From the
Glycogen synthase kinase-3 inactivates rabbit muscle glycogen
synthase by sequential phosphorylation of four COOH-terminal residues
Ser
The activity of rabbit muscle glycogen synthase (EC 2.4.1.11) is
under hormonal control mediated through multisite phosphorylation of
the enzyme by multiple protein
kinases
(1, 2, 3, 4) . Phosphorylation
tends to inactivate glycogen synthase although full activity can be
restored in the presence of glucose-6-P, leading to the use of the
-/+ glucose-6-P activity ratio as an index of the activation
state of the enzyme
(5) . The phosphorylation sites have been
localized (see Refs. 1-4), and the complete amino acid sequence
of the muscle isoforms of both human
(6) and rabbit
(7) enzyme is known. Two phosphorylation sites are near the
NH
To make a truncated protein, a 42-base primer was designed
to be complementary to the glycogen synthase cDNA from bases
2146-2172; it contains additional sequences that encode the TGA
stop codon to replace the GAC codon for Asp
To mutagenize both NH
Western blot analyses were performed using guinea
pig anti-glycogen synthase antibodies kindly provided by Dr. John C.
Lawrence, Jr. (Washington University, St. Louis, MO). After separation
on 7.5% SDS-PAGE, immunoblotting was performed as described
previously
(20) .
Analysis of in Vivo Phosphorylation of
Glycogen Synthase Mutants-The [
Despite studies spanning many years, the exact mechanism by
which the multisite phosphorylation of glycogen synthase controls
enzyme activity in cells is still not fully understood. For the rabbit
muscle enzyme, phosphorylation in vivo has been demonstrated
at each of the nine distinct sites indicated in
Fig. 1(9) . A rather precise mechanism had been proposed
to explain how the enzymes casein kinase II and glycogen synthase
kinase-3 bring about an ordered sequential phosphorylation in the
region of sites 3, 4, and 5 (3). Our first effort to test this
hypothesis, which utilized expression of rabbit muscle glycogen
synthase in COS cells, led to the unexpected result that mutagenesis,
intended to disrupt the sequential phosphorylation, did not hinder
inactivation via the two key regulatory sites 3a and 3b
(15) .
The result implied that there could be an alternate mechanism for the
phosphorylation of sites 3a and/or 3b independent of the prior
COOH-terminal phosphorylations such as at site 5. In the present study,
we demonstrated directly that sites 3a and 3b are indeed phosphorylated
in COS cells in the absence of other phosphorylations in this region of
the molecule.
Rabbit muscle glycogen synthase expressed in COS cells
associates with insoluble structures in cell homogenates
(20) .
Co-expression with glycogenin significantly increases the proportion of
soluble enzyme (20), but there is still a tendency for the more active
glycogen synthase mutants to distribute more in the pellet fraction
(, Ref. 15). Moreover, glycogen synthesized in COS cells
by spontaneously active glycogen synthase mutants accumulated only in
the pellet fraction. Our explanation is that the new glycogen formed is
a particulate glycogen that can precipitate with low speed
centrifugation (4). In fact, application of low speed centrifugation
(16,000
The objective of the present study was to investigate the importance
of sites 3a and 3b in the control of glycogen synthase and demonstrate
that their phosphorylation in COS cells could occur independently of
COOH-terminal phosphorylations. Through use of the
Wild type or endogenous COS cell glycogen synthase had an
extremely low activity ratio (), consistent with earlier
results and indicative of a high degree of phosphorylation. Ser
Wang and Roach
(8) analyzed phosphorylation of recombinant
glycogen synthase by casein kinase-II and glycogen synthase kinase-3
in vitro. Site-directed mutagenesis followed by
phosphorylation allowed comparison of enzyme phosphorylated at sites
3a, 3b, 3c, 4, and 5 (wild type), at sites 3b, 3c, 4, and 5 (site 3a
mutated), or at sites 3c, 4, and 5 (site 3b mutated). The results
implicated phosphorylation of site 3a, and to a lesser degree site 3b,
in inactivation of glycogen synthase. However, the data did not
distinguish whether phosphorylation only at site 3a and/or 3b would be
sufficient to cause inactivation or whether inactivation required all
five sites to be phosphorylated
(8) . In the present study, we
have clearly demonstrated that phosphorylation of glycogen synthase at
site 3a and/or 3b can inactivate in the absence of phosphate at sites
3c, 4, and 5. Although incorporation of
[
The results strongly
suggest that the inactivation of glycogen synthase via COOH-terminal
phosphorylation can occur by more than one mechanism. The mechanism
first proposed involved the concerted action of casein kinase II and
glycogen synthase kinase-3
(16, 17) . From the results of
this study, an alternative mode of inactivation exists, since
independent and direct phosphorylation of both site 3a and site 3b can
occur. The data suggest the existence of site 3a and site 3b kinases,
at least in cultured cells.
