From the Diabetes Unit and Medical Services,
Massachusetts General Hospital and Harvard Medical School, Boston,
Massachusetts 02129 and the § Biosignal Research Center,
Kobe University, Kobe 657, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study identifies the operation of a
signal tranduction pathway in mammalian cells that provides a
checkpoint control, linking amino acid sufficiency to the control of
peptide chain initiation. Withdrawal of amino acids from the nutrient
medium of CHO-IR cells results in a rapid deactivation of p70 S6 kinase and dephosphorylation of eIF-4E BP1, which become unresponsive to all
agonists. Readdition of the amino acid mixture quickly restores the
phosphorylation and responsiveness of p70 and eIF-4E BP1 to insulin.
Increasing the ambient amino acids to twice that usually employed
increases basal p70 activity to the maximal level otherwise attained in
the presence of insulin and abrogates further stimulation by insulin.
Withdrawal of most individual amino acids also inhibits p70, although
with differing potency. Amino acid withdrawal from CHO-IR cells does
not significantly alter insulin stimulation of tyrosine
phosphorylation, phosphotyrosine-associated phosphatidylinositol
3-kinase activity, c-Akt/protein kinase B activity, or
mitogen-activated protein kinase activity. The selective inhibition of
p70 and eIF-4E BP1 phosphorylation by amino acid withdrawal resembles
the response to rapamycin, which prevents p70 reactivation by amino
acids, indicating that mTOR is required for the response to amino
acids. A p70 deletion mutant, p702-46/
CT104, that is resistant
to inhibition by rapamycin (but sensitive to wortmannin) is also
resistant to inhibition by amino acid withdrawal, indicating that amino
acid sufficiency and mTOR signal to p70 through a common effector,
which could be mTOR itself, or an mTOR-controlled downstream element,
such as a protein phosphatase.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brief starvation engenders a decrease in protein synthesis, particularly in skeletal muscle, which is rapidly reversed on refeeding (1). The contribution of the nutrients themselves to the regulation of protein synthesis as compared with the concomitant hormonal responses has been the object of considerable study. Insulin, e.g., is well known to stimulate protein synthesis in skeletal muscle; however, much evidence indicates that a major portion of the increase in the skeletal muscle protein synthesis in vivo seen on refeeding is independent of changes in insulin and perhaps attributable to the nutrients themselves (2). Nevertheless, in vivo it is difficult to isolate the contributions of amino acids and other nutrients from those of insulin, insulin-like growth factors, and other regulators whose availability in vivo is altered by nutrients.
Mammalian cells in culture exhibit an inhibition of overall protein
synthesis with depletion of medium amino acids, which is quantitatively
substantial, but rapidly reversible (3). As with short term fasting
in vivo (4), amino acid withdrawal in vitro is
characterized by a loss of polysomes and an increase in monomeric
ribosomes pointing to a block in peptide chain initiation (5, 6). As
regards the mechanism of translational inhibition, attention focused
initially on the role of eIF-2 phosphorylation. Phosphorylation of
eIF-2
at Ser-51 enhances its affinity for eIF-2B, resulting in
sequestration of this critical, low abundance guanyl nucleotide
exchange factor, and thereby inhibiting regeneration of the eIF-2/GTP
necessary to bind Met-tRNAi (7, 8). In Saccharomyces
cerevisiae, the depletion of amino acids leads to the activation
of the eIF-2
kinase known as GCN2, presumably through the
accumulation of uncharged tRNA (9). The occurrence of increased
eIF-2
phosphorylation has been well documented in a variety of
mammalian cells deprived of amino acids, and in
CHO1 lines that contain
mutant aminoacyl tRNA synthetases (reviewed in Ref. 3).
