Diabetes Research Laboratory (L.R., N.D.) Winthrop University
Hospital Mineola, New York 11501
School of Medicine
(N.B.) State University of New York Stony Brook, New York
11794
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ABSTRACT |
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INTRODUCTION |
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It is well known that the substrate specificity and subcellular localization of PP-1 is dictated by its targeting subunits. In mammals three tissue-specific proteins have been identified that target PP-1 to glycogen (5). PP-1G (also known as RG1) encodes a 124-kDa protein product that is expressed in skeletal muscle and heart (5, 6). GL encodes a 33-kDa protein that is expressed exclusively in liver (7). PTG encodes a protein that is expressed in all tissues except the testis, being most abundant in skeletal muscle, liver, adipose tissue, and heart (8).
The glycogen-associated form of PP-1 derived from skeletal muscle is the best characterized phosphatase to date. The PP-1 holoenzyme consists of a highly conserved 37-kDa catalytic subunit (PP-1C) and a 124-kDa regulatory subunit (PP-1G, migrating as a 160-kDa subunit on SDS-PAGE) (5, 9). The PP-1G subunit binds to glycogen with high affinity and directs PP-1C to glycogen protein particles, thereby enhancing dephosphorylation of glycogen-bound PP-1 substrates such as glycogen synthase (GS), glycogen phosphorylase kinase, and glycogen phosphorylase a (5, 9, 10). Phosphorylation at site 1 (serine46) of the PP-1G subunit in response to insulin enhances the activity of the holoenzyme toward glycogen-bound substrates, while phosphorylation of PP-1G at site-2 (serine65) in response to adrenalin causes inhibition of glycogen synthesis and stimulation of glycogenolysis (5, 9, 10, 11). Thus, the PP-1G subunit plays a key role in the control of glycogen synthesis by insulin and counterregulatory hormones, thereby participating in nonoxidative glucose disposal (5, 9, 11, 12).
We have recently demonstrated that insulin rapidly activates the glycogen-associated form of PP-1 in cultured L6 rat skeletal muscle cells (13). PP-1 activation is accompanied by an increased phosphorylation of PP-1G (13). Modulation of the levels of this subunit either by overexpression of recombinant PP-1G or by depletion of endogenous PP-1G using an antisense RNA strategy results in increased activation or inhibition of insulin-stimulated glucose uptake and glycogen synthesis (14). These alterations in insulin responsiveness are due to activation or inhibition of the PP-1 catalytic subunit that is bound to PP-1G (13, 14). Also, studies on Pima Indians indicate that insulin resistance in these subjects is accompanied by marked reductions in basal and insulin-stimulated skeletal muscle PP-1 catalytic activities and impaired GS activation despite elevations in the contents of the PP-1C subunit and GS (15, 16).
Functional alterations of PP-1 may be responsible for impaired insulin-stimulated glycogen synthesis in skeletal muscle, which is characteristic of insulin-resistant individuals. Due to conflicting reports between Caucasian subjects with the Asp905Tyr polymorphism exhibiting insulin resistance and hypersecretion of insulin (17) and recent studies in a Japanese population indicating that this mutation is found in 70% of healthy individuals and, therefore, is not associated with insulin resistance (18), we attempted to evalute the impact of the PP-1G Asp905Tyr mutation on cellular responsiveness to insulin.
The results of the functional studies with L6 rat skeletal cells stably expressing Asp905Tyr mutation of PP-1G indicate that an Asp905Tyr mutation of PP-1G is indeed accompanied by increased sensitivity to cAMP agonists in terms of GS activation and glycogen synthesis.
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RESULTS |
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To test whether the Flag tag itself alters the conformation and
function of the fused protein, equal amount of cell lysates from
Flag-tagged wild-type PP-1G and mutant PP-1G
were immunoprecipitated with a monoclonal Flag antibody followed by
Western blot analyses of the immunoprecipitates with PP-1G
antibody. As shown in Fig. 1D, the Flag antibody specifically
immunoprecipitated a 160-kDa protein that reacted with
PP-1G subunit antibody (Fig. 1D
, lanes 3 and 4). This band
was absent in mock immunoprecipitates in which Flag antibody was
substituted by mouse IgG (Fig. 1D
, lane 2) as well as in parent L6 cell
extracts immunoprecipitated with a Flag antibody (Fig. 1D
, lane 1). The
results indicated that the Flag tag itself did not alter the
conformation of the recombinant PP-1G. Mutant
PP-1G clone 3 expressing the highest levels of Flag-tagged
PP-1G subunit was used as a representative cell line for
all of the insulin and cAMP dose-response studies on PP-1, GS
activation, and glycogen synthesis, and results were compared with
clone 211 overexpressing comparable levels of the recombinant
Flag-tagged wild-type PP-1G and control L6 cells.
