Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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The catabolic properties of glucocorticoid hormones are largely attributable to dual regulation of protein degradation and synthesis. With regard to the latter, glucocorticoids modulate the translational machinery, namely that component functional in translation initiation. This investigation revealed that in L6 myoblasts, dexamethasone, a synthetic glucocorticoid, deactivated the ribosomal protein S6 kinase (p70S6k) within 4 h, as evidenced by diminished phosphorylation of its physiological substrate, the 40S ribosomal protein S6. This deactivation correlated with dephosphorylation of p70S6k at Thr389, whereas phosphorylation of Ser411 was unaffected. Furthermore, glucocorticoid administration induced dephosphorylation of the cap-dependent translational repressor, eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), thereby facilitating conjunction of the inhibitor and eIF4E. The mechanism of action is reminiscent of classical transcriptional regulation by steroid hormone receptors in that these effects were preceded by a temporal lag and were sensitive to inhibitors of glucocorticoid receptor function as well as transcriptional and translational inhibition. Okadaic acid and calyculin A corrected the dexamethasone-induced dephosphorylation of p70S6k and 4E-BP1, implicating a PP1- and/or PP2A-like protein phosphatase(s) in the observed phenomena. Hence, glucocorticoids attenuate distal constituents of the phosphatidylinositol-3 kinase signaling pathway and thereby encumber the protein synthetic apparatus.
eIF4E binding protein; eIF4G; dexamethasone; translation initiation
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INTRODUCTION |
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THE PATHOPHYSIOLOGICAL SEQUELAE that derive from hypercortisolism, as observed in Cushing's syndrome or chronic glucocorticoid therapy, are often characterized by a marked loss of lean body mass caused by enhanced proteolysis (10, 27, 34) and/or diminished protein synthesis (29, 30). Moreover, glucocorticoids are inherently diabetogenic, in that hormonal excess renders peripheral tissues insulin resistant and thus refractory to the metabolic and anabolic processes governed by insulin despite associated hyperinsulinemia. At the cellular level, the mechanisms dictating the catabolic and antianabolic character of glucocorticoids are only now emerging.
Manifestation of the antianabolic effects of glucocorticoids is heralded by an initial series of biochemical events that predispose the cell to nitrogen loss. This acute phase occurs within 4 h and is distinguished in part by attenuation of the cell's protein synthetic apparatus. In particular, the eIF4 class of eukaryotic initiation factors, which coordinately regulate expression of the overwhelming majority of cellular mRNAs, i.e., 7-methylguanosylated mRNAs, is potently affected. Administration of dexamethasone induces dephosphorylation of eIF4E binding protein 1 (4E-BP1), which strengthens its affinity for eIF4E, competitively inhibits eIF4G-eIF4E coupling, and ultimately prevents assembly of the functional eIF4F holocomplex (33). Not surprisingly, the 70 kDa ribosomal protein S6 kinase (p70S6k), which lies downstream of phosphatidylinositol-3 kinase (PI-3 kinase) and appears to be subject to direct or indirect regulation by the mammalian target of rapamycin (mTOR) is simultaneously dephosphorylated in skeletal muscle challenged with dexamethasone. This serine/threonine protein kinase has been implicated in cell cycle progression across the G1 checkpoint (4, 9, 21) and in the translational selection of transcripts exhibiting a unique 5' terminal oligopyrimidine (TOP) signature (16).
Because glucocorticoids negatively modulate protein synthesis, in part via downregulation of key translation factors, an attempt was undertaken to characterize mechanistically those events that underlie and contribute to this process. Furthermore, the influence of acute glucocorticoid action on signal transduction pathways previously implicated in regulation of p70S6k and 4E-BP1 was assessed.