The gels in which fragments C1 and
C2
Full-length or
truncated glycogen synthases that were additionally mutated at various
phosphorylation sites were expressed in COS cells. Activities in the
presence and absence of glucose-6-P were analyzed in soluble and pellet
fractions of cell homogenate as described under ``Experimental
Procedures.'' Results are the means ± S.E. for the number
of experiments indicated in parentheses.
(site 4), Ser
(site 3c), Ser
(site 3b), and Ser
(site 3a). Effective recognition
of glycogen synthase by glycogen synthase kinase-3 occurs only after
the phosphorylation of Ser
(site 5) catalyzed by casein
kinase II. The present study addresses specifically the role of sites
3a and 3b in the regulation of glycogen synthase expressed in COS
cells. Simultaneous Ser
Ala substitutions at sites 3 a, b and c,
4, and 5 in the same protein molecule eliminated
P
labeling in the proteolytic fragment
Arg
-Lys
, which contains these sites. This
mutant enzyme (which also had a Ser
Ala substitution at site 2
in the NH
terminus) had a -/+ glucose-6-P
activity ratio of
0.8, similar to that of totally dephosphorylated
enzyme. Reinstating serine residues at either site 3a or site 3b
restored labeling in the Arg
-Lys
peptide
and caused a decrease in the activity ratio to 0.4-0.6. When both
sites 3a and 3b were reintroduced, there was complete inactivation of
the enzyme. Thus, sites 3a and 3b are sufficient for the inactivation
of glycogen synthase and act synergistically to control activity. This
investigation demonstrates the existence of an alternate mechanism for
the phosphorylation of sites 3a and 3b that does not depend on prior
phosphorylation of site 5.
terminus, and at least seven other sites are located in
the COOH-terminal 100 residues (Fig. 1). Phosphorylations at
different sites have different effects on enzyme activity. For example,
phosphorylation at site 3c (Ser
)
(8) , site 4
(Ser
)
(9) , site 5
(Ser
)
(10, 11) , or site 1b
(Ser
)
(12) has a minimal effect on activity ratio,
and phosphorylation at site 1a (Ser
) causes at best a
modest decrease
(12) . In vitro, phosphorylation at
sites 2 (Ser
) and 2a (Ser
)
(13, 14) or at sites 3a (Ser
) and 3b
(Ser
)
(8) correlates with substantial inactivation
of glycogen synthase. Similar conclusions were drawn from analysis of
mutagenized enzyme expressed in COS cells
(15) . Phosphorylation
of glycogen synthase in vitro is mediated by what has been
termed hierarchal phosphorylation
(3) . Thus, phosphate
introduced into site 2
(13, 14) appears to be important
for recognition by casein kinase I, which in turn phosphorylates site
2a. In the COOH terminus, the enzyme glycogen synthase kinase-3
phosphorylates sites in the order 4, 3c, 3b, and 3a, but in vitro glycogen synthase kinase-3 action requires that the glycogen
synthase has first been phosphorylated at site 5 by casein
kinase-II
(16, 17, 18) . It was proposed that
phosphate can serve as part of the recognition sequence for glycogen
synthase kinase-3 in the sequence motif SXXXS(P)
(19) .
However, we recently demonstrated that, for glycogen synthase expressed
in COS cells, disruption of the recognition sequence for glycogen
synthase kinase-3 by Ser
Ala substitution at site 3c, 4, or 5
did not preclude inactivation of the enzyme
(15) . In the present
study, we show that glycogen synthase expressed in COS cells could be
phosphorylated directly at sites 3a or 3b when all adjacent
phosphorylation sites were eliminated by mutagenesis.
