Activation of the various mitogen-activated protein (MAP) kinase pathways converts many different extracellular stimuli into specific cellular responses by inducing the phosphorylation of particular groups of substrates. One important determinant for substrate specificity is likely to be the amino-acid sequence surrounding the phosphorylation site; however, these sites overlap significantly between different MAP kinase family members. The idea is now emerging that specific docking sites for protein kinases are involved in the efficient binding and phosphorylation of some substrates. The MAP kinase-activated protein (MAPKAP) kinase p90(rsk) contains two kinase domains: the amino-terminal domain (D1) is required for the phosphorylation of exogenous substrates whereas the carboxy-terminal domain (D2) is involved in autophosphorylation. Association between the extracellular signal-regulated kinase (Erk) MAP kinases and p90(rsk) family members has been detected in various cell types including Xenopus oocytes, where inactive p90(rsk) is bound to the inactive form of the Erk2-like MAP kinase p42(mpk1). A new MAP kinase docking site has been identified that is located at the carboxyl terminus of p90(rsk). This docking site is required for the efficient phosphorylation and activation of p90(rsk) in vitro and in vivo and is also both necessary and sufficient for the stable and specific association with p42(mpk1). The sequence of the docking site is conserved in other MAPKAP kinases, suggesting that it might represent a new class of interaction motif that facilitates efficient and specific signal transduction by MAP kinases (Gavin, 1999).
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. This report shows 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, it was found 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, it was found that inactivating mutations in the RSK1 kinase domains disruptes 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 increases 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 (Roux, 2003).
Inhibitors of the oncogenic Ras-MAPK pathway have been intensely pursued as therapeutics. Targeting this pathway, however, presents challenges due to the essential role of MAPK in homeostatic functions. The phosphorylation and activation of MAPK substrates is regulated by protein-protein interactions with MAPK docking sites. Active ERK1/2 (extracellular signal-regulated kinase 1/2)-MAPKs localize to effectors containing DEF (docking site for ERK, (F)/(Y) -X-(F)/(Y) -P)- or D-domain (docking domain) motifs. The in vivo activity was examined of ERK2 mutants with impaired ability to signal via either docking site. Mutations in the DEF-domain binding pocket prevent activation of DEF-domain-containing effectors but not RSK (90 kDa ribosomal S6 kinase), which contains a D domain. Conversely, mutation of the ERK2 CD domain, which interacts with D domains, prevents RSK activation but not DEF-domain signaling. Uncoupling docking interactions does not compromise ERK2 phosphotransferase activity. ERK2 DEF mutants undergo regulated nuclear translocation but are defective for Elk-1/TCF transactivation and target gene induction. Thus, downstream branches of ERK2 signaling can be selectively inhibited without blocking total pathway activity. Significantly, several protooncogenes contain DEF domains and are regulated by ERK1/2. Therefore, disrupting ERK-DEF domain interactions could be an alternative to inhibiting oncogenic Ras-MAPK signaling (Dimitri, 2005).
Glutathione S-transferase (GST)-fusion proteins containing the carboxyl-terminal tails of three p90 ribosomal S6 kinase (RSK) isozymes (RSK1, RSK2, and RSK3) interact with extracellular signal-regulated kinase (ERK) but not c-Jun-NH2-kinase (JNK) or p38 mitogen-activated protein kinase (MAPK). Within the carboxyl-terminal residues of the RSK isozymes is a region of high conservation corresponding to residues 722LAQRRVRKLPSTTL735 in RSK1. Truncation of the carboxyl-terminal 9 residues, 727VRKLPSTTL735, completely eliminates the interaction of the GST-RSK1 fusion protein with purified recombinant ERK2, whereas the truncation of residues 731PSTTL735 has no effect on the interaction with purified ERK2. ERK1 and ERK2 co-immunoprecipitate with hemagglutinin-tagged wild type RSK2 (HA-RSK2). However, ERK does not co-immunoprecipitate with HA-RSK2(1-729), a mutant missing the carboxyl-terminal 11 amino acids, similar to the minimal truncation that eliminates in vitro interaction of ERK with the GST-RSK1 fusion protein. Kinase activity of HA-RSK2 increases 6-fold in response to insulin. HA-RSK2(1-729) has a similar basal kinase activity to that of HA-RSK2 but is not affected by insulin treatment. Immunoprecipitated HA-RSK2 and HA-RSK2(1-729) can be activated to the same extent in vitro by active ERK2, demonstrating that HA-RSK2(1-729) is properly folded. These data suggest that the conserved region of the RSK isozymes (722LAQRRVRKL730 of RSK1) provides for a specific ERK docking site approximately 150 amino acids carboxyl-terminal to the nearest identified ERK phosphorylation site (Thr573). Complex formation between RSK and ERK is essential for the activation of RSK by ERK in vivo. Comparison of the docking site of RSK with the carboxyl-terminal tails of other MAPK-activated kinases reveals putative docking sites within each of these MAPK-targeted kinases. The number and placement of lysine and arginine residues within the conserved region correlate with specificity for activation by ERK and p38 MAPKs in vivo (Smith, 1999).
Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the 'T-loop' of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1), which binds with high affinity to the PI 3-kinase lipid product phosphatidylinositol-3,4,5-trisphosphate, activates many AGC kinases in vitro by phosphorylating the T-loop residue, but whether PDK1 also phosphorylates the hydrophobic motif and whether all other AGC kinases are substrates for PDK1 is unknown. Mouse embryonic stem (ES) cells in which both copies of the PDK1 gene were disrupted are viable. In PDK1-/- ES cells, PKB, p70 S6 kinase and p90 Rsk are not activated by stimuli that induced strong activation in PDK1+/+ cells. Other AGC kinases – namely, protein kinase A (PKA), the mitogen- and stress-activated protein kinase 1 (MSK1) and the AMP-activated protein kinase (AMPK) – have normal activity or are activated normally in PDK1-/- cells. The insulin-like growth factor 1 (IGF1) induces PKB phosphorylation at its hydrophobic motif, but not at its T-loop residue, in PDK1-/- cells. IGF1 does not induce phosphorylation of p70 S6 kinase at its hydrophobic motif in PDK1-/- cells. It is concluded PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1-/- cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase (Williams, 2000).
G protein-coupled receptors (GPCRs) are essential for normal central CNS function and represent the proximal site(s) of action for most neurotransmitters and many therapeutic drugs, including typical and atypical antipsychotic drugs. Similarly, protein kinases mediate many of the downstream actions for both ionotropic and metabotropic receptors. Genetic deletion of p90 ribosomal S6 kinase 2 (RSK2) potentiates GPCR signaling. Initial studies of 5-hydroxytryptamine (5-HT)2A receptor signaling in fibroblasts obtained from RSK2 wild-type (+/+) and knockout (-/-) mice showed that 5-HT2A receptor-mediated phosphoinositide hydrolysis and both basal and 5-HT-stimulated extracellular signal-regulated kinase 1/2 phosphorylation are augmented in RSK2 knockout fibroblasts. Endogenous signaling by other GPCRs, including P2Y-purinergic, PAR-1-thrombinergic, beta1-adrenergic, and bradykinin-B receptors, was also potentiated in RSK2-deficient fibroblasts. Importantly, reintroduction of RSK2 into RSK2-/- fibroblasts normalized signaling, thus demonstrating that RSK2 apparently modulates GPCR signaling by exerting a 'tonic brake' on GPCR signal transduction. These results imply the existence of a novel pathway regulating GPCR signaling, modulated by downstream members of the extracellular signal-related kinase/mitogen-activated protein kinase cascade. The loss of RSK2 activity in humans leads to Coffin-Lowry syndrome, which is manifested by mental retardation, growth deficits, skeletal deformations, and psychosis. Because RSK2-inactivating mutations in humans lead to Coffin-Lowry syndrome, these results imply that alterations in GPCR signaling may account for some of its clinical manifestations (Sheffler, 2006).