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EXPERIMENTAL PROCEDURES |
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Materials. Enhanced chemiluminescence (ECL) detection kits and donkey anti-rabbit and sheep anti-mouse horseradish peroxidase-conjugated IgG were purchased from Amersham Life Sciences. Polyvinylidene difluoride (PVDF) membranes were purchased from Bio-Rad. [35S]Easytag express protein-labeling mix was acquired from NEN Research Products. Anti-p70S6k, anti-phospho-Ser411-p70S6k, anti-phospho-Thr389-p70S6k, and anti-4E-BP1 antibodies were purchased from Santa Cruz Biotechnology. Anti-PKB (protein kinase B), anti-p38MAPK (mitogen-activated protein kinase), anti-phospho-p38MAPK, anti-Erk, (extracellular signal-regulated kinase), and anti-phospho-Erk antibodies were purchased from New England Biolabs. Anti-mTOR antibody was a generous gift from Dr. Christopher Lynch (The Pennsylvania State University College of Medicine). Anti-phospho-S6 antibody was a gift from Dr. Morris Birnbaum (The University of Pennsylvania). Dexamethasone sodium phosphate, mifepristone, geldanamycin, actinomycin D, cycloheximide, emetine, puromycin, and rapamycin were purchased from Sigma. Okadaic acid was acquired from GIBCO. Calyculin A was purchased from Calbiochem.
Cell Culture.
L6 myoblasts (American Type Culture Collection) were seeded in
60- or 100-mm culture dishes in DMEM supplemented with 10% fetal calf
serum (HyClone Labs), 100 U/ml benzylpenicillin, and 100 µg/ml
streptomycin sulfate. Cells were grown to 70-80% confluence and
then induced to quiescence by replacement of the medium with DMEM
devoid of serum and antibiotics for 24 h. In some cases, the
medium was supplemented with pharmacological agents, as described in
the legend of each figure. Subsequently, cells were scraped and lysed
in buffer A [20 mM HEPES, pH 7.4, 100 mM KCl, 2.5% Triton X-100, 0.25% deoxycholate, 50 mM NaF, 0.2 mM EDTA, 2 mM EGTA, 1 mM
dithiothreitol, 50 mM -glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2 mM benzamidine, 0.8 µM leupeptin and 0.6 µM
pepstatin].
Measurement of protein synthesis. Cells seeded in 60-mm dishes were labeled with 5 µl of [35S]Easytag express protein-labeling mix (11 mCi/ml) for 30 min before harvest. Protein synthesis was assayed by monitoring the incorporation of [35S]methionine and [35S]cysteine into protein, as described elsewhere (19).
Analysis of Western blots. Protein immunoblots were developed via ECL and quantitated by scanning densitometry, as described previously (18, 20).
Phosphorylation of p70S6k.
An aliquot of L6 cell homogenate was added to an equivalent volume of
2× SDS sample buffer, heated at 95°C for 5 min and then separated
into multiple electrophoretic forms by SDS-PAGE. The proteins were then
transferred to PVDF membranes, which were subsequently incubated with
anti-p70S6k antibody or anti-phosphopeptide antibodies
directed against Thr389 or Ser411. It is
generally accepted that the electrophoretic mobility of p70S6k is inversely correlated with its degree of
phosphorylation (35). Because the most highly
phosphorylated form, and thus the species exhibiting the slowest
migration, is also the most active form of the protein
(36), mobility is typically a reasonable index of
p70S6k activation. However, this form of highly retarded
mobility is undetectable under conditions of glucocorticoid exposure.
Therefore, the electrophoretic species migrating slower than the
lowermost signal, designated herein as p70S6k-, were
densitometrically quantitated and expressed as the percentage of total
p70S6k in hyperphosphorylated (relative to
p70S6k-
) forms; thus the data are indicative of
p70S6k phosphorylation/activation.
Quantitation of phosphorylated and unphosphorylated 4E-BP1.
Quantitation of the phosphorylation state of 4E-BP1 was carried out
exactly as described elsewhere (18). Briefly, cell lysates were subjected to SDS-PAGE and then immunoblotted with anti-4E-BP1 antibody. Analogous to p70S6k, the rate of migration of
4E-BP1 is inversely related to increasing phosphorylation of the
protein. Three electrophoretic species, referred to as ,
, and
, readily resolve with SDS-PAGE; the most hyperphosphorylated form
is denoted
, the hypophosphorylated form being denoted
.