More recently, the eIF-4F complex has emerged as an important site of translational control (reviewed in Ref. 10). This complex contains the scaffold protein eIF-4G, which binds eIF-4E, the m7GTP-cap binding protein, eIF-4A, an RNA helicase and the RNA-binding protein eIF-4B. The eIF-4E component binds the 5' mRNA cap; 4A/4B then unwind the secondary structure in the 5'-untranslated segment, thereby enabling the 40 S-Met-tRNAi complex, aided by eIF-3, to efficiently scan the mRNA 5'-untranslated segment to the AUG start site. Both the assembly and activity of the 4F complex are regulated by phosphorylation. In mammalian cells, eIF-4E, -4B, and -4G are each phosphorylated in vivo in a manner that correlates positively with overall translational rate. The regulation of eIF-4E has been characterized most extensively (10). Phosphorylation of 4E, predominantly at Ser-209 (11, 12), occurs in response to insulin, mitogens (6, 10), or stress (13) and is accompanied by a 3-fold increase in 4E affinity for mRNA (14). In addition, the availability of 4E for incorporation into the 4F complex is independently regulated through the PHAS/4E-BP polypeptides. PHAS1 was purified as a heat-and acid-soluble polypeptide that exhibited rapid and multiple phosphorylation in vivo in response to insulin and mitogens (15), and was independently isolated as an eIF-4E-binding protein by interaction cloning (16). The 4E-BP1/PHAS1 polypeptide binds to 4E in a manner that is competitive with 4G (17); thus, the insulin/mitogen-stimulated phosphorylation of 4E-BP1, which inhibits 4E-BP binding to 4E (16, 18), makes 4E available for incorporation into the 4F complex. Although 4E and 4E-BPs are coordinately regulated by insulin and mitogens, examples of discordant regulation are common (13, 19), indicating that the phosphorylation of the two polypeptides is independently mediated.
Evidence from in vivo models points to an important role for the 4E-BPs in translational regulation by nutrients. Thus, the phosphorylation of 4E-BPs in mouse skeletal muscle is diminished by starvation and restored by refeeding. Concomitantly, the association of 4E with 4E-BP1 increases with starvation and diminishes with refeeding whereas the association of 4E with 4G, as expected responds in the reciprocal manner (20). As regards the relative importance of insulin versus nutrients in the control of 4E-BP1 phosphorylation, it is notable that the changes in phosphorylation of 4E-BP in response to fasting and feeding remain intact in NOD and ob/ob mice (20), diabetic models who fail to show any change in insulin concentration in response to these dietary maneuvers (2).
The phosphorylation of eIF-4E is not altered by fasting and refeeding; however, the phosphorylation of the p70 S6 kinase responds to fasting and refeeding in parallel to 4E-BP1 (20). The p70 S6 kinase participates in translational control in a manner quite distinct from eIF-4F. This insulin/mitogen-activated protein kinase, whose major known substrate is the 40 S ribosomal subunit protein S6 (21), is critical for the translation of a subclass of mRNAs whose 5'-untranslated region contains a short oligopyrimidine sequence immediately after the 5' cap (22-24). Nevertheless, despite their quite different roles in translational control, it appears that the phosphorylation of both 4E-BP and p70 S6 kinase, but not eIF-4E, is coordinately controlled not only by insulin and mitogens, but independently by nutrients, as reflected in vivo by the responses to fasting and feeding (20).