A signaling pathway has been elucidated whereby growth factors activate the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB), a critical regulator of immediate early gene transcription. Growth factor-stimulated CREB phosphorylation at serine-133 is mediated by the RAS-mitogen-activated protein kinase (MAPK) pathway. MAPK activates CREB kinase, which in turn phosphorylates and activates CREB. Purification, sequencing, and biochemical characterization of CREB kinase reveals that it is identical to a member of the pp90(RSK) family, RSK2. RSK2 mediates growth factor induction of CREB serine-133 phosphorylation both in vitro and in vivo. These findings identify a cellular function for RSK2 and define a mechanism whereby growth factor signals mediated by RAS and MAPK are transmitted to the nucleus to activate gene expression (Xing, 1996).
Although Ca2+-stimulated cAMP response element binding protein- (CREB-) dependent transcription has been implicated in growth, differentiation, and neuroplasticity, mechanisms for Ca2+-activated transcription have not been defined. Extracellular signal-related protein kinase (ERK) signaling is obligatory for Ca2+-stimulated transcription in PC12 cells and hippocampal neurons. The sequential activation of ERK and Rsk2 by Ca2+ leads to the phosphorylation and transactivation of CREB. The Ca2+-induced nuclear translocation of ERK and Rsk2 to the nucleus requires protein kinase A (PKA) activation. Interestingly, Ca2+-mediated CREB phosphorylation in wild-type PC12 cells is decreased by a selective PKA inhibitor. With a high efficiency transfection protocol, expression of dominant negative PKA also attenuates Ca2+-stimulated CREB phosphorylation. In addition, treatment with the PKA inhibitors also inhibits depolarization-mediated CREB phosphorylation in primary hippocampal neurons. These results suggest that in PC12 cells and hippocampal neurons, PKA activity is required for Ca2+-induced CREB phosphorylation (Impey, 1998).
Because the nuclear translocation of ERK may be necessary for ERK-activated transcription, and PKA is required for Ca2+ stimulation of CREB phosphorylation, nuclear translocation of ERK was monitored when PKA was inhibited. To efficiently induce the nuclear translocation of ERK by Ca2+, PC12 cells were treated with KCl and a direct activator of L-type Ca2+ channels. Depolarization induces the phosphorylation of ERK and its translocation to the nucleus in both PC12 cells and hippocampal neurons. The specific PKA inhibitors inhibited the nuclear translocation of Erk in PC12 cells and hippocampal neurons. Western blotting of cytosolic fractions shows that the inhibition of Erk translocation by treatment with PKA inhibitors is not the result of an effect on Erk activation. To verify that PKA is required for the nuclear translocation of ERK, the cytosolic-to-nuclear ratio of phospho-ERK in KCl-stimulated hippocampal neurons was also quantitated. A specific PKA inhibitor significantly inhibited the translocation of ERK to the nucleus. The importance of PKA activity for ERK nuclear translocation was confirmed by transiently transfecting PC12 cells with a dominant negative PKA fused to green fluorescent protein. Only cells that express dominant negative PKA-GFP show impaired nuclear translocation of phospho-ERK. These results suggest that PKA is required for the phosphorylation and transactivation of CREB by Ca2+, because PKA is required for the nuclear translocation of ERK. However, since Rsk2 [a member of the pp90(RSK) family] is a major Ca2+-activated CREB kinase in PC12 cells, inhibition of Erk translocation should also block the activation of nuclear but not cytosolic Rsk2. Accordingly, inhibition of PKA blocks the activation of Rsk2 in the nuclear fraction but not in the cytosolic fraction. Treatment with PKA inhibitors attenuates the nuclear translocation of Rsk2. This is not surprising, because it is known that both ERK and Rsk2 are tightly associated in vivo and that they cotranslocate to the nucleus. Collectively, these data indicate that PKA may be necessary for the phosphorylation and transactivation of CREB by Ca2+, because PKA is required for the nuclear translocation of ERK and subsequent nuclear activation of the CREB kinase Rsk2. Inhibition of PKA also significantly impairs the translocation of ERK to the nucleus in response to NGF. Interestingly, coexpression of dominant negative PKA attenuates NGF-stimulated Elk1 transcriptional activation. Evidently, the modulation of ERK translocation by PKA activity plays a general role in the activation of transcription by mitogens and neurotrophic factors. NGF does not detectably elevate intracellular cAMP, suggesting that basal PKA activity is sufficient for neurotrophic factors and other strong ERK activators to induce nuclear translocation of ERK. Nevertheless, in the case of depolarization, which activates ERK to a lesser degree, the concomitant depolarization-mediated increase in cAMP levels enhances ERK translocation. These results may explain why PKA activity is required for Ca2+-stimulated CREB-dependent transcription. Furthermore, the full expression of the late phase of long-term potentiation (L-LTP) and L-LTP-associated CRE-mediated transcription requires ERK activation, suggesting that the activation of CREB by ERK plays a critical role in the formation of long lasting neuronal plasticity (Impey, 1998).