Figure 1:
Arrangement of
phosphorylation sites in wild type and mutated glycogen synthase. The
line represents rabbit muscle glycogen synthase with
phosphorylation sites indicated by verticaltickmarks. The amino acid sequence of the region containing
phosphorylation sites (underlined) from 3a to 5 is given. For
the mutants, the amino acids in the positions corresponding to each
phosphorylation site are indicated. The mutants are identified by a
five-letter designation in which the letters (A or S)
show the amino acids present at sites 3a, 3b, 3c, 4, and 5, in that
order.
Construction of Expression Vectors and Site-directed
Mutagenesis
Vectors for expression of glycogenin (pCMV-GN) and
glycogen synthase (pCMV-GS) were constructed as described previously
(20). Four glycogen synthase mutants, S640A,S644A,S648A,S652A,S656A
(AAAAA), S644A,S648A,S652A,S656A (SAAAA), S640A,S648A,S652A,S656A
(ASAAA), and S648A,S652A,S656A (SSAAA), were constructed in which the
indicated Ser residues were changed into Ala by PCR(
)
mutagenesis (see also Fig. 1). Four different 78-base
mutagenic oligonucleotides were synthesized to replace the sequence
from bases 2020-2103, which contained a unique SacII
site (bases 2032-2037). The individual codon changes were TCT
GCT, TCG
GCG, TCG
GCG, AGC
GCC, and AGC
GCC for the Ser
Ala replacements at positions 640 (site
3a), 644 (site 3b), 648 (site 3c), 652 (site 4), and 656 (site 5),
respectively. The primers were used individually in combination with a
downstream 82-base primer complementary to the glycogen synthase
sequence at bases 2260-2332. This primer contains additional
sequence to generate an XbaI site immediately after the stop
codon. The PCR products were purified by agarose gel electrophoresis,
digested with SacII/XbaI, repurified, and used to
replace the corresponding wild type sequence in the pCMV-GS expression
vector.
and an
XbaI site located 3` to the stop codon. This primer was used
in conjunction with an upstream 25-base primer identical to the
sequence from bases 1975-1999. To construct a truncated protein
containing phosphorylation site mutations, fragments from mutants
AAAAA, SAAAA, ASAAA, or SSAAA were excised from pCMV-GS with
AflIII and used as a template for PCR. The PCR products were
digested with SacII/XbaI and ligated into the pCMV-GS
vector. The DNA sequence corresponding to the
SacII/XbaI regions in each mutant was confirmed by
DNA sequencing.
- and COOH-terminal
phosphorylation sites, the SacII/XbaI fragments were
religated into a pCMV-GS vector in which the codon for Ser
(site 2) was mutated to code for Ala
(15) .
Analysis of Expressed Glycogen Synthase
Mutants
Recombinant plasmids were purified twice by
CsCl/ethidium bromide equilibrium density gradient
centrifugation
(21) . COS M9 cells in 35-mm plates were
transfected with 0.5 µg pCMV-GS containing wild type or mutant cDNA
in the presence of 0.5 µg pCMV-GN using LipofectACE reagent (Life
Technologies, Inc.) as described previously
(15) . Cells were
lysed, homogenized, and centrifuged at 16,000 g for 15
min as described previously
(20) . The supernatants and
resuspended pellets obtained are designated as the soluble and pellet
fractions, respectively. Glycogen synthase activity was determined by
measuring incorporation of [
C]glucose from
UDP-[U-
C]glucose into glycogen with or without
7.2 mM glucose-6-P using the filter paper assay of Thomas
et al.(22) . The activity of the pellet fraction was
expressed in terms of the protein in the corresponding soluble
fraction; the -/+ glucose-6-P activity ratio was calculated
after subtraction of endogenous glycogen synthase activity of COS
cells
(15) .
P]phosphate
labeling and immunoprecipitation of glycogen synthase expressed in COS
cells were performed as described previously
(15) using guinea
pig anti-glycogen synthase antibodies
(23) different from those
used in Western blotting and Protein A-agarose beads (Pierce). After
immunoprecipitation, the beads bound to
[
P]glycogen synthase were resuspended in 100
mM ammonium bicarbonate, 25 mM
-mercaptoethanol
and a portion was boiled in SDS sample buffer and analyzed by 7.5%
SDS-PAGE according to Laemmli
(24) with subsequent
autoradiography. The remainder of the beads were incubated with 0.1
µg/ml chymotrypsin (Worthington) for 16 h at 30 °C. The beads
were then centrifuged at 12,000
g for 5 min,
resuspended in the same buffer, and centrifuged again. The
supernatants, which contain the COOH-terminal glycogen synthase
proteolytic fragment, were combined. The beads were treated with SDS
sample buffer, and uncleaved protein was separated from the
NH
-terminal proteolytic fragment by 7.5% SDS-PAGE.