Quantitation of eIF4E, 4E-BP1 · eIF4E, and eIF4G · eIF4E complexes.
Quantitation of the respective factors and complexes was carried out
exactly as outlined previously (18). eIF4E, 4E-BP1 · eIF4E, and eIF4G · eIF4E complexes were immunoprecipitated
from whole cell lysates by use of a mouse monoclonal anti-eIF4E
antibody. The immune complexes were isolated by incubation with a goat
anti-mouse BioMag IgG bead slurry (PerSeptive Diagnostics). In
preparation for incubation with antigen-antibody complexes, the beads
were blocked in 0.1% nonfat dry milk in buffer B [50 mM
tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.4, 150 mM NaCl, 5 mM
EDTA, 0.1% -mercaptoethanol, 0.5% Triton X-100, 50 mM NaF, 50 mM
-glycerophosphate, 0.1 mM PMSF, 1 mM benzamidine, and 0.5 mM sodium
orthovanadate] for 1 h at 4°C. The beads were captured by means
of a magnetic stand, washed twice with buffer B, and washed
once with buffer B containing 500 instead of 150 mM NaCl.
After incubation of the immune conjugates with magnetic beads, the
resultant complexes were eluted in SDS sample buffer and then boiled
for 5 min. The beads were pelleted by centrifugation, and the
supernatants were collected and subjected to SDS-PAGE. After proteins
were separated electrophoretically, they were transferred to PVDF
membranes. The membranes were incubated with a mouse monoclonal
anti-eIF4E antibody, an anti-4E-BP1 antibody, or a rabbit polyclonal
anti-eIF4G antibody overnight at 4°C. Finally, the blots were
developed with an ECL Western blotting kit.
Quantitation of phosphorylated and unphosphorylated eIF4E. Quantitation of the percentage of eIF4E in the phosphorylated and unphosphorylated forms was performed by means of slab gel isoelectric focusing with subsequent protein immunoblot analysis, exactly as previously described (37).
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RESULTS |
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The function of glucocorticoids in catabolic and antianabolic capacities has long been appreciated. To begin to address the underlying molecular phenomena associated with glucocorticoid-regulated protein metabolism, events involved in the assembly of eIF4F, the cellular machinery that facilitates recruitment of mRNA to the ribosome, was assessed. This mRNA binding complex is composed of several polypeptides, including eIF4E, eIF4G, eIF4A, and eIF4B; eIF4G, the largest subunit of the complex, serves as a docking platform for other components of the holoprotein and additional initiation factors, such as eIF3. The association of eIF4E, which recognizes the 5' cap of eukaryotic mRNA, with eIF4G is required for loading ribosomes onto the message. Therefore, the formation of the active eIF4F complex represents an important control point for the regulation of translation initiation.
The classical mechanism of glucocorticoid action involves migration of
the transformed glucocorticoid receptor into the nucleus, where it
physically recognizes specific DNA response elements and thereby
transactivates or transrepresses target genes. However, interference of
various transcriptional pathways by the liganded glucocorticoid
receptor can occur independently of the DNA binding properties of the
receptor. Such genomic regulation cumulatively serves to tailor
cellular phenotype to environmental demands and is typically preceded
by a temporal lag because of the time requirement of mRNA and protein
synthesis and/or degradation. Indeed, 2 h of incubation with
dexamethasone was required for significant redistribution of total
4E-BP1 into hypophosphorylated species; this occurred concomitantly
with disappearance of the - or hyperphosphorylated form of the
protein (Fig. 1A).
Furthermore, because 4E-BP1-
is the only form of the inhibitor
incapable of sequestering eIF4E, the loss of the
-form implies that
a larger proportion of total eIF4E should be bound by 4E-BP1, thereby
limiting its availability. Not surprisingly, co-immunoprecipitation of
4E-BP1 with eIF4E revealed that eIF4E was increasingly coupled to
4E-BP1 in direct relation to the period of incubation with
dexamethasone (Fig. 1B).
