(Received for publication, May 5, 1995; and in revised form, June 6, 1995)
From the
PHAS-I levels increased 8-fold as 3T3-L1 fibroblasts
differentiated into adipocytes and acquired sensitivity to insulin.
Insulin increased PHAS-I protein (3.3-fold after 2 days), the rate of
PHAS-I synthesis (3-fold after 1 h), and the half-life of the protein
(from 1.5 to 2.5 days). Insulin also increased the phosphorylation of
PHAS-I and promoted dissociation of the PHAS-I
The stimulation of protein synthesis by insulin is the net
result of changes in both transcriptional and translational processes
(for review, see (1) and (2) ). In liver, heart,
skeletal muscle, and adipose tissue, insulin increases total protein by
more than it increases total mRNA(2) , indicative of the
importance of increased translation in the stimulation of overall
protein synthesis by the hormone. The synthesis of certain proteins,
such as those encoding ribosomal proteins(3, 4) , is
increased disproportionately by insulin. A highly structured (G/C-rich)
5`-nontranslated region (5) or a polypyrimidine motif near the
5` cap (6) are two structural motifs that may contribute to the
selective increase in translation in response to insulin. The presence
of the cap, m Generally, the rate-limiting step
for translation is the initiation phase, which involves recognition of
capped mRNA, melting of secondary structure in the 5` region of the
mRNA, and binding to the 40 S ribosomal
subunit(9, 10, 11, 12) . These
processes require eIF-4F, a complex containing a large subunit (p220)
whose function is not fully defined, an ATP-dependent helicase
(eIF-4A), ( Recombinant
PHAS-I is an excellent substrate for MAP kinase(19) , and
phosphorylation of PHAS-I by MAP kinase in vitro dramatically
decreases binding of PHAS-I to eIF-4E(16) . Moreover, the major
site (Ser The
experiments described in this report were performed to investigate
mechanisms by which insulin controls the levels of PHAS-I and regulates
the phosphorylation of protein.
Two-dimensional electrophoresis, with isoelectric focusing in the
first dimension and SDS-PAGE in the second, was performed as described
by Blackshear et al.(38) . PHAS-I was detected by
immunoblotting, essentially as described above, except that antibody
binding was detected using horseradish peroxidase conjugated to goat
anti-rabbit IgG and a colorimetric stain as directed by the supplier
(Bio-Rad, horseradish peroxidase color development reagent).
Figure 1:
Nucleotide sequence of
mouse PHAS-I cDNA (A) and the deduced amino acid sequence of
PHAS-I protein from 3T3 L1 cells (B).
Figure 12:
Resolution of PHAS-I kinases by Mono Q
chromatography. Adipocytes were incubated for 10 min without additions
(
Figure 2:
Expression of PHAS-I and GLUT4 during
differentiation of 3T3-L1 fibroblasts into adipocytes. To induce
differentiation, the medium of confluent cultures of 3T3-L1 fibroblasts
was replaced (day 0) with growth medium (10% fetal bovine serum in
DMEM) supplemented with 0.5 mM isobutylmethylxanthine, 0.25
µM dexamethasone, and 350 nM insulin. This medium
was replaced after 2 days with growth medium, which was then changed
every other day. Cells were homogenized at increasing times after
addition of the differentiation medium. Homogenates were centrifuged at
100,000
3T3-L1 adipocytes were used to
investigate the regulation of PHAS-I synthesis and degradation in
vitro (Fig. 3). Treating adipocytes with insulin for 2 days
increased the amount of PHAS-I protein (measured by immunoblotting)
relative to total protein by 3.3 ± 0.5-fold (n =
3 experiments). To investigate the mechanism of the increase in PHAS-I,
[
Figure 3:
PHAS-I synthesis (A) and
degradation (B). A, 3T3-L1 adipocytes were incubated
in medium containing [
Pulse-chase experiments were performed to investigate
the rate of degradation of PHAS-I (Fig. 3B). Cells were
incubated with [
Figure 4:
Regulation of the phosphorylation of
PHAS-I and the association of PHAS-I and eIF-4E by insulin and
rapamycin. 3T3-L1 adipocytes were incubated without or with 20 nM rapamycin for 10 min. The cells were then incubated for increasing
times without insulin, with 20 nM insulin, or with the
combination of insulin plus rapamycin. Samples of extracts (20 µl)
were dissolved in SDS sample buffer. Other samples (100 µl) were
incubated with m
The effects of insulin on increasing
PHAS-I phosphorylation occurred rapidly, with maximum effects observed
after 10 min of incubation (Fig. 5). PHAS-I
Figure 5:
Time course of insulin action on PHAS-I in
the absence and presence or rapamycin. Experiments were conducted as
described in the legend to Fig. 4. Effects of treatments on
PHAS-I
The effects of insulin on
the MAP kinase isoforms, ERK-1 (Fig. 6A) and ERK-2 (Fig. 6B), and on ribosomal protein S6 kinase activity (Fig. 6C) also reached peaks after 10 min of
incubation. As noted previously(33) , the effects of insulin on
the MAP kinases declined with longer times of incubation with the
hormone, although activities were still approximately 2 times higher
than the control after an hour of incubation (Fig. 6, A and B). The effect of insulin on increasing ribosome
protein S6 kinase activity was more prolonged than the effect on MAP
kinase, and S6 kinase was more than 6-fold higher than the control
after 1 h with insulin (Fig. 6C). Thus, the time course
of PHAS-I phosphorylation in response to insulin correlated better with
that of S6 kinase activation than that of MAP kinase activation.
