From the School of Biochemistry and Genetics, Medical School, University of Newcastle, Newcastle Upon Tyne NE2 4HH, United Kingdom
Received for publication, June 5, 2000, and in revised form, September 6, 2000
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ABSTRACT |
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Insulin and a number of metabolic factors
stimulate glycogen synthesis and the enzyme glycogen synthase. Using
human muscle cells we find that glycogen synthesis is stimulated by
treatment of the cells with lithium ions, which inhibit glycogen
synthase kinase 3. Insulin further stimulates glycogen synthesis in the presence of lithium ions, an effect abolished by wortmannin and rapamycin. We report also that amino acids stimulate glycogen synthesis
and glycogen synthase, these effects also being blocked by rapamycin
and wortmannin. Amino acids stimulate p70s6k
and transiently inhibit glycogen synthase kinase 3 without
effects on the activity of protein kinase B or the mitogen-activated
protein kinase pathway. Thus, the work reported here
demonstrates that amino acid availability can regulate glycogen
synthesis. Furthermore, it demonstrates that glycogen synthase kinase 3 can be inactivated within cells independent of activation of protein
kinase B and p90rsk.
One of the key actions of insulin in the lowering of blood glucose
is the stimulation of glycogen synthesis in skeletal muscle, involving
activation of the enzyme glycogen synthase
(GS)1 (1). Phosphorylation
decreases GS activity, and the extent of this inactivation depends on
the sites phosphorylated and on the concentrations of allosteric
effector molecules such as glucose-6-phosphate. Activation of GS by
insulin results from dephosphorylation principally at a cluster of 3 serine residues collectively termed "site 3" located at the
carboxyl terminus of GS (2). Further evidence indicates that the
phosphorylation state of site 2 (or 2a or both) is also decreased in
response to insulin (3). Phosphorylation at site 3 is catalyzed
principally by glycogen synthase kinase 3 (GSK-3), and
dephosphorylation is catalyzed by the glycogen-bound form of protein
phosphatase 1 (PP1; Refs. 1, 4). Both PP1 and PP2A can dephosphorylate
sites 2 and 3 in vitro (2), and there is evidence for a
mechanism of the glycogen-bound form of PP1 activation by insulin that
is mediated by p90rsk (5, 6). This provides an attractive
theory for insulin exerting this stimulatory effect on GS by activating
the glycogen-bound form of PP1. Recent evidence, however, has
questioned this theory. Of particular significance is the finding that
PD98059, which blocks activation of p90rsk (7), has no
effect on insulin stimulation of glycogen synthesis and GS in various
cell types (8-10).
Attention has since focused on the effects of insulin on GSK-3. GSK-3
is inactivated in response to insulin or growth factors by
phosphorylation at a single serine residue close to its amino terminus
(11, 12). Three insulin-stimulated kinases, namely p70s6k,
p90rsk, and protein kinase B (PKB), have been shown to
phosphorylate and inactivate GSK-3 in vitro (12, 13).
However, agents that prevent the activation of both p70s6k and
p90rsk by insulin do not block the inactivation of GSK-3,
strongly suggesting that the kinase acting under these conditions is
PKB (14).
Despite accumulating evidence for a key role of PKB and GSK-3 in
controlling GS, there is evidence for one or more alternative pathways
contributing to the effect of insulin. First, GSK-3 does not
phosphorylate GS at site 2, which is also dephosphorylated in response
to insulin (3). Second, use of the immunosuppressive drug rapamycin,
which prevents the activation of p70s6k (15, 16), has provided
evidence for contribution of a rapamycin-sensitive pathway to the
activation of glycogen synthesis and GS by insulin in skeletal muscle,
adipocytes, and hepatoma cells (10, 17-19). In human muscle cells in
culture, the activation of glycogen synthesis and GS by both insulin
and epidermal growth factor is partly blocked by preincubation
of the cells with rapamycin, which, however, is without effect on the
inactivation of GSK-3 by these hormones (10, 20). This suggests that
two pathways are contributing to the activation of glycogen synthesis
in this system, the phosphoinositide-dependent kinase 1-PKB-GSK-3 pathway and a rapamycin-sensitive pathway, which can act downstream of, or independent of, GSK-3.