Another response shared by 4E-BPs (25-27) and the p70 S6 kinase (28, 29), but not by eIF-4E, is a susceptibility to dephosphorylation in vivo in response to the macrolide immunosuppressant, rapamycin. This effect of rapamycin is mediated indirectly, through the ability of rapamycin, in complex with FKBP12 to bind to the mTOR kinase and inhibit its activity (30). Mutant mTORs that are unable to bind rapamycin/FKBP12 can protect p70 S6 kinase and 4E-BP1 from rapamycin-induced dephosphorylation (30, 31). Recently, mTOR has been shown to phosphorylate 4E-BP1 directly in vitro, at sites corresponding to those phosphorylated in vivo during insulin stimulation (58, 59). Nevertheless, the kinase activity of mTOR is not altered by insulin treatment prior to extraction (at least as measured after mTOR immunoprecipitation, Refs. 31 and 32) and the physiologic regulators of the TOR kinase are not known. TORs are 280-kDa polypeptides whose kinase domain is most closely related to the checkpoint control kinases, ATM, TEL1, MEC1, and the DNA protein kinase (33). In S. cerevisiae, loss-of-function mutations in both TOR genes, or treatment with rapamycin, each cause a profound inhibition of overall translational initiation and elicit a phenotype characteristic of starved cells entering G0, the stationary phase (34). The biochemical steps through which the TORs regulate translation in yeast are not known (35, 36); however, they appear to participate in a nutrient-sensing pathway that controls protein phosphatase activity (37). The possibility that mTOR might participate in nutrient-sensing in mammalian cells was raised by the observation that hepatocytes incubated in the absence of amino acids exhibit a selective decrease in the phosphorylation of S6, that is rapidly reversed by addition of amino acids but blocked by rapamycin (38). These findings imply that amino acids, independent of insulin or mitogens, can regulate the activity of the p70 S6 kinase through an mTOR-dependent mechanism. We therefore set out to characterize the pathways that regulate the phosphorylation of p70 S6 kinase and eIF-4E BP1 in response to amino acid sufficiency, mTOR and receptor tyrosine kinases. Our results provide evidence that amino acids and mTOR signal to the translational apparatus through convergent pathways distinct from those controlled by the insulin receptor.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials-- Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were purchased from Sigma. Dulbecco's phosphate-buffered saline (D-PBS), amino acids, and Ham's F-12 medium were purchased from Life Technologies, Inc.
Antibodies--
A monoclonal antibody against influenza virus
hemagglutinin (12CA5) was purchased from Boehringer Mannheim; a
monoclonal anti-FLAG antibody (M2) was from Eastman Kodak Corp. The
monoclonal antibody 12CA5 was used to recover HA-tagged polypeptides.
Phosphospecific antibody against p70 S6 kinase were a gift from New
England Biolabs Inc. Phosphospecific and nonspecific antibody against
ERK1/2 were purchased from New England Biolabs Inc. A polyclonal
antiserum raised against a synthetic peptide corresponding to amino
acids of 337-352 of p701 S6 kinase was used for immunoblot of p70
(39) and a antiserum against GST fusion protein of C-terminal 104 amino acids of p70 was used for immunoprecipitation of endogenous p70. A
polyclonal antiserum against mTOR was raised against GST fusion protein
of amino-terminal 100 amino acids of mTOR expressed in Escherichia coli (31). A polyclonal antibody against eIF-4E BP1 was raised against a GST-eIF-4E BP1 fusion protein expressed in
E. coli and purified by chromatography using GST-eIF-4E
BP1.
Cell Culture-- HEK293 cells (40) and CHO-IR (41) cells were cultured as described earlier in DMEM or Ham's F-12 medium, respectively. For the starvation of amino acids, cells were first incubated in DMEM (293) or Ham's F-12 (CHO-IR) medium without FCS for 16 h, washed once with Dulbecco's phosphate-buffered saline (D-PBS, containing 0.1 g/liter CaCl2) and incubated in the same buffer for the times indicated for up to 2 h. Readdition of amino acids involved changing the medium to D-PBS containing individual amino acids or a mixture of amino acids as indicated. The concentration of each amino acids designated as 1× is as follows (in mg/liter): L-Arg, 84; L-Cys, 48; L-Glu, 584; L-His, 42; L-Ile, 105; L-Leu, 105; L-Lys, 146; L-Met, 30; L-Phe, 66; L-Thr, 95; L-Trp, 16; L-Tyr,72; L-Val, 94. A mixture of all these amino acids, each at this concentration is designated as the "1× amino acid mixture".
Transient transfection was performed by lipofection method using LipofectAMINE (Life Technologies, Inc.) as described previously (31).Cell Lysis and Immunoblot--
Cell treatments were terminated
by removal of the medium, followed by one wash with ice-cold
phosphate-buffered saline and freezing in liquid nitrogen; cells were
stored at 80 °C until lysis. Cells were lysed in ice-cold Buffer A
(50 mM Tris/HCl (pH = 8.0), 1% Nonidet P-40, 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 20 mM
-glycerophosphate, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin), and the extracts were centrifuged at 10,000 × g for 20 min.
