From the Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, Oregon 97201
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The tumor promoter palytoxin has been found to activate the stress-activated protein kinase/c-Jun NH2-terminal kinase 1 (SAPK/JNK1), and it also potentiates, as demonstrated here, the p38/HOG1 mitogen-activated protein kinase and the upstream activator of SAPK/JNK1, SEK1/MKK4. In search of possible mechanisms for both the cytotoxicity and the activation of stress kinases by palytoxin, we found that palytoxin is a potent inhibitor of cellular protein synthesis. The inhibition of translation by palytoxin does not result from its direct binding to the translational apparatus. We have previously demonstrated that ribotoxic stressors (Iordanov, M. S., Pribnow, D., Magun, J. L., Dinh, T.-H., Pearson, J. A., Chen, S. L.-Y., and Magun, B. E. (1997) Mol. Cell. Biol. 17, 3373-3381) signal the activation of SAPK/JNK1 by binding to or covalently modifying 28 S rRNA in ribosomes that are active at the time of exposure to the stressor. Palytoxin acted as a ribotoxic stressor, inasmuch as it required actively translating ribosomes at the time of exposure to activate SAPK/JNK1. Palytoxin has been shown to augment ion fluxes by binding to the Na+/K+-ATPase in the plasma membrane of cells. To determine whether altered fluxes of either Na+ or K+ could be responsible for the effects of palytoxin on translation and on activation of SAPK/JNK1, cells were exposed to palytoxin in modified culture medium in which a major portion of the Na+ was replaced by either K+ or by choline+. The substitution of Na+ by K+ strongly inhibited the ability of palytoxin both to inhibit protein translation and to activate SAPK/JNK1, whereas the substitution of Na+ by choline+ did not. These results suggest that palytoxin-induced efflux of cellular K+ mimics ribotoxic stress by provoking both translational inhibition and activation of protein kinases associated with cellular defense against stress.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Palytoxin is a non-peptide water-soluble marine toxin that is a
potent tumor promoter in the mouse skin carcinogenesis model (2-4).
Many tumor-promoting compounds that are effective in the skin of
carcinogen-initiated mice also produce an inflammatory reaction
(5-15). Inflammatory mediators such as interleukin-1 (IL-1)1 and tumor necrosis
factor- are potent activators of stress kinases such as SAPK/JNK1
and p38/HOG1 (for a review, see Ref. 16). Kuroki et al. (17)
have shown that Swiss 3T3 cells exposed to concentrations of palytoxin
as low as 0.1 nM display an abundant activation of the
stress-activated kinase SAPK/JNK1. Stimulation of SAPK/JNK1 has been
shown to lead to activation of AP-1, a dimeric transcription factor
composed of Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2)
family members (for a review, see Ref. 18). SAPK/JNK1 phosphorylates
c-Jun at serines 63 and 73 in its NH2 terminus, thereby
increasing the transcription activating potential of AP-1 (19, 20).
Stimulation of SAPK/JNK1 also leads to the phosphorylation and
activation of the transcription factor Elk-1 (21, 22), which operates
on the regulatory region of c-fos (for reviews, see Refs. 23
and 24), and to the phosphorylation of activating transcription
factor-2 (25, 26), which, as an activating transcription factor-2/c-Jun
heterodimer, operates on the promoter of c-jun (27). Thus,
SAPK/JNK1 increases AP-1 activity in the nucleus by activating
pre-existing AP-1 complexes and by transcriptionally inducing the
expression of the components of AP-1, c-Jun, and c-Fos.
The cellular effects of palytoxin, which include ionic disequilibria (28-30), increased production of prostaglandins from arachidonic acid (31-36), and alterations in the affinity of the EGF receptor (37-39), have been attributed to the ability of palytoxin to bind to the Na+/K+-ATPase situated in the plasma membrane. Direct binding of palytoxin to the Na+/K+-ATPase, also known as the sodium pump, transforms the pump into a permanently open ion channel that permits the outward flux of K+ and the inward flux of Na+ and that is independent of ATP hydrolysis (28-30). Conclusive demonstration that palytoxin acts through the sodium pump has come from heterologous expression of the sodium pump in Saccharomyces cerevisiae, which lose intracellular K+ following exposure to palytoxin (29, 30).
