Phosphorylation and inactivation of the pyruvate dehydrogenase from the anaerobic parasitic nematode, Ascaris suum. Stoichiometry and amino acid sequence around the phosphorylation sites.

Tryptic digestion of the fully phosphorylated Ascaris suum pyruvate dehydrogenase complex yielded a single tetradecapeptide containing 2 phosphorylated serine residues. Its amino acid sequence was Tyr-Ser-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Ser(P)-Tyr-Arg and was very similar to one of the tryptic phosphopeptides isolated from mammalian and yeast pyruvate dehydrogenases. At partial phosphorylation, three peptides were isolated which corresponded to the monophosphorylated (sites 1 and 2) and diphosphorylated tetradecapeptides. In contrast to results reported from mammalian complexes, phosphorylation of the ascarid complex paralleled inactivation, and no additional phosphorylation occurred after inactivation was complete. Complete inactivation of the complex was associated with the incorporation of 1.7-1.9 mol of phosphoryl groups/mol of alpha-pyruvate dehydrogenase subunit, and the strict preference of the pyruvate dehydrogenase kinase for site 1 was not observed. Whereas site 1 was initially phosphorylated more rapidly than site 2, at 50% inactivation, 41% of the incorporated phosphoryl groups were incorporated into site 2. In addition, substantial amounts of peptide monophosphorylated at site 2 also accumulated, suggesting that prior phosphorylation at site 1 was not necessary for phosphorylation at site 2. Phosphorylation also caused a marked decrease in the mobility of the alpha-pyruvate dehydrogenase subunit on sodium dodecyl sulfate-polyacrylamide gels and the apparent separation of mono- and diphosphorylated forms of the enzyme. The significance of these observations in the regulation of the unique anaerobic mitochondrial metabolism of A. suum is discussed.


Phosphorylation and Inactivation of the Pyruvate Dehydrogenase from the Anaerobic Parasitic Nematode, Ascaris suum
Tryptic digestion of the fully phosphorylated Ascaris suum pyruvate dehydrogenase complex yielded a single tetradecapeptide containing 2 phosphorylated serine residues. Its amino acid sequence was Tyr-Ser-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Ser(P)-Tyr-Arg and was very similar to one of the tryptic phosphopeptides isolated from mammalian and yeast pyruvate dehydrogenases. At partial phosphorylation, three peptides were isolated which corresponded to the monophosphorylated (sites 1 and 2) and diphosphorylated tetradecapeptides. In contrast to results reported from mammalian complexes, phosphorylation of the ascarid complex paralleled inactivation, and no additional phosphorylation occurred after inactivation was complete. Complete inactivation of the complex was associated with the incorporation of 1.7-1.9 mol of phosphoryl groups/mol of a-pyruvate dehydrogenase subunit, and the strict preference of the pyruvate dehydrogenase kinase for site l was not observed. Whereas site 1 was initially phosphorylated more rapidly than site 2, at 50% inactivation, 41% of the incorporated phosphoryl groups were incorporated into site 2. In addition, substantial amounts of peptide monophosphorylated at site 2 also accumulated, suggesting that prior phosphorylation at site 1 was not necessary for phosphorylation at site 2. Phosphorylation also caused a marked decrease in the mobility of the apyruvate dehydrogenase subunit on sodium dodecyl sulfate-polyacrylamide gels and the apparent separation of mono-and diphosphorylated forms of the enzyme. The significance of these observations in the regulation of the unique anaerobic mitochondrial metabolism of A. suum is discussed.
Mitochondrial energy metabolism in body wall muscle of the adult parasitic nematode, Ascaris s u m , is anaerobic and results in the accumulation of the organic acids acetate, propionate, succinate, 2-methyl butyrate, and 2-methyl valerate (1,2). The tricarboxylic acid cycle is not functional, and electron transport is antimycin-and cyanide-insensitive (3). Instead of oxygen, unsaturated organic acids are used as terminal electron acceptors, and the NADH-dependent reductions of fumarate and 2-methyl branched-chain enoyl-CoAs appear to be coupled to rotenone-sensitive, electron transport-associated ADP phosphorylations (4, 5).