Although p70
Figure 6:
Time courses of the activation of MAP
kinase and ribosomal protein S6 kinases by insulin in the absence and
presence of rapamycin. Cells were incubated with rapamycin and insulin
as described in the legend to Fig. 4. Activation of the ERK-1 (A) and ERK-2 (B) isoforms of MAP kinase was
monitored using an in-gel assay with myelin basic protein as substrate (42) . The activity of S6 protein kinases (C) was
measured using intact 40 S ribosomes as substrate. The x axes
represent the times of incubation with insulin (see legend to Fig. 5). The results are expressed as percentages of the
respective control values and are means ± S.E. from three
experiments.
Rapamycin appears to act
in cells by binding to one or more members of a family of FK506 binding
proteins (FKBPs)(23, 24, 25) . The inhibitory
action of rapamycin on p70
Figure 7:
Reversal of the effects of rapamycin on
PHAS-I and S6 protein kinases by FK506. 3T3L1 adipocytes were incubated
in duplicate without additions, with 20 nM rapamycin, with 12
µM FK506, or with the combination of rapamycin plus FK506
for 10 min. After adding insulin to half of the cells, the incubations
were continued for 10 min. A, samples were subjected to
SDS-PAGE, and the relative amounts of PHAS-I
Figure 8:
Effects of rapamycin and MEK inhibitor on
the activation of MAP kinase and ribosomal protein S6 kinases.
Adipocytes were incubated in medium without additions, with 20 nM rapamycin for 10 min, with 50 µM PD 098059 for 30 min
or with PD 098059 for 20 min, followed by rapamycin plus PD 098059 for
10 min. Insulin was then added to the medium, and incubations were
continued for 10 min. Activities of ERK-1 (A), ERK-2 (B), and ribosomal protein S6 kinases (C) are
expressed as a percentage of the respective control values. Means
± S.E. of six experiments are
presented.
Figure 9:
Effects of rapamycin and MEK inhibitor on
insulin-stimulated phosphorylation of ribosomal protein S6 and PHAS-I. A,
Consistent
with its failure to affect the
Figure 10:
Effects of rapamycin and MEK inhibitor on
PHAS-I. Cells were incubated as described in the legend to Fig. 8. Relative amounts of PHAS-I
Figure 11:
Resolution of multiple forms of PHAS-I
A kinase that acted on both free
[H The present findings demonstrate that the stimulation of
PHAS-I phosphorylation by insulin involves at least one pathway that is
independent of the MAP kinase signaling pathway. The complex
two-dimensional electrophoretic pattern indicates that several sites in
PHAS-I are phosphorylated in response to insulin (Fig. 11). As
MAP kinase phosphorylates only one site, Ser
When bound to rapamycin,
FKBP12 binds tightly to a much larger protein, designated TOR (target
of rapamycin)(23, 24, 25) . TOR was
originally identified in yeast, but mammalian counterparts, termed
FRAP(49) , RAFT1(57) , and mTOR (50) have been
recently cloned. TOR proteins have regions of homology with the lipid
kinase, phosphatidylinositol-3`-OH kinase, although it has not been
demonstrated that the TORs possess lipid kinase activity. Sensitivity
to rapamycin is generally viewed as indicative of the p70 Purified p70 Another possibility is that
rapamycin promotes dephosphorylation of PHAS-I, not by inhibiting a
PHAS-I kinase, but by stimulating a phosphatase. This has been
suggested as a mechanism for regulation of p70 The possibility that insulin inhibits a PHAS-I
phosphatase should also be considered. That insulin-stimulated PHAS-I
kinase activity was not detected when the
[H
In addition to its role in translation, eIF-4E has potent mitogenic
actions. Increasing eIF-4E in cells, either by cDNA transfection (31, 56) or by microinjecting the
protein(32) , not only stimulated cell growth but also caused
the cells to acquire a transformed phenotype. A final speculative idea
is that by inhibiting eIF-4E action, PHAS-I dephosphorylation might
contribute to the antiproliferative effects of rapamycin.
eukaryotic
initiation factor-4E (eIF-4E) complex, effects that were maximal within
10 min. With recombinant [H
]PHAS-I as substrate,
mitogen-activated protein (MAP) kinase was the only insulin-stimulated
PHAS-I kinase detected after fractionation of extracts by Mono Q
chromatography; however, MAP kinase did not readily phosphorylate
[H
]PHAS-I when the
[H
]PHAS-I
eIF-4E complex was the substrate.
Thus, while MAP kinase may phosphorylate free PHAS-I, it is not
sufficient to dissociate the complex. Moreover, rapamycin attenuated
the stimulation of PHAS-I phosphorylation by insulin and markedly
inhibited dissociation of PHAS-I
eIF-4E, without decreasing MAP
kinase activity. Rapamycin abolished the effects of insulin on
increasing phosphorylation of ribosomal protein S6 and on activating
p70
. The MAP kinase kinase inhibitor, PD 098059, markedly
decreased MAP kinase activation by insulin, but it did not change
PHAS-I phosphorylation or the association of PHAS-I with eIF-4E. In
summary, insulin increases the expression of PHAS-I and promotes
phosphorylation of multiple sites in the protein via multiple
transduction pathways, one of which is rapamycin-sensitive and
independent of MAP kinase. Rapamycin may inhibit translation initiation
by increasing PHAS-I binding to eIF-4E.
GPPPN (where N is any
nucleotide)(7) , appears to be required for insulin-stimulated
translation to occur(8) .
)and the mRNA cap binding protein, eIF-4E. The
activity of eIF-4E is controlled by PHAS-I(13) , a widely
expressed heat- and acid-stable protein that is phosphorylated in
response to insulin and growth
factors(14, 15, 16) . PHAS-I binds to eIF-4E
and inhibits translation of capped mRNA, both in vitro and in
intact cells(17, 18) . When phosphorylated in the
appropriate sites in response to insulin, PHAS-I does not bind to
eIF-4E(16, 17, 18) . Thus, release of eIF-4E
from inhibition by PHAS-I may increase eIF-4F activity.