Endoproteinase Lys-C (Boehringer Mannheim) was added to the
supernatants (final concentration, 30 µg/ml) for 16 h at 30 °C.
The COOH-terminal chymotryptic fragments, either without treatment or
after incubation with endoproteinase Lys-C, were analyzed by gel
electrophoresis in the Tricine-based system using a 10% spacer gel and
16.5% polyacrylamide gel with 6 M urea according to Schagger
and von Jagow
(25) . After electrophoresis, gels were dried and
autoradiograms prepared.
Other Methods
Protein was quantitated by the
method of Bradford
(26) , using bovine -globulin as
standard.
Expression of Glycogen Synthase Mutants in COS
Cells
Wild type rabbit muscle glycogen synthase expressed in COS
cells was associated predominantly with a low speed pellet
fraction
(20) . The proportion of the soluble glycogen synthase
was increased by co-expression with rabbit muscle glycogenin in all
experiments described below
(15, 20) . The glycogen
synthase was analyzed in both soluble and pellet fractions of COS cell
homogenates. Consistent with earlier work
(15) , wild type
glycogen synthase was expressed in COS cells as a polypeptide with
M
87,000 as estimated by SDS-PAGE
(Fig. 2). Simultaneous Ser
Ala mutations at sites 3a, 3b,
3c, 4 and 5, with the NH
-terminal site 2 left unchanged
(AAAAA) or with site 2 also mutated to Ala (2, AAAAA), led to
expression of enzyme with a significantly reduced apparent
M
of
84,000, consistent with the enzyme
having a reduced phosphorylation state
(8, 17) . In this
background of mutations, conversion of the residue at position 3a to
Ser (SAAAA or 2, SAAAA) had no effect on the electrophoretic mobility,
which remained at
84,000. However, the presence of serine at site
3b (ASAAA or 2, ASAAA) resulted in two discrete species with small
differences in their mobilities. With serines at both sites 3a and 3b
(SSAAA or 2, SSAAA), two species of glycogen synthase were resolved by
SDS-PAGE (Fig. 2). Mutation at site 2 had no effect on the
electrophoretic behavior of the enzymes containing COOH-terminal
mutations. These data indicate phosphorylation of site 3b by a
mechanism distinct from the ordered mechanism proposed for its
phosphorylation in vitro by glycogen synthase
kinase-3
(3) .
Figure 2:
Expression of glycogen synthase mutants in
COS M9 cells. Cells transfected with pCMV-GN and pCMV-GS DNA, encoding
wild type or mutated glycogen synthase, were analyzed by immunoblot.
Mutations were introduced at only COOH-terminal phosphorylation sites
(A and B) or in conjunction with the
NH-terminal site 2 (C and D). Lysed cell
suspensions were centrifuged to generate soluble (A and
C) and pellet (B and D) fractions. The
pellets were resuspended in the starting volume, and glycogen synthase
activities (+glucose-6-P) in both fractions were measured. The
samples were diluted to obtain equal amounts of enzyme activity and
were analyzed by SDS-PAGE followed by transfer to nitrocellulose, which
was probed with anti-glycogen synthase antibodies and
I-labeled protein A. The samples from control cells were
loaded on the gel without dilution. The migration of molecular mass
markers (in kDa) is indicated. W.T., wild
type.
COOH-terminal Phosphopeptide Mapping of Glycogen Synthase
Expressed in COS Cells
Transfected COS cells were labeled with
[P]phosphate, and the expressed enzyme was
isolated by immunoprecipitation. Treatment of immunoprecipitates with
0.1 µg/ml chymotrypsin released a labeled polypeptide with
M
22,400 as judged by electrophoresis in the
Tricine-based system (Fig. 3). A much weaker signal was observed
in control samples from untransfected cells, due to the endogenous COS
cell glycogen synthase.
