Regulation of the extracellular signal-regulated kinase pathway in adult myocardium: differential roles of G(q/11), Gi and G(12/13) proteins in signalling by alpha1-adrenergic, endothelin-1 and thrombin-sensitive protease-activated receptors

Using adenoviruses encoding RGS2, RGS4 and Lsc (regulator of G protein signalling (RGS) domain of p115 RhoGEF), we investigated the contributions of G(q/11), Gi and G(12/13) proteins to G protein-coupled receptor (GPCR)-mediated activation of the extracellular signal-regulated kinase (ERK) pathway in adult rat ventricular myocytes (ARVM). Exposure to phenylephrine, endothelin-1 (ET-1) or thrombin induced significant activation of ERK1/2 and their downstream target 90 kDa ribosomal S6 kinase (p90RSK), which was abolished by overexpression of RGS4 (inhibits signalling via G(q/11) and Gi) or RGS2 (inhibits signalling via G(q/11)). Pertussis toxin (inhibits signalling via Gi) only partially attenuated the activation of ERK1/2 and p90(RSK) by phenylephrine and ET-1, but abolished such activation by thrombin. Overexpression of Lsc (inhibits signalling via G(12/13)) did not affect the responses to phenylephrine and ET-1, but suppressed the activation of ERK1/2 and p90RSK by thrombin. We conclude that full activation of the ERK pathway in ARVM by alpha1-adrenergic, ET-1 and thrombin receptors requires the activation of distinct families of heterotrimeric G proteins.     

Stimulation of the plasma membrane Na+/H+ exchanger NHE1 by sustained intracellular acidosis. Evidence for a novel mechanism mediated by the ERK pathway

Activity of the Na+/H+ exchanger (NHE) isoform 1 (NHE1) is increased by intracellular acidosis through the interaction of intracellular H+ with an allosteric modifier site in the transport domain. Additional regulation is achieved via kinase-mediated modulation of the NHE1 regulatory domain. To determine if intracellular acidosis stimulates NHE1 activity solely by the allosteric mechanism, we subjected cultured neonatal rat ventricular myocytes (NRVM) with native NHE1 expression to intracellular acidosis (pHi approximately 6.6) for up to 6 min by transient exposure to NH4Cl and its washout in the presence of NHE inhibition (by zero [Na+]o or the NHE1 inhibitor cariporide) in HCO3- -free medium. After the desired duration of acidosis, NHE was reactivated (by reintroduction of [Na+]o or removal of cariporide), and the rate of recovery of pHi (dpHi/dt) was measured as the index of NHE activity. Regardless of the method used when intracellular acidosis was sustained for > or =3 min, subsequent NHE activity was significantly increased (>4-fold). Similar NHE stimulatory effects of sustained acidosis were observed in adult rat ventricular myocytes and COS-7 cells. Sustained (3 min) intracellular acidosis activated several NHE1 kinases in NRVM, in an in-gel kinase assay using as substrate a glutathione S-transferase fusion protein of the NHE1 regulatory domain. Detailed investigation of ERK and its downstream effector p90RSK, two putative NHE1 kinases, revealed time-dependent activation of both by intracellular acidosis in NRVM. Furthermore, inhibition of MEK1/2 by pretreatment of NRVM with two structurally distinct inhibitors, PD98059 (30 microM) or UO126 (3 microM), inhibited the activation of ERK and p90RSK and abolished the stimulation of NHE activity by sustained (3 min) intracellular acidosis. Our data show that not only the extent but also the duration of intracellular acidosis regulates NHE1 activity and suggest that the stimulatory effect of sustained intracellular acidosis occurs through a novel mechanism mediated by activation of the ERK pathway.     

A novel role for protein phosphatase 2A in receptor-mediated regulation of the cardiac sarcolemmal Na+/H+ exchanger NHE1

