©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of Sites 3a and 3b (Ser and Ser) in the Control of Rabbit Muscle Glycogen Synthase(*)

Alexander V. Skurat , Peter J. Roach

From the (1) Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Glycogen synthase kinase-3 inactivates rabbit muscle glycogen synthase by sequential phosphorylation of four COOH-terminal residues Ser (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.


INTRODUCTION

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 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.




EXPERIMENTAL PROCEDURES

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.

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 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.

To mutagenize both NH- 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) .

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 [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.


RESULTS

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 (AAAAA682). In contrast, phosphorylation at sites 3a (SAAAA682) and 3b (ASAAA682) 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 AAAAA682 indicates a level of phosphorylation in this region of 1.63 units. Phosphorylation at either site 3a or site 3b, in mutants SAAAA682 and ASAAA682 respectively, was only 4-9% as much as in 682. However, when both sites 3a and 3b were present (mutant SSAAA682), phosphorylation increased synergistically to 0.46 units, over twice the sum of the phosphorylation in the SAAAA682 and ASAAA682 mutants. The P content of the C2 peptide from the SSAAA682 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 ().


DISCUSSION

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 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.

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 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 AAAAA682 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.

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 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.

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 [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.

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.() 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

The gels in which 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

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.



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DK27221 (to P. J. R.) and Juvenile Diabetes Foundation International Grant 193186 (to A. V. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: PCR, polymerase chain reaction; Tricine, N-tris(hydroxymethyl)methylglycine; PAGE, polyacrylamide gel electrophoresis.

The exact sequence and isozyme type of COS cell glycogen synthase is not known, but the endogenous enzyme here behaves very similarly to the rabbit muscle enzyme with respect to the cleavages used. Both muscle and liver isoforms are highly conserved among species (7, 31), and even between muscle and liver isoforms the sequence identity is 70% (32). Glycogen synthases from muscle, heart, fat, kidney, and brain have been shown to have similar immunoreactivity in Western blot analyses (33).

A. V. Skurat and P. J. Roach, unpublished results.

In other studies, we made Ser 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).

Much of our work has so far utilized transient expression in COS cells, but in other studies we have stably transformed Rat1 fibroblasts with wild type glycogen synthase as well as several mutants. The key points regarding glycogen synthase activity ratio have been found also in Rat1 cells. For example, the glycogen synthase mutants 2, AAAAA and 2, SSAAA have high and low activity ratio, respectively. (A. V. Skurat and P. J. Roach, unpublished results).