) phosphorylated by MAP kinase in vitro is phosphorylated in response to insulin in
adipocytes(19) . However, other kinases are involved in
phosphorylating PHAS-I, as sites in addition to Ser
are
phosphorylated in the protein (19) . Insulin activates multiple
protein kinases in cells(20) . p70
is one such
kinase that is part of a signal transduction pathway that is distinct
from the MAP kinase signaling pathway(21, 22) .
Because MAP kinase and p70
are regulated by many of the
same hormones and growth factors, assigning specific functions to the
kinases has been difficult. Recently, reagents have become available
that allow discrimination between the pathways. The immunosuppressant,
rapamycin (for review, see (23, 24, 25) ),
blocks activation of p70
by insulin without affecting the
activation of MAP kinase or the downstream kinase,
p90
(26, 27, 28) . An
inhibitor (PD 098059) of MEK, the protein kinase that phosphorylates
and activates MAP kinase(29) , selectively inhibits the MAP
kinase signaling pathway (30) .
3T3-L1 Adipocytes
3T3-L1 fibroblasts were
cultured in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal calf serum on plastic culture dishes (10-cm
diameter) and converted to adipocytes by using differentiation medium
as described previously (33) . Unless otherwise stated,
experiments were performed on adipocytes 12-16 days after
withdrawal from differentiation medium. The growth medium was replaced
with Low P buffer containing 135 mM NaCl,
5.4 mM KCl, 1.4 mM CaCl
, 1.4 mM MgSO
, 5 mM glucose, 5 mg/ml bovine serum
albumin, 0.2 mM NaP
, and 10 mM HEPES (pH
7.4). In
P-labeling experiments, the Low P
buffer was supplemented with Na
P
(2
mCi/5 ml). The cells were homogenized after 3 h of incubation in Low P
buffer. Additions of insulin and other
agents were made at the appropriate time prior to the end of this
incubation period to give the desired treatment. To terminate the
incubation, the medium was aspirated, and the cells were rinsed twice
with phosphate-buffered saline and homogenized (1 ml of buffer/dish) in
a glass homogenization tube with a Teflon pestle driven at 1,000 rpm.
Phosphate-buffered saline contained 145 mM NaCl, 5.4 mM KCl, and 10 mM NaP
(pH 7.4). Homogenization buffer contained 1 mM EDTA, 5
mM EGTA, 10 mM MgCl
, 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10
µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM benzamidine,
10 mM KP
, and 50 mM
-glycerophosphate (pH 7.3). Homogenates were centrifuged at
10,000
g for 20 min, and the supernatants were
retained for analyses. The protein content was determined by using
bicinchoninic acid(34) .
Adipocytes were
rinsed twice and incubated for 1 h at 37 °C in methionine-free
DMEM. To measure the initial rate of incorporation of
[S Labeling
S]methionine into PHAS-I, the medium was
replaced with methionine-free DMEM supplemented with 10 µM
[
S]methionine (10 µCi/ml) and incubated for
increasing times (±20 nM insulin). In pulse-chase
experiments, cells were incubated with 10 µM [
S]methionine (10 µCi/ml) for 3 h at 37
°C and then rinsed twice with DMEM and incubated (±20 nM insulin) for increasing times in DMEM supplemented with 2 mM methionine. The cells were homogenized as described above.
Antibodies and Immunoprecipitations
PHAS-I
antibodies were generated by immunizing rabbits with a peptide
(CSSPEDKRAGGEESQFE) having a sequence derived from the COOH-terminal
region of PHAS-I. The antibodies were affinity-purified as described
previously(13) . Antiserum to eIF-4E was generated in rabbits
and was provided by Nahum Sonenberg (McGill University). GLUT-4
antibody was prepared by immunizing rabbits with a peptide having the
sequence of the last 12 residues in the COOH terminus of GLUT-4 and was
affinity-purified before use(35) . For immunoprecipitation,
PHAS-I antibodies were coupled to protein A-agarose beads (0.5 mg of
antibody/ml of beads) by incubation at 23 °C for 60 min in Homogenization buffer. The beads were then washed 3 times with Homogenization buffer. Samples (100 µl) of extract were
incubated with beads (10 µl) for 30 min at 23 °C with constant
mixing. After washing the beads 5 times, proteins were eluted with SDS
sample buffer.Electrophoretic Analyses
Samples were subjected to
SDS-PAGE using the method of Laemmli(36) . To determine the
relative S contents of proteins, gels were dried and
radioactivity was measured using a PhosphorImager (Molecular Dynamics).
For immunoblotting, proteins were electrophoretically transferred to
nylon membranes (Immobilon, Millipore). The membranes were immersed in
phosphate-buffered saline containing 5% powdered milk (Carnation) and
incubated for at least 1 h. Sheets were then incubated with PHAS-I
antibody (2 µg/ml) or GLUT-4 antibody (2 µg/ml) in
phosphate-buffered saline plus milk for 2 h and then washed as
described previously(37) . Antibody binding was detected by
enhanced chemiluminescence (Tropix system) using alkaline phosphatase
conjugated to either goat anti-rabbit IgG or protein A. The intensities
of the bands corresponding to PHAS-I were determined by two-dimensional
scanning using a laser densitometer (Molecular Dynamics).