Recently, we reported on the identification of the 28 S ribosomal RNA
as a specific sensor for stress induced by a subset of agents that
inhibit protein synthesis (1). Some inhibitors of translation are
strong activators of SAPK/JNK1, whereas other equally effective
inhibitors of translation are unable to activate SAPK/JNK1. The
translational inhibitors that activated SAPK/JNK1, termed ribotoxic
stressors (1), either bind to the 28 S rRNA in the peptidyl transferase
center (40) (e.g. anisomycin and blasticidin S) or cause
specific damage to 28 S rRNA (e.g. ricin A chain and
-sarcin) within a conserved loop involved in binding of the
elongation factors EF-1 and EF-2 (for a review, see Ref. 41).
Activation of SAPK/JNK1 and of its upstream activator SEK1/MKK4 by this
group of ribotoxins can occur only when ribosomes are actively
translating at the time of exposure to the ribotoxic stressor. Prior
inhibition of translation by nonactivating agents such as diphtheria
toxin, T-2 toxin, pactamycin, or emetine for as little time as 2 min
abrogate the ability of the ribotoxic stressors, but not of IL-1
or
osmotic stress, to stimulate SAPK/JNK1 activity. We concluded that the
sensors for ribotoxic damage are ribosomes, which can transduce signals
that activate SEK1/MKK4 and SAPK/JNK1 only when they are
translationally active at the time of induced ribotoxicity. Although
many stress signals ultimately converge to activate the stress kinases,
signals arising during ribotoxicity are initially conveyed through a
pathway distinct from those used by IL-1
and osmotic stress (1). The
transduction of stress signals through ribosomes is a feature that
eukaryotes share with prokaryotes, whose ribosomes respond to some
translational inhibitors by recapitulating cellular responses
characteristic of either heat shock or cold shock (42).
In the experiments described here, we found that, like ribotoxic stressors, palytoxin potently inhibited protein synthesis in the concentration range that leads to activation of the stress kinases SEK1/MKK4, SAPK/JNK1, and p38/HOG1. Similar to ribotoxic stressors, the ability of palytoxin to activate SAPK/JNK1 depended on the presence of actively translating ribosomes at the time of exposure to palytoxin. However, unlike ribotoxic stressors, palytoxin did not inhibit protein synthesis as a consequence of direct binding to the translational apparatus. To determine whether the effects of palytoxin on translation and SAPK/JNK1 activation resulted from altered ionic fluxes, a major portion of the Na+ in the culture medium was replaced by either K+ or choline+. The substitution of Na+ by K+, but not by choline+, inhibited the ability of palytoxin both to inhibit protein translation and to activate SAPK/JNK1, suggesting that the efflux of cellular K+ may be responsible for both translational inhibition and activation of SAPK/JNK1. Thus, loss of K+ from stressed cells may lead to the activation of stress kinases through ribosome-mediated signaling.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chemicals, Cytokines, and Ribotoxins--
Anisomycin, emetine,
D-sorbitol, and choline were from Sigma. IL-1
(recombinant mouse) was from Genzyme (Cambridge, MA). Palytoxin was
from Calbiochem. Anisomycin, emetine, and palytoxin were dissolved in
(H3C)2SO. In all the cases when
(H3C)2SO was used as a vehicle, corresponding
control cells received the same amount of the vehicle alone (typically
not more than 0.2% (v/v)). D-Sorbitol was dissolved in
Dulbecco's modified Eagle's medium (DMEM) as a 3 M stock
solution. All radiochemicals were from NEN Life Science Products.
