the initial signal. How distinct signals can converge on a unique signalling pathway that will nevertheless produce signal-specific responses provides a theoretical paradox that can be traced back early in evolution. In bilaterians, the CREB pathway connects diverse extra-cellular signals via cytoplasmic kinases to the CREB transcription factor and the CBP co-activator, regulating according to the context, cell survival, cell proliferation, cell differentiation, pro-apoptosis, long-term memory, hence achieving a “hub” function for cellular and developmental processes. In hydra, the CREB pathway is highly conserved and activated during early head regeneration through RSK-dependent CREB phosphorylation. We show here that the CREB transcription factor and the RSK kinase are co-expressed in all three hydra cell lineages including dividing interstitial stem cells, proliferating nematoblasts, proliferating spermatogonia and spermatocytes, differentiating and mature neurons as well as ectodermal and endodermal myoepithelial cells. In addition, CREB gene expression is specifically up-regulated during early regeneration and early budding. When the CREB function was chemically prevented, the early post-amputation induction of the HyBraI gene was no longer observed and head regeneration was stacked. Thus, in hydra, the CREB pathway appears already involved in multiple tasks, such as reactivation of developmental programs in an adult context, self-renewal of stem cells, proliferation of progenitors and neurogenesis. Consequently, the hub function played by the CREB pathway was established early in animal evolution and might have contributed to the formation of an efficient oral pole through the integration of the neurogenic and patterning functions.

The Transcriptional ETS2 Repressor Factor Associates with Active and Inactive Erks through Distinct FXF Motifs

The transcriptional ETS2 repressor factor (ERF) is phosphorylated by Erks both in vivo and in vitro. This phosphorylation determines the subcellular localization and biological function of ERF. Here, we show that active and inactive Erk2 proteins bind ERF with high affinity through a hydrophobic pocket formed by the alphaF and alphaG helices and the activation loop of Erk2. We have identified two FXF motifs on ERF that mediate the specific interaction with Erks. One of these motifs is utilized only by active Erks, whereas the other mediates the association with inactive Erks but also contributes to interaction with active Erks. Mutation of the phenylalanines of these motifs to alanines resulted in decreased association and phosphorylation of ERF by Erks both in cells and in vitro. ERF proteins carrying these mutations exhibited increased nuclear accumulation and increased inhibition of cellular proliferation. Expression of ERF regions harboring these motifs could inhibit Erk activity in cells. Our data suggest that, in the proper context, FXF motifs can mediate a strong and specific interaction not only with active but also inactive Erks and that these interactions determine protein function in vivo.

Nuclear actin and myosin I are required for RNA polymerase I transcription

The presence of actin and nuclear myosin I (NMI) in the nucleus suggests a role for these motor proteins in nuclear functions. We have investigated the role of actin and nuclear myosin I (NMI) in the transcription of ribosomal RNA genes (rDNA). Both proteins are associated with rDNA and are required for RNA polymerase I (Pol I) transcription. Microinjection of antibodies against actin or NMI, as well as short interfering RNA-mediated depletion of NMI, decreased Pol I transcription in vivo, whereas overexpression of NMI augmented pre-rRNA synthesis. In vitro, recombinant NMI activated Pol I transcription, and antibodies to NMI or actin inhibited Pol I transcription both on naked DNA and pre-assembled chromatin templates. Whereas actin associated with Pol I, NMI bound to Pol I through the transcription-initiation factor TIF-IA. The association with Pol I requires phosphorylation of TIF-IA at Ser 649 by RSK kinase, indicating a role for NMI in the growth-dependent regulation of rRNA synthesis.