To investigate this further, one approach is to activate the two
pathways independently. Recent work has established that activation of
p70s6k can be regulated by the availability of amino acids.
Amino acid starvation causes inactivation of p70s6k, which can
be reversed by the readdition of amino acids, a process that is
rapamycin sensitive and that does not involve activation of PKB or
p90rsk (21-23). Using this approach, we report here that the
rate of glycogen synthesis and the activity of GS are increased in
response to amino acids, indicating that the p70s6k pathway can
control these processes independent of other signaling pathways.
Materials--
All tissue culture trays were from Costar
(Cambridge, MA). Culture media, penicillin-streptomycin, and
trypsin-EDTA were from Life Technologies (Paisley, UK). Chick embryo
extract was obtained from ICN (Costa Mesa, CA).
D-[U-14C]Glucose (300 mCi/mmol) and uridine
diphospho-D-[6-3H]glucose (814 GBq/mmol) were
from Amersham Pharmacia Biotech (Buckinghamshire, UK), and
[
Anti-p70s6k, anti-phospho (Thr389) p70s6k,
and anti-phospho (Ser21 and Ser9) GSK-3 Cell Culture--
Human myoblasts were grown from needle biopsy
samples taken from the gastrocnemius muscle of healthy subjects with no
family history of type 2 diabetes and with normal glucose tolerance and normal insulin sensitivity as assessed using the short insulin tolerance test. Myoblasts were maintained in growth medium consisting of Ham's F-10 nutrient mixture containing 20% fetal calf serum, 1%
chick embryo extract, 100 units/ml penicillin, and 100 µg/ml streptomycin. All experiments were performed using cells between the
5th and 15th passage at >90% confluence. Before hormone treatment, cells were incubated for at least 2 h in serum-free Ham's F-10 medium or serum-free minimum Eagle's medium (MEM) salt solution containing 5.5 mM glucose (MEMS). For amino
acid readdition experiments MEMS was replaced with
MEMS plus amino acid (AA) medium containing 1× or
2× concentrations of amino acids. The concentrations of amino acids
designated 1× are as follows (in mg/liter): L-Arg, 126.4;
L-Cys, 24.02; L-Glu, 292; L-Ile,
52.46; L-Leu, 52.46; L-Lys, 73.06;
L-Met, 14.92; L-Phe, 33.02; L-Thr,
47.64; L-Trp, 10.2; L-Tyr, 36.22;
L-Val, 46.86; L-Ala, 8.9; L-Asn,
13.2; L-Asp, 13.3; L-Gln, 14.7;
L-Gly, 7.5; L-Pro, 11.5; and
L-Ser, 10.5.
Estimation of Glycogen Synthesis--
Glycogen synthesis was
determined as [14C]glucose incorporation into glycogen
over 1 h, as described previously (20). Results are expressed as
picomoles of glucose incorporated into glycogen per minute per
milligram of protein.
Assay of Glycogen Synthase--
After the indicated treatments,
cells were rapidly washed three times with ice-cold phosphate-buffered
saline and collected, by scraping, into GS extraction buffer (10 mM Tris-HCl, pH 7.8, 150 mM KF, 15 mM EDTA, 60 mM sucrose, 1 mM
2-mercaptoethanol, 10 µg/ml leupeptin, 1 mM benzamidine,
1 mM phenylmethylsulfonyl fluoride). Cells were then
disrupted by briefly sonicating using a Soniprep 150. Glycogen synthase
activity was determined in whole lysates as the incorporation of
[3H]glucose from uridine-5'-diphosphate
[U-3H]glucose into glycogen, as described previously
(25). Samples were incubated with reaction mixture (50 mM
Tris-HCl, pH 7.8, 20 mM EDTA, 25 mM KF, 1%
glycogen, 0.4 mM UDP-[3H]glucose (specific
activity, 3000 dpm/nmol), containing either 0.1 mM (active)
or 10 mM (total) glucose-6-phosphate) for 30 min at
30 °C. Results are expressed as fractional activities
(active/total).