REFERENCES
  1. Cohen, P.(1982) Nature 296, 613-620 [Medline] [Order article via Infotrieve]
  2. Cohen, P.(1986) in The Enzymes (Boyer, P. D., and Krebs, E. G., eds) Vol. 17, pp. 461-497, Academic Press, Inc., Orlando, FL
  3. Roach, P. J.(1990) FASEB J. 4, 2961-2968 [Abstract]
  4. Larner, J.(1990) Adv. Enzymol. Relat. Areas Mol. Biol. 63, 173-231 [Medline] [Order article via Infotrieve]
  5. Roach, P. J., Takeda, Y., and Larner, J.(1976) J. Biol. Chem. 251, 1913-1919 [Abstract]
  6. Browner, M. F., Nakano, K., Bang, A. G., and Fletterick, R. J.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1443-1447 [Abstract]
  7. Zhang, W., Browner, M. F., Fletterick, R. G., DePaoli-Roach, A. A., and Roach, P. J.(1989) FASEB J. 3, 2532-2536 [Abstract/Free Full Text]
  8. Wang, Y., and Roach, P. J.(1993) J. Biol. Chem. 268, 23876-23880 [Abstract/Free Full Text]
  9. Poulter, L., Ang, S. G., Gibson, B. W., Williams, D. H., Holmes, C. F. B., Caudwell, F. B., Pitcher, J., and Cohen, P.(1988) Eur. J. Biochem. 175, 497-510 [Abstract]
  10. DePaoli-Roach, A. A., Ahmad, Z., and Roach, P. J.(1981) J. Biol. Chem. 256, 8955-8962 [Abstract/Free Full Text]
  11. Cohen, P., Yellowlees, D., Aitken, A., Donella-Deana, A., Hemmings, B. A., and Parker, P. J.(1982) Eur. J. Biochem. 124, 21-35 [Medline] [Order article via Infotrieve]
  12. Embi, N., Parker, P. J., and Cohen, P.(1981) Eur. J. Biochem. 115, 405-413 [Medline] [Order article via Infotrieve]
  13. Flotow, H., and Roach, P. J.(1989) J. Biol. Chem. 264, 9126-9128 [Abstract/Free Full Text]
  14. Nakielny, S., Campbell, D. G., and Cohen, P.(1991) Eur. J. Biochem. 199, 713-722 [Abstract]
  15. Skurat, A. V., Wang, Y., and Roach, P. J.(1994) J. Biol. Chem. 269, 25534-25542 [Abstract/Free Full Text]
  16. Picton, C., Woodgett, J., Hemmings, B., and Cohen, P.(1982) FEBS Lett. 150, 191-196 [CrossRef][Medline] [Order article via Infotrieve]
  17. DePaoli-Roach, A. A., Ahmad, Z., Camici, M., Lawrence, J. C., Jr., and Roach, P. J.(1983) J. Biol. Chem. 258, 10702-10709 [Abstract/Free Full Text]
  18. Zhang, W., DePaoli-Roach, A. A., and Roach, P. J.(1993) Arch. Biochem. Biophys. 304, 219-225 [CrossRef][Medline] [Order article via Infotrieve]
  19. Fiol, C. J., Mahrenholz, A. M., Wang, Y., Roeske, R. W., and Roach, P. J.(1987) J. Biol. Chem. 262, 14042-14048 [Abstract/Free Full Text]
  20. Skurat, A. V., Cao, Y., and Roach, P. J.(1993) J. Biol. Chem. 268, 14701-14707 [Abstract/Free Full Text]
  21. Maniatis, T., Fritsch, E. F., and Sambrook, J.(1982) Molecular Cloning: A Laboratory Manual, pp. 1.42-1.43, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  22. Thomas, J. A., Schlender, K. K., and Larner, J.(1968) Anal. Biochem. 25, 486-499 [Medline] [Order article via Infotrieve]
  23. Lawrence, J. C., Jr., Hiken, J., DePaoli-Roach, A. A., and Roach, P. J. (1983) J. Biol. Chem. 258, 10710-10719 [Abstract/Free Full Text]
  24. Laemmli, U. K.(1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  25. Schagger, H., and von Jagow, G.(1987) Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  26. Bradford, M. M.(1979) Anal. Biochem. 72, 248-254 [CrossRef]
  27. Picton, C., Aitken, A., Bilham, T., and Cohen, P.(1982) Eur. J. Biochem. 124, 37-45 [Abstract]
  28. Kuret, J., Woodgett, J. R., and Cohen, P.(1985) Eur. J. Biochem. 151, 39-48 [Abstract]
  29. Woodgett, J. R.(1990) EMBO J. 9, 2431-2438 [Abstract]
  30. Farkas, I., Hardy, T. A., Goebl, M. G., and Roach, P. J.(1991) J. Biol. Chem. 266, 15602-15607 [Abstract/Free Full Text]
  31. Nuttall, F. Q., Gannon, M. C., Bai, G., and Lee, E. Y.(1994) Arch. Biochem. Biophys. 311, 443-449 [CrossRef][Medline] [Order article via Infotrieve]
  32. Bai, G., Zhang, Z., Werner, R., Nuttall, F. Q., Tan, A. W. H., and Lee, E. Y. C.(1990) J. Biol. Chem. 265, 7843-7848 [Abstract/Free Full Text]
  33. Kaslow, H. R., and Lesikar, D. D.(1984) FEBS Lett. 172, 294-298 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.