Cloning of 3T3L1 PHAS-I cDNA
A 3T3-L1 adipocyte
cDNA library (in Zap II) provided by Dr. Fred Fiedorek
(University of North Carolina) was used to obtain mouse PHAS-I cDNA by
expression cloning(39) . Briefly, phage (10
) were
grown at 42 °C until small plaques became visible. Nitrocellulose
membranes impregnated with
isopropyl-1-thio-
-D-galactopyranoside to induce
expression of proteins were applied, and the phage were allowed to grow
for 3 h at 37 °C. Membranes were incubated in blocking solution and
PHAS-I antibodies and then washed essentially as described for
detecting PHAS-I by immunoblotting. Clones that bound antibodies were
detected by enhanced chemiluminescence. After plaque-purification,
inserts were subcloned into pBluescript SK
(Stratagene) for nucleotide sequencing, which was performed using
an automatic sequencing apparatus and sequencing kit (part 901497) as
directed by the supplier (Applied Biosystems). Out of the 25 positive
clones isolated, 15 represented partial-length cDNAs that had sequences
identical to regions of the PHAS-I sequence (see Fig. 1), which
was derived separately from two independent clones.
Preparation of Recombinant
Proteins
``Histidine-tagged'' PHAS-I
([H]PHAS-I) was expressed in bacteria and
purified as described previously(19) . Human eIF-4E cDNA cloned
into pET-11d (Novagen) was provided by Dr. Rosemary Jagus (Maryland
Biotechnology Institute, Baltimore, MD). eIF-4E was expressed in Escherichia coli and purified as described by Stern et
al.(40) . [H
]PHAS-I
eIF-4E
complexes were generated by allowing eIF-4E to renature in the presence
of excess [H
]PHAS-I. This was accomplished by
slight modification of the eIF-4E purification method(40) . E. coli (BL21(DE3)pLysS strain, 800 ml) expressing
[H
]PHAS-I was pelleted by centrifugation (7,000
g for 15 min) and homogenized in 30 ml of 8 M urea, 50 mM dithiothreitol, and 50 mM HEPES (pH
7.6). After centrifugation at 39,000
g for 20 min, the
supernatant containing [H
]PHAS-I was added to 20
ml of a solution containing eIF-4E that had been extracted from
inclusion bodies (from 800 ml of bacteria) by using this buffer.
Subsequent steps, including renaturation by sequential dialysis to
remove urea and affinity chromatography with
m
GTP-Sepharose, were performed as described by Stern et
al.(40) . Because [H
]PHAS-I does not
bind directly to m
GTP-Sepharose(16) , only
[H
]PHAS-I bound to eIF-4E is recovered with the
cap affinity resin. To estimate the relative amounts of
[H
]PHAS-I and eIF-4E in the complex, samples were
subjected to SDS-PAGE, and proteins were stained with Coomassie Blue
(for example, see Fig. 12). Based on staining intensities
determined by optical density scanning, the preparation contained 0.5
mg of [H
]PHAS-I (predicted M
= 15,094) per mg of eIF-4E (predicted M
= 25,117), which is similar to the proportion of 0.6 mg of
[H
]PHAS-I per mg of eIF-4E that would be expected
of a 1:1 complex of [H
]PHAS-I and eIF-4E.
), with 20 nM EGF (▴), or with 20 nM insulin (▪). Extracts were applied to a Mono Q column, and
proteins were eluted with an increasing gradient of NaCl. PHAS-I kinase
activities were measured using [
-
P]ATP and
either 50 µg/ml [H
]PHAS-I
eIF-4E (A) or 25 µg/ml [H
]PHAS-I (B) as substrates. After incubating for 20 min at 30 °C,
samples were subjected to SDS-PAGE, and amounts of
P
incoroporated into PHAS-I were determined by scintillation counting of
gel slices. A Coomassie Blue-stained gel of the preparations of
[H
]PHAS-I (lane1) and
[H
]PHAS-I
eIF-4E (lane2)
used is shown as an inset in B. Protein kinase
activities in fractions 56-60 were also assayed in the absence
and presence of heparin (from porcine intestine, 0.1 mg/ml) with
phosvitin (1.5 mg/ml) as substrate (C). Protein kinase
activities (A-C) are expressed as pmol of
phosphate incorporated into either PHAS-I or phosvitin/min/ml column
fraction.
Anion-exchange Chromatography
Extracts (5 ml) were
diluted with HO (5 ml) and applied at 21 °C to a Mono Q
HR 5/5 column (Pharmacia Biotech Inc.) equilibrated with Buffer A (1 mM EGTA, 0.1 mM
Na
VO
, and 50 mM
-glycerol
phosphate, pH 7.3) as described previously(41) . The flow rate
was maintained at 1 ml/min, and fractions were collected each min.
Proteins were eluted by increasing Buffer B (Buffer A
plus 800 mM NaCl) to produce the following gradient of NaCl: 0
mM for 15 min, 0-350 mM in 50 min, and
350-800 mM in 5 min.
Measurements of Protein Kinase Activities
The
activities of the ERK-1 and ERK-2 isoforms of MAP kinase activity were
measured using [-
P]ATP and myelin basic
protein as substrates in the gel renaturation method described by Wang et al.(42) . Ribosomal protein S6 kinase activity was
measured using intact 40 S ribosomes as substrate as described
previously(41) .
Other Materials
Porcine insulin (27 units/mg) was
supplied by Eli Lilly Co. PD 098059 was provided by Parke-Davis. EGF
was purchased from Boeringer Mannheim.
[-
P]ATP and
[
S]methionine were from DuPont NEN. Rapamycin
and FK506 were obtained from Calbiochem and Fujisawa Pharmaceuticals,
respectively. Most commonly used chemicals were from Sigma.
Oligonucleotides and peptides were synthesized by the Molecular Biology
Core Laboratory of the Washington University Diabetes Center.
Mouse PHAS-I
PHAS-I cDNA from 3T3-L1 adipocytes (Fig. 1) was highly homologous to rat PHAS-I cDNA(13) .