In body wall muscle of adult A. mum, pyruvate is formed intramitochondrially by malic enzyme, and its subsequent oxidation by the pyruvate dehydrogenase complex (PDC)' is an important regulatory site in the helminth's unique anaerobic mitochondrial metabolism. Pyruvate oxidation generates not only the reducing power, but also the thioester linkages needed to drive branched-chain fatty acid synthesis through a reversal of @-oxidation (6, 7). The subunit composition of ascarid PDC is similar to complexes isolated from mammalian tissues, and its activity also is regulated by an intrinsic pyruvate dehydrogenase kinase and Mg2+,Ca2+-dependent pyruvate dehydrogenase phosphatase, which catalyze the reversible phosphorylation of the cy-pyruvate dehydrogenase subunit (E1,) (8,9). However, many of the kinetic parameters of the ascarid complex differ markedly from mammalian PDCs and appear to be modified for the unique reducing environment present within the adult ascarid mitochondria (8,14). Recently, we have observed that phosphorylation of ascarid El, is proportional to the inactivation of the complex, and no additional phosphorylation is observed after inactivation is complete. In addition, phosphorylation of ascarid El, markedly reduces its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (9). These results contrast with those reported for mammalian PDCs, where three distinct phosphorylation sites have been identified, only one of which is primarily responsible for inactivation and where phosphorylation does not appear to markedly alter the mobility of El, during SDS-PAGE (10-12). This study identifies specific phosphorylation sites in ascarid El, and characterizes the stoichiometry of site-specific phosphorylation and inactivation.

Materials
Pyruvate dehydrogenase complex (3-5 pmol of NADH produced per min/mg of protein) containing pyruvate dehydrogenase kinase activity was isolated from frozen A. mum muscle strips as described previously (9). Ascarids were obtained from Routh Packing (Sandusky, OH). Partially purified bovine kidney pyruvate dehydrogenase phosphatase was a generous gift of Dr. T. Roche (Kansas State University, Manhattan, KS) or was prepared according to Pratt et al. (13). [y-32P]ATP was obtained from Du Pont-New England Nuclear. Trifluoroacetic acid was from Aldricb, and phenyl isothiocyanate was from Pierce Chemical Co. HPLC reagents were from Fisher. All other chemicals were of reagent-grade and were purchased from Sigma.
Isolation of Phosphotryptic Peptide from A. suum PDC-Ascarid tryptic phosphopeptides were isolated essentially as described previously for the isolation of the tryptic phosphopeptide from yeast PDC (16). A. s u m PDC (21 mg of protein) was phosphorylated to 98% inactivation with [y3'P]ATP as described above. Phosphorylated PDC then was twice precipitated with trichloroacetic acid (lo%, w/ v) and resuspended in 20 ml of 0.2 M NH4HC03, and 1 mg of L-1tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin was added. Following incubation for 4 h at 23 "C, an additional 1 mg of trypsin was added to ensure complete digestion. After 20 h, the incubation mixture was centrifuged (3000 X g, 10 min) to remove insoluble material (~2 % of the total radioactivity), lyophilized, and stored at -20 "C prior to phosphopeptide isolation.
The lyophilized tryptic peptides were resuspended in 2 M urea and filtered (0.45-pm pore size). Initially, the digest was chromatographed on a Sephadex G-50-50 column (1.6 X 89 cm) and eluted (18 ml/h) with 0.2 M NHdHC03 (pH 8.2) to remove [y-32P]ATP. Radioactive peptide fractions eluted in a broad peak (11 ml; retention time of 5.5 h) and were pooled and lyophilized for further purification. Lyophilized peptide was resuspended in 0.2 M NH4HC03, further purified on a Waters reverse-phase Cls pBondapak column (100 X 8 mm), and eluted (60 min, 1.5 ml/min) with a linear gradient of acetonitrile (0-60%) in 0.1% trifluoroacetic acid. Radioactive fractions eluted in a single peak were pooled and lyophilized. In subsequent peptide purifications, the Sephadex chromatography step was omitted. A small peak of [y-32P]ATP was sometimes observed in the void volume during reverse-phase HPLC. Lyophilized phosphopeptide was resuspended in 20% acetonitrile, further purified by anion-exchange HPLC on a Dynamax AX anion-exchange column (250 X 4.1 mm, Rainin Instrument Co. Inc.), and eluted (60 min, 1 ml/min) with a linear gradient of ammonium acetate (0-1 M) in 20% acetonitrile. Fractions containing radioactivity were again pooled and lyophilized. Recovery of radioactivity from all columns was greater than 80%, and the overall recovery of radioactive peptide was ahout 50%.