Cell Culture-- Rat-1 cells were maintained as described previously (43). The derivative cell line FC2-Rat1 has been described by Rodland et al. (44). All experiments presented here were performed using confluent, quiescent cultures obtained through serum deprivation for typically 24 h. DMEM and MEM were from Life Technologies, Inc.
Immunoprecipitation of SAPK/JNK1 and Extracellular Signal-regulated Kinase and Immunocomplex Kinase Assays-- All immunoprecipitations and immunocomplex kinase reactions were performed as described for SAPK/JNK1 in Ref. 1. For immunoprecipitation of SAPK/JNK1, the antibody sc-474 was used, and for immunoprecipitation of extracellular signal-regulated kinase 1, the antibody sc-93-G was used (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Western Blot Analysis of SEK1/MKK4 Phosphorylation-- The analysis of threonine 223 phosphorylation of SEK1/MKK4 was performed with the antibody 9151S, and the analysis of tyrosine 182 phosphorylation of p38/HOG1 was performed with the antibody 9211S (both from New England BioLabs Inc., Beverly, MA) as described in Ref. 1.
Measurement of Protein Synthesis-- Incorporation of [3H]leucine was performed as described in Ref. 1. Determination of protein synthesis using FC2-Rat1 cells and a chloramphenicol acetyltransferase (CAT) assay has been described by Rodland et al. (44). The measurement of luciferase mRNA translation was performed with the Rabbit Reticulocyte Lysate System (catalog number L4960) as described by the manufacturer (Promega, Madison, WI).
Measurement of Na+/K+-ATPase Activity-- The activity of the Na+/K+-ATPase in membranal preparations from Rat-1 cells was determined as described by Brotherus et al. (51) with modifications. Briefly, S100 microsomal pellets (20 µg of total protein/experimental point) were resuspended in 0.6 ml of assay solution (pH 7.2; 0.6 mM EGTA, 156 mM NaCl, 24 mM KCl, 3.6 mM MgCl2, 3.6 mM ATP, 60 mM imidazole, 10 mM Na3N; with or without 0.5 mM ouabain) in the presence of varying concentrations of palytoxin. After 60 min of incubation at 37 °C, the reactions were stopped by the addition of 1.5 ml of ice-cold stopping solution (made by the sequential addition on ice of 21.3 ml of 1 M HCl, 18.3 ml of H2O, 1.29 g of L-ascorbic acid, 2.13 ml of 10% ammonium molybdate, and 3.3 ml of 20% SDS). After 10 min of incubation on ice, the color development was achieved by the addition of 1.5 ml of a solution containing 2% (w/v) sodium meta-arsenite, 2% (w/v) sodium citrate, and 2% (v/v) glacial acetic acid. After incubation for 10 min at room temperature, the absorption of the samples was measured at 850 nm, and the amount of Pi in each sample was determined using a standard containing 50 µM Na3PO4.
PhosphorImager and Statistical Analyses--
The quantification
of 32PO43 transferred from
ATP onto GST-Elk-1 in experiments measuring SAPK/JNK1 kinase activity
was performed using the PhosphorImager apparatus and the IPLab
GelTM software from Molecular Dynamics (Sunnyvale, CA). Statistical
analyses were performed using the StatViewTM software from Abacus
Concepts, Inc. (Berkeley, CA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation of SAPK/JNK1, SEK1/MKK4, and p38/HOG1 by Palytoxin-- The addition of palytoxin to serum-deprived Rat-1 cells potently induced the activation of SAPK/JNK1 when examined 30 min later (Fig. 1, A and B). As reported previously by Kuroki et al. (17), the ability of palytoxin to activate SAPK/JNK1 was biphasic. At concentrations greater than 1 nM, cells displayed a decreased responsiveness to palytoxin; concentrations of palytoxin greater than 10 nM were ineffective in activating SAPK/JNK1.