The keratinocyte growth factor receptor (KGFR) is a member of the fibroblast growth factor receptor (FGFR) superfamily. The proximal signaling molecules of FGFRs are much less characterized compared with other growth factor receptors. Using the yeast two-hybrid assay, ribosomal S6 kinase (RSK) was identified as a protein that associates with the cytoplasmic domain of the KGFR. The RSK family of kinases controls multiple cellular processes, and these studies show association between the KGFR and RSK. Using a lung-specific inducible transgenic system, protective effects of KGF on the lung epithelium has been demonstrated, as well as KGF-induced activation of the prosurvival Akt pathway. A kinase inactive RSK mutant blocks KGF-induced Akt activation and KGF-mediated inhibition of caspase 3 activation in epithelial cells subjected to oxidative stress. RSK2 recruits PDK1, the kinase responsible for both Akt and RSK activation. When viewed collectively, it appears that the association between the KGFR and RSK plays an important role in KGF-induced Akt activation and consequently in the protective effects of KGF on epithelial cells (Pan, 2004).
Receptor tyrosine kinase (RTK) signals regulate the specification of a varied array of tissue types by utilizing distinct modules of proteins to elicit diverse effects. The RSK proteins are part of the RTK signal transduction pathway and are thought to relay these signals by acting downstream of extracellular signal-regulated kinase (ERK). Ribosomal S6 kinase 4 (Rsk4) is an inhibitor of RTK signals. Among the RSK proteins, RTK inhibition is specific to RSK4 and, in accordance, is dependent upon a region of the RSK4 protein that is divergent from other RSK family members. Rsk4 inhibits the transcriptional activation of specific targets of RTK signaling as well as the activation of ERK. Developmentally, Rsk4 is expressed in extraembryonic tissue, where RTK signals are known to have critical roles. Further examination of Rsk4 expression in the extraembryonic tissues demonstrates that its expression is inversely correlated with the presence of activated ERK 1/2. These studies demonstrate a new and divergent function for RSK4 and support a role for RSK proteins in the specification of RTK signals during early mouse development (Myers, 2004)
Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. This study shows that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 ribosomal S6 kinase 1 (S6K1). The mitogen-activated protein kinase (MAPK)-activated kinase, p90 ribosomal S6 kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin/hamartin complex, resulting in increased mTOR signaling to S6K1. Together, these data unveil a regulatory mechanism by which the Ras/MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function (Roux, 2004).
The mechanism by which LKB1 is regulated in cells is not known. Stimulation of Rat-2 or embryonic stem cells with activators of ERK1/2 or of cAMP-dependent protein kinase induces phosphorylation of endogenously expressed LKB1 at Ser(431). Pharmacological and genetic evidence is presented that p90(RSK) mediates this phosphorylation in response to agonists that activate ERK1/2, and cAMP-dependent protein kinase mediates this phosphorylation in response to agonists that activate adenylate cyclase. Ser(431) of LKB1 lies adjacent to a putative prenylation motif, and full-length LKB1 expressed in 293 cells is prenylated by addition of a farnesyl group to Cys(433). These data suggest that phosphorylation of LKB1 at Ser(431) does not affect farnesylation and that farnesylation does not affect phosphorylation at Ser(431). Phosphorylation of LKB1 at Ser(431) does not alter the activity of LKB1 to phosphorylate itself or the tumor suppressor protein p53 or alter the amount of LKB1 associated with cell membranes. The reintroduction of wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed growth, but mutants of LKB1 in which Ser(431) was mutated to Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting growth. In contrast, a mutant of LKB1 that cannot be prenylated is still able to suppress the growth of cells (Sapkota, 2001).