Cyanogen Bromide Digestion-Peptide (5 nmol) was incubated in 100 p1 of cyanogen bromide (7 mg/ml) in 0.1 M HC1 at room temperature according to Gross and Whitkop (18). The digested samples then were diluted with 100 pl of HzO and lyophilized three times. Cleavage products were separated by reverse-phase HPLC as described for the initial tryptic digest. Peptide fragments were collected, counted for radioactivity, and lyophilized for amino acid analysis.
Amino Acid Analysis-Amino acids were analyzed by two different methods. In the first, highly purified peptide (1-2 nmol) was hydrolyzed for 24 h in constant-boiling HCI in evacuated tubes flushed with nitrogen at 110 "C, and amino acid analysis was performed on a Beckman System 6300 high performance analyzer. To reduce the destruction of tyrosine residues, 1% phenol in water was added prior to hydrolysis. The second method involved the precolumn derivatization of amino acids with phenyl isothiocyanate (19). Peptides (2-5 nmol) were hydrolyzed under nitrogen in 6 M HCl at 105 "C for 24 h, followed by lyophilization. The samples then were dissolved in 100 pl of coupling buffer (acetonitrile:pyridine:triethylamine:HzO (105:2:3)). Following a second lyophilization, samples were redissolved in 100 p1 of coupling buffer, and 5 pl of phenyl isothiocyanate was added. After a 5-min incubation at room temperature, the solution was lyophilized and dissolved in 15 p1 of 0.05 M ammonium acetate (pH 6.8). Phenylthiocarbamyl-derivatives were separated by reverse-phase HPLC (19). Both methods gave similar results.
Peptide Sequencing-Automated Edman degradations were per-formed with an Applied Biosystems Model 470A gas-phase protein sequenator. Phenylthiohydantoin (PTH)-derivatives recovered from the sequenator were dried under vacuum, redissolved in 20 pl of acetonitri1e:methanol (l:l), and separated by HPLC (20). Most of the PTH-derivatives were identified by their absorbance at 254 nm. PTH-6-Ser and PTH-6-Thr were detected at 313 nm. Gel Electrophoresis-Samples containing 32P-labeled PDC (8 pg of protein) were mixed with an equal volume of 4% sodium dodecyl sulfate and 2% 2-mercaptoethanol and heated at 95 "C for 5 min. SDS-PAGE was performed on 10% gels according to Laemmli (21). Autoradiographs were obtained after exposure of X-Omat x-ray film to the dried gels for 5 h at -70 "C.

RESULTS
Characterization of Tryptic Phosphopeptides from A. mum Pyruvate Dehydrogenase Complex-A. suum PDC was fully phosphorylated with [y3*P]ATP and digested with trypsin, and tryptic phosphopeptides were isolated by HPLC as described recently for the isolation of tryptic phosphopeptide from yeast PDC phosphorylated with a purified mammalian pyruvate dehydrogenase kinase (17). A single radioactive peak eluted by reverse-phase HPLC at about 15% acetonitrile from the complete tryptic digest of both fully and partially phosphorylated PDCs (data not shown). One major radioactive peptide (TA12; 97% radioactivity injected) was recovered from anion-exchange HPLC from tryptic peptides prepared from fully phosphorylated PDC (Fig. lA). In contrast, when tryptic phosphopeptides were prepared from partially phosphorylated PDC (less than 50% activity), three radioactive peptides (TA1, TA2, and TA12) were separated by anionexchange HPLC (Fig. 1B). The amino acid compositions of peptides TA1, TA2, and TA12 were identical (Table I). However, they differed in the amount of covalently bound phosphoryl groups. Peptides TA1 and TA2 contained one phosphoryl group, whereas peptide TA12 contained two phosphoryl groups. The amino acid sequence analysis of peptide TA12 is presented in Table 11. No PTH-derivatives were detected at cycles 5 and 12, which is consistent with the inability of phosphorylated serine residues to form PTHderivatives (22). It appears that the ascarid tryptic phosphopeptide contains 2 phosphorylated serine residues and is very similar to a phosphorylated tetradecapeptide isolated from both yeast and mammalian pyruvate dehydrogenase complexes (10)(11)(12)16).