G(q) protein-coupled receptor stimulation increases sarcolemmal Na(+)/H(+) exchanger (NHE1) activity in cardiac myocytes by an ERK/RSK-dependent mechanism, most likely via RSK-mediated phosphorylation of the NHE1 regulatory domain. Adenosine A(1) receptor stimulation inhibits this response through a G(i) protein-mediated pathway, but the distal inhibitory signaling mechanisms are unknown. In cultured adult rat ventricular myocytes (ARVM), the A(1) receptor agonist cyclopentyladenosine (CPA) inhibited the increase in NHE1 phosphorylation induced by the alpha(1)-adrenoreceptor agonist phenylephrine, without affecting activation of the ERK/RSK pathway. CPA also induced significant accumulation of the catalytic subunit of type 2A protein phosphatase (PP2A(c)) in the particulate fraction, which contained the cellular NHE1 complement; this effect was abolished by pretreatment with pertussis toxin to inactivate G(i) proteins. Confocal immunofluorescence microscopic imaging of CPA-treated ARVM revealed significant co-localization of PP2A(c) and NHE1, in intercalated disc regions. In an in vitro assay, purified PP2A(c) dephosphorylated a GST-NHE1 fusion protein containing aa 625-747 of the NHE1 regulatory domain, which had been pre-phosphorylated by recombinant RSK; such dephosphorylation was inhibited by the PP2A-selective phosphatase inhibitor endothall. In intact ARVM, the ability of CPA to attenuate the phenylephrine-induced increase in NHE1 phosphorylation and activity was lost in the presence of endothall. These studies reveal a novel role for the PP2A holoenzyme in adenosine A(1) receptor-mediated regulation of NHE1 activity in ARVM, the mechanism of which appears to involve G(i) protein-mediated translocation of PP2A(c) and NHE1 dephosphorylation.     

ERK1/2-p90RSK-mediated phosphorylation of Na+/H+ exchanger isoform 1. A role in ischemic neuronal death

The function and regulation of Na(+)/H(+) exchanger isoform 1 (NHE1) following cerebral ischemia are not well understood. In this study, we demonstrate that extracellular signal-related kinases (ERK1/2) play a role in stimulation of neuronal NHE1 following in vitro ischemia. NHE1 activity was significantly increased during 10-60 min reoxygenation (REOX) after 2-h oxygen and glucose deprivation (OGD). OGD/REOX not only increased the V(max) for NHE1 but also shifted the K(m) toward decreased [H(+)](i). These changes in NHE1 kinetics were absent when MAPK/ERK kinase (MEK) was inhibited by the MEK inhibitor U0126. There were no changes in the levels of phosphorylated ERK1/2 (p-ERK1/2) after 2 h OGD. The p-ERK1/2 level was significantly increased during 10-60 min REOX, which was accompanied by nuclear translocation. U0126 abolished REOX-induced elevation and translocation of p-ERK1/2. We further examined the ERK/90-kDa ribosomal S6 kinase (p90(RSK)) signaling pathways. At 10 min REOX, phosphorylated NHE1 was increased with a concurrent elevation of phosphorylation of p90(RSK), a known NHE1 kinase. Inhibition of MEK activity with U0126 abolished phosphorylation of both NHE1 and p90(RSK). Moreover, neuroprotection was observed with U0126 or genetic ablation or pharmacological inhibition of NHE1 following OGD/REOX. Taken together, these results suggest that activation of ERK1/2-p90(RSK) pathways following in vitro ischemia phosphorylates NHE1 and increases its activity, which subsequently contributes to neuronal damage.     

Molecular interpretation of ERK signal duration by immediate early gene products

The duration of intracellular signalling is associated with distinct biological responses, but how cells interpret differences in signal duration are unknown. We show that the immediate early gene product c-Fos functions as a sensor for ERK1 (extracellular-signal-regulated kinase 1) and ERK2 signal duration. When ERK activation is transient, its activity declines before the c-Fos protein accumulates, and under these conditions c-Fos is unstable. However, when ERK signalling is sustained, c-Fos is phosphorylated by still-active ERK and RSK (90K-ribosomal S6 kinase). Carboxy-terminal phosphorylation stabilizes c-Fos and primes additional phosphorylation by exposing a docking site for ERK, termed the FXFP (DEF) domain. Mutating the DEF domain disrupts the c-Fos sensor and c-Fos-mediated signalling. Other immediate early gene products that control cell cycle progression, neuronal differentiation and circadium rhythms also contain putative DEF domains, indicating that multiple sensors exist for sustained ERK signalling. Together, our data identify a general mechanism by which cells can interpret differences in ERK activation kinetics.     

Sustained intracellular acidosis activates the myocardial Na/H exchanger independent of amino acid Ser and p90