The Ras-mitogen-activated protein (Ras-MAP) kinase pathway regulates various cellular processes, including gene expression, cell proliferation, and survival. Ribosomal S6 kinase (RSK), a key player in this pathway, modulates the activities of several cytoplasmic and nuclear proteins via phosphorylation. The cytoskeletal protein filamin A (FLNa) is a membrane-associated RSK target. The N-terminal kinase domain of RSK phosphorylates FLNa on Ser(2152) in response to mitogens. Inhibition of MAP kinase signaling with UO126 or mutation of Ser(2152) to Ala on FLNa prevents epidermal growth factor (EGF)-stimulated phosphorylation of FLNa in vivo. Furthermore, phosphorylation of FLNa on Ser(2152) is significantly enhanced by the expression of wild-type RSK and antagonized by kinase-inactive RSK or specific reduction of endogenous RSK. Strikingly, EGF-induced, FLNa-dependent migration of human melanoma cells is significantly reduced by UO126 treatment. Together, these data provide substantial evidence that RSK phosphorylates FLNa on Ser(2152) in vivo. Given that phosphorylation of FLNa on Ser(2152) is required for Pak1-mediated membrane ruffling, these results suggest a novel role for RSK in the regulation of the actin cytoskeleton (Woo, 2004).
Evidence is presented that RSK1 (ribosomal S6 kinase 1), a downstream target of MAPK (mitogen-activated protein kinase), directly phosphorylates nNOS (neuronal nitric oxide synthase) on Ser847 in response to mitogens. The phosphorylation thus increases greatly following EGF (epidermal growth factor) treatment of rat pituitary tumour GH3 cells and is reduced by exposure to the MEK (MAPK/extracellular-signal-regulated kinase kinase) inhibitor PD98059. Furthermore, it is significantly enhanced by expression of wild-type RSK1 and antagonized by kinase-inactive RSK1 or specific reduction of endogenous RSK1. EGF treatment of HEK-293 (human embryonic kidney) cells, expressing RSK1 and nNOS, led to inhibition of NOS enzyme activity, associated with an increase in phosphorylation of nNOS at Ser847, as is also the case in an in vitro assay. In addition, these phenomena were significantly blocked by treatment with the RSK inhibitor Ro31-8220. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and decrease of NOS activity. Within minutes of adding EGF to transfected cells, RSK1 associated with nNOS and subsequently dissociated following more prolonged agonist stimulation. EGF-induced formation of the nNOS-RSK1 complex was significantly decreased by PD98059 treatment. Treatment with EGF further revealed phosphorylation of nNOS on Ser847 in rat hippocampal neurons and cerebellar granule cells. This EGF-induced phosphorylation was partially blocked by PD98059 and Ro31-8220. Together, these data provide substantial evidence that RSK1 associates with and phosphorylates nNOS on Ser847 following mitogen stimulation and suggest a novel role for RSK1 in the regulation of nitric oxide function in brain (Song, 2007).
The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. This study demonstrates that the p90 ribosomal protein S6 kinase (RSK; see Drosophila) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3 (Shahbazian, 2006).
The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. This study shows that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, Ser-289 was identified as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. These results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells (Anjum, 2005).
Extracellular signal-regulated kinase (ERK) signaling is important for neuronal synaptic plasticity. The protein kinase ribosomal S6 kinase (RSK)2, a downstream target of ERK, uses a C-terminal motif to bind several PDZ domain proteins in heterologous systems and in vivo. Different RSK isoforms display distinct specificities in their interactions with PDZ domain proteins. Mutation of the RSK2 PDZ ligand does not inhibit RSK2 activation in intact cells or phosphorylation of peptide substrates by RSK2 in vitro but greatly reduces RSK2 phosphorylation of PDZ domain proteins of the Shank family in heterologous cells. In primary neurons, NMDA receptor (NMDA-R) activation leads to ERK and RSK2 activation and RSK-dependent phosphorylation of transfected Shank3. RSK2-PDZ domain interactions are functionally important for synaptic transmission because neurons expressing kinase-dead RSK2 display a dramatic reduction in frequency of AMPA-type glutamate receptor-mediated miniature excitatory postsynaptic currents, an effect dependent on the PDZ ligand. These results suggest that binding of RSK2 to PDZ domain proteins and phosphorylation of these proteins or their binding partners regulates excitatory synaptic transmission (Thomas, 2005).