Effect of Asp905Tyr Mutant PP-1G
Expression on Basal and Insulin-Stimulated PP-1 Catalytic Activities in
the Extracts and PP-1G
Immunoprecipitates
To determine whether expression of the mutant PP-1G
results in alterations in PP-1 activity in the basal and
insulin-stimulated state, we first measured the activity of bound PP-1C
in PP-1G immunoprecipitates. In L6 neo controls, insulin
treatment caused a 70% increase in immunoprecipitated PP-1 catalytic
activity over basal values, whereas overexpression of the wild-type
PP-1G resulted in a 150% increase in insulin-stimulated
PP-1 enzymatic activity along with a small increase in basal PP-1
activity (Table 1). In contrast, despite
a 3-fold increase in PP-1G subunit content, cells
expressing mutant PP-1G exhibited a 2040% decrease in
basal PP-1 activity when compared with L6 neo controls and
PP-1Gwild, respectively (Table 1
). Treatment with 10
nM insulin for 5 min caused a 66% increase in PP-1
catalytic activity in the immunoprecipitates of mutant cells (Table 1
),
which is 2-fold less than the stimulation observed in cells with
wild-type PP-1G. The observed decrease in bound PP-1
catalytic activity in mutant cells was not due to any difference in the
efficiency of PP-1G immunoprecipitation as Western
blot analysis of the immunoprecipitates with PP-1G subunit
antibody detected comparable amounts of this subunit in cells
overexpressing wild-type PP-1G and mutant PP-1G
but rather due to reductions in the amount of PP-1C that is bound to
PP-1G in mutant cells vs. wild-type
PP-1G overexpressors (see also Fig. 2C
).
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Given the knowledge that the PP-1G subunit plays a major
role in the regulation of PP-1 catalytic activity in response to
insulin as well as counterregulatory hormones, we next examined whether
the Asp905Tyr mutation of PP-1G affects cellular
responsiveness of PP-1 to cAMP agonists. Serum-starved cells were
pretreated with 1 mM (Bu)2cAMP (a
cell-permeable analog of cAMP) for 30 min followed by insulin treatment
(10 nM) for 5 min and assayed for PP-1 enzymatic activity.
In cells expressing recombinant Flag-tagged wild-type PP-1G
as well as L6 controls, cAMP agonist had little effect on the basal
PP-1 activity but decreased insulin-mediated PP-1 activation by
4050% (Fig. 2B). In contrast, the Asp905Tyr mutation of
PP-1G caused a 60% decrease in the basal PP-1 activity
upon exposure to (Bu)2cAMP. Furthermore, cAMP agonist
completely blocked insulins effect on PP-1 activation and decreased
insulin-stimulated PP-1 activity below the basal values in these mutant
cells (Fig. 2B
). Western blot analysis of glycogen pellets isolated
from control and mutant cells indicated that despite a 3-fold increase
in PP-1G content, the amount of PP-1C that was bound to
PP-1G in mutant cell lines was markedly less when compared
with cells expressing Flag-tagged wild-type PP-1G (Fig. 2C
). Insulin and cAMP did not significantly alter the amount of PP-1C
that was associated with PP-1G in all the three cell types
studied (Fig. 2C
). This is in contrast to earlier reports from Cohens
laboratory documenting that cAMP treatment results in dissociation of
catalytic subunit from the regulatory subunit (10). The reason for the
discrepancy between our in vivo studies and those reported
earlier is not clear at present.
Kinetic studies on cAMP-mediated inhibition of insulins effect on
PP-1 activity is shown in Fig. 3. In
mutant cells, half-maximal inhibition in insulins effect on PP-1
activity was observed at a concentration of 0.01 mM
(Bu)2cAMP. Complete inhibition of insulins effect on PP-1
occurred at a cAMP concentration of 0.1 mM. In contrast,
cells expressing wild-type PP-1G as well as control L6
cells exhibited a 20% inhibition at a concentration of
0.1
mM. A 50% decrease in insulin-stimulated PP-1 activity was
observed with 1 mM (Bu)2cAMP in these control
cells.
