Phosphorylase Phosphatase

Vol. 259, No. 9, Issue of May 10, pp. 5857-5863, 19W Printed m U.S A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1984 hy The American Society of Biological Chemists, Inc

Phosphorylase Phosphatase INTERCONVERSIONOFACTIVE

AND INACTIVEFORMS* (Received for publication, September 27, 1983)

Emma Villa-MoruzziS, Lisa M. BallouQ, and Edmond H. FischerlI From the Department of Biochemistry SJ-70, University of Washington School of Medicine, Seattle, Washington 98195

the M,= 70,000 complex could also be activated by FA in a There is much evidence suggesting that the physiological Mg-ATP-dependent reaction resulting in the phosphorylation regulation of phosphorylase phosphatase is mediated in part of the inhibitory subunit (Ballou et al., 1983). The inactive through the action of two heat-stable inhibitors (forreviews, complex described here has several characteristics in common see Lee et al., 1980; Li, 1982; Ingebritsen and Cohen, 1983). with the Fc system and it has been suggested that they may Inhibitor 1, first described by Huang and Glinsmann (1975), be the same enzyme (Ballou et al., 1983). is active only after phosphorylationby CAMP-dependent proThe purpose of this study was to investigate the changes in tein kinase (HuangandGlinsmann, 1976a; Nimmoand conformation brought about by Mn”, FA, and inhibitor 2, Cohen, 1978) of a specific threonine residue (Aitken et al., using boththenative complex andthe isolated catalytic 1982). Hormones such as adrenalin and insulininfluence the subunit. ~~

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EXPERIMENTALPROCEDURES

* This work was supported by Grant AM 07902 from the National Institutes of Health and by a grant from the Muscular Dystrophy Association. Similar data havebeen obtained by S. Jurgensen, E. Shacter, C. Y. Huang, P. B. Chock, S.-D. Yang, J. R. Vandenheede, and W. Merlevede, and are reported in theaccompanying paper. The costs of publication of thisarticle were defrayed in part by the payment of page charges.Thisarticlemustthereforehe hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of an international fellowship from the American Association of University Women. On leave of absence from the Institute of General Pathology, University of Pisa, Pisa, Italy. S Supported in part by Molecular and Cellular Biology Training (;rant GM07270 from the National Institutes of Health. ‘To whom correspondence should be addressed.

Materials-Rabbit muscle phosphorylase b was purified according to Fischer and Krebs (1958) as modified by DeLange et al. (1968) and phosphorylase kinase according to Cohen (1973). The catalytic suhunit of CAMP-dependent protein kinase was isolated by the method of Peters et al. (1977). Rabbitmuscle phosphorylase phosphatase was prepared as described by Ballou et al. (1983), and FA was partially purified by a modification ofthe method of Vandenheede et al. (1980) as described by Ballou et al.(1983). Phosphatase inhibitor 2 was isolated by the procedure of Yang et al. (1981b) as modified by Hemmings et al. (1982). Phosphorylase a was prepared as described by Krebs et al. (1964). Radioactive 54Mn2+(7000 mCi/mmol) and 17“‘PIATP (3000 Ci/mmol) were from New England Nuclear. Tosylphenylalanylchloromethylketone-treatedtrypsinand limabean trypsin inhibitor were from Worthington, ATP and bovine serum