Controlling ERK Signaling Location

Certain stimuli, such as growth factors, trigger the Ras-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway to elicit responses that affect processes such as cell growth and survival. Activated ERK can act on cytoplasmic targets, such as the serine-threonine kinase RSK, or it can translocate to the nucleus and phosphorylate substrates including the transcription factor Elk-1. A putative membrane protein called Sef (similar expression to fgf genes) inhibits the Ras-ERK signaling cascade in response to growth factors, but its point of action has not been clear. Torii et al. report that in response to growth factor stimulation, human Sef (hSef) binds to activated MEK within the MEK-ERK complex in the cytoplasm. Activated MEK phosphorylates, and hence activates, associated ERK, a modification that releases activated ERK to translocate to the nucleus. The study shows that in unstimulated cells, overexpressed hSef localized mainly to the Golgi apparatus. After stimulation with growth factor, some hSef translocated to the plasma membrane. Activated MEK and ERK colocalized with hSef at the Golgi and plasma membrane. Overexpression of hSef blocked growth factor-stimulated nuclear translocation of ERK, whereas knockdown of hSef expression by RNA interference increased ERK nuclear localization and expression of target genes in response to growth factor treatment. In growth factor-treated cells overexpressing both hSef and MEK, hSef was isolated in a complex with activated MEK. Although hSef inhibited phosphorylation of the nuclear target Elk-1, phosphorylation of the cytoplasmic target RSK was unaffected. The authors propose that hSef is a signaling-dependent regulator of the Ras-ERK pathway and restricts where ERK activation is localized within the cell. S. Torii, M. Kusakabe, T. Yamamoto, M. Maekawa, E. Nishida, Sef is a spatial regulator for Ras/MAP kinase signaling. Devel. Cell 7 , 33-44 (2004). [Online Journal]

Inhibition of Ligand-independent ERK1/2 Activity in Kidney Proximal Tubular Cells Deprived of Soluble Survival Factors Up-regulates Akt and Prevents Apoptosis

Mouse kidney proximal tubular epithelial (MK-PT) cells die by apoptosis over 7-10 days when deprived of all survival factors. We show here that withdrawal of all survival factors from MK-PT cells is associated with a progressive increase in the activity of extracellular signal-regulated kinase-1 and -2 (ERK1/2) and a progressive decrease in phosphorylated Akt, a kinase critical to cell survival. Pharmacological inhibition of MEK1/2, the immediate upstream kinase for ERK1/2, not only prevented the decrease in phosphorylated Akt, but also prolonged MK-PT cell survival. Inhibition of ERK1/2, by itself, in the absence of any other known survival factors, was as potent as epidermal growth factor in maintaining MK-PT cell viability. ERK1/2 co-immunoprecipitated with Akt in a multimolecular assembly of signaling molecules, containing at a minimum ERK1/2, Akt, Rsk, and 3-phosphoinositide dependent kinase 1 (PDK1). We hypothesize that the kinase Rsk, whose activation requires phosphorylation by both ERK1/2 and PDK1, acts as a bridge bringing ERK1/2 into proximity with PDK1-associated Akt. Although a number of interactions between the Raf-MEK-ERK and PI3K-Akt signaling pathways have been described, our results are the first to show modulation of Akt activity by signaling events originating with ERK1/2. Spontaneous activation of ERK1/2 occurs via MEK1/2 and appears to depend on oxidant stress, accompanying induction of the default pathway of apoptosis. Together, these data suggest that the spontaneous activation of ERK1/2, in the absence of known extracellular stimuli, represents a previously unrecognized major regulatory pathway determining the fate of cells destined to die by the default pathway of apoptosis.