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the myocardium and other tissues. It is an important mediator of the myocardial damage that occurs after ischemia-reperfusion injury and is implicated in heart hypertrophy. Regulation of NHE1 has been proposed as a therapeutic target for cardioprotection. We therefore examined mechanisms of control of NHE1 in the myocardium. Several different amino acids have been implicated as a being critical to NHE1 regulation in a number of tissues including Ser⁷⁰³, Ser⁷⁷⁰, and Ser⁷⁷¹. In the myocardium, NHE1 is activated in response to a variety of stimuli including activation by an ERK-dependent sustained intracellular acidosis. In this study, we determined whether Ser⁷⁰³ and p90^rsk activity are critical in activation of NHE1 by sustained intracellular acidosis. In vitro phosphorylation of NHE1 C-terminal fusion proteins determined that ERK-dependent phosphorylation of the cytoplasmic region was not dependent on Ser⁷⁰³; however, phosphorylation by p90^rsk required Ser⁷⁰³. A Ser703Ala mutation decreased basal NHE1 activity in CHO cells but not in cardiomyocytes. NHE1 with a Ser703Ala mutation was activated in response to sustained intracellular acidosis in CHO cells. In addition, sustained intracellular acidosis also activated the Ser703Ala mutant protein in isolated cardiomyocytes and phosphorylation levels were also increased by acidosis. The presence of a dominant-negative p90^rsk kinase also did not prevent activation and phosphorylation of NHE1 by sustained intracellular acidosis in isolated cardiomyocytes. We conclude that Ser⁷⁰³ and p90^rsk are not required for activation by sustained intracellular acidosis and that p90^rsk phosphorylation of Ser⁷⁰³ is independent of this type of activation.     

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.     

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.     

90-kDa ribosomal S6 kinase is phosphorylated and activated by 3-phosphoinositide-dependent protein kinase-1.

90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation of the linker region at Ser(369), catalyzed by extracellular signal-regulated kinase (ERK), and at Ser(386), catalyzed by the C-terminal kinase, after its activation by ERK. In addition, the N-terminal kinase must be phosphorylated at Ser(227) in the activation loop by an as yet unidentified kinase. Here, we show that the isolated N-terminal kinase of RSK2 (amino acids 1-360) is phosphorylated at Ser(227) by PDK1, a constitutively active kinase, leading to 100-fold stimulation of kinase activity. In COS7 cells, ectopic PDK1 induced the phosphorylation of full-length RSK2 at Ser(227) and Ser(386), without involvement of ERK, leading to partial activation of RSK2. Similarly, two other members of the RSK family, RSK1 and RSK3, were partially activated by PDK1 in COS7 cells. Finally, our data indicate that full activation of RSK2 by growth factor requires the cooperation of ERK and PDK1 through phosphorylation of Ser(227), Ser(369), and Ser(386). Our study extend recent findings which implicate PDK1 in the activation of protein kinases B and C and p70(S6K), suggesting that PDK1 controls several major growth factor-activated signal transduction pathways.     

Truncated MEK1 is required for transient activation of MAPK signalling in G2 phase cells

The primary endpoint of signalling through the canonical Raf–MEK–ERK MAP kinase cascade is ERK activation. Here we report a novel signalling outcome for this pathway. Activation of the MAP kinase pathway by growth factors or phorbol esters during G2 phase results in only transient activations of ERK and p90RSK, then suppression to below control levels. A small peak of ERK and p90RSK activation in early G2 phase cells was identified, and inhibition of this delayed entry into mitosis. The previously identified, proteolytically cleaved form of MEK1 termed tMEK (truncated MEK1), is also induced with G2 phase MAPK pathway activation. We demonstrate that addition of recombinant mutants of MEK1 with an N-terminal truncation similar to that of tMEK also inhibited ERK and p90RSK activations and delayed progression into mitosis. Only catalytically inactive forms of tMEK were capable of these effects, but surprisingly, phosphorylation on the activating Ser218/222 sites was also required. A lack of MEK1 or ability to accumulate tMEK resulted in the absence of the feedback inhibition of ERK and p90RSK activations. tMEK is a novel output from the canonical MAP kinase signalling pathway, acting in a MAPK signalling-regulated dominant negative manner to inhibit ERK and p90RSK activations, acting as a dampening mechanism to reduce the magnitude or duration of MAPK pathway signalling in G2/M phase.     

Characterization of Regulatory Events Associated with Membrane Targeting of p90 Ribosomal S6 Kinase 1