Kinase Assay--
p70 S6 kinase activity was determined by
immunocomplex assay by using 40 S ribosomal subunits as substrate (42).
PI 3-kinase activity coimmunoprecipitated with anti-phosphotyrosine
antibody (PY-20) was determined as described previously (43). c-Akt/PKB kinase activity was measured by immunocomplex assay using myelin basic
protein as substrate. CHO-IR cells transfected with HA-c-Akt/PKB were
lysed in ice-cold buffer A and immunoprecipitated with anti-HA antibody. The immunoprecipitates were washed twice with buffer A
containing 0.5 M NaCl, and twice with the buffer composed
of 20 mM Tris (pH = 7.4) and 1 mM DTT. The
kinase reaction was started by adding the reaction mixture (50 mM Tris (pH = 7.4), 10 mM
MgCl2, 1 mM DTT, 50 µM ATP, 1 µM protein kinase inhibitor (PKI) peptide, 1 mM EGTA, 1 mg/ml myelin basic protein, and 2 µCi of
[-32P]ATP), incubated at 30 °C for 30 min, and
stopped by adding SDS sample buffer. The mixture was separated by
SDS-PAGE, transferred onto PVDF membrane, and analyzed by
autoradiography.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amino Acids Regulate the Phosphorylation and Activation of p70 S6
Kinase--
Withdrawal of amino acids from the medium leads to a
rapid, reversible deactivation of the p70 S6 kinase and partial
dephosphorylation of eIF-4E BP1. This response was observed in the
three cell types examined: CHO-IR, HEK293, and Swiss 3T3 cells. As
regards p70, the already low activity of endogenous p70 in
serum-deprived CHO-IR cells (henceforth called the "basal"
activity) declines further during the 2 h following amino acid
withdrawal, and the ability of insulin to stimulate activity is
virtually abolished (Fig. 1, A
and B). This inhibition of p70 activity is fully reversible; reintroduction of the mixture of amino acids at the concentrations used
in DMEM (a concentration henceforth called 1× (see "Experimental Procedures"), and used as a reference level for all responses examined), restores both basal and insulin/serum-stimulated p70 kinase
activity within 30 min (Figs. 1 (A and B) and
2). A similar response is seen in 293 cells, examining either endogenous p70 (Fig. 1D) or
transiently expressed recombinant p701 (Fig. 1C and
6A). The decline in p70 activity that occurs after
withdrawal of amino acids is not due solely to the loss of
insulin/serum stimulation, because the p70 activity in
insulin/serum-deprived cells also diminishes greatly. This is best
appreciated in 293 cells, where the basal activity of p70 is 30-60%
of the maximally stimulated activity (Fig. 1D); amino acid
withdrawal, after a slight lag, results in a marked decrease in the
already high basal p70 activity that is reversible on readdition of
amino acids (Figs. 1D and 6A). The changes in p70
activity are specific for amino acid withdrawal and addition; omission
and reintroduction of glucose or the vitamins found in DMEM do not
alter p70 activity. The ability of p70 to be activated by serum or
insulin is fully restored by concentrations of amino acids far below
those that maximally increase basal activity (Fig. 2). In CHO-IR cells,
full restoration of the response to insulin and serum is evident after
readdition of the complete amino acid mixture at 0.25× concentration,
and the absolute magnitude of p70 activity after addition of
insulin/serum is not altered as the concentration of the amino acid
mixture is raised to 2×. In contrast, the basal activity of p70 rises progressively as the concentration of the amino acid mixture is raised,
so that at the 2× concentration, p70 kinase activity is near maximal
and shows little or no further increase on addition of insulin. The
response of p70 to 10% undialyzed serum parallels the response to
insulin, allowing for the amino acid content of the serum itself (Fig.
2). The effects of medium amino acids on the activity of p70 are due to
overall amino acid sufficiency, although the effects of individual
amino acids are not equal (Table I).
