Regulation of Liver Hydroxymethylglutaryl-CoA Reductase by a Bicyclic Phosphorylation System*

(Received for publication, December 21, 1979, and in revised form, October 7, 1980)

Thomas S. Ingebritsen$, Rex A. Parker, and David M. Gibson From the Department o f Biochemistry, Indiana University School of Medicine, Indianapolis, Ind ium 46223

Protein phosphatase C was purified 1500-fold from rat liver by a six-step procedure including a fractiona- tion step with 80% ethanol at room temperature and two successive chromatographic separations on DEAE- Sephadex. This preparation restored hydrox~ethyl - glutaryl-CoA (HMG-CoA) reductase activity in liver microsomes pretreated with MgATP and also inacti- vated HMG-CoA reductase kinase.

The relative rates of activation of HMG-CoA reduc- tase, inactivation of reductase kinase, and dephospho- rylation of phosphorylase a were constant throughout each step in the purification. Each of the reactions catalyzed by the purified phosphatase were inhibited in parallel by sodium fluoride (Kj = 3 to 4 mM),

Inactivated reductase kinase was fully reactivated by MgATP and an enzyme, termed HMG-CoA reductase kinase kinase, which was separated from HMG-CoA reductase kinase by chromatography on DEAE-cellu- lose.

These new studies support the thesis (Ingebritsen, T. S., Lee, H.-S., Parker, R. A., and Gibson, D. M. (1978) Biochem. Biophys. Res. Commun. 81, 1268-1277) that liver HMG-CoA reductase is controlled by a bicyclic system in which both HMG-CoA reductase and HMG- CoA reductase kinase are regulated by reversible phos- phorylation.

More than 80% of the HMG-CoA reductase kinase and HMG-CoA reductase kinase kinase activities in rat liver extracts were found in the cytosol in contrast to HMG- CoA reductase which is firmly bound to liver micro- somes. The activities of HMG-CoA reductase kinase and HMG-CoA reductase kinase kinase were not influ- enced by CAMP or the specific heat-stable inhibitor of CAMP-dependent protein kinase. Similarly, high con- centrations of CAMP-dependent protein kinase (in the presence of CAMP) were unable to catalyze either the inactivation of HMG-CoA reductase or the reactivation of HMG-CoA reductase kinase. These results are dis- cussed in the light of the recent observation (Ingebrit- sen, T. S., Geelen, M. J. H., Parker, R. A., Evenson, K. J., and Gibson, D. M. (1979) J. Biol. Chern. 254, 9986- 9989) that HMG-CoA reductase was inactivated, and HMG-CoA reductase kinase activity was activated in isolated hepatocytes in response to glucagon and CAMP.

* This research was supported by grants from the National Insti- tutes of Health (AM19199 and AM21278), the American Heart As- sociation, Indiana Affiliate, and the Grace M. Showalter Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

dee, DDl4HN, Scotland, United Kingdom. $. Present address, Department of Biochemistry, University of Dun-

Cholesterol biosynthesis in mammalian liver is regulated principally through the microsomal enzyme hydroxymethyl- glutaryl-CoA reductase (NADPH, EC 1.1.1.34), which cata- lyzes the rate-limiting reaction in this pathway (1-4). Previous studies have shown that reductase' can be interconverted in vitro between an active and an inactive form (1, 2, 5-8). Inactivation requires ATP and Mg" and an enzyme termed reductase kinase present in both cytosol and microsomes.

We recently presented evidence that reductase kinase is also regulated by reversible phosphorylation (1, 2, 8). A mi- crosomal preparation of reductase kinase became inactive a t 37°C unless the protein phosphatase inhibitor sodium fluoride was present. The inactivated reductase kinase was partially reactivated by incubation wit,h ATP and Mg"' in a low ionic strength buffer. It was not clear, however, whether this was catalyzed by HMG-COA' reductase kinase (autophosphoryia- tion) or by another kinase enzyme. A partially purified protein phosphatase (termed protein phosphatase C (9)) from rat liver was capable of catalyzing both the inactivation of reductase kinase and the reactivation of HMG-CoA reductase. This suggested that both of these reactions involved a dephospho- rylation mechanism. These experiments did not, however, eliminate the possibility that the two reactions were catalyzed by contaminating enzymes in the phosphatase preparat,ion.