|
Translational Inhibition by Palytoxin-- Previously we reported that some adverse agents that activate SAPK/JNK1 require ribosomes actively engaged in translation at the the moment of exposure to do so, while simultaneously inhibiting protein synthesis. These agents were termed ribotoxic stressors (1). To determine if it could be acting as a ribotoxic stressor, palytoxin was tested for its ability to inhibit the incorporation of [3H]leucine into proteins in Rat-1 cells. Indeed, palytoxin inhibited the incorporation of [3H]leucine in a dose-dependent manner (Fig. 1B). The ability of palytoxin to both activate stress kinases (Fig. 1, A and B) and repress [3H]leucine incorporation (Fig. 1B) became apparent at concentrations of the toxin that were inhibitory for the Na+/K+-ATPase in membranal preparations isolated from Rat-1 cells (Fig. 1B; see "Experimental Procedures," and see below). Because palytoxin is known to act on membrane-associated functions, we considered the possibility that transport of [3H]leucine into cells is impaired following exposure to palytoxin. To avoid this potential complication, we applied a method for measuring cellular protein synthesis that is independent of transport and incorporation of amino acids. A derivative of the parental Rat-1 cells, the cell clone FC2-Rat1 bears a stably integrated CAT reporter gene under the control of a 1-kilobase promoter sequence of the human c-fos gene (44). The expression of CAT mRNA is kept at very low levels in quiescent cells, but it can be induced up to 100-fold upon stimulation with EGF in the presence of cycloheximide (not shown). The presence of cycloheximide prevents the translation of the CAT mRNA into CAT protein. Release from the cycloheximide-induced translational arrest (Fig. 1C, t = 0 min) allows efficient translation of the accumulated CAT mRNA. The accumulation of CAT protein increased linearly within 2 h following the washout of cycloheximide as measured by a CAT activity assay (Fig. 1C). Since EGF was also removed from the medium together with cycloheximide, the increase of CAT activity resulted solely from translation of the CAT mRNA that had accumulated before the washout. Treatment of the cells with palytoxin 60 min after the release from translational arrest resulted in a substantial decrease in CAT activity detectable both 30 and 60 min after the addition of palytoxin (Fig. 1C). We therefore conclude that exposure of cells to palytoxin indeed interferes with the process of translation.
Many inhibitors of protein synthesis alter ribosomal activity by binding directly to ribosomes (49, 50). The actions of these inhibitors are readily detected following their addition to reticulocyte lysates that contain ribosomes and all of the necessary ingredients to allow the initiation and elongation of translation in vitro. To test whether palytoxin could inhibit protein synthesis by directly modifying the functionality of ribosomes in vitro, we added palytoxin to a reticulocyte lysate preparation engaged in protein synthesis (Fig. 2). The addition to the reticulocyte lysate of mRNA for luciferase reporter protein (Fig. 2, t = 0 min) and subsequent monitoring of luciferase activity demonstrated the effectiveness of the lysate in promoting both initiation and elongation. The addition of the elongation inhibitor emetine at 8 min completely blocked the further accumulation of luciferase activity. Palytoxin (30 nM) was completely ineffective in inhibiting translation of luciferase when added prior to the addition of luciferase mRNA. These data demonstrate that neither translational initiation nor translational elongation were affected by the addition of palytoxin to a cell-free translational system. We therefore conclude that the inhibition of translation induced by palytoxin in vivo is unlikely to be mediated by direct interaction between palytoxin and the translational apparatus.
|
Translational Inhibitors Interfere with Palytoxin-induced Activation of SAPK/JNK1-- The demonstration that palytoxin is a potent in vivo inhibitor of protein synthesis prompted us to test whether palytoxin shares some of the properties of ribotoxic stressors, which both activate SAPK/JNK1 and inhibit protein translation (1). We previously showed that the activation of SAPK/JNK1 by ribotoxic stressors is rapidly suppressed in cells whose protein synthesis had been previously blocked by translational inhibitors that are incapable of activating SAPK/JNK1 (1). Mediation of SAPK/JNK1-activating signals could only occur in ribosomes that are active at the time of exposure of ribotoxic stressors. The following experiments test whether activation of SAPK/JNK1 by palytoxin similarly depends on the presence of active ribosomes.