Thus, little or no restoration of p70 activity occurs with readdition
of any single amino acid. Readdition of a complete mixture of amino
acids at 1×, but lacking single amino acids provides the most
informative data (Table I). This removal of Arg or Leu is most
inhibitory (70-90%), Lys or Tyr omission next in potency (approximately 30-50% inhibition), whereas Cys or Glu omission are
well tolerated, and His, Iso Met, Phe, Thr, Trp, and Val omission give
moderate, variable inhibition under the conditions examined. Despite
the potent inhibition caused by Arg or Leu omission, readdition of
either individually does not stimulate p70 activity at all, and the two
together enable only slight reactivation.
|
|
|
|
Amino Acids Regulate the Phosphorylation of eIF-4E-BPs-- Withdrawal of medium amino acids leads to a reversible dephosphorylation of eIF-4E-BPs. Endogenous eIF-4E-BP poly- peptides can be detected in extracts of CHO IR cells by immunoblot, using a polyclonal anti-eIF-4E BP1 antiserum, and are visualized after SDS-PAGE as a ladder of bands. Exposure of the cells to insulin diminishes the abundance of the more rapidly migrating bands and increases the abundance of the slowest migrating bands, a response shown previously to be due to increased eIF-4E-BP phosphorylation (25-27, 31, 32). Omission of medium amino acids is accompanied by the disappearance of the most slowly migrating eIF-4E-BP polypeptide band, and a downshift of eIF-4E-BP polypeptides into two rapidly migrating bands; a similar downshift in eIF-4E-BP mobility occurs in response to rapamycin and wortmannin (Fig. 4A). Addition of insulin in the absence of amino acids fails to cause an upshift in the mobility of eIF-4E-BPs; readdition of amino acids restores the most slowly migrating eIF-4E-BP polypeptide, as well as the response to insulin (Fig. 4A). A similar pattern is observed for recombinant eIF-4E-BP1 expressed transiently in 293 cells. Removal of medium amino acids after stimulation by 10% FCS, leads, after a slight lag, to the disappearance of the most slowly migrating eIF-4E-BP1 polypeptides (Fig. 4B). Readdition of amino acids results in a time-dependent reappearance of the hyperphosphorylated eIF-4E-BP1 polypeptides. As with p70, the response to serum is restored by amino acids before the basal phosphorylation of eIF-4E-BP1 is fully recovered (Fig. 4C).
|
Amino Acids Do Not Regulate Insulin Receptor Kinase, PI 3-Kinase, c-Akt/PKB, or MAPK Activities-- Inasmuch as the withdrawal of amino acids from CHO-IR cells reversibly blocks the ability of insulin to stimulate the phosphorylation and activation of p70 S6K and the eIF-4E-BPs, we examined the effects of amino acid withdrawal on several steps in insulin signal transduction upstream of, or parallel with the activation of the p70 S6 kinase (Fig. 5, A-D). Amino acid withdrawal has no effect on insulin-stimulated receptor tyrosine autophosphorylation, or on the tyrosine-specific phosphorylation of endogenous IRS proteins (Fig. 5A). The insulin-stimulated increase in PI 3-kinase immunoprecipitated by anti-phosphotyrosine antibodies, which is mostly bound to the IRS proteins (41), is slightly (~20%) decreased after 2 h of amino acid withdrawal, a decrease that is not corrected by readdition of amino acids (Fig. 5B). Nevertheless, the very potent insulin activation of recombinant c-Akt/PKB, expressed transiently in CHO-IR cells, is unaffected by amino acid withdrawal (Fig. 5C). The insulin-stimulated phosphorylation at the activating sites on the endogenous p42/p44 MAPKs, monitored by anti-P-peptide immunoblot, is slightly diminished after amino acid withdrawal, but is not restored by amino acid readdition (Fig. 5D). In addition, neither amino acid withdrawal nor readdition altered the activation loop phosphorylation of stress-activated protein kinase (SAPK) or p38 endogenous to the CHO-IR cells (data not shown). Thus, the effects of amino acid withdrawal/readdition on basal and insulin-activated Ser/Thr phosphorylation of p70 S6 kinase and eIF-4E BP1 reflects the operation of a signal transduction response clearly distinguished from those recruited by insulin or generalized stress.