5857

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Phosphorylase phosphatase is isolatedas an inactive phosphorylation state of the protein (Foulkes et al., 1980, M , = 70,000 complex made up of a catalytic and a 1982; Khatra et al., 1980). Inhibitor 2, originally detected in regulatory subunit (inhibitor2). Separation of the two rabbit muscle phosphatase preparations (Huang and Glinscomponents yields the free catalytic subunit in a com- mann, 1976a, 1976b) has since been identified as a modulator pletely inactive state. It can be activated by Mn2+or protein co-purifying with a phosphatase termed FC (Yang et Co2+, not Mg2+or Ca2+. No metal ion is incorporated al., 1980, 1981a, 1983). Fc is completely inactive until treated use of 64Mn2+.The with a factor F A in the presence of Mg-ATP (Goris et al., during thisprocess, as shown by the inactive complex, but not the isolated catalytic subunit, 1979; Yang et al., 1980); inhibitor 2 is required for thisprocess can be activated by the protein kinase F A (Vanden- (Yang et al., 1981a; Vandenheede et al., 1981). F A possesses heede, J. R., Yang, S.-D., Goris, J., and Merlevede, W. protein kinase activityand is thoughtto beidentical to (1980) J. Biol. Chem. 255, 11768-11774), which glycogen synthase kinase 3 (Vandenheede et al., 1980; Cohen causes the simultaneous phosphorylation of inhibitor 2 et al., 1982). It has recently been reported that activation of and conversion of the catalytic subunit to an active the enzyme by FA is accompanied by a phosphorylation of conformation. The activated enzyme undergoes autodephosphorylation to produce a complex that is inac- inhibitor 2 (Hemmings et al., 1982; Ballou et al., 1983). We previously reported (Ballou et al., 1983) the purification tive even though the catalytic subunit is still in the of active form; in a slower step, it returns to its original an M , = 70,000 phosphorylase phosphatase consistingof a complex between a catalytic subunit ( M , = 38,000) and inhibinactive state.No such conversion occurs in the absence is 2 (Mr= 31,000). The enzyme was isolated in a completely of inhibitor 2, indicating that the regulatory subunit itor inactive form that could be fully activated by trypsin treatrequired for both the activation and inactivation reDuringthis reaction the actions. Complexes were preparedby adding inhibitor ment in the presence ofMn’+. 2 to various isolated catalytic subunits. Only the one regulatory subunit was rapidly destroyed, while the catalytic subunit was partially degraded to a species of M , = 33,000 reconstituted with the FA-activated species behaved like the native enzyme in that the catalytic subunit that was remarkably stable to further proteolysis.Limited underwent transformation to the inactive form, then tryptic attack in the absence of Mn2+produced an enzyme could be reactivated by FA. These data suggest that the that was still inactive until exposed to the divalent cation two subunitsmust interact in a highly specific manner (Brautigan et al., 1982). It was proposed that Mn2+served to to allow the structuralchanges accompanying the ac- expose the active site either by inducinga conformational tivation-inactivation process. Models are proposed for change in the catalytic subunit or by causing the release of the changesin conformation inducedby Mn2+or FA. an inhibitory fragment still bound to the enzyme. Like Fc,

5858

Control of Phosphorylase Phosphatase

RESULTS

Separation of the Inactive Catalytic Subunit and Its Activation by Mn'+-The earlier observation (Brautigan et al., 1982) that tryptic attack of the native' phosphatase in the absence of Mn'+ yields an inactive enzyme could be interpreted in three ways: ( a ) limited proteolysis inactivates the catalytic subunit, which can then be reactivated by Mn'+; ( b ) a fragment of the inhibitor remains bound to the active site and is released upon addition of the divalent metalion; or (c) the catalytic subunit in the native complex is in an inactive state that requires Mn2+ foractivity. Without a means to separate the two subunits, we could not distinguish among these possibilities. This problem was resolved by the use of FPLC; since the procedure can be carried out in 20-25 min, minimal losses of activity were encountered even at acidic pH. The two subunits of the M, = 70,000 inactive phosphatase were separated by ion exchange chromatography on a Polyanion SI column using FPLC at pH 6.0. The column profile in Fig. 1 shows that the catalytic subunit eluted ahead of the major inhibitory fractions. The inhibitory activity in fractions

' The abbreviations used are: FPLC, fast proteinliquid chromatography; MOPS, 4-morpholinepropanesulfonicacid; SDS, sodium dodecyl sulfate; 1-2, phosphatase inhibitor 2; Ei and E., inactive and activeconformations of thephosphatasecatalyticsubunit; EaMn, catalytic subunit activated by Mn'+; EaFA,catalytic subunit activated by FA; EaTr"", catalytic subunit activated by trypsin and Mn". is used to describe the M, = 'The term "native phosphatase" 70,000 inactive complex isolated according to our purification procedure. It does not imply that it is the form in which the enzyme exists in crude muscle extracts.

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IO 20 30 40 50 Fraction number FIG. 1. Ion exchange chromatographyof the inactive native phosphatase by FPLC.The enzyme (73 pg; 490 units after trypsin-

0

Mn'' activation) was applied to a Polyanion SI column and eluted with pH 6.0 buffer as described under "Experimental Procedures." Phosphatase activitywas assayed either directly (0)or in the presence of 0.5 mM Mn'+ (0).Inhibitory activity (A)was assayed as described under "Experimental Procedures" after dialyzing each fraction for 2 h at 4 "Cagainst 10 mM MOPS buffer, pH 7.0, containing 0.01% Brij35 and 15 mM 2-mercaptoethanol. The bar indicatespooled fractions; -" , NaCl concentration. The inset shows a silver-stained SDSpolyacrylamide gel (10%) containing 1 pg of native enzyme before FPLC ( l a n e 0) and catalytic subunit from pool A (lamA ) .