In the present communication, data are presented which show that both the reactivation of reductase and the inacti- vation of reductase kinase by the protein phosphatase C preparation are indeed catalyzed by protein phosphatase. Inactive reductase kinase is shown to be fully reactivated by a second protein kinase termed reductase kinase kinase which is distinct from reductase kinase. Neither of the kinase en- zymes can be identified with cA~P-dependent protein kinase.

MATERIALS AND METHODS

Animals Male Wistar rats were used in all studies. Animals employed in the

preparation of HMG-CoA reductase were maintained for at least 2 weeks on a controlled lighting schedule in which the room was illuminated from 1500 to 0300 daily. Rats were killed a t 0900 ( i e . a t the peak of the reductase diurnal cycle).

Preparation of Different Forms of HMG-CoA Reductase, Reductase Kinase, and Reductase Kinase Kinase

Active ~ i c r o s o m a ~ H M ~ ~ ~ o A Reducstase Deficient in Reductuse Kinase-Microsomes were isolated from rat liver (6) and resuspended in Buffer A (1 mM EDTA, 250 mM NaC1, 5 mM dithiothreitol, and 50 mM imidazole, pH 7.4) at a protein concentration of 5 to 10 mg/ml. The suspension was incubated for 2 h a t 37'C to inactivate reductase kinase (6-8) and then centrifuged a t 100,OOO X g for 60 min. The microsomal pellet was resuspended in Buffer A (5 to 10 mg of protein/

_" ' HMG-CoA reductase is referred to as reductase. e The abbreviation used is: HMG-CoA, hydroxymeth~lglutaryl-

CoA.

1138

HMG-CoA Reductase Kinase, Kinase Kinase, and Phosphatase Enzymes 1139

ml), and the extraction procedure was repeated two more times to remove inactive reductase kinase. The final microsomal pellet was resuspended ( 5 to 10 mg of protein/ml) in Buffer B (1 mM EI3TA, 250 mM NaCI, 5 mM dithiothreitol, and 50 mM orthophosphate, pH 7.4). frozen in liquid nitrogen, and stored at -70°C until used. This preparation was routinely employed as a substrate in the reductase kinase assay.

Inactive Microsomai HMG-CoA Reductase-Microsomes were isolated from rat liver (6) and resuspended in Buffer B (1 to 2 mg of p r o ~ e i n / m ~ ~ . T h e suspension was incubated with 4 mM MgCL and 2 mM ATP for 20 min a t 37”C, resulting in an 80 to 95% decrease in reductase activity. The incubation mixture was then centrifuged at 100,OOO x g for 1 h, and the resulting microsomal pellet was resus- pended in Buffer A (5 to 10 mg of protein/ml). The inactive micro- somal reductase was frozen in liquid nitrogen and stored at -70°C until use in the assay for reductase phosphatase.

Active Cytosolic Reductase Kinase--Rat liver was homogenized in 3.5 volumes of a solution containing 300 mM sucrose, 10 nlM 2- mercaptoethanol, and 50 mM NaF. The homogenate was centrifuged twice at 12,OOO X g for 20 min each time, and the final supernatant was further centrifuged at 100,OOO X g for 1 h. The resulting super- natant (cytosol) was frozen in liquid nitrogen and stored a t -70°C as active cytosolic reductase kinase.

Extraction of Active Reductase Kinase from Microsomal MPm- branes-Rat liver was homogenized and fractionated as described for the preparation of active cytosolic reductase kinase (above). The microsomes obtained as a by-product of this procedure were resus- pended in Buffer A supplemented with 50 mM NaF (5 to 10 mg of protein/ml). Microsomes were extracted two times with this buffer as described for the preparation of reductase kinase-deficient micro- somal HMG-CoA reductase. The two extracts were comhined, con- centrated 10-fold by ultrafiltration using an Amicon PMlO membrane, frozen in liquid nitrogen, and stored at -70°C. This preparation was routinely used as a substrate in the reductase kinase phosphatase assays (see below).

Inactive Reductase Kinase-This form of the enzyme was obtained as a by-product during the preparation of active microsomal reductase deficient in reductase kinase (see above). The first two microsomal extracts obtained in this procedure were combined, concentrated 10- fold by ultrafiltration using an Amicon PMlO membrane, frozen in liquid nitrogen, and stored at -70°C until used in the assays for reductase kinase kinase.