RSK is a serine/threonine kinase containing two distinct catalytic domains. Found at the terminus of the Ras/extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) kinase cascade, mitogen-stimulated ribosomal S6 kinase (RSK) activity requires multiple inputs. These inputs include phosphorylation of the C-terminal kinase domain activation loop by ERK1/2 and phosphorylation of the N-terminal kinase domain activation loop by phosphoinositide-dependent protein kinase-1 (PDK1). Previous work has shown that upon mitogen stimulation, RSK accumulates in the nucleus. Here we show that prior to nuclear translocation, epidermal growth factor-stimulated RSK1 transiently associates with the plasma membrane. Myristylation of wild-type RSK1 results in an activated enzyme in the absence of added growth factors. When RSK is truncated at the C terminus, the characterized ERK docking is removed and RSK phosphotransferase activity is completely abolished. When myristylated, however, this myristylated C-terminal truncated form (myrCTT) is activated at a level equivalent to myristylated wild-type (myrWT) RSK. Both myrWT RSK and myrCTT RSK can signal to the RSK substrate c-Fos in the absence of mitogen activation. Unlike myrWT RSK, myrCTT RSK is not further activated by serum. Only the myristylated RSK proteins are basally phosphorylated on avian RSK1 serine 381, a site critical for RSK activity. The myristylated and unmyristylated RSK constructs interact with PDK1 upon mitogen stimulation, and this interaction is insensitive to the MEK inhibitor UO126. Because a kinase-inactive CTT RSK can be constitutively activated by targeting to the membrane, we propose that ERK may have a dual role in early RSK activation events: preliminary phosphorylation of RSK and escorting RSK to a membrane-associated complex, where additional MEK/ERK-independent activating inputs are encountered.     

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.     

Role of epidermal growth factor receptor (EGFR)-signaling versus cellular acidosis via Na+/H+ exchanger1(NHE1)-inhibition in troglitazone-induced growth arrest of breast cancer-derived cells MCF-7.

PURPOSE:We previously showed that troglitazone (TRO) induces a profound cellular acidosis in MCF-7 cells as a result of inhibiting Na(+)/H(+) exchanger (NHE)1-mediated acid extrusion and this was associated with a marked reduction in cellular proliferation. The present study focuses on TRO-activated signaling pathways versus TRO-mediated NHE1-inhibition in reducing DNA synthesis. EXPERIMENTAL DESIGN: TRO activation of the signaling pathway involving epidermal growth factor receptor (EGFR)/MAPK/ERK kinase (MEK) 1/2/extracellular signal-regulated kinase (ERK) 1/2 was studied by Western blotting and phospho-specific antibodies. TRO induction of cellular acidosis and inhibition of NHE1 activity were measured using (2, 7)-biscarboxyethyl-5 (6)-carboxyfluorescein (BCECF) assay and NH4(+)/NH(3) pulsing. Cellular proliferation was assessed as DNA synthesis by (3)H-thymidine incorporation. RESULTS: TRO simultaneously reduces pH(i) and elevates phosphorylated-extracellular signal-regulated kinase (p-ERK). These responses reflected inhibition of acid extrusion and EGFR activation respectively and were sustained over 18h associated with a large decrease in DNA synthesis. Preventing TRO-induced ERK activation did not restore DNA synthesis or cellular pH. CONCLUSIONS: TRO activates two parallel pathways: I] EGFR/MEK1/2/ERK1/2 and II] NHE1 inhibition/cellular acidosis. Elimination of I] did not prevent the inhibition of DNA synthesis consistent with TRO-induced growth arrest dependent upon II] in tumorigenic non-metastatic breast cancer derived MCF-7 cells.     

Rapid and transient activation of the ERK MAPK signalling pathway by macrophage migration inhibitory factor (MIF) and dependence on JAB1/CSN5 and Src kinase activity

Macrophage migration inhibitory factor (MIF) is a 12.5 kD polypeptide that serves as a critical regulator of cell functions such as gene expression, proliferation or apoptosis. However, the signal transduction pathways through which MIF takes part in cellular regulation are only incompletely understood. MIF leads to CD74-dependent "sustained" activation of ERK1/2 MAPK, but MIF's role in "transient" ERK activation and the involved upstream pathways are unknown. Here we report that the transient ERK pathway was markedly activated by MIF. This effect involved the phosphorylation and activation of Raf-1, MEK, ERK, and Elk-1. Of note, rapid and transient ERK phosphorylation by MIF was measurable in MIF-deficient cells, suggesting that MIF acted in a non-autocrine fashion. Applying the inhibitor genistein, a tyrosine kinase (TPK) activity was identified as a critical upstream signalling event in MIF-induced transient ERK signalling. Experiments using the Src kinase inhibitor PP2 indicated that the involved TPK was a Src-type tyrosine kinase. A role for an upstream Src kinase was proven by applying Src-deficient cells which did not exhibit transient ERK activation upon treatment with MIF, but in which MIF-induced ERK signalling could be restored by re-expressing Src. Intriguingly, JAB1/CSN5, a signalosome component, cellular binding protein of MIF and regulator of cell proliferation and survival, had a marked, yet dual, effect on MIF-induced ERK signalling. JAB1 overexpression inhibited sustained, but not transient, ERK phosphorylation. By contrast, JAB1-knock-down by siRNA revealed that minimum JAB1 levels were necessary for transient activation of ERK by MIF. In conclusion, MIF rapidly and transiently activates the ERK pathway, an effect that has not been recognized previously. This signalling pathway involves the upstream activation of a Src-type kinase and is co-regulated by the cellular MIF binding protein JAB1/CSN5. Our study thus has unravelled a novel MIF-driven signalling pathway and an intricate regulatory system involving extra- and possibly intracellular MIF, and which likely critically participates in controlling cell proliferation and survival.     