9 through 19 was not due to 1-2 since it was resistant to trypsin. The isolated catalytic subunit emerged in a completely inactive form referred to as Ei, consistent with the hypothesis that it is present as an inactive species in the original complex. Ei could be activated by Mn'' or Co2+,the latter being slightly more effective, but not by up to 10 mM M e or Ca2+; the Mn2+-activated form is designated EaMn. The activation was immediate a t 30 "C a t Mn'' concentrations of 0.1 to 1.0 mM; a t lower concentrations of the metal ion, fullactivation was not achieved (half-maximum response at 40 p~ Mn"). A model accounting for these effects is presented under "Discussion." The inset of Fig. 1 shows that thepooled enzyme fractions contained no inhibitor 2 and that the major protein present was of the samesize as the catalytic subunit (M, = 38,000) in the nativecomplex. Separation of the two subunits was more efficient at pH5.0, but the inhibitor remained so tightly bound to thecolumn that itcould not be eluted except after extensive washing with 2 M salt. Very little separation was achieved at pH 7.0 unless 1-2 was phosphorylated by FA as described later, indicating that there was no significant dissociation of the native complex to free catalytic subunits thatwould be activated by Mn'+. In fact, incubation of the complex at pH 5.0 or 7.0 for 1 h a t 30 "C or overnight a t 0 "C in the presence of 1 mM Mn'+ generated no activity. Any activity seen in the presence of the divalentmetal ion was taken as anindication that theM, = 70,000 enzyme had sustained some proteolysis. Actiuation by FA-h addition to the trypsin-Mn'+ activation described above, the nativeenzyme can alsobe activated by the protein kinase FA, as first described by Yang et al. (1980). The regulatory subunit becomes phosphorylated during this process (Hemmings et al., 1982; Ballou et al., 1983). The relationship between the appearance of enzyme activity and 1-2 phosphorylation is illustratedin Fig. 2. Upon addition of FA, phosphatase activity and 32Pincorporation increased simultaneously and reached a plateau after 15 to 20 min, a


albumin from Sigma, and Brij-35 from Pierce. The FPLC' system and anion exchanger (Polyanion SI)were from Pharmacia. Phosphorylnse Phosphatase Actioity-This wasmeasured by release of "P from phosphorylase a as described by Brautigan et al. (1980) and Ballou et al. (1983). The assay buffer contained 20 mM imidazole, pH 7.5, 20 mM glucose, 5 mM theophylline, 1 mM dithiothreitol, and 1 mg/ml of bovine serum albumin. Assays were carried out a t 30 "C under several conditions to measure the variousenzyme species: (a) direct assay (control) to determine the level of active phosphatase; ( b ) in the presence of 0.5 mM Mn'+ that activates the free inactive catalytic subunit; (c) after trypsin treatment (20 pg/ml followed 5 min laterby a 6-fold excessof lima bean trypsin inhibitor) that destroys inhibitor 2 and allows expression of all catalytic subunits in the active conformation; ( d ) after trypsin-Mn'+ treatment (as in c but in the presence of 1 mM Mn2+),to measure the maximal phosphatase activity; and (e) aftera 10-min incubation with 1 pg/ml of FA, 0.5 mM ATP, and 1 mM M e . Phosphatase activated by FA could then be assayed after any of the pretreatmentsdescribed above. A unit of phosphatase activity is defined as 1 nmol of Pi released per min. Inhibitor 2-This was determined by adding heat-treated material (3 min a t 100 'C) to 100-150 milliunits of trypsin-Mn'+-activated phosphatase. The phosphatase assaywas initiated 5 min later. Ion Exchange FPLC-This was performed on a column (0.8 X 4.8 cm) of Polyanion SI equilibrated in 10 mM MOPS, 0.1 mM EDTA, 0.01% Brij-35, 0.1% ethanol, and 1 mM 2-mercaptoethanol at the desired pH. The column was eluted with a 0-2 M NaCl gradient a t a flow rate of 1 ml/min, and fractions of 0.5 ml were collected. When the chromatography was carried out at pH 5.0, the fractions were immediately neutralized by collection into tubes containing 60pl of 200 mM MOPS, pH 8.3. The pooled fractions were dialyzed a t 4 "C against 20 mM MOPS, pH 7.0,0.01% Brij-35, 50% glycerol, and 1 mM dithiothreitol, then storeda t -20 "C. Polyacrylamide Gel Electrophoresis-Thiswasperformed in the presence of SDS according to Laemmli (1970) and Studier (1973). The slab gels were stained by the silver procedure of Merrit et al. (1981a, 1981b). Densitometric analyses of autoradiographs were performed on a Helena Quick Scan gel scanner. Protein was determined according to Bradford (1976) using bovineserum albumin as a standard.