(
)
These results are
consistent with earlier studies indicating that chymotrypsin at low
concentrations selectively cleaves the Tyr
-Arg
bond in rabbit muscle glycogen synthase
(27) to generate
the C2 chymotryptic fragment, which contains all the COOH-terminal
phosphorylation sites, including sites 1a and 1b (27, 28). The C2
fragment contains only two lysine residues, Lys
and
Lys
. Lys
is located between sites 5 and 1a,
whereas Lys
lies between sites 1a and 1b (Fig. 4).
The lysine-specific proteinase has been successfully used for cleavage
of the C2 fragment of rabbit muscle glycogen synthase
(28) , and
we applied the same approach in the present study. At low
concentration, endoproteinase Lys-C generated three phosphorylated
polypeptides with apparent M
of 9200, 5200, and
4400 from C2, suggesting cleavage of at least two sites (Fig. 3).
The two larger species are also faintly seen in the analysis of the
endogenous COS cell glycogen synthase from the control cells. Since we
could not generate enough of the phosphorylated peptides for sequence
analysis, we took advantage of site-directed mutagenesis to help in
their identification. A mutant form of glycogen synthase truncated at
Lys
(
682) was constructed so that phosphorylation at
sites 1a and 1b would be eliminated. The truncation also coincides with
one of the predicted endoproteinase Lys-C cleavage sites in the C2
peptide. After incubation of COS cells expressing the
682 mutant
with [
P]phosphate, immunoprecipitation, and
digestion with chymotrypsin, only two radioactive peptides were
detected (Fig. 3). One species (22.4 kDa) is detected in the same
amounts in samples from untransfected control cells and thus represents
the C2 fragment of endogenous wild type glycogen synthase. The smaller
polypeptide (5.2 kDa) has the same size as one of the fragments
generated by treatment of C2 from wild type enzyme with endoproteinase
Lys-C. Treatment of C2
from truncated glycogen synthase by
endoproteinase Lys-C did not change the electrophoretic mobility of
this fragment (Fig. 3). Therefore, we conclude that the 5.2-kDa
fragment is the L1 peptide (Fig. 4), which is generated by
treatment of C2 from wild type glycogen synthase by endoproteinase
Lys-C. This peptide stretches from Arg
to Lys
and contains sites 3, 4, and 5. The other two labeled
polypeptides, of 9.2 kDa and 4.4 kDa, were not analyzed further, but
because of their absence in the digest of the
682 mutant, these
peptides must contain phosphorylation sites that are COOH-terminal to
Lys
such as sites 1a and/or 1b. We can exclude
phosphorylation of Thr
because phosphoamino acid analysis
of expressed wild type glycogen synthase indicated the presence only of
phosphoserine residues (data not shown). Whether other serine residues
in this region besides sites 1a and 1b are phosphorylated is not
established by this study.
Figure 3:
Identification of peptide containing
phosphorylation sites 3, 4, and 5. Control cells or COS cells
expressing wild type (w.t.) or truncated (682) glycogen
synthase were incubated with [
P]phosphate.
Immunoprecipitates were incubated in the presence of 0.1 µg/ml
chymotrypsin for 16 h at 30 °C. The released 22.4-kDa (C2) or
5.2-kDa fragment (C2
) was separated from the immune complex by
centrifugation at 12,000
g for 10 min. Samples were
divided into two groups and incubated for 16 h at 30 °C without any
additions (-) or in the presence of 30 µg/ml endoproteinase
Lys-C (+). The mixtures were subjected to electrophoresis in the
Tricine-based system in the presence of urea. The gel was dried and
exposed to x-ray film. Molecular masses of fragments are indicated in
kDa.
Figure 4:
Location
of phosphorylation sites in proteolytic fragments of glycogen synthase.
Glycogen synthase protein is depicted as a solidhorizontalline. Chymotrypsin at very low
concentration cleaves rabbit muscle glycogen synthase at two places
(verticalsolidarrows) generating C1 and C2
fragments (a) from wild type enzyme (27) or C1 and C2
fragments (b) from truncated protein. C1 fragments from both
wild type and truncated enzyme have the same size and contain sites 2
and 2a (see Fig. 1). Fragment C2 contains all COOH-terminal
phosphorylation sites, whereas fragment C2
is shorter and does not
have sites 1a and 1b. Two sites for endoproteinase Lys-C inside the C2
fragment are indicated (dashedarrows). One Lys-C
peptide, L1, has an identical amino acid sequence to C2
and
contains sites 3, 4, and 5.