RSK2 Activity Is Regulated by Its Interaction with PEA-15

The ERK MAP (mitogen-activated protein) kinase cascade modulates many cellular processes including transcription, adhesion, growth, survival, and proliferation. One target substrate of ERK involved in regulating transcription is the p90 ribosomal S6 kinase (RSK) isozyme, RSK2. Here we demonstrate that a small death effector domain-containing protein called PEA-15 binds RSK2. RSK2 and PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) co-precipitated from cells and were colocalized in the cytoplasm. Furthermore, purified PEA-15 bound in vitro translated RSK2, suggesting that these proteins interact directly. PEA-15 does not bind to RSK1 and therefore exhibits some binding specificity. RSK2 binds the COOH terminus of PEA-15 and does not interact with its NH2-terminal death effector domain. We show that this interaction has functional consequences including the inhibition of RSK2-dependent CREB transcription. PEA-15 expression also blocks histone H3 phosphorylation, an RSK2-dependent event that may contribute to effects on gene expression. These results can be attributed to two effects of PEA-15 on RSK2. First, PEA-15 blocks nuclear accumulation of RSK2 after epidermal growth factor stimulation. Second, PEA-15 inhibits RSK2 kinase activity by 50%. A mutant of PEA-15 that binds RSK2 but is localized to the nucleus had no effect on RSK2-dependent transcription. Interestingly, this mutant also did not affect RSK2 kinase activity. This may indicate that cytoplasmic retention of RSK2 is also required for PEA-15 to impair kinase activity. PEA-15 does not alter ERK phosphorylation of RSK2 and is not itself a substrate of RSK2. Hence the effects of PEA-15 on RSK2 represent a novel mechanism for the regulation of RSK2-mediated signaling.

Cascade of Distinct Histone Modifications during Collagenase Gene Activation

Gene activation in eukaryotes requires chromatin remodeling, in part via histone modifications. To study the events at the promoter of a mitogen-inducible gene, we examined the induction of expression of the collagenase gene. It has been established that the collagenase gene can be activated by c-Jun and c-Fos and that the transcriptional coactivator p300 is involved in the activation. As expected, we found histone acetyltransferase activity at the collagenase promoter during activation. Interestingly, we also found histone methyltransferase and kinase activity. Strikingly, the first modification observed is methylation of histone H3 lysine 4, which correlates with the binding of the SET9 methyltransferase and the assembly of a complex consisting of c-Jun, c-Fos, TATA binding protein, and RNA polymerase II. The assembly of the preinitiation complex also shows an ordered binding of the acetyltransferase p300, the RSK2 kinase, and the SWI/SNF component Brg-1. Our results suggest that collagenase gene activation involves a dynamic recruitment of different factors and that in addition to acetylation, histone H3 lysine 4 di- and trimethylation and histone H3 serine 10 phosphorylation are important steps in the activation of this gene.

A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation.

The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBalpha, MSK1 and SGK1 contain a similar phosphate-binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism in which the phosphorylated hydrophobic motif and activation loop act on the alphaC-helix of the kinase structure to induce synergistic stimulation of catalytic activity. Sequence conservation suggests that this mechanism is a key feature in activation of >40 human AGC kinases.

Cannabinoids Protect Astrocytes from Ceramide-induced Apoptosis through the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway

Cannabinoids, the active components of marijuana and their endogenous counterparts, exert many of their actions on the central nervous system by binding to the CB(1) cannabinoid receptor. Different studies have shown that cannabinoids can protect neural cells from different insults. However, those studies have been performed in neurons, whereas no attention has been focused on glial cells. Here we used the pro-apoptotic lipid ceramide to induce apoptosis in astrocytes, and we studied the protective effect exerted by cannabinoids. Results show the following: (i) cannabinoids rescue primary astrocytes from C(2)-ceramide-induced apoptosis in a dose- and time-dependent manner; (ii) triggering of this anti-apoptotic signal depends on the phosphatidylinositol 3-kinase/protein kinase B pathway; (iii) ERK and its downstream target p90 ribosomal S6 kinase might be also involved in the protective effect of cannabinoids; and (iv) cannabinoids protect astrocytes from the cytotoxic effects of focal C(2)-ceramide administration in vivo. In summary, results show that cannabinoids protect astrocytes from ceramide-induced apoptosis via stimulation of the phosphatidylinositol 3-kinase/protein kinase B pathway. These findings constitute the first evidence for an "astroprotective" role of cannabinoids.