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The PI-3 kinase signal transduction module that regulates, through
phosphorylation, the efficacy of 4E-BP1 mutually affects the
phosphorylation and thus the activation state of p70S6k.
Predictably, in cells administered dexamethasone, p70S6k
was markedly dephosphorylated; however, the glucocorticoid required 2
h to manifest significant effect. In cells deprived of serum for
24 h, p70S6k resolves into multiple readily detectable
electrophoretic forms after SDS-PAGE, wherein increasing
phosphorylation retards protein mobility. Presumably, it is the most
slowly migrating species that represents the heavily phosphorylated and
thus highly active form of the kinase (36). Although four
electrophoretic species are readily apparent in serum-deprived cells,
the form of slowest mobility represents only a small fraction of the
entire p70S6k pool. In dexamethasone-administered cells,
total p70S6k redistributed into relatively dephosphorylated
species, as evidenced by an accumulation of the bulk (85%) of the
enzyme into the most rapidly migrating form, designated
, whereas
immunoreactivity corresponding to the highly phosphorylated enzyme was
lost (Fig. 1C). The close temporal correlation between
dephosphorylation of 4E-BP1 and p70S6k underscores the
nature of coordinate regulation of these factors.
Because the proportion of p70S6k residing in the uppermost band, i.e., the heavily phosphorylated form, represented only a small fraction of the total kinase, the physiological significance of reduced basal activation by glucocorticoids seemed questionable. To address this issue, S6 phosphorylation was determined with the use of an anti-phospho-S6 antibody. Interestingly, the levels of phospho-S6 declined in parallel with the dephosphorylation of p70S6k observed after addition of glucocorticoids. Within 4 h of drug administration, phosphorylated S6 was reduced to ~50% of control values (Fig. 1D). Thus basal p70S6k activity maintains S6 in a nominally phosphorylated state that is further diminished by glucocorticoid-induced dephosphorylation/inactivation of the kinase. Although this is a reasonable explanation, the possibility that the decrement in immunoreactive phospho-S6 is caused by another S6 kinase or S6 phosphatase cannot be excluded. Whereas in serum-deprived CTLL-20 cells, 3 h of exposure to dexamethasone reduces p70S6k activity by 50%, in quiescent NIH3T3 fibroblasts, the drug is completely ineffectual, emphasizing the cell selectivity of p70S6k regulation by glucocorticoids (22).
Because glucocorticoids are known to modulate the expression of
particular genes, it seemed plausible that glucocorticoid-induced dephosphorylation of both p70S6k and 4E-BP1 could reflect
reduced expression of candidate upstream regulators. However, neither
the expression of mTOR nor that of PKB/Akt proteins was affected by
dexamethasone administration (Fig. 2).
Because in serum-deprived cells phosphorylation of PKB at
Ser473, which is essential for PKB activation, was
undetectable, a comparison of the activation state of PKB in control
and dexamethasone-treated cells was not possible. However, whereas
glucocorticoids acutely attenuate insulin-like growth factor
(IGF)-I-stimulated phosphorylation of p70S6k and 4E-BP1,
IGF-I-stimulated phosphorylation of PKB at Ser473 is
refractory to inhibition by glucocorticoids, suggesting that the
influence of these hormones is exerted downstream of or parallel to PKB
(unpublished results). Furthermore, phosphorylation of eIF4E by the MAP
kinase-integrating kinases (Mnks) is believed to enhance the affinity
of eIF4E for the 5' cap of mRNA and thus to facilitate translation
initiation. Neither the expression nor the activation of the Erks and
the p38MAPKs, which activate Mnks and thereby promote eIF4E
phosphorylation, were significantly influenced by glucocorticoids (Fig.
2, B-D).