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DISCUSSION |
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While the Asp905Tyr polymorphism of PP-1G was first linked to insulin resistance in a subset of Danish population with type 2 diabetes (17), recent studies in a Japanese population found that 70% of normal healthy individuals carried this variant of PP-1G (18). These studies suggest that racial differences and genetic background of an individual may play a major role in the pathogenesis of insulin resistance in addition to this mutation. In addition, recent studies on Pima Indians have shown that this Asp905Tyr mutation of PP-1G is prevalent in this population and does not correlate with diabetes (20). These authors suggest that a polymorphism in an mRNA-stabilizing element in the 3'-noncoding region of the PP-1G subunit best correlates with insulin resistance (20). The exact reason for these seemingly discordant results is unclear at present.
Nonetheless, several lines of evidence presented in this study suggest that an Asp905Tyr mutation of the PP-1G subunit is accompanied by altered sensitivity of PP-1 and other key regulatory enzymes of glycogen metabolism to cAMP agonists. First, treatment with (Bu)2cAMP was accompanied by marked reductions in basal PP-1 activity and a complete inhibition of insulin-mediated PP-1 activation. Second, cAMP agonist not only prevented insulins effects on PP-1 activation, GS, and glycogen synthesis but also reversed insulins stimulatory effects on these processes when added after stimulation of cells with insulin. Third, dose-response studies with cAMP agonist on GS inactivation, phosphorylase a activation, and glycogen breakdown indicate that L6 cells harboring mutant PP-1G exhibit a 2- to 5-fold increase in sensitivity toward cAMP agonists when compared with neo controls and cells expressing wild-type PP-1G. Finally, increased breakdown of glycogen in response to low concentrations of cAMP agonist in cells expressing mutant PP-1G was due to the failure of insulin to effectively block phosphorylase a activation and GS inactivation, and this may be due to cAMP-induced reductions in PP-1 activity. In the absence of cAMP, cellular responsiveness of GS and glycogen synthesis to insulin was comparable between control L6 cells and mutant cells, but markedly diminished when compared with cells overexpressing wild-type PP-1G even though the expression of the mutant PP-1G subunit resulted in a 3-fold increase in PP-1G subunit content.
Earlier studies from this laboratory have shown that insulin rapidly activates PP-1 in L6 cells, which is accompanied by increased phosphorylation of the PP-1G subunit (14). Treatment with cAMP agonist alone increased PP-1G phosphorylation but abolished insulin-mediated PP-1 activation as well as phosphorylation. Based on the in vitro studies on purified PP-1G, it was suggested that site-2 phosphorylation by PKA promotes dissociation of the PP-1C subunit and its translocation from glycogen-protein particles to the cytosol (5, 9, 10, 11). The released C subunit is 5- to 8-fold less effective than PP-1G holoenzyme in dephosphorylating GS and phosphorylase kinase. Thus, phosphorylation of the PP-1G subunit by cAMP-dependent kinase results in an immediate inhibition of glycogen synthesis and stimulation of glycogenolysis (5).
Insulin stimulates glycogen synthesis and inhibits glycogenolysis in skeletal muscle and this is mediated by the activation of PP-1G (14) as a result of the phosphorylation of site-1 on the G subunit (10, 13, 14) catalyzed by an insulin-stimulated protein kinase, Rsk2 (10, 21). However, recent studies using inhibitors of the mitogen-activated protein kinase-signaling pathway have demonstrated that this phosphorylation cascade is not involved in the regulation of glycogen synthesis (22, 23).
Asp905 of the PP-1G subunit is not located near any of the
known phosphorylation sites in the primary structure of the protein. In
addition, we did not observe any difference in the phosphorylation
status of the PP-1G subunit immunoprecipitated from cells
expressing mutant PP-1G and wild-type L6 cells, either in
the basal state or after treatment with insulin and or cAMP agonist
(data not shown). Thus, increased sensitivity to cAMP agonist is not
due to differences in the extent of PP-1G phosphorylation
in response to insulin and or cAMP agonist. Neither cAMP treatment
caused dissociation of the catalytic subunit from the regulatory
subunit (Fig. 2C) as suggested by the in vitro studies of
Dent et al (10). Given that PP-1 inactivation can be
mediated by inhibitors 1 and 2 and inhibitor 1 is activated by
cAMP-mediated phosphorylation, we cannot exclude the possibility of
increased phosphorylation and activation of inhibitor 1 in these mutant
cells vs. L6 control and Flag-tagged PP-1Gwild.