|
A p70 Mutant Resistant to Rapamycin Is Also Resistant to Amino Acid Withdrawal-- The selective inhibition of p70 S6 kinase and eIF-4E BP1 phosphorylation that occurs consequent to amino acid withdrawal corresponds closely to the cellular response seen on addition of rapamycin. Moreover, like rapamycin, amino acid withdrawal inhibits p70 activity in response to essentially all cell growth factors, phorbol esters, heat shock, vanadate and calyculin, as well as to cotransfected modulators, including V12 Ras and a constitutively active PI 3-kinase. In addition, as with rapamycin, the substantial activation of p70 S6 kinase activity caused by cycloheximide, an inhibitor of translational elongation, and anisomycin, an inhibitor of translational initiation, are both inhibited by amino acid withdrawal (Table II).
|
|
Rapamycin Inhibits p70 S6 Kinase Activation by Amino Acids-- Rapamycin promotes the dephosphorylation of p70 and eIF-4E-BPs through an inhibitory action on the mTOR kinase (30, 31). Rapamycin blocks the ability of amino acids to (re)activate p70 S6 kinase activity (Fig. 7) and to restore the phosphorylation of eIF-4E BP1 (data not shown), indicating that the mTOR-directed input is required for the response to amino acids. Consequently, if amino acids and mTOR control a common regulator of p70, this amino acid/mTOR-sensitive p70 regulator either has an absolute requirement for dual, independent inputs from both amino acids and mTOR or, alternatively, it receives a single input, with mTOR situated downstream of the amino acid signal.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amino Acid Sufficiency Signals to the Translational
Apparatus--
The results demonstrate that the availability of amino
acids controls the phosphorylation and activity state of two proteins that participate in the regulation of translation, i.e. the
p70 S6 kinase and eIF-4E BP1, an inhibitor of eIF-4E function. This response is specific for the amino acids and is not reproduced by
changes in the availability of glucose or in overall energy sufficiency. The changes in p70 and eIF-4E BP1 phosphorylation appear
to be part of a "general" control response, inasmuch as they are
elicited by depletion of virtually any single amino acid, although the
responses to omission of some amino acids, e.g. arginine and
leucine, are exceptionally strong. The inability of arginine alone to
restore activation of p70 even partially argues strongly against a
contribution from NO generation. The responses to amino acid omission
described here are probably part of a physiologic program directed at
the down-regulation of protein synthesis. The extent of translational
inhibition has not been measured directly, but might be estimated by
considering the effects of rapamycin on overall protein synthesis,
inasmuch as rapamycin leads to a similar inhibition of p70 S6 kinase
and eIF-4E BP1 phosphorylation. In several mammalian cell types,
rapamycin inhibits overall protein synthesis by 10-15%, although the
translation of mRNAs containing 5'-terminal oligopyrimidine tracts
such as those encoding ribosomal proteins and EF-2, is inhibited much
more strongly (22-24). Thus, it is likely that the deactivation of p70
and dephosphorylation of eIF-4E BP1 per se do not contribute
in a quantitatively substantial way to the marked inhibition of protein
synthesis known to be engendered by amino acid depletion. It is
probable that other changes such as in eIF-2 or eIF-4E
phosphorylation, or in the rate of polypeptide elongation, are of
greater quantitative importance (3, 6, 10). Nevertheless, the
dephosphorylation of p70 and eIF-4E BP1 reflect the operation of a
signaling apparatus that provides for the orderly shutdown of
translation in the face of substrate insufficiency. Conceivably, this
amino acid-regulated signal transduction pathway might also control the
increase in endogenous protein breakdown that occurs in many cells in
response to amino acid depletion. Thus, in isolated rat hepatocytes,
ambient amino acids control the extent of S6 phosphorylation in
situ, and negatively regulate autophagic proteolysis (38).