Control ofPhosphatase Phosphorylase EDTA

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FIG. 2. Phosphatase activation and inhibitor 2 phosphorylation by F A . Native phosphatase (22 pg/ml) was incubated a t 30 "C

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IO 20 30 40 Fraction number

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after destruction of ATP by addition of hexokinase and glucose (data not shown). However, while phosphatase activity returned almost to zero, there was only a 35% decrease in bound phosphate. Readdition of excess M e brought about a reactivation of the phosphatasewith some further increase in 1-2 phosphorylation. In this particular experiment, the extent of phosphatase activation was only 20% as compared to the total activity measured after trypsin-Mn2+ treatment. The level of phosphate incorporated into the regulatory subunit was similarly low. The reason why dephosphorylation of 1-2 does not correlate with loss of activity is unclear; one possibility is that more than one sitebecomes phosphorylated, thoughonly one of these affects the activityof the enzyme. Activation of the phosphatase following 1-2 phosphorylation cannot be explained simply on thebasis of a dissociation of the complex, since this would release the catalytic subunit in its inactive state. Obviously, a conversion of the inactive subunit to its active form (Ei to E.) must occur during the phosphorylation reaction. This conversion was demonstrated by two independent means: first, by direct isolation of the catalytic subunit by FPLC, and second, by its release in free form following trypsin treatment. In the first approach, the inactive complex (1160 units after maximal activation by trypsin-Mn*+ treatment)was activated 40% (480 units) by FA and then subjected to FPLC at pH6.0 (Fig. 3). As expected, the free catalytic subunit emerged in a addition of 1 mM Mn2+caused completely active form (EaF*.); no further activation, but rather a slight inhibition that is always observed with the FA-activatedenzyme. The catalytic subunit recovered in pool A was nearly homogeneous as judged by SDS-gel electrophoresis (Fig. 3, inset, lane A ) ; on the other hand, the inhibitory fractions(pool B) were contaminated by other proteins, including some catalytic subunit as shown in

lane B. Onceagain, theinhibitory activityinfractions 7 through 19 was not due to 1-2 since it was trypsin-resistant. While 480 units were applied to the column, 1120 units were recovered this suggested that whereas the catalytic subunit had been converted entirely to an active conformation, 60% of the activityremainedin an unexpressed form. A likely explanation for the presence of such high levels of unexpressed EA is that autodephosphorylation had taken place and that some of the enzyme was now inhibited by dephosphoinhibitor 2. Identical results were obtained when the chromatography was carried out at pH 5.0, indicating that the low pH in itself did notinactivatethe catalytic subunit. The subunits of theFA-activated enzyme, unlike those in the unphosphorylated complex, could be separated by FPLC at pH 7.0. In the second approach, the catalytic subunit in the FAactivated complex was released following destruction of 1-2 by trypsin, thus allowing expression of all the E. present, including any that might be masked by the inhibitor. Fig. 4 shows that the nativecomplex a t 0 min contained no active catalytic subunit, since no activity was detected after trypsin treatment. Within 5 min after addition of FA, a considerable conversion of Ei to E, hadoccurred, even though only a fraction of the totalactivity was expressed (compare speckled to solid bars).While the level of phosphatase activity remained low, almost all of the catalytic subunithad been converted to E. within a half-hour. Unexpectedly, the maximal activity as measured after trypsin-Mn" treatment (open bars) also increased during FA activation; the reason for this increase is not yet understood. After addition of excess EDTA, the system slowly returned to an inactive state; separation of the catalytic subunit by FPLC at pH5.0 confirmed that most of it had been reconverted to it's original inactive form.