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To determine whether downregulation of eIF4F and p70S6k was
truly caused by specific effects mediated by the glucocorticoid
receptor, cells were coadministered substances known to perturb
glucocorticoid receptor function. Pretreatment of cells with the heat
shock protein 90 (hsp90)-specific compound geldanamycin completely
prevented the glucocorticoid-induced effects on 4E-BP1 (Fig.
3A) and corrected dephosphorylation of p70S6k (Fig. 3B). This
benzoquinone ansamycin antibiotic, via interaction with the hsp90
component of glucocorticoid receptor heterocomplexes, hinders the
steroid binding capacity of the receptor, as well as facilitating its
degradation (32). Mifepristone (RU486), a glucocorticoid
analog that binds the glucocorticoid receptor with higher affinity than
dexamethasone but fails to efficiently transform the receptor,
exhibited antiglucocorticoid properties similar to geldanamycin:
it entirely abrogated dephosphorylation of 4E-BP1 while returning the
phosphorylation state of p70S6k to that observed in cells
administered only mifepristone (Fig. 3, A and B).
An effect of p70S6k dephosphorylation after exposure to
both geldanamycin and mifepristone was noted.
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Besides a prerequisite temporal lag, sensitivity to
pharmacological translational and transcriptional inhibitors is a
defining characteristic of gene induction by steroid hormones.
Therefore, emetine, puromycin, and cycloheximide, each of which impairs
the translational apparatus, were evaluated for their ability to
abrogate dephosphorylation of 4E-BP1 and p70S6k in response
to dexamethasone. Although the inhibitors prevented the
glucocorticoid-induced dephosphorylation of 4E-BP1 and
p70S6k, each compound promoted phosphorylation of these
factors when administered in the absence of hormone (Fig. 4,
A and B,
cycloheximide not shown). Moreover, the same observation was made after
treatment with the transcriptional inhibitor actinomycin D (Fig. 5,
B-E and Fig. 6, A
and B). To assess potential
nontranslational influences of these inhibitors on p70S6k
and 4E-BP1 phosphorylation, a temporal correlation, or lack thereof, between effector phosphorylation state and protein synthesis inhibition was sought. Indeed, inhibition of protein synthesis by cycloheximide occurred concomitantly with phosphorylation of p70S6k and
4E-BP1 (Fig. 5, A-E); even the more gradual decline in
protein synthesis engendered by actinomycin D temporally paralleled the phosphorylation state of these effectors. Modulated phosphorylation of
4E-BP1 and p70S6k appears to be secondary to translational
and not transcriptional inhibition per se, because actinomycin D, which
presumably inhibits transcription as a prelude to translational
inhibition, did not affect p70S6k and 4E-BP1
phosphorylation states before inhibition of protein synthesis.
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It is noteworthy that dephosphorylation of p70S6k subsequent to treatment with glucocorticoids correlated with dephosphorylation of Thr389, thus supporting the premise that modification of this site accounts for the observed diminution in S6 phosphorylation (Figs. 6D and 1D). It is generally believed that phosphorylation of four key residues clustered in the carboxy-terminal autoinhibitory pseudosubstrate domain of p70S6k (Ser411, Ser418, Thr421, and Ser424), which relieves intrasteric occlusion of the kinase's catalytic pocket, is induced after mitogen stimulation (12). These residues are flanked by a proline in the +1 position, rendering these sites, in vitro, sensitive to phosphorylation by enzymes of the MAP kinase superfamily, as well as other kinases exhibiting proline-directed substrate specificity. Curiously, phosphorylation of Ser411 was unchanged after exposure to glucocorticoids. Alternatively, actinomycin D (Fig. 6C) and okadaic acid (not shown), which promote phosphorylation of the kinase, induced a mild dephosphorylation of this site (Fig. 6C) concomitant with phosphorylation of Thr389 (Fig. 6D). Furthermore, phosphorylation of Ser411 was refractory to dephosphorylation by rapamycin (Fig. 6C), whereas neighboring residues Thr421 and Ser424 are reportedly much more sensitive to the macrolide (36). Importantly, dexamethasone does not reduce the phosphorylation status of Thr389 in cells treated with either actinomycin D (Fig. 6D) or okadaic acid (not shown) below that observed in cells administered inhibitor alone.