Overlapping regions of the G/C-rich 5`-nontranslated regions were
identical in the rat and mouse cDNAs, and the deduced amino acid
sequences of the proteins from the two species were 97% identical. As
in the rat protein, 27 out of 117 amino acids in mouse PHAS-I are
Ser/Thr residues (Fig. 1), which represent potential sites of
phosphorylation. The major site (Ser) phosphorylated by
MAP kinase is conserved in mouse PHAS-I, as is the consensus PEST motif
between Asp
and Ser
.
Control of PHAS-I Levels in 3T3-L1 Cells
Fat
contained the highest levels of PHAS-I mRNA (13) and protein (16, 43) of several rat tissues analyzed, suggesting
that PHAS-I was induced during the formation of fat cells. To
investigate this possibility, levels of PHAS-I were measured by
immunoblotting at different times as 3T3-L1 fibroblasts differentiated
into adipocytes (Fig. 2). PHAS-I increased markedly as the cells
differentiated. The increase in PHAS-I was first noted on the fourth
day after adding differentiation medium. This rise preceded by 2 days
the first detectable increase in levels of the insulin-responsive
glucose transporter, GLUT4, which was monitored as a marker of the
terminally differentiated state. By 8-days, PHAS-I reached a maximum,
representing an increase of approximately 8-fold over the level of the
protein found in fibroblasts.
g for 45 min. Samples (25 µg of protein)
of the supernatants and pellets were subjected to SDS-PAGE, and
immunoblots were prepared for PHAS-I and GLUT4, respectively. Levels of
the proteins are expressed as percentages of the maximum level
detected, and are mean values ± half of the range of two
experiments.
S]methionine-labeling experiments were
performed. Relative rates of synthesis of PHAS-1 were determined by
incubating 3T3-L1 adipocytes in the absence or presence of insulin for
increasing times with [
S]methionine before
PHAS-I was immunoprecipitated (Fig. 3A). Incorporation
of [
S]methionine into PHAS-I increased in a
linear manner for 3 h. Insulin increased the initial rate of
incorporation of [
S]methionine by approximately
3-fold (Fig. 3A). This effect of insulin was most
likely due to increased translation of PHAS-I mRNA, as it was observed
after incubating with the hormone for only 1 h, a time in which we have
been unable to detect any change in PHAS-I mRNA. (
)
S]methionine in the
absence and presence of 20 nM insulin for increasing times.
PHAS-I was immunoprecipitated and subjected to SDS-PAGE. The gels were
dried, and
S was detected by using a PhosphorImager. The
amount of
S in PHAS-I is expressed in arbitrary units,
where 1.0 represents the
S content of PHAS-I found in
control cells after 6 h of incubation with
[
S]methionine. The results are mean values
± S.E. from three experiments. B, 3T3-L1 adipocytes
were incubated in medium containing
[
S]methionine in the absence and presence of 20
nM insulin for 3 h. The cells were then rinsed to remove
[
S]methionine, incubated (with or without
insulin) in DMEM containing 2 mM methionine, and homogenized
after increasing times. [
S]PHAS-I was
immunoprecipitated and subjected to SDS-PAGE before relative amounts of
radioactivity were determined using a PhosphorImager. The results are
expressed in arbitrary units, with 1.0 representing the
S
content of PHAS-I recovered in PHAS-I immediately after the 3-h
incubation with [
S]methionine. Mean values
± half of the range of two experiments are
presented.
S]methionine for 3 h to label
the endogenous pool of PHAS-I. The cells were then rinsed and incubated
in the absence and presence of insulin in medium supplemented with
excess unlabeled methionine to limit reutilization of
[
S]methionine released as result of the
degradation of
S-labeled proteins. Assuming the
disappearance of [
S]PHAS-I to be a first order
process, values for t of [
S]PHAS-I in
control and insulin-treated cells were approximately 1.7 and 2.6 days,
respectively. In the presence of insulin, the t of PHAS-I was
approximately 20 times higher than that of insulin receptor substrate-1
(2-3 h) (44) , another insulin-stimulated phosphoprotein (20) that contains multiple PEST domains(44) . The
relatively slow rate of degradation of PHAS-I indicates that the PEST
motif in PHAS-I does not target it for rapid degradation in these
cells.
Control of PHAS-I Phosphorylation
PHAS-I (M
12,400) migrates anomalously when
subjected to SDS-PAGE (apparent M
=
20,000-24,000)(19) , probably because of its high content
of proline and glycine(13) . Moreover, PHAS-I from most tissues
appears as three bands, denoted
,
, and
(Fig. 4), representing protein phosphorylated to differing
extents(16) . Because phosphorylation decreases the
electrophoretic mobility of the protein, the shift to species of higher
apparent M
is indicative of increased
phosphorylation(16) . In nonstimulated cells, approximately 20%
of the PHAS-I was present as the nonphosphorylated (
) form, which
binds tightly to eIF-4E. As previously observed (16, 17, 18) , PHAS-I
was the
predominate form found bound to the initiation factor when complexes
were isolated from extracts of control cells by affinity purification
with m
GTP-Sepharose (Fig. 4). However, some PHAS-I
was also recovered with the cap-affinity resin (Fig. 4),
indicating that at least one site in PHAS-I may be phosphorylated
without loss of eIF-4E binding.