in 5 mM MOPS, pH 7.0, 15 mM 2-mercaptoethanol, 0.5 mM ATP, FIG. 3. Ion exchange chromatography of FA-activatedM. = and 1 mM M e ; after 5min ( t = O), FA was added to afinal 70,000 phosphatase by FPLC. Native enzyme (172 pg; 1160 units concentration of 6.2 pg/ml. Aliquots were diluted 20-fold and assayed after trypsin-Mn2+ treatment) was incubated for 30 min a t 30 "Cwith immediately (m). Excess EDTA (2mM final concentration) was added 163 pg of partiallypurified FA in 5 mM MOPS buffer, pH 7.0, a t 31 min, and M e (3 mM final concentration) was re-added a t 66 containing 15 mM 2-mercaptoethanol, 0.5 mM ATP, and 1 mM M e . min. In an identical experiment using [y-"PIATP (210 cpm/pmol), The activated enzyme (480 units) was applied to aPolyanion SI aliquots containing 0.55 pg of phosphatase were subjected to SDS- column and eluted with pH 6.0 buffer as described under "Experipolyacrylamide gel electrophoresis. The "P content of inhibitor 2 (0) mental Procedures." Phosphatase activity was assayed directly (m) was determined by scanning autoradiographs of the gel. or in the presence of 0.5 mM Mn2+ (0).Inhibitory activity (A)was assayed as described in the legend of Fig. 1. The bars indicate pooled rate depending on FA concentration. Addition of excess EDTA fractions containing catalytic subunit ( A ) and inhibitor 2 (€0; - - -, to block the kinase reaction resulted in an immediate dropin NaCl concentration. Theinset shows a silver-stained SDS-polyacrylgel (10%) containing: 1pg of native enzyme before FPLC (lane activity due to autodephosphorylationof the inhibitor by the amide 0); 1 pg of catalytic subunitfrom pool A (lane A ) ; and 1 pg of inhibitor activated phosphatase.A similar loss of activity was observed 2 from pool B (lane B ) .

5860


of Mn2+. Half-livesfor the various species at 30 "C were approximately 2-3 min for Ei, 1 h for EaMnin the presence of 1 mM Mn2+ (16min with 0.3 mM Mn2+), and35 min for E>. 250 EaMnwas much more stable at 0 "C, with half-lives of 40 h and 7 days in the presence of 0.3 and 1.0 mM Mn2+,respectively. Likewise, Ei was readily destroyed by trypsin, whereas 200 EaMn andE a Fwere ~ activated approximately 20% after 5 min a t 30 "C with a 5-fold excessof the protease. The presence of 150 1 mM Mn2+ duringproteolysis always increased the stability of the enzyme. There is strong evidence that EaMnis different from E a F ~ . 100 First, when EaMn was subjected to FPLC at pH 5.0, it emerged in an inactive form that could be reactivated by Mn2+. This is in contrast to E>, which remained fully active under the 50 same conditions. It is difficult to ascribe this inactivation to removal of a divalent metal ion from the enzyme since FPLC at pH 7.0 inbuffer containing 0.1 mM EDTA yielded a completelyactive subunit. Furthermore, the trypsin-Mn'+0 5 27 45 62 activatedphosphatase (EaTr"") remained50%active after Minutes FPLC at pH5.0 in buffer containing EDTA and 100% active ~ FIG. 4. Formation of expressed and unexpressed active at pH 6.0. The second difference between EaMn andE a F is that enzyme freshly activated by Mn2+ could be inhibited up forms of phosphorylase phosphatase following FAtreatment. The native phosphatase (21 pg/ml) was incubated a t 30 "C in the to 90% by a 2-fold excess of EDTA, whereas no inhibition of phosphatase assay buffer containing 0.5 mM ATP and 1 mM Mg2+. EaFA or of the trypsin-Mn'+-activated enzyme was observed. After 15 s, FA wasadded to a final concentration of 31 pg/ml to This EDTA inhibitionwas lost after prolonged incubation in initiatetheactivation. After 30 min,excess EDTA (3 mM final the presence of Mn'+, supportingthe view previously exconcentration) was addedto block thekinasereaction. Aliquots removed at the time points indicated were diluted 20-fold and assayed pressed (Brautigan et al., 1980, 1982) that the enzyme is not immediately under the following conditions (see also "Experimental a metalloprotein. Additional evidence in this regard was obProcedures"): open bars, aftertrypsin-Mn2+treatment, giving the tained by the use of radioactive Mn2+.Purified inactive catamaximal phosphatase activity; speckled bars, after incubation with lytic subunit (50 pmol) was incubated with 1 mM s4Mn2+(190 trypsin alone, to measure all catalytic subunits in the active conforcpmjpmol) for 2 min a t 30 "C. The fully activated enzyme mation (expressed andunexpressed);and solid bars, directassay was then subjected to gel filtration on Sephadex G-25 (fine). (control), giving the level of active phosphatase. The eluted material was still 80% active but contained less than 0.03 mol of Mn2+/mol of catalytic subunit. Similar data were obtained when the purified native complex was activated with trypsin in thepresence of '*Mn2+.A possible mechanism for the effects of Mn2+ and EDTA is presented under "Discussion." Conversion of the Catalytic Subunit to the Inactive Conformation in the Presence of Inhibitor 2-The differences between thethree active catalyticsubunits EaF~,EaMn,and EaTr"" were even more striking when their interaction with 1-2 was examined. There are two ways by which the activity of the phosphatase can be suppressed, direct inhibition by I2 and conversion to the inactive conformation (E, to E;). It was of interest to see whether these two processes are interrelated. As indicated earlier, none of the isolatedactive species spontaneously convertsto the inactive form E;; however, this 0 10 20 conversiondoes occur in theFA-activatednative complex upon dephosphorylation, implying an involvement of 1-2 in Minutes theinactivation process. To confirm thisassumption,the FIG. 5. Heat stability of phosphatase catalytic subunits. Iso- active catalytic species were incubated with the regulatory lated catalytic subunits were incubated a t 30 "C and assayed without subunit at 30 "C; in each case, an immediate inhibition of 75 dilution. E,, purified as described in the legend of Fig. 1, was either to 90% was observed. The reconstitutedcomplexes, exhibiting incubated in the absence of Mn2' and then assayed in the presence sizes similar to that of the native enzyme ( M , = 70,000) by of 0.5 mM Mn2+ (0)or convertedto EaM" andincubatedinthe legend gel filtration, were then assayed after trypsin treatment to presence of 1 mM Mn2+(0).EaFA, prepared as indicated in the reveal any conversion of E, to E, (Fig. 6). The datashow that of Fig. 3, was incubated without divalent metal ions(W). thetrypsin-Mn2+-activatedcatalyticsubunit remained entirely in the active conformation, since all the activity was Properties of the Inactive and Active Catalytic Subunitsrecovered after trypsin treatment. This is in marked contrast The inactive catalyticsubunit E; is extremely unstable at t o E a Fwhich ~, was completely converted to Ei in the presence 30 "C as compared to theMn"- or FA-activated subunits(Fig. of inhibitor 2, with a tl,xof approximately 12 min. EaMn,on the 5 ) . The loss of activity observed with the activated species other hand, was only partially (about 55%) converted to the was not due to conversion to Ei, but probably to anirreversible inactive form after 90 min. The inactive complexes obtained after 60 min were also denaturation since no reactivation occurred in the presence EDTA