There is an increasing body of circumstantial evidence implicating a protein phosphatase in the mediation of signals propagated by mTOR [(15, 36) and reviewed in (5)]. Therefore, the protein phosphatase-1 and -2A (PP1 and PP2A) inhibitors, okadaic acid and calyculin A, were evaluated for their ability to correct the dephosphorylation of p70S6k and 4E-BP1 induced by dexamethasone. Indeed, both okadaic acid and calyculin A sufficiently reversed dephosphorylation of p70S6k (Fig. 7) and 4E-BP1 (not shown) in response to glucocorticoids. In fact, okadaic acid alone potently induced phosphorylation of these effectors (not shown), implying that a PP1 and/or a PP2A-type phosphatase(s) either directly or indirectly regulates the phosphorylation states of p70S6k and 4E-BP1. Although both okadaic acid and calyculin A completely reversed the glucocorticoid-induced dephosphorylation of p70S6k and 4E-BP1, this was not due to inhibition of newly induced conventional phosphatases, because the relative expression levels of PP1 and PP2Ac, the catalytic subunit of PP2A, was not appreciably altered by glucocorticoids (not shown).
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DISCUSSION |
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The translational effectors p70S6k and eIF4E comprise a translational unit distinguished by its sensitivity to both the fungal metabolite wortmannin and the immunosuppressive antibiotic rapamycin, which inhibit the phosphotransferase activities of PI-3 kinase and mTOR, respectively. The synthesis of D3-phosphorylated phosphoinositides at the plasma membrane by PI-3 kinase recruits pleckstrin homology domain-containing proteins such as the phosphoinositide-dependent kinase 1 (PDK1) and PKB. It is believed that this colocalization at the membrane promotes efficient phosphorylation of PKB by PDK1, whose activity appears to be constitutive. PDK1 also phosphorylates p70S6k at a site that is analagous to that phosphorylated on PKB (1, 26) and that is indispensable for S6 kinase activity (24). Although initial studies utilizing a membrane-localized form of PKB implied that this kinase was upstream of p70S6k, recent evidence suggests that such PKB-mediated activation of p70S6k is artifactual and in fact derives from constitutive membrane association rather than from bona fide substrate selectivity (8). The phosphorylation status of 4E-BP1, however, does appear to lie downstream of PKB, because a dominant-interfering PKB variant prevents the phosphorylation of 4E-BP1 but not p70S6k (8). Moreover, PKB phosphorylates mTOR on Ser2448, a site phosphorylated in response to insulin (23). Therefore, because both PKB and mTOR kinase activities are enhanced in response to insulin, and because mTOR-mediated 4E-BP1 phosphorylation is enhanced upon selective activation of a PKB/estrogen receptor chimera (31), a signal propagated via a PI-3 kinase/PKB/mTOR pathway is likely to regulate 4E-BP1 and thus eIF4E. However, it should be emphasized that accumulating evidence suggests that direct regulation of 4E-BP1 by mTOR is insufficient to explain 4E-BP1 phosphorylation observed in vivo (11, 13). At least in vitro, mTOR can phosphorylate p70S6k on both Thr389 and on the carboxy-terminal cluster of proline-directed sites and does so cooperatively with PDK1 (14). Evidence has also emerged that mTOR may regulate PP2A and thereby exert an indirect effect on the phosphorylation status of p70S6k (25). Although TOR control of PP2A is better understood in yeast (15), evidence for a similar mechanism in mammalian cells remains largely circumstantial.