Inhibitor 1 activation might cause inactivation of PP-1 and activation
of phosphorylase a.
It should be noted that overexpression of the mutant PP-1G
subunit did not result in an increase in the amount of PP-1C that was
associated with the G-subunit (see Fig. 3C). The simplest
interpretation of these results is that this mutation of the
PP-1G subunit decreases its ability to associate with the C
subunit due to alterations in tertiary structure. Further
structure/function studies are warranted to understand the potential
binding defects.
The ability of insulin to effectively prevent cAMP-mediated phosphorylase a activation in control L6 cells and cells expressing wild-type PP-1G suggest that the process of glycogenolysis is more sensitive to insulin and the PP-1G subunit plays a dominant role in insulin-mediated inhibition of glycogenolysis. These results coincide with earlier observations in rat skeletal muscle demonstrating that glycogenolysis is more sensitive to insulin than glucose transport and glycogen synthesis (24).
In summary, the results of the present study suggest that the PP-1G subunit not only plays a dominant role in the control of PP-1 activation and glycogen synthesis but also in the control of glycogen breakdown. An Asp905Tyr mutation of the PP-1G subunit is accompanied by increased sensitivity to cAMP agonist because of PP-1 inhibition resulting in the failure of insulin to suppress glycogen breakdown in response to the cAMP agonist.
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MATERIALS AND METHODS |
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Site-Directed Mutagenesis of PP-1G
cDNA
The PP-1G cDNA was cloned from the rabbit skeletal
muscle cDNA library as described in our recent publication (14). As
detailed in this study (14), rabbit skeletal muscle PP-1G
cDNA was used for site-directed mutagenesis because of its close
similarity to human PP-1G. Substitution of an aspartate
with a tyrosine at codon 904 of rabbit PP-1G cDNA was
performed with the Chameleon site-specific mutagenesis kit, according
to the manufacturers instructions using the following primer.
5'-GCC TTT AAC TCA TAC ACG AAC AGA GC-3'
The mutation was confirmed by nucleotide sequence analyses.
Construction of Expression Vectors Carrying Flag Epitope-Tagged
Wild-Type and Mutant PP-1G
The mammalian expression vector pcDNA3 containing the
recombinant Flag epitope-tagged wild-type and mutant PP-1G
constructs were prepared by standard molecular biology techniques.
Briefly, the wild-type and the mutant form of PP-1G
constructs were cleaved from the Bluescript plasmid using
EcoRI, tagged at the 5'-end with a Flag epitope by
subcloning in-frame into a Flag expression vector (IBI).
Finally, the epitope-tagged PP-1G constructs were subcloned
into the mammalian expression vector, pcDNA3 using NdeI and
BamHI restriction sites.
Transfection and Selection of Stable Cell Lines Expressing
Recombinant Flag Epitope-Tagged Wild-Type and Mutant
PP-1G Subunits
The spontaneously fusing rat skeletal muscle cell line, L6, was
a kind gift from Dr. Amira Klip (The Hospital for Sick Children,
Toronto, Ontario, Canada). L6 myoblasts at second passage were grown
and maintained in -MEM containing 10% FBS. Cells were transfected
with the expression vectors carrying the epitope-tagged wild-type and
mutant PP-1G subunit cDNA as detailed in our recent
publication (14). Briefly, cells at approximately 30% confluence were
transfected with a mixture of 15 µg DNA + 50 µl Lipofectamine
reagent for 15 h. At approximately 72 h from the start of
transfection the cells were passaged 1:5 into medium containing 2 mg/ml
G418 for selection. Single stable clones were picked up after 56 days
and passaged into several 24-well plates for initial amplification.
After two further rounds of amplification, the clones were screened for
the expression of Flag epitope-tagged PP-1G. Initial
screening was performed by immunoblot analysis of cell extracts
prepared from myotubes (1214 days in culture) using anti-Flag
antibody described in Materials and Methods. Control cells
(referred to as L6 throughout the manuscript) were transfected with the
empty expression vector. Transfection per se did not affect
the extent of differentiation of L6 cells as monitored by analysis of
myogenin protein and cell morphology. Screening with the Flag antibody
led to the identification of one wild-type clone
(Flag-PP-1Gwild, clone 211) and two mutant clones (mutIV
nos. 3 and 11) out of the 200 G418 resistant clones that were screened
for Flag-tagged wild-type and mutant PP-1G, respectively. A
single mutant clone (mutIV no. 3) expressing the highest amounts of
Flag-PP-1G was amplified and used for all the dose-response
experiments described below along with a single wild-type
PP-1G clone (no. 211, referred to as
Fag-PP-1Gwild throughout the manuscript) overexpressing
comparable levels of recombinant wild-type PP-1G.