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TABLEI Reactivation of various reconstituted catalytic subunit-inhibitor2 complexes by FA ~Phosphatase species" Assay conditions

~

_

_

EaT'M n

+ 1-2

27

4

+ 1-2

_

E*F"

E.M"

+ 1-2

Ei

+ 1-2

the active conformation. Remarkably, the regulatory subunit conferred considerable stability to the system, since free E, would havebeen completely denatured under these conditions. Phosphorylation of Inhibitor 2 by CAMP-dependent Protein Kinase-Treatment of the native complex (0.6 pg/ml) with a 10-fold excess of the pure catalytic subunit of CAMP-dependent protein kinase for 15 min resulted in no activation of the phosphatase. Furthermore,when the phosphorylatedcomplex was subjected to FPLC at pH 7.0, all the catalytic subunit was recovered in the inactive conformation.There was a slightly better separation of the two subunits as compared to the native enzyme. On the other hand, nodissociation of the phosphorylated complex was detected in solution, since no activity could be measured after prolonged incubation in the presence of Mn". The level of phosphate introduced into the regulatory subunit was greater than that observed with FA, probablybecause noautodephosphorylation occurred. The sites phosphorylated by the two kinases must be different in view of the differences in enzymatic responseand because the amount of '"P introduced was additive. No phosphorylation was observed in the presence of excess phosphorylase kinase. DISCUSSION

Resolution of the phosphorylase phosphatase complex into its two components under nondenaturingconditions has confirmed that the catalytic and regulatory subunits have molecular weights of 38,000 and 31,000, respectively. More importantly, it has also been confirmed that the catalytic subunit indeed exists in an inactive state in the nativecomplex. As of now, only one treatment, namely exposure to Mn2+or Co'+, is known to activate the free inactive catalytic subunit Ei. The behavior of the enzyme in the presence of Mn'+ and the variable levels of inhibition caused by EDTA suggest that the reaction might proceed in two steps, as follows,

" "

treatment No (control) After trypsin After FA, Mg-ATP After FA,Mg-ATP then trypsin After trypsin-Mn2+

98 38 92

4 24 84

11 36 20 21

1

2 7 15

Mn2+ Ei

.