Preparation of Myoblast Extracts--
After incubation with
appropriate hormones, growth factors, or other agents, cells were
washed three times with ice-cold phosphate-buffered saline, and excess
liquid was removed. Cells were then scraped into extraction buffer (100 mM Tris-HCl, pH 7.4, 100 mM KCl, 2 mM EDTA, 25 mM KF, 0.1% (v/v) Triton X-100, 1 mM benzamidine, 0.1 mM
Na3VO4, 1 µg/ml pepstatin, 1 µg/ml
antipain, and 1 µg/ml leupeptin), transferred to 1.5-ml Eppendorf
tubes, and immediately frozen in liquid nitrogen. Before analysis,
samples were thawed and dispersed by sonication for 1 min (Sonibath;
Dawe). The protein concentration was determined by a dye binding method
(26).
Western Blot Analysis--
Cell extracts were resolved by 10%
SDS-polyacrylamide gel electrophoresis and transferred to
polyvinylidene difluoride membranes for 2.5 h at 250 mA in 10 mM 3-[cyclohexlamino]-1-propanesulphonic acid (pH 11.0)
containing 10% methanol using a Hoefer minigel transfer unit.
Membranes were probed with anti-GSK-3 Determination of GSK-3 and PKB Activities--
GSK-3 activity
was determined as described previously (27) with modifications. Samples
were assayed in a final volume of 50 µl containing 10 µl of total
cell extract (~10 µg of protein) and 40 µl of assay mixture (5 µM protein kinase A inhibitor, 0.1 mM EGTA,
0.1 mM [ In initial experiments, the effect of the inhibitors wortmannin,
rapamycin, and lithium on insulin-stimulated glycogen synthesis was
investigated in cultured human myoblasts (Fig.
1). Insulin stimulated glycogen synthesis
from extracellular glucose by ~3-fold. This activation was completely
blocked by wortmannin, a selective inhibitor of phosphatidylinositol
3-kinase, whereas only 40% inhibition was observed with rapamycin,
which prevents the activation of p70s6k. Rapamycin alone had
little effect on basal glycogen synthesis, decreasing it by ~10%
(data not shown). Wortmannin alone, however, induced a decrease of
~70%, implying that a wortmannin-sensitive mechanism is involved in
basal glycogen synthesis. This is consistent with previous work from
this laboratory (10, 20).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (148 TBq/mmol) was obtained from ICN. The
GSK-3 substrate phospho-eukaryotic initiation factor 2B peptide
(RRAAEELDSRAGS(p)PQL; Ref. 24) was a kind gift from Prof. C. G. Proud (University of Dundee, Dundee, UK). The PKB peptide substrate
Crosstide (GRPRTSSFAEG) and protein kinase A inhibitor
(TTYADFIASGRTGRRNAIHD) were synthesized in the University of Newcastle
Upon Tyne Facility for Molecular Biology. Wortmannin and rapamycin were
from Sigma (Poole, UK). Actrapid insulin was from Novo Nordisc
(Copenhagen, Denmark).
and
-
antibodies were obtained from New England Biolabs (Beverly, MA).
Anti-GSK-3
and anti-PKB
-(PH domain) antibodies were obtained from
Upstate Biotechnology (Lake Placid, NY).
and -
or anti-p70s6k phospho-specific antibodies (1:1000) or with
anti-GSK
(0.5 µg/ml) antibodies. Membranes were then incubated
with anti-rabbit or anti-sheep peroxidase-labeled secondary antibodies
(1:2000) and visualized using enhanced chemiluminescence.
-32 P]ATP (~500 cpm/pmol), 10 mM MgAc, 50 mM Tris-HCl, pH 7.5, 0.1% 2-mercaptoethanol or 0.2 mg/ml GSK phospho-eIF2B peptide, 50 mM
-glycerophosphate, pH 7.5, in the presence or absence
of 50 mM LiCl). After a 15-min incubation at 30 °C, 40 µl were spotted onto Whatman P81 phosphocellulose paper squares.