Pretreatment of cells for 15 min with emetine, an inhibitor of translational elongation, suppressed the palytoxin-induced activation of SAPK/JNK1 measured 30 min after the addition of palytoxin (Fig. 3, lanes 2 and 3). A similar suppression of SAPK/JNK1 activation was observed when cells were preincubated with pactamycin, a specific inhibitor of translational initiation (data not shown). Emetine also suppressed the activation of SAPK/JNK1 by the ribotoxic stressor anisomycin (1) (Fig. 3, lanes 4 and 5), but not by sorbitol, an osmotic stressor that does not act through ribosomal toxicity (1) (Fig. 3, lanes 6 and 7).
|
|
Effects of Palytoxin on Translation and Signaling in Media Containing Modified [Na+], [K+], or [Choline+]-- Several lines of evidence indicate that the biological effects of palytoxin are mediated through its interaction with the Na+/K+-ATPase (28-30). The most convincing evidence derives from the introduction of a mammalian Na+/K+-ATPase into yeast, which demonstrated that palytoxin interacts directly with this molecule to facilitate the simultaneous entry of Na+ and exit of K+ from cells (29, 30). Since it appeared that the ribotoxic activity of palytoxin could not be related to direct interaction with the translational machinery, we tested whether the ribotoxic actions of palytoxin could have resulted from ionic disequilibria produced in cells as a consequence of poisoning of the Na+/K+-ATPase. To test this possibility, we conducted experiments in which 75% of the 154 mM NaCl present in MEM culture medium was replaced by either KCl (116 mM KCl; K-MEM) or choline chloride (116 mM; Cho-MEM). Experiments were conducted to determine whether the modified isotonic MEM containing either K+ or choline+ would reduce the ability of palytoxin to inhibit protein synthesis or to activate SAPK/JNK1. Cells were far more sensitive to inhibition of protein synthesis by palytoxin in MEM, compared with K-MEM, which shifted the dose-response curve 2 decades to the right (Fig. 5). By contrast, cells were 3-fold more sensitive in Cho-MEM than in MEM. The ability of increased extracellular K+, but not choline+, to diminish substantially the ability of palytoxin to inhibit translation is consistent with the conclusion that efflux of K+ may be responsible for the effects of palytoxin. The inability of extracellular choline+ to reduce the translational inhibition by palytoxin is consistent with the notion that influx of Na+ is unlikely to be the cause of the effects of palytoxin.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ability of palytoxin to both inhibit protein synthesis and stimulate SAPK/JNK1 activity (Fig. 1) led us to investigate whether palytoxin acts as a ribotoxic stressor. Previously identified ribotoxic stressors share the following characteristics: (i) they inhibit translational elongation by interacting directly with 28 S rRNA in the region of the peptidyl transferase center or the S/R loop; (ii) they rapidly induce the activation of the stress kinases SEK1/MKK4, SAPK/JNK1, and p38/HOG1; and (iii) they induce the activation of stress kinases only in cells that contain actively translating ribosomes (1). Other nonribotoxic stressors that activate stress kinases, such as proinflammatory cytokines and osmotic stress, are distinguished from ribotoxic stressors by their ability to induce signaling to the stress kinases even in the absence of translationally active ribosomes. Although experiments demonstrated that palytoxin was ineffective as a translational inhibitor when added directly to actively translating ribosomes in vitro (Fig. 2), palytoxin nevertheless required translating ribosomes to transduce signals that activate SAPK/JNK1 in vivo. The rapid inhibition of SAPK/JNK1 activation by emetine, which substantially blocked the activation when added just 1 min prior to palytoxin (Fig. 3), was also observed when emetine was added just prior to ribotoxic stressors such as anisomycin and ricin (1). The rapid action of emetine in this context suggests that actively translating ribosomes are required for palytoxin-initiated signals to be transduced to the kinase(s) upstream of SEK1/MKK4 and SAPK/JNK1. That cells exposed to palytoxin became refractory to emetine when emetine was added 3 min after palytoxin suggests that the critical events of ribosome-mediated transduction are completed by 3 min. A similar time course was observed for anisomycin (1).2 Since the effects of palytoxin include translational inhibition, it appears plausible that the signaling is self-terminating and that the involvement of ribosomes in this process is completed within this short period of time.