Cell Culture
Transfected cell lines overexpressing Flag-tagged wild-type
PP-1G (clone 211) and mutant PP-1G (mutIV)
subunits were grown and maintained in -MEM containing 2% FBS, 400
µg/ml G418, and 1% antibiotic/antimycotic mixture in an atmosphere
of 5% CO2 at 37 C as previously described (14). Unless
otherwise stated, myotubes were used for all experiments after a 15-h
starvation in serum free
-MEM containing 5 mM
glucose.
Extraction and Assay of PP-1
Serum-starved cells were fed with serum-free medium containing 5
mM glucose. Identical dishes in triplicate were incubated
in the absence and presence of insulin (0.1100 nM) for
120 min. In some experiments, cells were pretreated with various
concentrations of (Bu)2cAMP (0.015 mM) or SpcAMP
(10-4 M) for 30 min before insulin exposure.
At the end of the incubation period, the medium was removed and the
cells rinsed three times with ice-cold PBS followed by extraction with
PP-1 extraction buffer as detailed in our recent publications (13, 14).
Assay of PP-1 Activity
The assay was performed as previously described (13, 14) using
32P-labeled glycogen phosphorylase a as a substrate (13).
Okadaic acid (OA) at 1 nM concentration was included in the
assay to inhibit PP-2A. As detailed in our earlier studies (13, 14, 25), at this concentration, OA inhibits only PP-2A activity and the
remaining activity represents PP-1.
Immunoprecipitation and Assay of PP-1 Catalytic Activity
Control and insulin-treated cells were harvested in ice-cold
lysis buffer containing 50 mM Tris, pH 7.4, 1
mM EDTA, 0.5 mM EGTA, 0.1 mM
PhMeSO2F, 10 µg/ml each of leupeptin, aprotinin,
antipain, soybean trypsin inhibitor, and pepstatin A, 100
mM NaCl, and 1% Triton X-100. The cell lysates were
centrifuged at 14,000 x g for 10 min to remove cell
debris. Cell lysate protein (100 µg) was diluted to 1 ml with lysis
buffer and precleared by incubation with rat or mouse IgG (5 µg/ml,
coupled to protein-A Sepharose) at 4 C for 30 min. The supernatants
were immunoprecipitated with 10 µg/ml anti PP-1G antibody
for 1 h at 4 C, followed by treatment with 50 µl protein A/G
Agarose (50% vol/vol) for 1 h. This antibody reacts very well
with both phosphorylated and nonphosphorylated PP-1G as
evidenced by equal amounts of PP-1G protein in the
immunoprecipitates detected by Western blot analyses (13, 14). To
prevent dephosphorylation during immunoprecipitation, 100
nM OA was added to cell lysates. The pellets were washed
four times with 1 ml wash buffer and resuspended in the phosphatase
assay buffer to the original volume. PP-1G bound to the
antibody was released by incubation with excessive antigenic peptide
(15 µg/ml) at 4 C for 1 h. PP-1 activity was measured on 5 µl
of immunodepleted supernatants and the immunoprecipitates as detailed
in our recent publication (14). To relieve the inhibition of
phosphatases due to OA, the immunoprecipitates were diluted to
1:100 with phosphatase assay buffer, and 10 µl aliquots were
used for the assay of PP-1 as detailed earlier (13, 14). In a duplicate
experiment, the immunoprecipitates were separated by SDS/PAGE followed
by immunoblot analyses of PP-1G subunit. PP-1 catalytic
activity in the immunoprecipitates was normalized for variation in the
contents of PP-1G in the immunoprecipitates.
Subcellular Localization of PP-1G
and Its Association with the Catalytic Subunit
Control and insulin- and cAMP-treated cells were sonicated in
300 µl extraction buffer (13, 14, 25) and centrifuged at 10,000
x g for 5 min at 4 C to remove nuclei and cell debris. The
supernatants were removed to fresh tubes and assayed for proteins, and
equal amounts of proteins (200 µg) were centrifuged for 25 min at
100,000 x g in a mini ultracentrifuge to sediment the
glycogen pellet. The supernatant was called cytosol. The glycogen
pellet was resuspended to the original volume in PP-1 extraction
buffer. Equal amounts of proteins were separated by SDS-PAGE followed
by Western blotting with PP-1G subunit and
PP-1C subunit antibodies, respectively.