Since all three of the phosphopeptides identified in this study had identical amino acid compositions and peptide TA12 contained twice as many phosphoryl groups as peptides TA1 and TA2, it appeared that peptides TA1 and TA2 might be monophosphorylated at different serine residues (5 or 12), as had been observed previously with mammalian PDCs (10)(11)(12). To test this hypothesis, each phosphopeptide was cleaved with CNBr, and cleavage products were separated by HPLC (Fig. 2). Over 90% of the original radioactivity was recovered in four peptides (Cl-C4). The amino acid compositions of these four peptides are presented in Table I. Peptides Cl-C3 have similar amino acid compositions and appear to consist of residues 1-6, whereas peptide C4 consists of residues 7-14. The recovery of multiple peptides with amino acid compositions identical (Cl and C2) or similar (C3) to residues 1-6 is reproducible. Their origin is not completely clear, but they could result from a number of factors, such as an equilibrium of homoserine/homoserine lactone or microheterogeneity not detected previously. Interestingly, the substitution of a glycine residue for a serine residue observed in cleavage peptide C3 is observed at position 2 in the phosphopeptide derived from yeast PDC ( Table 11). All of the radioactivity in peptide TA2 was recovered in cleavage peptide C4, whereas all of the radioactivity in peptide TA1 was recovered in cleavage pep- Elution profiles of tryptic phosphopeptides from phosphorylated PDC on anion-exchange high pressure liquid chromatography. Elution of the peptides was performed as described under "Experimental Procedures" using a linear gradient of ammonium acetate (0-1 M in 20% acetonitrile). Fully phosphorylated PDC (12.5 nmol of peptide injected) yielded one major radioactive peptide (TA12) which eluted at about 210 mM NHdOAc ( A ) . Partially phosphorylated PDC (7.5 nmol of peptide injected) yielded three radioactive peptides (TA1, TA2, and TA12) which eluted at about 50, 75, and 210 mM NHrHOAc, respectively ( B ) . Recovery of radioactivity was always about 95%.
tides Cl-C3 (Table 111). Radioactivity in peptide TA12 was equally distributed between cleavage peptides C4 and Cl-C3. These results indicate that peptide TA12 is diphosphorylated and that peptides TA1 and TA2 were monophosphorylated at serine residues 5 and 12, respectively.
Inactivation and Site-specific Phosphorylation of A. suum PDC-When A. suum PDC was incubated with [Y-~'P]ATP, it was rapidly inactivated, and phosphorylation correlated directly with inactivation (Table IV and Fig. 3). No additional phosphorylation was observed after inactivation was complete, and about 9 nmol of 32P/mg of protein was incorporated when phosphorylation was complete (8.9 f 0.6 nmol for 12 different preparations of PDC). The initial rate of phosphorylation was more rapid (2-3-fold) in MOPS (pH 7.0) than in Tris-HC1 (pH 7.5), and 60 mM KC1 stimulated (2-3-fold) the initial rate of phosphorylation in both buffers. However, total 32P incorporation and the linear relationship between phosphorylation and inactivation were the same under all incubation conditions (data not shown). Pyruvate dehydrogenase is a tetramer with a subunit composition of a2p2 (23). Based on an estimate of 20 pyruvate dehydrogenase tetramers/ complex, about 3.5-3.8 mol of 32P/mol of pyruvate dehydrogenase was incorporated. The role of phosphorylation at sites 1 and 2 of El, and the inactivation of the complex are not clear since the complete phosphorylation of both sites occurred during inactivation, and phosphorylation of neither site correlated directly with inactivation (Table IV and Fig.  4). Initially, phosphorylation at site 1 was more rapid than at site 2; and at 30% inactivation, 64% of the total 32P incorporated was present in site 1. However, phosphorylation at site 2 was still substantial, and 20% of the 32P incorporated was present as monophosphorylated site 2, suggesting that prior phosphorylation at site 1 was not necessary for phosphorylation at site 2 to occur. At 50% inactivation, 50% of the total 32P had been incorporated into the complex, and 59% was present in site 1 and 41% in site 2.