After washing in 175 mM phosphoric acid with four changes,
the papers were dried, and phosphate incorporation was determined by
liquid scintillation counting. Enzyme activity was defined as that
which catalyzes the incorporation of 1 nmol of phosphate into peptide
substrate in 1 min. GSK-3 activity is that in the absence of
Li+ minus that in its presence. PKB
was assayed after
immunoprecipitation as described previously (28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of inhibitors on the regulation of
glycogen synthesis by insulin and lithium. Myoblasts were
incubated in serum-free Ham's F-10 medium for 2 h before
incubation with radioactive [U-14C]glucose for 1 h
in the absence (B) or presence of 100 nM insulin
(I) or 50 mM lithium (Li) with no
inhibitors present or including 100 nM wortmannin
(W) or 100 nM rapamycin (R). Extracts
were prepared, and the quantity of radioactive glucose incorporated
into glycogen was determined. Results are mean ± S.E., at least
n = 4, from at least three subjects. Values are
expressed as picomoles per minute per milligram of protein. Statistical
significance compared with the value in the presence of insulin is
indicated by one star (p < 0.05) or
two stars (p < 0.01).
Work from several laboratories has indicated that the inactivation of GSK-3 is primarily responsible for dephosphorylation and activation of GS in response to insulin (10-12, 29, 30). Lithium is an allosteric inhibitor of GSK-3, mimicking the effect of insulin without affecting the activity of the mitogen-activated protein (MAP) kinase pathway (31). Treatment of cells with lithium chloride for 1 h caused a 3-fold increase in basal glycogen synthesis (Fig. 1), whereas no significant difference was observed in cells treated with an equal concentration of sodium chloride to correct for osmolarity changes (data not shown). This increase was not reduced significantly by wortmannin or by rapamycin (data not shown). In the presence of lithium, insulin stimulated glycogen synthesis a further 1.5-fold, an effect completely blocked by rapamycin (Fig. 1) or wortmannin (data not shown). Taken together, these data provide further support for a key role of GSK-3 and the presence of a rapamycin-sensitive, insulin-stimulated pathway in the control of glycogen synthesis.
To investigate the role of this rapamycin-sensitive pathway further,
amino acids were used to activate p70s6k selectively (21-23),
and the activity of GS and glycogen synthesis was measured (Fig.
2). Myoblasts starved of amino acids for
5 h were then incubated in MEMS containing 2×
concentrations of amino acids for various times. GS activity was
measured, and the values are shown as a ratio of the GS fractional
activity in the presence over the absence of amino acids in the culture
medium. GS activity increased 2.5- and 2.0-fold in response to 2×
concentrations of amino acids for 1 and 2 h, respectively.
Glycogen synthesis increased ~1.5-fold in response to 2×
concentrations of amino acids for 2 h. Both of these responses
were completely inhibited by wortmannin and rapamycin. PD98059 had no
significant effect on the amino acid stimulation, inhibiting basal and
stimulated values to similar extents (data not shown).
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To evaluate a possible role for p70s6k in the stimulation of
glycogen synthesis, the phosphorylation of this protein was measured under identical conditions. Fig.
3A shows that after 5 h
of amino acid deprivation, readdition of 1× or 2× concentrations of
amino acids resulted in the phosphorylation of p70s6k at
Thr389, the phosphorylation of which is most closely
related to the activation of p70s6k in vivo (32).
This phosphorylation was further increased in response to insulin. This
phosphorylation in response to amino acids was completely blocked by
rapamycin, indicating that the amino acid activation of p70s6k
involves the mammalian target of rapamycin (mTOR). A threshold level of
amino acids is also required for the insulin-stimulated phosphorylation
of p70s6k, because the increase in phosphorylation in response
to insulin was reduced by ~72% in cells incubated in the absence of
amino acids compared with cells in the presence of amino acids (Fig. 3A), consistent with earlier findings (23, 33).
Phosphorylation of p70s6k in response to amino acids is time
dependent, with a significant increase in phosphorylation appearing
after 10 min and persisting for at least 2 h (Fig. 3B).