Mn2+ EDTA

. [E. Mns+Iaf

EaMn

The first step involves the formation of an active enzymeMn'+ complex that can be disrupted by EDTA, thereby ex100 (so)* 100 (126)* 100 (59)* 100 (69)b plaining the90% inhibition observed shortly after additionof "Incubation was carriedout for 60 minundertheconditions Mn2+. Ina slower second step, the catalytic subunit undergoes described in the legend of Fig. 6; Mn2+(0.5 mM) was present only in a change inconformation to yield the active species EaMn, the EaMn reaction mixture. Values are expressedas per centof activity which is no longer susceptible to EDTAinhibition. This form obtained after trypsin-Mn2+ treatment. is also much more stable to trypsin and toprolonged incubaNumbers in parentheses represent the per cent activity remaining a t 60 min as compared to the enzyme activity before addition of tion at 30 "C. The change in conformation can be reversed inhibitor 2. either by subjecting the catalytic subunit toFPLC at pH 5.0 or by incubating it at 30 "C with inhibitor 2. Trypsin treattreated with FA to see if reactivation might occur (Table I). ment in the presence of Mn2+ converts the phosphatase into The complex containing EaT'"" underwent very poor reacti- a partially proteolyzed species EaT'"" that is not inhibited by vation, even thoughtheinhibitor became phosphorylated. EDTA. The irreversible activation by Mn2+described earlier While the complex originally made with E? was reactivated (Brautigan et al., 1980) was undoubtedly due to the presence only about 2076, a subsequent tryptic attack showed that the of a similar form. kinase treatment had restored the catalytic subunit almost The hypothesis that Mn2+ activation results from a conforentirely to its active conformation. The data obtained with mational change is based on the observation that no metal EM"are more difficult to interpret. On the one hand, there ion is required to maintain the enzyme in the active form; was little apparent reactivation of the phosphatase; on the earlier data of Brautigan et al. (1980) showing no incorporaother hand, the two values obtained after F A treatment are tion of s4Mn2+intotheactivatedphosphatase have been well below those obtained after trypsin treatment. This can confirmed using the highly purified EaT'"" and EaMnspecies be attributed in part to the fact that EaMn is inhibited by the described here. On the other hand, we cannot exclude the adenine nucleotides present in the reaction mixture. possibility that Mn2+causes a covalent modification,perhaps Table I also shows the effect of inhibitor 2 on the properties involving sulfhydryl groups of the catalytic subunit. Such a of the inactive catalytic subunit Ej.Very little reactivation of reaction hasbeen proposed by Yan and Graves (1982). the reconstituted complex was observed after FA treatment, The interconversion of inactive and active conformations and only about 15%of the catalytic subunitwas converted to is also observed during activation of the phosphatase by FA, ~

~

" " " "


FIG. 6. Conversion of active catalytic subunits to the inactive form Ei by inhibitor 2. Approximately 0.5 pg (9-11 units) of (a), EaM"(01,or EaF*(m) was incubated a t 30 "C with 1.4 pg of 1-2 in 225 pI of 5 mM MOPS, pH7.0, and 15 mM 2-mercaptoethanol. The E F solution also contained 0.5 mM MnZ+.Aliquots were diluted withphosphataseassay buffer andassayedaftertreatment with trypsin (E.) or trypsin-Mn2+ (Ebm,). The E./E,,I ratios were normalized tothe values obtained a t 0 min. E,F* was prepared as described in the legend of Fig. 3; EaMnwas produced by exposing Ei (prepared as described in the legend of Fig. 1) to 0.5 mM Mn2+. The trypsin-Mn'+-activated enzyme was preparedby incubating 170 pg of the native phosphatase with 1 mM Mn2+ and 10 pg/ml of trypsin for 10 min a t 30 "C; EaTr~Mn was then isolated by FPLC at pH 6.0 as described under "Experimental Procedures."

5861

5862


I

II

m

symbols represent the catalytic subunit in its inactive (circle) and active (square) conformations; inhibitor2 is represented by the filled circle. Form I is the native inactiveenzyme.

Acknowledgments-We thank Curtis Diltz and Richard Olsgaard for their expert technical assistance and Pamela Holbeck for typing the manuscript.