Reductase Kinase Kinase-This enzyme was prepared in two ways. Since the majority of liver reductase kinase kinase activity is in the cytosol (see under “Results”), the active cytosolic reductase kinase preparation (see above) was used as one source of reductase kinase kinase. A particulate preparation of reductase kinase kinase (containing microsomes and glycogen) was employed in several stud- ies. Although the latter preparation had a low specific activity (0.04 unitsimg) it was virtually devoid of reductase kinase activity. The method for obtaining this preparation (previously termed the “gly- cogen.protein complex” has been described elsewhere (1, 10).

Other Protein Preparations

Protein Kinase Inhibitor-The specific heat-stable protein inhib- itor of CAMP-dependent protein kinase was purified from rabbit skeletal muscle using the procedure of Nimmo and Cohen (11) up to and inc~uding the DEA~-celIulose step.

Protein Phospha~ase C-The enzyme was purified from rat liver using the method of Brandt et al. (9) up to and including the second DEAE-Sephadex chromatography step. The purified phosphatase was stored at -28’C in a buffer containing 2 mM EDTA, 0.2 mM dithiothreitol, 60% glycerol, and 20 mM imidazole, pH 7.4. A summary of the purification is given in Table I. The definition of the phospho- rylase unit is that of Brandt et a1 (9): 1 unit of phosphatase activates 0.2 mg of phosphorylase a (1 nmol of dimer) per min a t 37OC.

Subcellular Fractionation of Reductase Kinase a n d Reductase Kinase Kinase-In order to determine the subcellular distribution of reductase kinase, rat liver was homogenized and fractionated as described above for the preparation of active cytosolic reductase kinase and for the extraction of active reductase kinase from the microsomes. The fractions obtained (see Table 11) were assayed for reductase kinase activity and for histone kinase activity (see below). In the latter assay fractions were incubated in the presence of either CAMP (2 p l ) or an excess of the protein kinase inhibitor. The difference between these two values for histone kinase activity was designated cAMP-dependent protein kinase activity.

For the subcellular distr~bution of reductase kinase kinase, the liver

TABLE I Co-purification of phosphorylase phosphatase, reductase

phosphatase, a n d reductase kinase phosphatase acticities Protein phosphatase C was purified from 305 g of rat liver by the

method of Brandt et al. (9). A 1500-fold purification of the phospha- tase was attained. The indicated ratios are either reductase phospha- tase divided by phosphorylase phosphatase or reductase kinase phos- phatase divided by phosphorylase phosphatase. Reductase phospha- tase and reductase kinase phosphatase activities could not he reliably quantitated in the crude hoInogexlat,e because of the presence o f factors which severely inhibit HMG-CoA reductase (14. 15). Defini- tion of units is found under “Materials and Methods.”

Phosphorylase Reductase ki -

phosphatase phosphatase nase phospha- Fraction .- ”~ - -

tase

Total IJnits/ IJnits/ (!nits/ unltq me Il lZ me

Homogenate 15,600 0.23 S u p e ~ a t a n t , pH 3,960 0.17 0.017 0.10 0.11 0.6

Ammonium sulfate 4,070 0.25 0.VB 0.1 1 0.26 1.0

Ethanol precipita- 7,559 3.04 0.33 0.11 2.63 0.9

Ammonium sulfate 4,435 2.88 0.30 0.10 2.59 0.9

DEAE-Sephadex, 1,746 91.9 9.05 0.10 92.7 1.0

DEAE-Sephadex, 970 350.3 29.6 0.09 325.9 0.9

5.8

pellet, 0-70’55

tion

pellet, 40-75%!.

Column I

Column 2

TABLE I1 Comparison of the subcellular distributions of reductase kinuse

and reductase kinase kinase with that of CAMP-dependent protein kinase

Rat liver was subjected to subcellular fractionation as described under “Materials and Methods.” Activities are expressed as a per- centage of the activity in the 1 2 , W X g supernatant. Total activities in this fraction were: CAMP-dependent protein kinase, 4450 units/g of liver; reductase kinase, 51.0 units/g of liver; and reductase kinase kinase, 14.0 units/g of liver. These activities represented 98%>, 704, and 52’55, respectively, of the activities in the crude homogenate.