Despite the obvious complexity of regulation of p70S6k and 4E-BP1, the data reported herein support reasonable conclusions regarding the modulation of this translational component by glucocorticoids. If glucocorticoids are transcriptionally affecting distal PI-3 kinase effectors, then the event(s) that determines their respective phosphorylation states is caused by induction or repression or both. At present, the nature of such transcriptional phenomena is uncertain. It is clear, however, that abrogation of the influences of glucocorticoids by actinomycin D, cycloheximide, emetine, and puromycin is related to translational inhibition. Although the transcriptional inhibitor actinomycin D augments both p70S6k and 4E-BP1 phosphorylation, it does not do so until protein synthesis is attenuated (Fig. 5), suggesting that the resultant p70S6k and 4E-BP1 phosphorylation states are not due to artifactual activation of upstream signal transduction pathways; rather, a signal emitted by the crippled translational machinery may negatively, but futilely, feed back to p70S6k and 4E-BP1. Indeed, the notion of translational regulation by feedback mechanisms has been suggested. For instance, overexpression of eIF4E in a tetracycline-inducible background reduces p70S6k and 4E-BP1 phosphorylation in proportion to the degree of eIF4E expression (17). Because the activation state of PKB, an upstream regulator of mTOR, was unaffected by eIF4E induction, it was proposed that a sensor for translational homeostasis lies downstream of PKB. Nevertheless, correction of glucocorticoid-induced dephosphorylation of p70S6k and 4E-BP1 by transcriptional and translational inhibitors appears to be contingent on protein synthetic inhibition.
Because dexamethasone treatment results in accumulation of
p70S6k-, it is reasonable to speculate that this may be
caused by inhibition of the initial series of phosphorylation events
occurring within the carboxy terminus of the kinase. All of these
phosphorylation sites are situated in Ser/Thr, Pro motifs and,
as such, are readily phosphorylated by MAP kinase and cyclin-dependent
kinase family members in vitro. Whereas mTOR can also phosphorylate
these residues in vitro (14), it is unlikely to be the
physiologically relevant serum-activated kinase, because in an
amino-terminal truncation background, phosphorylation of these
clustered sites in response to serum is unabated by rapamycin
(7). Nevertheless, dephosphorylation of p70S6k
in response to dexamethasone is dissociable from dephosphorylation at
Ser411 (Fig. 7C),
because phosphorylation of this site is similar between control and
dexamethasone-administered cells. Moreover, rapamycin does not
influence the phosphorylation state of Ser411 in
serum-deprived myoblasts. This finding is consistent with an earlier
report demonstrating that in quiescent Swiss 3T3 cells, phosphorylation
of this site is largely rapamycin insensitive (12),
although serum-induced phosphorylation of Ser411 is
abrogated by rapamycin pretreatment in the same cell system (24). Furthermore, although insulin enhances
phosphorylation of Ser411, this site remains substantially
phosphorylated, even under conditions of serum starvation
(36). Interestingly, the same investigation revealed that
cohort proline-directed sites, i.e., Thr421 and
Ser424, which lie within intimate proximity of
Ser411, are phosphorylated to a lesser extent in the basal
condition and are more robustly phosphorylated after insulin
stimulation. Thus, although putative proline-directed phosphorylation
at Ser411 is unaffected by glucocorticoids, this does not
preclude the possibility that glucocorticoids induce dephosphorylation
of other neighboring residues in the enzyme's carboxy-terminal tail.