Analysis of the Phosphorylation at Sites 3a and 3b in
Rabbit Muscle Glycogen Synthase Expressed in COS Cells
The
COOH-terminal Ser Ala mutants (see Fig. 1) were expressed
in COS cells labeled with [
P]phosphate and
analyzed by immunoprecipitation and peptide mapping as described in the
preceding section. When analyzed by SDS-PAGE using the Tricine-urea
system
(25) , the
P-labeled C2 peptide had
essentially the same mobility for all of the mutants examined
(Fig. 5A). We had previously shown that mutations at
sites 3, 4, and 5 resulted in electrophoretic heterogeneity of the
COOH-terminal cyanogen bromide fragment (CB-2) when analyzed by
standard SDS-PAGE
(15) . CB-2 extends only 23 amino acids beyond
the NH
terminus of the C2 fragment analyzed in the present
study and might have been expected to display a similar heterogeneity.
The difference in behavior is therefore most likely due to the
different gel systems used, with the Tricine-urea system less sensitive
to the presence of covalent phosphate in this molecular weight range
than the Laemmli system.
Figure 5:
Electrophoretic analysis of COOH-terminal
proteolytic fragments of glycogen synthase mutated at phosphorylation
sites 3, 4, and 5. Full-length glycogen synthases with the same
mutations as described in Fig. 1 were expressed in COS cells, labeled
with [P]phosphate and immunoprecipitated. The C2
fragments generated by treatment of the immune complex with
chymotrypsin were subsequently digested by endoproteinase Lys-C as
described in the legend to Fig. 3. Fragments generated by chymotrypsin
(A) and endoproteinase Lys-C (B) were subjected to
electrophoresis in the presence of urea. Gels were dried and exposed to
x-ray film. W.T., wild type.
The C2 peptide can be digested by
endoproteinase Lys-C to generate three phosphopeptides, of which the L1
peptide contains sites 3, 4, and 5 (Fig. 5B). As might
be predicted, none of the mutations in the region of sites 3, 4, and 5
affected the electrophoretic mobility of the 9.2-kDa and 4.4-kDa
peptides, which contain sites COOH-terminal of residue 682. In
contrast, the mobility of the L1 peptide was reduced as the number of
potential phosphorylation sites was decreased by mutagenesis in the
SAAAA, ASAAA, and SSAAA mutants. For a small hydrophilic peptide like
L1, the presence of phosphate must have a greater proportional effect
on electrophoretic mobility in this system than with the large C2
peptide. Because of these effects on electrophoretic mobility, it was
unclear whether mutagenesis of all five sites in the AAAAA mutant
eliminated phosphorylation of L1 or resulted in a phosphorylated
fragment that was not resolved from the 9.2-kDa peptide. In fact, L1
derived from the SAAAA and ASAAA mutants was only just resolved from
the 9.2-kDa peptide (Fig. 5B). To solve these problems,
mutation of phosphorylation sites was combined with a mutation to
truncate the protein at Lys. The truncated glycogen
synthase expressed in COS cells was readily distinguished from the
endogenous COS cell enzyme because of its significantly increased
mobility and, as for the full-length protein, mutations of
phosphorylation sites affected the exact electrophoretic mobility in
the Laemmli system (Fig. 6A). After treatment of the
immunoprecipitate with chymotrypsin to cleave the
Tyr
-Arg
bond, the large C1 fragment
remained associated with the immunoprecipitate whereas the small
C2
fragment was released (Fig. 6, B and
C). Labeled C1 from endogenous glycogen synthase and from all
the truncation mutants had a similar electrophoretic mobility
corresponding to a polypeptide with M
68,000
(Fig. 6B). Noncleaved protein in the immunoprecipitates
was visible as a minor band with lower mobility
(Fig. 6B). Fragments C1 contain the
NH
-terminal sites 2 and 2a, which are phosphorylated in
rabbit muscle glycogen synthase expressed in COS cells
(15) and
which can account for the labeling of these species. The C2 fragment
from the endogenous COS cell glycogen synthase (apparent
M
22,400) was present in all samples and was
well separated from the chymotryptic fragments (C2
) derived from
the truncation mutants (Fig. 6C). Note that the
mobilities of C2
were the same as those of L1 from the full-length
phosphorylation site mutants, consistent with the choice of the site of
truncation (compare Fig. 5B and 6C). No
phosphorylation at C2
was detected if sites 3, 4, and 5 had been
mutated (AAAAA
682). In contrast, phosphorylation at sites 3a
(SAAAA
682) and 3b (ASAAA
682) was evident even though all
other COOH-terminal sites were mutated (Fig. 6C).