mings et al. (1982) showing a continued increase in enzyme activity while the 32Pcontent of inhibitor 2 decreases. Surprisingly,activation with FA appears to increase the total phosphatase activity as measured after trypsin-Mn'+ treatment (see Fig. 4). One possible explanation is that the native enzyme is partially inactivated by the protease before it has had time tobe converted by Mn'+ to the trypsin-stable active conformation. By contrast, EaF*produced during FA activation does not undergo this loss of activity. If this were the case, trypsin-Mn'+ treatment would not truly express the total phosphatase activitywhen applied to the nativecomplex or to any species containing E;. The differences in behavior observed between the enzyme as isolated and the reconstituted complexes (see Fig. 6 and Table I) suggest that the catalytic and inhibitory subunitsin the native phosphatase must interactin a very specific manner. The reconstituted [Ei.I-2]complex, for instance, cannot be activated by FA, as if 1-2 were bound to a different site that does not allow activation of the enzyme even though phosphorylation takes place. An additional piece of evidence supporting thisconclusion is that the FA-activated native complex can be inhibited by a second molecule of inhibitor 2 (data not shown).Whereasthisinhibition could be attributedto a displacement of the phosphorylated inhibitor by the dephosphoprotein,such apossibilityseemsunlikelybecause the inhibition is immediate and cannotbe reversed by FA. Therefore, a more probableexplanation is that the second molecule of inhibitor binds toa different site on the catalytic subunit. Thereconstituted [EaFA.I-2] complex, ontheotherhand, behaves like thenative enzyme inthat (a) thecatalytic subunit can return to the inactive conformation and ( b )it can be readily reactivated by FA.These resultswould indicate that the regulatory subunit can interact with the active conformation of the catalytic subunit at the correct site, which may be inaccessible in isolated E;. However, not all active conformations will necessarily interact properly with the inhibitor, since EaMn was not converted completely to Ei andFA did not reactivate thisinactive complex. It is interesting to note that the enzyme thathassustained limitedproteolysis during trypsin-Mn" treatment can no longer be reconverted to the inactive form by incubation with inhibitor2. The -M, = 5000 fragment (Ballou et al., 1983) that has been clipped during the reaction could either participate in the inactivation process or be directly involved in shielding the catalyticsite. In conclusion, the picture that emerges from this study is thatthenativephosphataseis subjected to two levels of inhibition: first, it is bound to inhibitor 2, and second, the catalytic subunit exists in an inactive conformation. In that sense, the enzyme is unlike the CAMP-dependent protein kinase, in which dissociation of the inactive complex brings about the liberation of active catalyticsubunits.Whyare there two superimposed levels of inhibition? One possibility is that it could provideafail-safemechanism toprevent activation of the enzyme, for instance, after the accidental destruction of the inhibitory subunit by intracellularproteases. The phosphatase is a hydrolytic enzyme that would create havoc within the cell if allowed to operate unchecked. Alternatively, this double level of inhibition could be part of a higher order of control in which hormones such as insulin inactive could participate. Insulin could cause the activation ofFA, could block the dephosphorylation of the activated complex, or could allow expression of the potential activity hidden in FIG. 7. Proposed model for the activation-inactivation cycle the inactive form I11 of Fig. 7. of the M. = 70,000 phosphatase during FAtreatment. The open

as illustrated by the model in Fig. 7 . The regulatory subunit of the native inactiveenzyme (form I) is phosphorylatedat a specific site by F A ; during thisreaction the Ei toE, transition takes place and the phosphatasebecomes active (form 11). We have no evidence that E, dissociates from 1-2 at this step, as suggested by Hemmings et al. (1982). Since addition of phosphorylated inhibitor2 to Ei causes no activation of the enzyme (data not shown), it can be assumed that every time 1-2 is phosphorylated it undergoes a conformational change that is transferred to the catalytic subunit. There is no evidence that it acts simply by displacing a proteolytic fragment of 1-2 still bound to the catalytic subunit and lacking the phosphorylation site (Resink et al., 1983). According to this model, FA cannot activateisolated E,, and thisis indeed what is observed. The active form I1 undergoes autodephosphorylation to produce form 111, an inactive complex in which thecatalytic subunit is still in the active conformation. Thelevel of 111 can be measured by the use of trypsin, which destroys the regulatory subunit and releases the active catalytic subunit. Complex I11 can either be reactivated by phosphorylation or it can return to the native inactive stateby a slow conversion of E. to E;. The latter stepoccurs only in the presence of 1-2. Since the E, to Ei transition is slow, form I11 accumulates; this situation prevails at low levels ofFA, where the rate of formation of I1 is decreased. Under these conditions, while only a fraction of the total phosphatase activity is expressed, the proportion of catalyticsubunitinthe active conformation increases with each phosphorylation-dephosphorylationcycle until nearly all of it may be converted to theactive form E,. The requirement for inhibitor 2 for both activation of the enzyme and its return to the inactive conformation supports the view expressed by Yang et al. (1981a) that the regulatory subunit plays a modulator role. The slow return of the active to the inactive conformation of the catalytic subunit (form 111 -+ I) indicates that phosphorylase phosphatase displays hystereticcharacteristics. The same might apply to the regulatory subunit. Inhibitor 2 might remain in a noninhibitory state for some time after dephosphorylation. If this were the case, the enzyme would form a transient intermediate (I1 -+ 11* "+ 111) which would remain active even though the regulatory subunit would be dephosphorylated. This might also explain the data of Hem-

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