In all experiments endogenous levels of p70s6k protein did not
change, as determined by immunoblotting (data not shown).
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To investigate whether amino acid deprivation and readdition were
affecting GSK-3, its activity and phosphorylation status were
established under identical conditions. Fig.
4 shows that GSK-3 was inactivated by
~30% in response to amino acids, an effect seen within 10 min of
amino acid addition and which was reversed after 30 min. Following a
recent publication showing the inactivation of GSK3 in response to
exercise without corresponding phosphorylation (34), the
phosphorylation status of GSK-3 was also measured. Corresponding
Western blots show a direct correlation of inactivation with
phosphorylation (Fig. 4). During this experiment there was no
significant change in GSK-3 protein expression. Although these effects
are consistent with p70s6k causing inactivation of GSK-3 and
activation of GS, it is important to exclude the involvement of PKB or
the MAP kinase pathway. Importantly, there was no significant change in
PKB activity under matching conditions (Fig.
5), and the phosphorylation status of MAP
kinase did not alter after amino acid withdrawal and readdition (data not shown). Wortmannin completely blocked the amino acid-induced inactivation and phosphorylation (Fig. 6)
of GSK-3, whereas PD98059 was without effect (data not shown).
Rapamycin essentially blocks the amino acid-induced phosphorylation and
inactivation of GSK-3 (Fig. 6), indicating that the mTOR-p70s6k
pathway is principally responsible for the effects on GSK-3.
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DISCUSSION |
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Insulin stimulates glycogen synthesis by both increasing glucose uptake and activating GS. Stimulation of GS by insulin involves the net dephosphorylation of specific serine residues, termed sites 2 and 3 (35). Inactivation of GSK-3 has been shown to be principally responsible for dephosphorylation of site 3, but the factor regulating site 2 dephosphorylation remains unidentified. Glycogen synthesis can also be stimulated in an insulin-independent manner, for example, after exercise, a process associated with glycogen depletion (36). After glycogen depletion in cultured human muscle cells, readdition of glucose leads to the stimulation of GS by an as yet unknown mechanism but one that apparently does not involve phosphorylation and inactivation of GSK-3.2 Therefore, the role of GSK-3 in controlling glycogen synthesis was further investigated using its allosteric inhibitor lithium ions. Incubation of human myoblasts with lithium resulted in a significant increase in glycogen synthesis in these cells, which is consistent with a major role for GSK-3 in controlling glycogen synthesis. However, the addition of insulin resulted in a further, rapamycin-sensitive increase in glycogen synthesis, indicating a contribution by an alternative insulin- and rapamycin-sensitive pathway. The obvious candidate is the p70s6k signaling pathway, which fulfills both these criteria and for which we and others have obtained evidence previously (10, 17-19). To investigate this further we have used the findings that it is possible to activate p70s6k in a rapamycin-sensitive manner using amino acids, without the parallel activation of PKB or MAP kinase that occurs with insulin stimulation (21-23). We show here that amino acid depletion and subsequent readdition also lead to the stimulation of GS and glycogen synthesis (Fig. 2, A and B), effects that are completely blocked by both wortmannin and rapamycin. A key question is whether this activation involves inhibition of GSK-3. As shown in Fig. 4, GSK-3 is phosphorylated and inactivated in response to amino acids.
Three kinases, namely p70s6k, p90rsk, and PKB, have been shown to phosphorylate and inactivate GSK-3 in vitro (12, 13). Importantly, the present work and previous data (21-23) show that amino acids failed to activate PKB or the MAP kinase pathway. However, p70s6k is phosphorylated and presumably activated by amino acids (Fig. 3). Furthermore, GSK-3 inhibition is completely blocked by both wortmannin and rapamycin, implying that the p70s6k pathway is primarily responsible for the inactivation of GSK-3 in response to amino acids. Very recent work on amino acid transport in rat L6 myotubes has shown that leucine inactivates GSK-3 in a rapamycin-sensitive manner without activation of either PKB or the MAP kinase pathway (37).