Assay of GS Activity
Cells were treated with and without insulin (10 nM)
for 10 min followed by (Bu)2cAMP (0.015 mM) for 30 min.
In some experiments, cells were pretreated with cAMP (1 mM)
for 30 min followed by insulin for 10 min. The cells were extracted
with GS extraction buffer (14) and assayed for GS activity in the
presence of low (0.1 mM) and high concentrations (50
mM) of glucose-6 phosphate (Glc6P), using 0.7
mM uridine
diphospho-D-[U-14C]glucose
([U-14C]UDP-glucose) as a substrate as detailed in our
recent publication (14). Insulin-stimulated GS activity (picomoles of
[U-14C]UDP-glucose incorporated into glycogen/min/mg
protein) was expressed as percent fractional activity measured in the
presence of low Glc6P divided by the activity measured in
the presence of high Glc6P.
Assay of Phosphorylase a Activity
Phosphorylase a was assayed by monitoring the conversion of
[14C]glucose-1 phosphate (Glc-1-P) into glycogen
according to previously published protocols (26). Briefly, cells were
treated with and without insulin and cAMP as detailed in GS assay and
extracted in a homogenization buffer containing 10 mM
Tris-HCl, pH 7.0, 150 mM NaF, 15 mM EDTA, 15
mM 2-mercaptoethanol, 10 µg/ml each of leupeptin,
antipain, aprotinin, pepstatin A, 1 mM benzamidine, and 1
mM phenymethylsulfonyl fluoride. The final assay mixture
contained 75 mM Glc-1-P, 125 mM NaF, 0.6%
glycogen, and labeled Glc-1-P at 0.08 µCi/assay. Glycogen
phosphorylase a activity was determined in the presence and absence of
5 mM AMP. Caffeine (1 mM) was added to the
assay mixture in tubes without any AMP to inhibit phosphorylase b
activity.
Glucose Incorporation into Glycogen
Glucose incorporation into glycogen was measured using
D-[U-14C]glucose as described previously (14, 27). To examine the effect of cAMP agonist on insulin-stimulated
glycogen synthesis, cells were treated with 10 nM insulin
for 10 min, and then (Bu)2cAMP (0.015 mM) was
added for 30 min followed by the addition of
[U-14C]glucose for 90 min.
Immunoblot Analysis of PP-1G and
PP-1 Catalytic Subunits
Cells were washed four times with ice-cold PBS followed by the
addition of 200 µl cell lysis buffer containing 50 mM
Tris-HCl, pH 7.6, 2.0 mM EDTA, 2.0 mM EGTA,
1.0% SDS, 1.0 mM benzamidine, 2.0 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml each of leupeptin,
aprotinin, antipain, soybean trypsin inhibitor, and pepstatin A. The
cells were scraped and the cell lysate was sonicated and centrifuged at
2000 x g for 5 min. Extraction resulted in complete
recovery of proteins in the supernatant. Typically, 20 µg of protein
were mixed with Laemmli sample buffer containing 0.1% bromophenol
blue, 1.0 M NaH2PO4, pH 7.0, 50%
glycerol, 10% SDS, boiled for 5 min, and loaded on a 7.5% SDS
polyacrylamide gel (28). The proteins were transferred to
polyvinylidene difluoride (PVDF) membrane, the membranes were probed
with 1) PP-1G subunit antibody, 2) Flag antibody, and 3)
PP-1Cß antibody. This was followed by incubation with
[125I]protein A (0.2 µCi/ml, for PP-1G) or
horseradish peroxidase-conjugated goat IgG followed by enhanced
chemiluminescence and autoradiography. The intensity of the
signal was quantitated by densitometric analysis of the
autoradiograms.
Protein Assay
The protein content of the cell extracts was determined with
either bicinchoninic acid (29) or Bradford reagent (30).
Statistics
The Students t test or ANOVA was used to evaluate
the significance of the effect of insulin and (Bu)2cAMP on
PP-1 activity, GS activity, and glycogen synthesis. Results are
expressed as mean ± SEM of three to four different
experiments each performed in triplicate.
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FOOTNOTES |
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This work was supported in part by a research grant from the American Diabetes Association and medical education funds from Winthrop University Hospital.
Received for publication September 15, 1998. Revision received June 9, 1999. Accepted for publication June 29, 1999.
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REFERENCES |
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