GTP-Sepharose to determine the relative
amounts of PHAS-I that copurified with eIF-4E. After washing the
resins, proteins were eluted with SDS sample buffer. Representative
immunoblots of extracts (Extract) and of proteins that bound
m
GTP-Sepharose (m
GTP) in the regions containing
PHAS-I are presented.
and
were
decreased by 60% after this time (Fig. 5, A and B), and the more highly phosphorylated PHAS-I
was
increased 8-fold (Fig. 5C). The effects of insulin
persisted for 60 min, although some dephosphorylation of PHAS-I
occurred with the longer periods of incubation, as indicated by an
increase in the
form (Fig. 5B). Insulin also
rapidly decreased the amount of PHAS-I bound to eIF-4E (Fig. 5D). The maximum effect was observed after 5 min
and represented a decrease of approximately 60% in the amount of PHAS-I
bound to eIF-4E. Partial reversal of the insulin effect was noted after
60 min (Fig. 5D) and was associated with increased
binding of PHAS-I
(Fig. 4).
(A), PHAS-I
(B), and PHAS-I
(C) were determined from differences in the optical densities
of the appropriated bands, which were measured by scanning laser
densitometry. Changes in PHAS-I binding to eIF-4E (D)
represent differences in the amounts of PHAS-I (
plus
forms)
recovered when eIF-4E was partially purified using
m
GTP-Sepharose. The abscissa (A-D)
represents the time of incubation after the addition of insulin. Note
that because rapamycin was added 10 min before insulin, the actual time
of incubation with rapamycin (in both the Rapamycin or Rapamycin + Insulin treatments) was 10 min longer than
indicated by the scale on the x axes. The results are
expressed as percentages of the respective control values and are means
± S.E. from three experiments.
itself does not phosphorylate PHAS-I in
vitro(19) , these findings suggest that other elements in
the p70
pathway might be involved in regulating PHAS-I.
Attenuation of Insulin-Stimulated Phosphorylation by
Rapamycin
Results obtained with rapamycin also suggest a role of
the p70 pathway in regulating the phosphorylation of
PHAS-I. Incubating cells with rapamycin alone decreased the
phosphorylation of PHAS-I, as indicated by decreased PHAS-I
(Fig. 5C), and increased PHAS-I
(Fig. 5A). Rapamycin also attenuated the effects of
insulin on increasing the phosphorylation of PHAS-I. In the presence of
rapamycin, insulin did not decrease the level of PHAS-I
below the
control level, even after an hour of incubation (Fig. 5A). As might be expected, rapamycin markedly
inhibited the effect of insulin on decreasing the amount of PHAS-I
bound to eIF-4E, although an effect of the hormone was observed with
longer times of incubation (Fig. 5D). After 60 min,
PHAS-I binding to eIF-4E was decreased by approximately 25% in cells
incubated with the combination of insulin plus rapamycin. Moreover, in
the presence of rapamycin, insulin still increased PHAS-I
by
3-4-fold (Fig. 5C), indicating that the hormonal
effect on PHAS-I phosphorylation was not completely inhibited. In
contrast, rapamycin abolished the increase in ribosomal protein S6
kinase activity (Fig. 6C). This finding indicates that
under the conditions of these experiments, essentially all of the
ribosomal protein S6 protein kinase activity measured in extracts was
due to p70
, as rapamycin inhibits activation of this
kinase without affecting
p90
(26, 27, 28) . MAP
kinase activity was also affected by rapamycin, but the direction of
the effect depended on the time of incubation with insulin. In cells
incubated for 5 min with insulin, prior treatment with rapamycin
increased the amount of ERK-1 and ERK-2 activity; however, with longer
times of incubation with insulin, the activities of the MAP kinase
isoforms were slightly less in extracts of rapamycin-treated cells (Fig. 6, A and B).
can be partially reversed by
the structurally related drug,
FK506(26, 27, 28) , which competes with
rapamycin for binding to FKBPs(23, 24, 25) .
Consistent with previous findings with
p70
(26, 27, 28) , FK506
attenuated the inhibitory effect of rapamycin on the activation of
ribosomal protein S6 kinase by insulin (Fig. 7B). FK506
was without effect on the amount of PHAS-I
in either the absence
or presence of insulin; however, FK506 abolished the effects of
rapamycin on PHAS-I
in both the presence and absence of the
hormone (Fig. 7A). These results indicate that the
effects of rapamycin on PHAS-I phosphorylation are mediated by FKBPs.
were determined by
immunoblotting. The results are expressed as a percentage of the
control and are mean values + S.E. from three experiments. B, ribosomal protein S6 kinase was measured in cell extracts.
The results are expressed as pmol of phosphate incorporated into
protein S6 per min/culture, and are mean values ± S.E. of three
experiments.
Failure of the MEK Inhibitor, PD 098059, to Prevent
Insulin-stimulated Phosphorylation of PHAS-I
PD 098059 acts
noncompetitively with respect to ATP to inhibit MEK1 and MEK2 in
vitro. The inhibitor blocks the activation of MEK by
growth factors in a variety of cells and inhibits the hormonal
stimulation of MAP kinase activity without inhibiting other protein or
lipid kinases(30) .
Incubating 3T3L1 adipocytes
with 50 µM PD 098059 decreased the effects of insulin on
ERK-1 and ERK-2 by approximately 80% (Fig. 8, A and B). Experiments were conducted with
P-labeled
adipocytes to investigate the effects of the MEK inhibitor on PHAS-I
phosphorylation (Fig. 9).
P-Labeled PHAS-I
immunoprecipitated from extracts appeared as two bands corresponding to
PHAS-I
and
(Fig. 9A). Incubating cells with
the inhibitor was without effect on the
P-content of
PHAS-I. Rapamycin attenuated the insulin-stimulated phosphorylation of
PHAS-I (Fig. 9A), confirming the results from the
immunoblotting analyses (Fig. 5C). Rapamycin abolished
phosphorylation of the M
= 31,000 extract
protein (Fig. 9A), previously shown to be ribosomal
protein S6(45) . Phosphorylation of S6 was also slightly
decreased by the MEK inhibitor, as was insulin-stimulated ribosomal
protein S6 kinase activity (Fig. 9B).