The transient nature of the inhibition of GSK-3 (maximal after 15 min) does not coincide with the prolonged activation of GS and p70s6k, which persists up to 2 h (Fig. 2A). It is possible that transient inactivation of GSK-3 triggers the activation of GS, which could then be maintained by a further rapamycin-sensitive pathway, perhaps acting on GS without involvement of GSK-3. This is supported by the data in Fig. 1, which indicate that when GSK-3 is inhibited by lithium ions, insulin can further stimulate glycogen synthesis in a rapamycin-sensitive manner. This would be consistent also with earlier work on insulin effects in human muscle cells (10, 20) and with work (38) that showed that rapamycin has no effect on the activation of GS in skeletal muscle up to 5 min after stimulation by insulin but has a significant inhibitory effect at longer time points.
The amino acid-stimulated activation of p70s6k observed in the human muscle cell lines is wortmannin and rapamycin sensitive. Rapamycin acts by associating with a cellular protein, FKBP12, and this complex then binds to mTOR, thus inhibiting its kinase activity. This would therefore indicate that mTOR is required for the response to amino acids, either directly or through the regulation of a downstream element, such as p70s6k or a protein phosphatase. Although mTOR has been shown to phosphorylate p70s6k directly in vitro (39), recent evidence points to the existence of a phosphatase, which is activated by rapamycin, via the inhibition of mTOR (23, 40). Phosphatidylinositol 3-kinase is also implicated because of its wortmannin sensitivity, but it has previously been reported that wortmannin can selectively inhibit mTOR directly by binding to the catalytic domain at concentrations of 0.1-1 µM (41, 42). Therefore, it is possible that phosphatidylinositol 3-kinase does not play a role in this mechanism despite the wortmannin-sensitive nature of the process. It has also recently been reported that phosphoinositide-dependent kinase 1 can phosphorylate p70s6k in vivo (43), but there is no evidence to date to suggest that phosphinositide-dependent kinase 1 kinase activity is increased in response to amino acids, especially because PKB activity does not increase (Fig. 5).
Because of the sustained amino acid-stimulated activation of p70s6k and the sensitivity to both wortmannin and rapamycin, this leads to the reasoning that this enzyme is part of the signal mechanism involved in the amino acid-stimulated increase in GS and glycogen synthesis activities. It is important to point out that p70s6k itself may not play a role, and events may stem directly from mTOR. It has recently been discovered that mTOR is involved in the regulation of PP2A (40), a phosphatase known to act on GSK-3 in vitro. This could provide evidence to support a theory whereby GSK-3 is regulated by this phosphatase in response to amino acid availability. PP2A also has the ability to dephosphorylate GS at sites 2 and 3, leading to its activation; therefore, this phosphatase may play a coordinating role in the overall process.
In summary, the work reported here demonstrates that amino acid
availability can regulate glycogen synthesis. Furthermore, it
demonstrates that GSK-3 can be inactivated within cells independent of
activation of PKB and p90rsk.
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ACKNOWLEDGEMENTS |
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We thank Dorothy Fittes for excellent technical assistance and Dr. Mark Walker and Prof. Doug Turnbull for performing the muscle biopsies.
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FOOTNOTES |
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* This work was supported by the British Diabetic Association.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.
Holds a studentship from the Biotechnology and Biological Sciences
Research Council, United Kingdom.
§ To whom correspondence should be addressed: School of Biochemistry and Genetics, Medical School, University of Newcastle, Framlington Place, Newcastle Upon Tyne NE2 4HH, UK. Tel: 44-191-222-7433; Fax: 44-191-222-7424; E-mail: s.j.yeaman@ncl.ac.uk.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M004812200
2 J. L. Armstrong, S. M. Bonavaud, R. Halse, and S. J. Yeaman, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: GS, glycogen synthase; GSK-3, glycogen synthase kinase 3; PP, protein phosphatase; PKB, protein kinase B; MAP, mitogen-activated protein; mTOR, mammalian target of rapamycin; MEMS, serum-free minimum Eagle's medium salt solution containing 5.5 mM glucose; AA, amino acid.
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