M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF), which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. The C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. In turn, p90(rsk) phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. These results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B (Palmer, 1998).
During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90Rsk, a MAP kinase target, were used. Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes fail to form metaphase I spindles, fail to reaccumulate cyclin B to a high level and fail to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes enter S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90Rsk are able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. It is concluded that the MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90Rsk, is sufficient to mediate these effects during oocyte maturation (Gross, 2000).
The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint. This study shows that xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity. It is concluded that Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk) (Schwab, 2001).
The cell cycle in oocytes generally arrests at a particular meiotic stage to await fertilization. This arrest occurs at metaphase of meiosis II (meta-II) in frog and mouse, and at G1 phase after completion of meiosis II in starfish. Despite this difference in the arrest phase, both arrests depend on the same Mos-MAPK (mitogen-activated protein kinase) pathway, indicating that the difference relies on particular downstream effectors. Immediately downstream of MAPK, Rsk [p90 ribosomal S6 kinase, p90(Rsk)] is required for the frog meta-II arrest. However, the mouse meta-II arrest challenges this requirement, and no downstream effector has been identified in the starfish G1 arrest. To investigate the downstream effector of MAPK in the starfish G1 arrest, a neutralizing antibody was used against Rsk and a constitutively active form of Rsk. Rsk was activated downstream of the Mos-MAPK pathway during meiosis. In G1 eggs, inhibition of Rsk activity released the arrest and initiated DNA replication without fertilization. Conversely, maintenance of Rsk activity prevented DNA replication following fertilization. In early embryos, injection of Mos activated the MAPK-Rsk pathway, resulting in G1 arrest. Moreover, inhibition of Rsk activity during meiosis I led to parthenogenetic activation without meiosis II. It is concluded that immediately downstream of MAPK, Rsk is necessary and sufficient for the starfish G1 arrest. Although CSF (cytostatic factor) was originally defined for meta-II arrest in frog eggs, distinguishing 'G1-CSF' for starfish from 'meta-II-CSF' for frog and mouse is proposed. The present study thus reveals a novel role of Rsk for G1-CSF (Mori, 2006).
A mechanism by which the Ras-mitogen-activated protein kinase (MAPK) signaling pathway mediates growth factor-dependent cell survival has been characterized. The neurotrophin BDNF (brain-derived neurotrophic factor) and its receptor TrkB regulate the survival of newly generated granule neurons within the developing cerebellum. BDNF promotes the survival of cultured rat cerebellar granule neurons; upon BDNF withdrawal, these neurons die by apoptosis. BDNF induces phosphorylation of MAPK. Inhibition of MAPK activity by PD098059, a pharmacological agent that blocks MEK activity, diminishes the effect of BDNF on the survival of cerebellar granule cells. Likewise, the introduction of a dominant interfering form of MEK (MEK-KA97) blocks BDNF-enhancement of neuronal survival. These results indicate that activation of MAPK is required for BDNF-induced survival of cerebellar granule neurons (Bonni, 1999).
Like BDNF, insulin-like growth factor 1 (IGF-1) (or a high concentration of insulin that stimulates the IGF-1 receptor) promotes the survival of cerebellar granule neurons. Both BDNF and IGF-1 activate phosphatidylinositol 3-kinase (PI-3K) and the protein kinase Akt (PKB: Drosophila homolog Akt1) cascade in cerebellar granule neurons. Although the PI-3K-Akt signaling pathway mediates the survival-promoting effects of BDNF and IGF-1, inhibition of MAPK in cerebellar neurons has no effect on IGF-1 receptor-mediated cell survival. These results suggest that BDNF and IGF-1 promote cell survival at least in part by distinct mechanisms (Bonni, 1999).