P-Labeled adipocytes were subjected to the
treatments indicated in the legend to Fig. 8. Samples of extract
and of immunoprecipitated PHAS-I were subjected to SDS-PAGE. An
autoradiogram of the dried gel of extract samples is presented. S6
denotes
P-labeled ribosomal protein S6, which has an
apparent M
= 31,000. The picture (PHAS-I IP) of immunoprecipitated PHAS-I was obtained using a
PhosphorImager. B, samples of extracts from cells incubated
without
P were subjected to SDS-PAGE. Other samples were
incubated with m
GTP-Sepharose. After washing, the resin
proteins were eluted with SDS-sample buffer and subjected to
electrophoresis. PHAS-I immunoblots are
presented.
P content of PHAS-I, PD
098059 did not change the electrophoretic pattern of PHAS-I determined
by immunoblotting (Fig. 9B). Quantitation of results
from six such experiments indicated that the MEK inhibitor was without
effect on the proportions of PHAS-I
(Fig. 10A),
(Fig. 10B), or
(Fig. 10C).
Moreover, the inhibitor did not attenuate the effect of insulin on
dissociation of the PHAS-I
eIF-4E complex assessed by
m
GTP binding (Fig. 9B and 10D),
although in some experiments PD 098059 itself decreased the amount of
PHAS-I recovered with the m
GTP resin.
(A), PHAS-I
(B), and PHAS-I
(C) forms were determined
by optical density scanning, and are expressed as a percentage of the
total PHAS-I present. The relative amount of PHAS-I bound to eIF-4E (D) was assessed by determining how much PHAS-I (
and
forms) was recovered when eIF-4E was partially purified using
m
GTP. Results in D are expressed as a percentage
of the control. The results presented (A-D) are
mean values ± S.E. from six
experiments.
Multiple Forms of PHAS-I
To investigate the
complexity of PHAS-I phosphorylation, extracts from control and
insulin-treated cells were analyzed by two-dimensional electrophoresis (Fig. 11). Consistent with increasing PHAS-I phosphorylation,
insulin promoted a general shift to species of lower pI and
electrophoretic mobility. PHAS-I ,
, and
Identified by Two-dimensional Electrophoresis
from control cells appeared as a
doublet, containing the spots labeled
1 and
2. As the
band did not appear to be labeled with
P in extracts of
control cells (Fig. 9),
1 and
2 probably
represent PHAS-I species that differ in covalent modification other
than phosphorylation. Consequently, the doublets containing
1 and
2 and
7 and
8, and the minor doublet containing
4
and
5 may represent corresponding pairs differing by such a
modification, although it is not feasible to determine if this is the
case. The relatively large shifts in mobility produced by insulin are
almost certainly due to phosphorylation. A total of eight major spots
and six minor spots were detected by immunoblotting. Even allowing for
other covalent modifications, it seems clear that several sites in
PHAS-I are phosphorylated in response to insulin.
,
, and
by two-dimensional electrophoresis. Adipocytes
were incubated without (A, control) or with (B) 20
nM insulin for 10 min. Extract proteins were resolved by
isoelectric focusing in the first dimension and SDS-PAGE in the second
dimension. The composite drawing (C) identifies spots detected
in gels of the control and insulin-treated
samples.
Insulin-stimulated PHAS-I Kinases
To attempt to
identify PHAS-I kinases, extracts from 3T3L1 adipocytes that had been
incubated with insulin or EGF were fractionated by Mono Q
chromatography before PHAS-I kinase activity was measured using
recombinant [H]PHAS-I or a complex of
[H
]PHAS-I and eIF-4E as substrates. In agreement
with previous results obtained with native recombinant
PHAS-I(41) , both insulin and EGF increased
[H
]PHAS-I kinase activities that eluted in the
positions of ERK-1 and ERK-2 (Fig. 12B). In contrast,
neither insulin nor EGF increased kinase activity toward
[H
]PHAS-I bound to eIF-4E (Fig. 12A). We were also unable to detect insulin- or
EGF-stimulated PHAS-I kinase activity in unfractionated extracts when
the [H
]PHAS-I
eIF-4E complex was used as
substrate.
]PHAS-I and [H
]PHAS-I
bound to eIF-4E eluted at relatively high concentrations of NaCl
(fraction 58). This enzyme phosphorylated phosvitin and was inhibited
by heparin (Fig. 12C). These properties(46) ,
and the position of elution from Mono Q(47) , are suggestive of
casein kinase II, which has been shown previously to phosphorylate
PHAS-I(19, 48) .
, at a
reasonable rate in vitro(19) , other kinases must be
involved in the insulin response to account for multisite
phosphorylation of PHAS-I. Moreover, by using rapamycin and PD 098059,
the activation of MAP kinase could be dissociated from PHAS-I
phosphorylation. Rapamycin markedly reduced the effect of insulin on
phosphorylating PHAS-I without inhibiting MAP kinase (Fig. 4-6), and the MEK inhibitor markedly decreased MAP
kinase activity under conditions in which the phosphorylation of PHAS-I
was not detectably changed (Fig. 8-10). Thus, it seems
clear that MAP kinase is not the only mediator of PHAS-I
phosphorylation.