” ””

Activity -_

Fraction IiPduc- Reductase p ~ ~ ~ ~ ~ ~ i . tase ki- kinase ki-

nase naSe nase

12,OOO X g Supernatant 100 100 100 Cytosol 86 78 83 Microsomes 12 17 4 Combined microsomal extracts I 2 13 3 Extracted microsomes 1 1 1

””

~.

was homogenized and fractionated as described for reductase kinase, except that the microsomes were extracted and finally resuspended in 1 mM EDTA, 30 mM 2-mercaptoethanol, and 5 mM Tris, pH 7.4.

Enzyme Assays

HMG-CoA Reductase Assay-The method for assaying this en- zyme has previously been described in detail (6, IO). The assay was started by addition of 0.05 ml of a co-factor-substrate mixture con- taining sufficient EDTA and NaCl to bring the final concentrations to 30 mM and 250 mM, respectively. One unit of HMG-CoA reductase was defined as that amount which catalyzed the formation of I nmol of mevalonic acid per min a t 37°C.

HMG-CoA Reductase Kinase Assay-In this assay, kinase-defi- cient microsomal reductase (200 to 400 milliunits per assay) was incubated with fractions containing the kinase in a solution (0.1 ml, final volume) consisting of Buffer B supplemented with 4 mM MgClr, 2 mM ATP, and 50 mM NaF. The react,ion was initiated by the addition of MgATP 2nd terminated by adding the reductase assay co-factor-substrate .nixture. The reaction was linear with time and reductase kinase concentration (up to 50% inactivation of the reduc- tase) (Fig. iA). The apparent K,#, for ATP was found to be 0.2 mM in

1140 HMG-GOA Reductase Kinase, Kinase Kinase, and Phosphatase Enzymes

A

0 2 V 6 8 1 0 0 100 100 300

R K Ius1 PHOSPnORYLASE PHOSPNATASE I m u l

C

0 10 20

PHOSPHATASE (mu1

FIG. 1. Dose-response curves for the assay of HMG-CoA reductase kinase (RK), HMG-GOA reductase phosphatase, and HMG-CoA reductase kinase phosphatase. A, reductase kinase activit,y in microsomal extracts was assayed using a 5-min incubation in the standard reductase kinase assay. The ordinate shows the decrease in HMG-CoA reductase activity (milliunits per tube) due to added reductase kinase (expressed as micrograms of protein). Control reductase activity (no reductase kinase) was 330.6 millunits per tube. B, increase in reductase activity (milliunits per tube) due to added protein phosphatase C (expressed as phosphorylase phosphatase ac-

the presence of 10 mM MgC12. At 2 mM ATP maximal activities were obtained when the total Mg"+ concentration was in excess of the total concentrations of EDTA and ATP. One unit of reductase kinase is that amount which decreased the activity of reductase by 1 unit per min at 37°C.

Reductase Kinase Kinase Assay-The enzyme was assayed by incubating an appropriate aliquot with inactive reductase kinase (0.2 mg) in a medium (0.1 ml, total volume) containing 50 mM NaCI, 60 mM NaF, 4 mM MgCI?, 2 mM ATP, 0.2 mM EDTA, I mM dithiothreitol, and 10 mM imidazole, pH 7.4. MgATP was added to start the reaction. After 10 rnin, 0.02 ml of 5 X concentrated Buffer B was added, and the mixture was further diluted with Buffer B containing 50 mM NaF and assayed for reductase kinase activity by the standard procedure. Further increases in reductase kinase activity were prevented in this assay by the high ionic strength in which it was performed (see under "Results"). Control incubations were carried out either in the absence of added inactive reductase kinase or reductase kinase kinase to correct for a degree of cross-contamination in these preparations.

Histone Kinase Assay-Three assay methods were used. In each the activity was assessed by measuring the incorporation of ,"P from [y-,'2P1ATP into histone type IIA. Method 1 was the filter paper assay of Corbin and Reimann (12). In Methods 2 and 3 the assays were carried out using, respectively, reductase kinase or reductase kinase kinase assay conditions in which histone type IIA replaced either reductase or reduct.ase kinase as the assay substrate. In Method I, the final histone concentration was 7.1 mg/ml, while in Methods 2 and 3 the concentration was 6.0 mg/ml. In all three assays, the reaction was stopped by transferring 0.05 ml of the incubat,ion mixture to a filter paper disk. Further processing of the samples and the definition of histone kinase units are as described by Corbin and Reimann (12).