The MAPK-activated kinases, the Rsks, catalyze the phosphorylation of the pro-apoptotic protein BAD at serine 112 both in vitro and in vivo. The Rsk-induced phosphorylation of BAD at serine 112 suppresses BAD-mediated apoptosis in neurons. Rsks also are known to phosphorylate the transcription factor CREB (cAMP response element-binding protein) at serine 133. Activated CREB promotes cell survival, and inhibition of CREB phosphorylation at serine 133 triggers apoptosis. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism comprising the posttranslational modification and inactivation of a component of the cell death machinery and the increased transcription of pro-survival genes (Bonni, 1999).
To determine whether CREB contributes to BDNF's ability to enhance cerebellar granule cell survival, the effects of two distinct dominant interfering forms of CREB on the BDNF survival response were tested. K-CREB, in which Arg287 is converted to Leu, forms dimers with endogenous CREB proteins via its leucine zipper domain. K-CREB inhibits the binding of endogenous CREB to the promoters of CREB-responsive genes. M1-CREB, in which Ser133 is converted to Ala, competes with endogenous CREB proteins for binding to the promoters of CREB-responsive genes. However, once bound to DNA, M1-CREB does not activate transcription. When transfected into cerebellar granule neurons, either K-CREB or M1-CREB inhibits the effect of BDNF on cell survival. However, the dominant interfering forms of CREB do not inhibit IGF-1-mediated cerebellar granule cell survival; this finding suggests that these proteins act specifically to block the BDNF response. In addition, M1-CREB does not lead to inhibition of Rsk function because its expression in 293T cells does not inhibit the MEK-induced phosphorylation of BAD at Ser112 (Bonni, 1999).
CREB has been implicated in mediating adaptive responses of neurons to trans-synaptic stimuli. These findings indicate that CREB may also have a function in the regulation of neuronal survival in the developing central nervous system. Mice in which the CREB gene has been disrupted die perinatally before the majority of cerebellar granule neurons are generated However, analysis of the CREB-/- mouse embryos has revealed a number of abnormalities in brain development that may reflect the contribution of CREB to the regulation of the survival of neurons. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism that modulates the cell death machinery directly by phosphorylating and thereby inhibiting the pro-apoptotic protein BAD, and by inducing the expression of pro-survival genes in a CREB-dependent manner. Suppression of BAD-mediated cell death by Rsk occurs relatively early after the removal of extracellular survival factors, whereas the contribution of CREB-mediated cell survival is detected significantly later. Therefore, the two arms of the MAPK-Rsk-regulated mechanism might act with different kinetics or at different times in developing neurons (Bonni, 1999).
Growth factors activate an array of cell survival signaling pathways. Mitogen-activated protein (MAP) kinases transduce signals emanating from their upstream activators: MAP kinase kinases (MEKs). The MEK-MAP kinase signaling cassette is a key regulatory pathway promoting cell survival. The downstream effectors of the mammalian MEK-MAP kinase cell survival signal have not been previously described. Identified here is a pro-survival role for the serine/threonine kinase S6 kinase p90 ribosomal S6 kinase Rsk1, a downstream target of the MEK-MAP kinase signaling pathway. In cells that are dependent on interleukin-3 (IL-3) for survival, pharmacological inhibition of MEKs antagonize the IL-3 survival signal. In the absence of IL-3, a kinase-dead Rsk1 mutant eliminates the survival effect afforded by activated MEK. Conversely, a novel constitutively active Rsk1 allele restores the MEK-MAP kinase survival signal. Experiments in vitro and in vivo have demonstrated that Rsk1 directly phosphorylates the pro-apoptotic protein Bad at the serine residues that, when phosphorylated, abrogate Bad's pro-apoptotic function. Constitutively active Rsk1 causes constitutive Bad phosphorylation and protection from Bad-modulated cell death. Kinase-inactive Rsk1 mutants antagonize Bad phosphorylation. Bad mutations that prevent phosphorylation by Rsk1 also inhibit Rsk1-mediated cell survival. These data support a model in which Rsk1 transduces the mammalian MEK-MAP kinase signal in part by phosphorylating Bad (Shimamura, 2000).
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