Phosphorylation and inactivation of PDC were accompanied by a marked decrease in the mobility of El, during SDS-PAGE (Fig. 5A). During inactivation, the complete conversion of El, from a higher to a lower mobility form was observed. The altered mobility of El, was reversible. Incubation of fully phosphorylated A. s u m PDC with partially purified bovine kidney pyruvate dehydrogenase phosphatase, 10 mM MgC12, and 0.1 mM CaC12 for 60 min restored about 80% of the original PDC activity and converted most of the lower mobility form of El, to the higher mobility form. The bovine kidney pyruvate dehydrogenase phosphatase used in this study was about 20-fold less active with ascarid PDC than with bovine kidney PDC incubated under similar conditions (data not shown).
Autoradiography of PDC separated by SDS-PAGE during the time course of phosphorylation-inactivation revealed a band corresponding to the lower mobility form and, during the initial stages of phosphorylation, a second radioactive band intermediate in apparent molecular weight between the higher and lower mobility forms (Fig. 5B). At greater than 75% inactivation, this second radioactive band was not observed, even with different exposure times or the use of slower x-ray film (data not shown).

DISCUSSION
This study demonstrates that 2 serine residues in El, of the A. suum pyruvate dehydrogenase complex are phosphorylated by its endogenous pyruvate dehydrogenase kinase. The amino acid sequence of the tryptic phosphopeptide containing these phosphorylation sites has been established and is very similar to phosphorylation sites 1 and 2 identified in mammalian pyruvate dehydrogenase complexes (10)(11)(12). In mammalian pyruvate dehydrogenase complexes, three distinct phosphorylation sites have been identified, and inactivation is associated with the phosphorylation of site 1 (11). The ratio of site occupancy for up to 90% inactivation is 90:3:1 for sites 1-3, respectively; and phosphorylation of site 2 requires prior phosphorylation at site 1 (24). In the facultatively anaerobic yeast Saccharomyces cerevisiae, PDC does not appear to be phosphorylated in vivo, and pyruvate dehydrogenase kinase activity has never been detected (17,25). However, when S.
cerevisiae PDC is incubated with purified bovine kidney pyruvate dehydrogenase kinase, a single serine residue corresponding to site 1 is phosphorylated and results in the inactivation of the complex (17). In contrast, the role of the two

Tyr-Ser-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Ser(P)-Tyr-Arg
Mammalian"  Elution profile of cyanogen bromide cleavage peptides from tryptic phosphopeptide TA12 on reverse-phase high pressure liquid chromatography. Highly purified A. suum tryptic phosphopeptide (TA12; 5 nmol), derived as described for Fig.  1, was digested with cyanogen bromide as described under "Experimental Procedures" and separated by reverse-phase high pressure liquid chromatography using a linear gradient of ammonium acetate (0-60% in 0.1% trifluoroacetic acid). phosphorylation sites in the inactivation of ascarid PDC is not as clear. Initially, site 1 is phosphorylated more rapidly than site 2, but substantial amounts of monophosphorylated site 2 also are formed. More importantly, at 80% inactivation, P incorporation at sites 1 and 2 is equal, and no additional 32

TABLE 111
Cyanogen bromide digestion of tryptic phsphopeptides derived from the A. suum pyruvate dehydrogenase complex The A. suum pyruvate dehydrogenase complex (18 mg of protein) was inactivated to about 50% activity with [-y-32P]ATP. Partially phosphorylated complex was then completely digested with trypsin, and tryptic phosphopeptides (TA1, TA2, and TA12) were isolated by high pressure liquid chromatography as described for Fig. 3. Purified phosphopeptides were then digested with cyanogen bromide, and cleavage peptides were separated by reverse-phase high pressure liquid chromatography as described for Fig. 4. Individual peaks (Cl-C4) were collected and counted for radioactivity and analyzed for amino acid composition. phosphorylation is observed after inactivation is complete.

Phosphopeptide
No evidence for additional phosphorylation sites in the ascarid pyruvate dehydrogenase complex was ever observed. Either site 3 is not present in the ascarid complex, or, less likely, it is already phosphorylated in the purified complex. During isolation, the ascarid complex is routinely incubated with 10 mM MgC12 and 0.1 mM CaC& to achieve full activation while it still contains endogenous pyruvate dehydrogenase phosphatase activity. The close agreement between 32P content and amino acid analysis suggests that, at least for sites 1 and 2, few protein-bound phosphoryl groups remain after this activation step.