Comparison of
P labeling in different mutants is
complicated by potential differences in expression level
(15) .
Therefore, phosphorylation of C2
fragments was quantitated through
the use of a
-scanner (AMBIS radioanalytic imaging system) and
normalized to the amount of radioactivity detected in the C1 fragment
(containing wild type NH
-terminal phosphorylation sites)
from the same sample (). Comparison of C2
phosphorylation in the mutant
682 with AAAAA
682 indicates a
level of phosphorylation in this region of 1.63 units. Phosphorylation
at either site 3a or site 3b, in mutants SAAAA
682 and
ASAAA
682 respectively, was only 4-9% as much as in
682.
However, when both sites 3a and 3b were present (mutant SSAAA
682),
phosphorylation increased synergistically to 0.46 units, over twice the
sum of the phosphorylation in the SAAAA
682 and ASAAA
682
mutants. The
P content of the C2
peptide from the
SSAAA
682 mutant was 28% of that found with the
682 species.
Figure 6:
Electrophoretic analysis of proteolytic
fragments of truncated glycogen synthase containing mutations at
COOH-terminal phosphorylation sites. P-labeled glycogen
synthase, immunoprecipitated from COS cells, which was digested with
chymotrypsin, and fragments C1 and C2
were separated by
centrifugation as described in the legend to Fig. 3. Controls were not
digested. Beads containing uncleaved glycogen synthases (A)
and the C1 fragment (B) were boiled in SDS sample buffer, and
the released proteins were analyzed by electrophoresis according to
Laemmli (24) in a 7.5% gel. Fragments C2
(C) were
analyzed by electrophoresis according to Schagger and von Jagow (25) in
16.5% gel in the presence of urea. Gels were dried and exposed to x-ray
film.
Analysis of Glycogen Synthase Activity in Expressed
Mutants
Expression of wild type or mutated glycogen synthase
significantly increased the total enzyme activity (+glucose-6-P)
in COS cells. Consistent with our earlier data
(15) , the
activities of expressed enzymes were distributed between the soluble
and pellet fractions of COS cell homogenates, and the mutants with the
highest activity ratios tended to associate more with the pellet
fraction (). Truncation at Lys did not
significantly affect the expression and distribution of glycogen
synthase activity. No substantial increase in the -/+
glucose-6-P activity ratio of the expressed mutants was observed if
mutagenesis was limited to either the NH
terminus or the
COOH terminus of glycogen synthase, confirming that phosphorylation at
only one of these termini is enough for inactivation
(15) . To
study the relative contribution of COOH-terminal phosphorylation sites
to the inactivation of glycogen synthase, the inactivating effect of
NH
-terminal phosphorylations can be eliminated by
introducing a Ser
Ala mutation at site 2
(15) . Mutation
of site 2 in conjunction with all five COOH-terminal sites (2, AAAAA,
) led to expression of glycogen synthase with a very high
activity ratio, 0.78-0.86, close to the enzyme activity ratio of
unphosphorylated wild type glycogen synthase expressed in
Escherichia coli(8, 18) . Restoration of serine
residues at either the site 3a or the site 3b position (2, SAAAA and 2,
ASAAA) partially inactivated the enzyme. However, establishing
phosphorylation sites at both positions (in the mutant 2, SSAAA) led to
almost complete inactivation of glycogen synthase ().
g for 15 min) to isolate particulate glycogen
from cells expressing hyperactive mutants results in a co-purification
of the glycogen synthase.
(
)
In this and previous
studies
(15) , we found that any given mutation of glycogen
synthase would alter the activity ratio in both pellet and soluble
fractions in the same sense, but the activity ratio was always lower in
the pellet (, Ref. 15). For simplicity, we will use the
pellet values for activity ratio in the following discussion although
the arguments are not really changed by using the soluble values.