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It is also possible that glucocorticoids induce dephosphorylation of p70S6k by hindering phosphorylation events that occur subsequent to those in the carboxy terminus. Of other sites associated with p70S6k activation, i.e., Thr229, Thr367, Ser371, Thr389, and Ser404, phosphorylation of Thr229, which is situated in the catalytic activation loop, and of Thr389, which lies within a hydrophobic segment interjacent to the catalytic loop and the carboxy terminus, cooperatively evokes full activation of the kinase (7, 36). Moreover, deletion of the amino and carboxy termini, which renders the kinase resistant to inhibition by rapamycin, also reveals that phosphorylation of Thr229 persists in the basal state, whereas phosphorylation of Thr389 occurs only after mitogen stimulation (6). The Thr229 kinase has been identified as PDK1 (26), whose constitutive activation may explain perpetual phosphorylation of this site when the steric constraints of the amino and carboxy termini are alleviated by truncation. It is also noteworthy that phosphorylation of either Thr229 or Thr389 alone is associated with only 2-5% p70S6k activity, whereas phosphorylation at both sites synergistically evokes complete activation. Under conditions of serum depletion, the hyperphosphorylated form of p70S6k remains mildly discernible (Fig. 6B), as does basal phosphorylation of Thr389 (Fig. 6D); glucocorticoids render the corresponding signals undetectable. Furthermore, dexamethasone-induced dephosphorylation of Thr389 correlates with dephosphorylation of the endogenous p70S6k substrate, ribosomal protein S6. Targeted deletion of p70S6k in murine embryonic stem cells completely abolishes S6 phosphorylation, suggesting that p70S6k is largely responsible for S6 phosphorylation in vivo (16). Therefore, dephosphorylation of S6 in response to glucocorticoids is likely to be mediated by dephosphorylation of p70S6k at Thr389.
The mechanism of dexamethasone-induced dephosphorylation of 4E-BP1 remains cryptic. It is clear, however, that the observed dephosphorylation is sufficient to promote sequestration of eIF4E by 4E-BP1 (Fig. 1B). The kinase activity associated with or intrinsic to mTOR phosphorylates 4E-BP1 in vitro and in transiently transfected HEK293 cells (2). Moreover, Thr37 and Thr46 are specifically targeted by mTOR, albeit the functional consequence of phosphorylation at these sites remains contentious (3, 11). Nonetheless, mTOR is a likely candidate for regulation of 4E-BP1 phosphorylation by glucocorticoids; if regulation of mTOR kinase activity does indeed occur, it is not via downregulation of the mTOR gene (Fig. 2A).
Glucocorticoids induce a clear redistribution of 4E-BP1 into - and
-electrophoretic forms (Fig. 1A), both of which retain the capacity to associate with eIF4E (Fig. 1B). Because
additional phosphorylation at other serum-responsive residues (e.g.,
Ser65, Ser70,
Ser83, and Ser112)
requires previous phosphorylation of Thr37 and
Thr46, the latter sites are proposed to prime 4E-BP1 for
subsequent phosphorylation (11). It has been demonstrated
that all three electrophoretic species of 4E-BP1 are detectable with
the use of antiphosphopeptide antibodies specific for Thr37
and Thr46, although the antibodies cannot discriminate
between either site (11). Because 4E-BP1 exists
predominantly in
- and
-forms after glucocorticoid
administration, and because both
- and
-forms are phosphorylated
at Thr37 and/or Thr46, it seems plausible that
glucocorticoids may attenuate the same serum-activated effectors that
regulate the carboxy-terminal, serum-sensitive phosphorylation sites.
The distal element of the PI-3 kinase signal transduction cassette, comprised of mTOR, 4E-BP1, and p70S6k, is recognized as a critical site of translational control in mammalian cells and, as such, has been the focus of widespread investigation. Herein, we report that glucocorticoids negatively regulate eIF4E and p70S6k function in undifferentiated rat L6 skeletal myoblasts in a manner that appears characteristic of classical steroid hormone action in that the process requires several hours to emerge and is sensitive to transcriptional and translational inhibition. Furthermore, the close temporal correlation between modulation of 4E-BP1 and p70S6k suggests that this mutual regulation may involve a common mechanism. Dephosphorylation of ribosomal protein S6 is secondary to dephosphorylation of p70S6k at Thr389, but not at Ser411, suggesting that glucocorticoids deactivate p70S6k through site-selective dephosphorylation.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Health Grants DK-15658 (L. S. Jefferson) and T32-GM-08619 (O. J. Shah).
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. S. Jefferson, Dept. of Cellular and Molecular Physiology, The Pennsylvania State Univ. College of Medicine, P. O. Box 850, Hershey, PA 17033 (E-mail: jjefferson{at}psu.edu).
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. §1734 solely to indicate this fact.
Received 14 October 1999; accepted in final form 27 January 2000.
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