Sensed by a DEF Domain

How activation of one pathway, the mitogen-activated protein kinase (MAPK) ERK pathway, can stimulate such opposite cellular responses as proliferation or differentiation is one of the fundamental questions in cell biology (see Assoian). Murphy et al . suggest that the DEF domain in such immediate early gene products as Fos, Fra-1, Fra-1, Jun, Myc, and others may be a critical component to interpreting the transient or sustained activation of the ERK pathway. Analysis of the phosphorylation of c-Fos and mutants in positions Ser 374 and Ser 362 (the site phosphorylated by the kinases ERK and RSK) showed that phosphorylation of those two sites stimulated the phosphorylation of other residues (Thr 325 and Thr 331 ) by ERK. The ability of ERK to phosphorylate residues other than Ser 374 depended on the integrity of an ERK-binding motif called DEF (amino acids F 343 TYP in c-Fos). Phosphorylation of Thr 325 correlated with the stimulation of cell proliferation in response to sustained ERK signaling. Mutation of Thr 325 and Thr 331 or the DEF domain blocked the induction of transformation by c-Fos and inhibited AP-1-stimulated transcription. The authors propose the following model for how DEF-domain-containing proteins serve as ERK sensors. Growth factors stimulate immediate early gene transcription through the ERK pathway. If the signal is transient, then the gene products are unstable. If the ERK signal persists, then the initial phosphorylation of the proteins exposes the DEF domain, to which ERK binds and causes phosphorylation of other sites, stabilizing the protein. R. K. Assoian, Common sense signalling. Nat. Cell Biol. 4 , E187-E188 (2002). [Online Journal] L. O. Murphy, S. Smith, R.-H. Chen, D. C. Finger, J. Blenis, Molecular interpretation of ERK signal duration by immediate early gene products. Nat. Cell Biol. 4 , 556-564 (2002). [Online Journal]

The Ribosomal S6 Kinases, cAMP-responsive Element-binding, and STAT3 Proteins Are Regulated by Different Leukemia Inhibitory Factor Signaling Pathways in Mouse Embryonic Stem Cells

Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.

A systematic approach to the complete study of a signaling molecule: ribosomal p90 rsk as an example

Ribosomal p90 rsk is a kinase of central importance in transducing mitogenic signals from an activated receptor to the cell nucleus and for protein synthesis. Here, we analyze the optimal steps to fully describe this kinase in both normal neutrophils and leukemic cell lines. These are: (i) immunological analyses (immunoblotting and immunoprecipitation); (ii) enzyme activity assays (in vitro and “in-gel”); and (iii) immunobiochemical combination methods (immunoprecipitation/kinase assay, immunoprecipitation/“in-gel” assay and ion exchange chromatography/immunoblotting). For the enzyme assays, we describe a novel method to measure ribosomal p90 rsk kinase activity “in-gel”, based on a renatured-protein method that allows for the direct quantitation of enzyme activity. Finally, we present an algorithm that can be readily implemented to the quantification of the extent of stimulation of a kinase in response to a particular extracellular stimuli. In our case, it was found that activation of p90 rsk was higher in proliferating leukemic cells than in mature neutrophils, indicating that a suppression of key signal transduction links could contribute to the maturational arrest typical of acute leukemia. All the techniques and strategies described here for p90 rsk could be easily extrapolated to the study of any signal transduction molecule, provided it has a phosphotransferase activity.

Assays for monitoring p70 S6 kinase and RSK activation

Publisher Summary This chapter focuses on the assays for monitoring p70S6 kinase (p70S6k) kinase and p90 RSK (more commonly referred to as RSK) activation. A hallmark of the mitogenic response is an increase in protein synthesis. This increase arises from the modulation of multiple components of the translational machinery, including phosphorylation of the S6 polypeptide of the 40S ribosomal subunit. Much has been learned about the signaling pathways that mediate activation of p70S6k and RSK. One common activator of both enzymes is p21 ras . However, p21 ras utilizes distinct downstream effectors to elicit their activation. The development of specific antibodies against p70S6k and RSK has allowed monitoring of their activation in vivo . The availability of clean immunoblotting antibodies that detect mobility shifts is particularly useful for examining stimulation of transfected forms ofp70S6k by candidate activators. Because p70S6k protein levels can vary from sample to sample during transfections, such antibodies provide an additional control by demonstrating p70S6k activation through changes in the ratio of the four phosphorylated bands.

Negative Regulation of the Serine/Threonine Kinase B-Raf by Akt

Negative Regulation of the Serine/Threonine Kinase B-Raf by Akt