682 truncation,
we were able unequivocally to identify the phosphopeptide (L1 from wild
type or C2
from the truncation mutants) containing phosphorylation
sites 3, 4, and 5. No
P labeling corresponding to this
peptide was observed in the AAAAA mutant, suggesting that there are no
other phosphorylation sites in this region (or more precisely, if other
sites exist, their recognition has been destroyed by the mutations).
Using the AAAAA or AAAAA
682 mutants as the reference, reinstating
a Ser at either site 3a or site 3b (SAAAA or ASAAA) resulted in the
detection of a phosphorylated peptide, thus proving that direct
phosphorylation of these sites could occur in COS cells. However, when
both sites were reverted to Ser residues in the
682 truncation,
the relative phosphorylation of the C2
peptide was more than
predicted from the results with each single mutant. In other words,
sites 3a and 3b acted synergistically with respect to
P
labeling.
Ala mutations mimic dephosphorylation under these conditions, and we
had shown that appropriate combinations of such mutations could
significantly activate the expressed glycogen synthase. The most
effective combination in the earlier study was enzyme-mutated at site 2
and site 3a, with a pellet activity ratio of
0.4
(15) . In
the present study, we mutated sites 3a, 3b, 3c, 4, and 5 to Ala, and
the resulting enzyme still had very low activity ratio. As before,
additional mutation of site 2, to eliminate NH
-terminal
phosphorylation, was necessary to cause significant activation, to an
activity ratio of
0.8, close to that of dephosphorylated enzyme
in vitro. Now, with an essentially fully active 2, AAAAA
mutant as the reference, the effects of reinstating phosphorylation at
sites 3a and 3b on activity could be evaluated. Introduction of either
site 3a or 3b correlated with a detectable decrease in activity ratio,
to 0.38 or 0.56, respectively, but introduction of both resulted in a
fully inactivated enzyme with activity ratio of 0.06. Therefore,
phosphorylation at sites 3a and 3b is sufficient to inactivate glycogen
synthase. Phosphate groups rather than simply negative charges are
required for inactivation of the enzyme.
(
)
In
addition, there is a synergism to the inactivation of glycogen synthase
mediated via sites 3a and 3b echoing that observed for the
phosphorylation of these sites in the
682 truncation. We should
stress that measurements of activity were made with full-length enzyme,
whereas quantitation of phosphorylation utilized the
682
truncation. However, it seems reasonable to propose a similar synergism
in the phosphorylation of sites 3a and 3b in full-length enzyme.
P]phosphate at site 3a was less than at site 3b
(), the inactivation due to site 3a was greater
(), consistent with earlier hypotheses of a predominant
role of site 3a in regulation of glycogen synthase activity (8, 15).
Note, though, that phosphorylation at both sites 3a and 3b is required
for full inactivation of glycogen synthase.
(
)
However, since
sites 3a and 3b undergo synergistic phosphorylation, there could be
ordered phosphorylation at the level of these two sites. Glycogen
synthase kinase-3 could still be a site 3a kinase, as in the original
model, but now acting subsequent to a distinct site 3b kinase whose
identity is currently unknown. In this model, site 3b would serve both
to affect activity directly and to influence the phosphorylation of
site 3a. It is interesting that in glycogen synthase from
Saccharomyces cerevisiae, homologs of sites 3a and 3b are
important for control, but their modification by the casein kinase
II/glycogen synthase kinase-3 mechanism cannot occur since equivalents
of sites 3c, 4, and 5 are not present
(30) . Therefore, perhaps
in yeast only one of the control mechanisms is operative, whereas in
mammals alternative means of regulation are available.
Table:
Analysis of phosphorylation of glycogen synthase
in fragments C1 and C2
were separated are shown in Fig. 6, B and C,
respectively. These were analyzed by the AMBIS radioanalytic imaging
system. Radioactivities of the fragments C1 were calculated after
subtraction of the radioactivity in the control sample (63 cpm).
Radioactivities of the fragments C2
were obtained after
subtraction of background (47 cpm).
Table:
Effect of phosphorylation site mutations on the
activity of glycogen synthase expressed in COS cells
Glu or Ser
Asp mutations at all five phosphorylation sites (in
conjunction with a Ser
Ala substitution at site 2). The
resulting enzyme was fully active (activity ratio 0.7-0.8),
indicating that substitution of residues with acidic side chains did
not mimic the effect of phosphorylation on the glycogen synthase
activity (A. V. Skurat, Y. Wang, and P. J. Roach, unpublished results).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.