Bigendothelin-1 via p38-MAPK-dependent mechanism regulates adult rat ventricular myocyte contractility in sepsis

We tested the hypothesis that exogenous administration of the ET-1 precursor, bigET-1, would regulate adult rat ventricular myocyte (ARVM) contractility in a p38-mitogen activated protein kinase (p38-MAPK)-dependent mechanism during sepsis. Ventricular myocytes from adult rat hearts (both sham and septic) were stimulated to contract at 0.5 Hz and mechanical properties were evaluated using an IonOptix Myocam system. Immunoblot analysis was used to determine the phosphorylation of p38-MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2). ARVMs were treated with vehicle, bigET-1 and inhibitors for 24 h and then subjected to functional and biochemical estimations. Septic ARVM displayed a distorted cell membrane and irregular network within the cells along with increased cell contractility as evidenced by elevated peak shortening (PS), maximal velocity of shortening (+dL/dt) and relengthening (-dL/dt) in comparison to sham ARVM. BigET-1 treatment caused ARVM enlargement in both sham and sepsis groups. BigET-1 (100 nM) produced an increase in ARVM contractility in sham group as compared to vehicle treatment. However, septic ARVM treated with bigET-1 exhibited unaltered ARVM contractility, and upregulated ET(B) receptors as compared to respective sham group. BigET-1 increased the concentration of ET-1 and upregulated phosphorylation of p38-MAPK but not of ERK1/2 in sham and septic ARVM. Furthermore, inhibition of p38-MAPK by SB203580 (10 microM) increased ARVM contractility in sham but not in sepsis group. BigET-1 reversed SB203580-induced increase in PS in sham group but accentuated it in sepsis group. BigET-1 also reversed SB203580-induced inhibition of p38-MAPK phosphorylation in sham but not in septic ARVM. SB203580 pretreatment followed by bigET-1 administration significantly decreased p38-MAPK phosphorylation and downregulated ET(B) receptor expression as compared to bigET-1 treatment per se in sepsis group but not in sham. We concluded that a bigET-1-induced non-responsive effect on septic ARVM contractile function could be due to upregulation of p38-MAPK phosphorylation and ET(B) receptor expression.     

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.     

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.     

Mitogen-activated protein kinase-dependent activation of the Na+/H+ exchanger is mediated through phosphorylation of amino acids Ser770 and Ser771

We investigated regulation of the type 1 isoform of the Na(+)/H(+) exchanger by phosphorylation. Four specific groups of serine and threonine residues in the regulatory carboxyl-terminal tail were mutated to alanine residues: group 1, S693A; group 2, T718A and S723A/S726A/S729A; group 3, S766A/S770A/S771A; and group 4, T779A and S785A. The proteins were expressed in Na(+)/H(+) exchanger-deficient cells, and the activity was characterized. All of the mutants had proper expression, localization, and normal basal activity relative to wild type NHE1. Sustained intracellular acidosis was used to activate NHE1 via an ERK-dependent pathway that could be blocked with the MEK inhibitor U0126. Immunoprecipitation of (32)P-labeled Na(+)/H(+) exchanger from intact cells showed that sustained intracellular acidosis increased Na(+)/H(+) exchanger phosphorylation in vivo. This was blocked by U0126. The Na(+)/H(+) exchanger activity of mutants 1 and 2 was stimulated similar to wild type Na(+)/H(+) exchanger. Mutant 4 showed a partially reduced level of activation. However, mutant 3 was not stimulated by sustained intracellular acidosis, and loss of stimulation of activity correlated to a loss of sustained acidosis-mediated phosphorylation in vivo. Mutation of the individual amino acids within mutant 3, Ser(766), Ser(770), and Ser(771), showed that Ser(770) and Ser(771) are responsible for mediating increases in NHE1 activity through sustained acidosis. Both intact Ser(770) and Ser(771) were required for sustained acidosis-mediated activation of NHE1. Our results suggest that amino acids Ser(770) and Ser(771) mediate ERK-dependent activation of the Na(+)/H(+) exchanger in vivo.