Protein Phosphatase Assays-~hosphorylase phosphatase, reduc- tase phosphatase, and reductase kinase phosphatase assays were all carried out a t 37°C using similar incubation conditions. In the phos- phorylase phosphatase assay, the incubation was carried out in 5 mM EDTA, 5 mM theophylline, 5 mM dithiothreitol, 0.5 mg/ml of bovine serum albumin (as stabilizer), and 50 mM imidazole, pl i 7.4 (total volume, 0.1 ml). The reductase phosphatase assay also contained 50 mM NaCl (added with the reductase), while in the reductase kinase phosphatase assay theophylline was omitted because it was found to inhibit reductase kinase. The assays were initiated by addition of 0.02

tivity) after a 10-min incubation in the standard reductase phospha- tase assay. C, net decrease in reductase kinase activity (milliunits per tube) due to added protein phosphatase C (expressed as phosphoryl- ase phosphatase activity) after a 10-min incubation in the standard reductase kinase phosphatase assay. Active reductase kinase ex- tracted from the microsomes (see under "Materials and Methods") was used as substrate. In the control incubations without added protein phosphatase C total reductase kinase activity a t IO min was 192.2 miiliunits per tube.

ml of substrate: phosphorylase a (1 mg/ml) in Buffer C (1 n m EDTA, 5 mM dithiothreitol, and 50 mM imidazole, pH 7.4), inactive micro- somal reductase (20 mg of protein/ml), or active reductase kinase (2.5 to 5.0 mg of protein/mtf. Prior to assay, active reductase kinase was passed through a column of Sephadex G-25 equilibrated with Buffer C in order to remove NaF.

Phosphatase activities were assessed by measuring changes in activity of each substrate. In the phosphorylase phosphatase assay, the reaction was stopped, and the decrease in phosphorylase a activity was estimated (9). The reductase phosphatase assay was terminated

start the reductase assay. Since NADPH and HMG-CoA inhibit by addition of 0.05 mi of the reductase co-factor-substrate mixture to

reductase activation (2, 13), no further increase in activity was ob- served during the reductase assay. The reductase kinase phosphatase assay was terminated by the addition of 0.15 mI of 2 X concentrated Buffer B containing sufficient NaF to give a final concentration of 50 mM. A 0.02-ml aliquot was then assayed for reductase kinase activity by the standard procedure.

In each assay, the dose of phosphatase and the incubation times were adjusted such that 20 to 508 of t.he maximal change in activity occurred during the assay. The assays for reductase phosphatase and reductase kinase phosphatase activity were linear with respect to added phosphatase as long as the total change in reductase or reduc- tase kinase activity, respectively, in the incubation was less than 509 of the maximal change (Fig. 1, B and C ) . Control incubations without added phosphatase were carried out to correct for the small amount of phosphatase activity endogenous in the inactive reductase and the active reductase kinase preparations.

Unless otherwise specified, protein phosphatase C purified through the second DEAE-Sephadex step was used. Prior to assay, the enzyme was subjected to gel filtration on Sephadex G-25 equilibrated with 5 mM EDTA, 0.5 mM dithiothreitol, and 50 mM imidazole, pH 7.4, to remove glycerol, since this inhibits reductase phosphatase activity (6).

Units of phosphorylase phosphatase were as defined by Brandt et al. (9). One unit of reductase phosphatase was that amount which increased HMG-CoA reductase activity by 1 unit per min a t 37°C. One unit of reductase kinase phosphatase was that amount which decreased reductase kinase activity by 1 unit in I min at 37°C.

Protein Assays-Proteins were assayed by either the Lowry method (30) or, if indicated, the Bio-Rad protein assay (31).