Moreover, the ability of increased medium amino acids to suppress
proteolysis is partially antagonized by rapamycin, leading to the
suggestion that S6 phosphorylation and autophagic proteolysis are
regulated, at least in part, by common, amino acid-sensitive signal
transduction pathways.
Amino Acid Withdrawal and Rapamycin Inhibit a Common Set of
Signaling Elements--
The striking similarity of the cellular
response to amino acid depletion to that caused by rapamycin led us to
inquire as to whether amino acid sufficiency and mTOR regulated a
common signal transduction pathway. Both treatments induce selective dephosphorylation of p70 and eIF-4E BP1 without altering the insulin activation of PI 3-kinase, c-Akt/PKB, or MAPK. Moreover, the
p702-46/
CT104 variant, which is highly resistant to the
dephosphorylation engendered by amino acid withdrawal, is comparably
resistant to rapamycin-induced dephosphorylation. The possibility that
amino acid sufficiency signals through a pathway that includes TOR is
further reinforced by the similarity in the response of S. cerevisiae to nitrogen depletion as compared with its response to
rapamycin or TOR 1/2 deletion (34). Both rapamycin and TOR1/2 deletion
cause S. cerevisiae to arrest in early G1 in an unbudded
state which resembles closely the phenotype of cells arrested by
nutrient deprivation (i.e., G0 or stationary
phase). In particular, cells accumulate glycogen and become
thermotolerant; overall translation is inhibited by 90%, but certain
mRNAs, e.g. UB14 (ubiquitin), continue to be vigorously
translated.
The Proximate Effectors Mediating Amino Acid and Insulin Regulation of the p70 S6 Kinase and eIF-4E BP1-- Amino acid readdition, like insulin, activates p70 in situ through a multisite phosphorylation, and also promotes the multiple phosphorylation of eIF-4E BP1. What are the proximate events accounting for these changes in phosphorylation in response to amino acid sufficiency? Although the p70 kinase kinases have not yet been fully defined, it is clear that the sites phosphorylated during activation are situated in a variety of amino acid sequence contexts, and reflect the independent operation of several different protein kinases (40, 42, 44-49, 57). The initial modification involves the multiple phosphorylation of a cluster of (Ser/Thr) Pro sites, situated in the psuedosubstrate inhibitory domain of the p70 carboxyl-terminal tail (Ser-434, -441, -447, and -452; Thr-444) (44, 47) and probably Ser-394 (58), situated in a 65-amino acid segment immediately carboxyl-terminal to the catalytic domain, that is conserved in sequence and location among p70, c-Akt/PKBs, PKCs, and Rsks (49). These sites can be phosphorylated in vitro by an array of proline-directed kinases including erk1, erk2, cdc2, and other as yet unidentified proline-directed protein kinases (57). Notably, the five sites of insulin-stimulated phosphorylation on eIF-4E BP1 identified thus far are all (Ser/Thr) Pro sites (59), and mTOR has been shown recently to phosphorylate these sites directly in vitro (60). The phosphorylation of p70 at these (Ser/Thr) Pro sites, although insufficient to activate the kinase (57) facilitates the further PI 3-kinase-regulated phosphorylation of p70 Thr-412 in the catalytic domain carboxyl-terminal extension (45, 48), and p70 Thr-252 in activation loop of catalytic subdomain VIII (40, 45-48). Phosphorylation of the latter two sites, acting together in a strongly cooperative fashion, serves to activate the p70 catalytic function; modification of either Thr-252 or Thr-412 singly, at least in vitro, is insufficient to generate more than 2-5% of the maximal p70 activity (46). Recent evidence indicates that phosphorylation of p70 Thr-252 is catalyzed 3-phosphoinositide-dependent protein kinase 1 (PDK1) (46, 47) a kinase that also activates c-Akt/PKB by phosphorylation of c-Akt/PKB Ser-308, a site homologous with p70 Thr-252 (61); p70 Thr-412 is phosphorylated by a different, as yet unidentified, 3-phosphoinositide-regulated protein kinase.