Role of MAP Kinase
Our results do not eliminate MAP
kinase as a mediator of PHAS-I phosphorylation in cells. Supporting a
role of MAP kinase is an increasing list of agents that both activate
MAP kinase and increase the phosphorylation of PHAS-I in adipocytes. In
addition to insulin, this list includes
EGF(14, 16, 38) , platelet-derived growth
factor(14, 38) , phorbol 12-myristate
13-acetate(38) , and vanadate. However, these
agents also activate other kinases. Moreover, in considering the role
of the MAP kinase in regulating the association of PHAS-I and eIF-4E,
it is necessary to address the inability of ERK-1 and ERK-2 to
phosphorylate PHAS-I when the protein was bound to eIF-4E (Fig. 12). Although the rate of dissociation of
nonphosphorylated PHAS-I and eIF-4E has not been measured directly in
cells, it appears to be relatively slow, as complexes of PHAS-I and
eIF-4E can be isolated from cell extracts. The stability of the complex
implies that the bound form of PHAS-I would have to be phosphorylated
to account for the rapid dissociation of PHAS-I and eIF-4E observed in
response to insulin ( Fig. 4and Fig. 5). Phosphorylation
of the free PHAS-I might still serve an important function, as
phosphorylation of Ser
should prevent reassociation of the
protein with eIF-4E(16) . Thus, MAP kinase might function in
this capacity. It is also possible that MAP kinase acts on the
phosphorylated form of PHAS-I found in the complex. To test this
hypothesis, it will be necessary to determine how to generate the
PHAS-I
eIF-4E complex.
Potential Mechanisms Involved in Rapamycin-sensitive
Regulation of PHAS-I
An important finding is that the control of
PHAS-I phosphorylation involves a rapamycin-sensitive pathway. The
dephosphorylation of PHAS-I in response to rapamycin was associated
with increased PHAS-I binding to eIF-4E (Fig. 5D),
indicative of the functional importance of this pathway. Rapamycin
appears to exert its action in cells by binding to FKBP12, a protein
with peptidyl-prolyl isomerase
activity(23, 24, 25) . Rapamycin and FK506
compete for binding to the same site in FKBP12, and both drugs inhibit
the isomerase activity. This activity is not involved in the effects of
rapamycin on PHAS-I phosphorylation, since FK506 did not affect PHAS-I
phosphorylation (Fig. 7A). However, FK506 abolished the
effects of rapamycin on PHAS-I phosphorylation (Fig. 7A), consistent with the interpretation that the
drug effects were mediated by FKBP12. pathway, since rapamycin blocks activation of the kinase by
insulin and growth factors. Activation of p70
is also
blocked by wortmannin, a potent inhibitor of phosphatidylinositol-3`-OH
kinase(51) . We have found that wortmannin potently inhibits
the phosphorylation of PHAS-I in response to insulin.
Thus,
effects of both wortmannin and rapamycin are suggestive a role of the
p70
pathway in mediating the phosphorylation of PHAS-I.
from cycloheximide-treated rat liver did
not phosphorylate recombinant PHAS-I in vitro(19) .
Moreover, no insulin-stimulated PHAS-I kinase activity was detected in
the elution position (see (41) ) of p70
when
extracts of 3T3-L1 adipocytes were fractionated by Mono Q (Fig. 12). Therefore, it is unlikely that direct phosphorylation
of PHAS-I by p70
explains PHAS-I phosphorylation in
cells. However, other elements in the p70
pathway are
candidates. Activation of p70
is associated with
phosphorylation of four clustered sites having a Ser/Thr-Pro
motif(52) . Interestingly, PHAS-I contains seven such motifs (Fig. 1), and one of these (Ser
) has been shown to
be phosphorylated in response to insulin in rat
adipocytes(19) . Thus, PHAS-I and p70
might be
phosphorylated by the same kinase.
as
rapamycin caused dephosphorylation of sites in p70
distinct from those phosphorylated in response to
mitogens(53) . A rapamycin-stimulated phosphatase would provide
a means for increasing phosphorylation independently of
p70
. It is important to keep this in mind when using
rapamycin sensitivity to implicate the p70
pathway in the
phosphorylation of proteins such as PHAS-I and ribosomal protein S6.
However, there is still no direct evidence that rapamycin increases
phosphatase activity.
]PHAS-I
eIF-4E complex was used as
substrate would be consistent with phosphatase regulation, although
there are other explanations for the inability to detect
insulin-stimulated PHAS-I kinase activity. For example, the stimulated
activities of kinases regulated by noncovalent mechanisms might not
persist after homogenization. Nevertheless, inhibition of a phosphatase
could allow PHAS-I kinases not activated by insulin to contribute to
the regulation of PHAS-I. Casein kinase II and protein kinase C are the
only kinases other than MAP kinase that have been shown to
phosphorylate recombinant PHAS-I(19) . Purified casein kinase
appears to phosphorylate PHAS-I in the
[H
]PHAS-I
eIF-4E complex at least as well as
it phosphorylates free [H
]PHAS-I.
Implications of the Dephosphorylation of PHAS-I in
Response to Rapamycin
In lymphoid cells, rapamycin selectively
inhibits translation of mRNAs encoding elongation factors and ribosomal
proteins(54) . Specificity for the inhibitory effect of
rapamycin on translation initiation appears to be a polypyrimidine
motif located just downstream of the 5` cap site(6) . The
correlation between this effect and the inhibition of ribosomal protein
S6 phosphorylation by rapamycin in Swiss mouse 3T3 cells led to the
suggestion that S6 phosphorylation was involved in the increased
translation of these messages(6) . Our findings indicate that
the effect of rapamycin on inhibiting translation initiation involves
dephosphorylation of PHAS-I and inhibition of eIF-4E. The initiation
factor promotes selective translation of certain mRNAs, particularly
those having highly structured 5`-nontranslated regions(55) .
While experiments are needed to determine whether eIF-4E increases
translation of mRNAs containing the polypyrimidine motif, it might be
noted that the motif is located near the cap site where eIF-4E binds.
= 12,000; MAP kinase,
mitogen-activated protein kinase; MEK, MAP kinase kinase; TOR, target
of rapamycin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.