Phosphorylation of ascarid El, decreased its mobility during SDS-PAGE, so that during the inactivation of the complex by phosphorylation, the complete conversion of E l , from a higher mobility to a lower mobility form was observed. This correlation has not been described previously for mammalian  Aliquots were removed at the times indicated and either assayed spectrophotometrically for pyruvate dehydrogenase complex activity (0) or counted for protein-bound 32P (0) as described under "Experimental Procedures." Inset, relationship between percentage inactivation ( y axis) and nmol of 32P incorporated per mg of protein ( y axis) (0). El, or yeast El, phosphorylated with purified bovine kidney pyruvate dehydrogenase kinase, although some workers (17, 26-28) have described El, doublets on autoradiographs of phosphorylated brain homogenates separated by SDS-PAGE. In contrast, phosphorylation of the mammalian branchedchain a-ketoacid dehydrogenase does decrease its mobility during SDS-PAGE, and two radiolabeled bands have been identified by autoradiography which correspond to mono-and diphosphorylated dehydrogenase subunits (29). A similar situation appears to exist in ascarid PDC. Initially, two radiolabeled bands were observed which correspond to mono-and diphosphorylated El, subunits. As phosphorylation proceeds, 25 mM NaF, and 60 mM KC1 in a final volume of 6 ml. Aliquots were withdrawn and assayed as described under "Experimental Procedures" for enzymatic activity and total protein-bound phosphoryl groups (0) and the degree of phosphorylation at site 1 (0) and site 2 (0), as calculated from the amount of radioactivity in mono-and diphosphorylated tryptic peptides isolated as described for Fig. 1. A. suum complex was phosphorylated as described for Fig. 3, and aliquots were removed, treated, electrophoresed, and exposed to xray film as described under "Experimental Procedures." Lane 1, dephosphorylated pyruvate dehydrogenase complex; lanes 2-10, aliquots removed from the incubation mixture at 0.33, 0.67, 1, 2, 3, 5, 10, 15, and 30 min, respectively. Ela, dephosphorylated a-pyruvate dehydrogenase. Molecular weight standards (arrowheads) were: phosphorylase b (92,000), bovine serum albumin (67,000), ovalbumin (45,000), and carbonic anhydrase (31,000). the calculation of 3.5-3.8 mol of 32P incorporated per mol of pyruvate dehydrogenase.
The physiological significance of the differences in phosphorylation noted between ascarid and mammalian PDCs remains to be defined in studies using intact mitochondria, but the apparent lack of preference of the ascarid pyruvate dehydrogenase kinase for the inactivating phosphorylation site (Ser') may relate to the potential problem of maintaining PDC activity under the reducing conditions present in adult A. mum mitochondria. Whereas third-stage larval ascarids are aerobic and possess a functional tricarboxylic acid cycle, adult ascarids, which reside in the microaerophilic lumen of the small intestine, are predominantly anaerobic and rely on PDC to fuel their unique mitochondrial metabolism (2, 30). The elevated NADH/NAD and acetyl-CoA/CoASH ratios present in mitochondria of the adult favor the inactivation of PDC, even though it appears that stimulation of the ascarid pyruvate dehydrogenase kinase is less sensitive to these elevated ratios than the corresponding mammalian kinase (9,31). To maintain PDC activity under physiological conditions favoring inactivation, total PDC activity in adult ascarid body wall muscle is much greater than values reported from any other organism, in spite of the fact that the final specific activity of purified A. mum PDC is lower ((5 pmol of NADH formed per min/mg of protein) than values reported for yeast or mammals (10-20 pmol of NADH formed per min/mg of protein). Indeed, PDC in isolated A. mum muscle mitochondria is only 20% active, but because of its elevated amounts, substantial activity is still present even under conditions favoring pyruvate dehydrogenase kinase and substantial inactivation.' The reason for the lower specific activity of the ascarid complex is unclear (9,17,32). It is recovered in high yield, and the results of this study suggest that it is fully dephosphorylated during activation.