The evidence presented in this study indicates that amino acid sufficiency does not alter significantly insulin activation of PI 3-kinase or the (Ser/Thr) kinases that act on p70 kinase. Thus, erk1 and erk2 are not significantly affected by amino acid depletion. In addition, the lack of inhibition of c-Akt/PKB by amino acid withdrawal is especially significant, inasmuch as c-Akt/PKB activation requires the PtdIns (3,4,5)P3-regulated phosphorylation at two sites entirely homologous to p70 Thr-252 and Thr-412, namely c-Akt/PKB Ser-308, a phosphorylation catalyzed by PDK1 (60), and c-Akt/PKB Thr-473. A strong indication that amino acids do not influence the p70 Thr-412 kinase is provided by consideration of regulation of the p70Conclusions--
We offer the following model for the amino acid
regulation of p70 S6 kinase and eIF-4E BP1 phosphorylation, and its
relation to insulin/growth factor-regulated inputs (Fig.
8). We propose that amino acid
sufficiency acts through a signal arising from the translational
apparatus, which reflects the availability of substrate for protein
synthesis. This signal is generated downstream of amino acid transport,
but upstream of the steps inhibited by anisomycin and cycloheximide,
translational inhibitors known to activate the p70 S6 kinase. The
chemical identity of the signal is unknown; however, examples of
candidate signaling molecules whose abundance is altered by amino acid
insufficiency include uncharged tRNAs, adenosine tetraphosphate, and in
bacteria, guanosine 5',3'-bispyrophosphate. We propose that this signal
regulates in situ the activity of a protein phosphatase that
selectively dephosphorylates p70 S6 kinase, eIF-4E BP1, and perhaps
other elements that control translation. Moreover, based on the close similarity in the pattern of responses to rapamycin and amino acid
withdrawal, and especially the parallel insensitivity of p702-46/
CT104 to amino acid depletion and rapamycin, we propose that the putative amino acid-responsive phosphatase is also regulated by mTOR. Rapamycin, by inhibiting mTOR in situ, activates
the phosphatase. The p70
2-46/
CT104 variant is resistant to amino acid depletion and rapamycin because of a diminished susceptibility to
the putative phosphatase; however, because it requires an
insulin-stimulated, PI 3-kinase-dependent phosphorylation
of Thr-252 and Thr-412, it remains susceptible to inhibition by
wortmannin. We emphasize that the existence of such a phosphatase is
entirely speculative; an enzyme with the expected regulatory behavior
(i.e. responsive to amino acid sufficiency and mTOR) and
specificity has not been identified.
|
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. Comb of New England Biolabs for the anti-p70 phosphopeptide antibodies; Y. Tsujishita, M. Nanahoshi, and T. Nishiuma for preparation of the anti-eIF-4E BP1 antibodies; J. Woodgett for the c-Akt/PKB cDNA; and J. Prendable for preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK17776 and CA73818 (to J. A.) and by a grant from the Juvenile Diabetes Foundation International (to K. Y.).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.
¶ Supported by a fellowship from Juvenile Diabetes Foundation, International.
Present address: Biosignal Research Center, Kobe University,
Kobe, Japan.
** To whom correspondence should be addressed: Diabetes Research Laboratory, Dept. of Molecular Biology, Massachusetts General Hospital, 50 Blossom St., Boston, MA 02114. Tel.: 617-726-6909; Fax: 617-726-5649; E-mail: avruch{at}helix.mgh.harvard.edu.
1 The abbreviations used are: CHO, Chinese hamster ovary; D-PBS, Dulbecco's phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; PVDF, polyvinylidene difluoride; DTT, dithiothreitol; BP, binding protein; PAGE, polyacrylamide gel electrophoresis; eIF, eukaryotic initiation factor; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; PKB, protein kinase B; PKC, protein kinase C; PtdIns, phosphatidylinositol.
2
Subtract 23 for the amino acid number in
p702.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|