The binding of palytoxin to Na+/K+-ATPase
results in an "open" ion channel that permits free passage of
Na+ and K+ into or out of cells, depending on
the ion gradient on both sides of the cell membrane (28-30). Kuroki
et al. (17) reported that in Swiss 3T3 fibroblasts DMEM in
which Na+ was replaced by K+ suppressed the
ability of palytoxin to activate SAPK/JNK1; from these data they
concluded that palytoxin-activated Na+ influx was
responsible for the activation of SAPK/JNK1. Our data in Rat-1
fibroblasts are in agreement with those of Kuroki et al.
(17) and furthermore demonstrated that replacement of Na+
by K+ strongly suppressed the translational inhibition by
palytoxin (Figs. 5 and 6). However, neither SAPK/JNK1 activation nor
translational inhibition was diminished when Na+ was
replaced by choline+, a nonpenetrating cation used to
maintain osmotic balance, and in fact both SAPK/JNK1 activation and
translational inhibition were increased in the
choline+-containing medium. These data suggest that
palytoxin-induced efflux of K+, rather than influx of
Na+, was responsible for both SAPK/JNK1 activation and
translational inhibition. The inability of K-MEM to suppress the
activation of SAPK/JNK1 mediated by other agents such as anisomycin,
sorbitol, or IL-1 suggests that replacement of Na+ by
K+ did not generally inhibit the transduction of
stress-generated signals from other ribotoxic or nonribotoxic
stressors. Additionally, the activation of SAPK/JNK1 and suppression of
translation that occurred in palytoxin-treated cells following the
removal of K-MEM and replacement by palytoxin-free MEM demonstrates
that the exposure of cells to K-MEM did not impair the ability of the
cells to respond when placed in an appropriate ionic environment.
Palytoxin is the most potent nonproteinaceous toxin that has been identified. Although the ability of palytoxin to alter cation fluxes has been well documented, to date there has been no explanation for its severe toxicity. The demonstration in this paper that palytoxin can inhibit protein synthesis at picomolar concentrations (IC50 = 1 pM, Fig. 1B) places palytoxin among the most potent translational inhibitors, and this may account for its potent toxicity.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge the technical assistance of Thanh-Hoai Dinh, Jennifer Magun, and Jean Pearson.
![]() |
FOOTNOTES |
---|
* This work was supported by U.S. Public Health Service Grants CA-39360 and ES-08456.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Tel.: 503-494-7811;
Fax: 503-494-4253; E-mail: magunb{at}OHSU.edu.
1 The abbreviations used are: IL, interleukin; SAPK/JNK, stress-activated protein kinase/c-Jun NH2-terminal kinase; p38/HOG1, mammalian homolog of the S. cerevisiae high osmolarity glycerol response kinase-1; SEK1/MKK4, SAPK/extracellular signal-regulated kinase-1/mitogen-activated protein kinase kinase-4; AP-1, activator protein-1; GST-Elk-1, glutathione S-transferase/Elk-1 fusion protein; MEM, modified Eagle's medium; EGF, epidermal growth factor.
2 M. S. Iordanov, D. Pribnow, J. L. Magun, J. A. Pearson, T.-H. Dinh, and B. E. Magun, submitted for publication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|