HMG-CoA Reductase Kinase, Kinase Kinase, and Phosphatase Enzymes

Materials Reagents were obtained from the following sources. 3-Hydroxy-3-

[3-"C]methylglutaryl-CoA (50 Ci/mol) was from New England Nu- clear, ~~-[2-~'H]mevalonic acid and adenosine 5'-[y-"'P]triphosphate (3 Ci/mmol) were from Amersham, Sephadex (3-25 and DEAE-Seph- adex A-50 were from Pharmacia, DEAE-cellulose (DE52) was from Whatman, and histone type IIA and all other biochemicals were from Sigma. Phosphorylase a (KC 2.4.1.1, twice crystallized), 28 u n i ~ / m g of protein from rabbit skeletal muscle, and CAMP-dependent protein kinase from bovine heart (peak 11) and from rabbit skeletal muscle (peak I) were from Sigma.

RESULTS

Co-purification of Reductase Phosphatase and Reductase Kinase Phosphatase with Protein Phosphatase C-In a pre- vious communication (8), we showed that a partially purified preparation of protein phosphatase C was capable of catalyz- ing both the reactivation of HMG-CoA reductase and the inactivation of reductase kinase. In these studies, however, the possibility that the two reactions were catalyzed by a contaminating activity(ies) in the phosphatase preparation was not excluded. The studies presented below were, there- fore, undertaken to examine this possibility. As seen in Table I, the relative rates of reactivation of HMG-CoA reductase, of inactivation of reductase kinase, and of dephosphory~ation of phosphorylase a were constant after each of the six steps in the purification of protein phosphatase C. Furthermore, in studies carried out with the purified phosphatase, NaF was found to inhibit all three activities in parallel with an apparent K, of 3 to 4 mM in each reaction (Fig. 2). In control incubations, NaF had no effect on either reductase or reductase kinase activity, per se. These studies indicate that protein dephos- phorylation is associated with both the reactivation of reduc- tase and the inactivation of reductase kinase.

Reactivation of Reductase Kinase-Inactive reductase ki- nase, extracted from the microsomes (see under "Materials and Methods"), was slowly reactivated by incubating the preparation with Mg' and ATP in a low ionic strength buffer (8) (Fig. 3) . In the absence of either Mg2+ or ATP, no reacti- vation occurred (not shown). When cytosol was added to the incubation, the rate of reactivation was greatly enhanced in a dose-dependent manner (Fig. 3) . The reactivation of reductase kinase was blocked when the incubation was carried out in the Buffer €3 used to assay reductase kinase either in the absence (8) or presence of cytosol (data not shown). This effect was presumably due to the high ionic strength of the

10 20 30 10 5 0 NaF ImHl

FIG. 2. Parallel inhibition of reductase phosphatase, reduc- tase kinase phosphatase, and phosphorylase phosphatase by NaF. Reductase phosphatase (VI, reductase kinase phosphatase (O), and phosphorylase phosphatase (8) activities are expressed as the percentage of the maximal activity (without NaF) at each con- centration of NaF.

/ I i C

1141

0 . 1 . 2 . 3

CYTOSOL fmgl

FIG. 3. Dose-responee curve for reactivation of reductase kinase by cytosolic reductase kinase kinase. The indicated quan- tity of rat liver cytosol was incubated in the presence (0) or absence (0) of inactive reductase kinase for 10 min in the standard reductase kinase kinase assay. Inactive reductase kinase was a soluble prepa- ration extracted from the microsomes (see under "Materials and Methods"). Total reductase kinase activity in the reductase kinase kinase assay is plotted on the ordinate.

medium. However, inhibition by orthophosphate has not been ruled out. This property made it possible to distinguish be- tween reductase kinase activity and reductase kinase activa- tion (i.e. reductase kinase kinase activity) using assays in which the inactivation of HMG-CoA reductase was the ulti- mate end point.

Liver cytosol contains both reductase kinase and reductase kinase phosphatase. As seen in Fig. 4, inactivation of reductase kinase activity by endogenous phosphatase is blocked with 50 mM NaF. The inactivated cytosolic reductase kinase was fully reactivated by carrying out a second incubation with added reductase kinase kinase in the presence of M&+ and ATP (Fig. 4). Incubation of active reductase kinase (preincubated in the presence of 50 mM NaF to block inactivation) with reductase kinase kinase under the same conditions did not increase reductase kinase activity.

Separation of Reductase Kinase from Reductase Kinase Kinase by Gradient Elution from DEAE-Cellulose. Reduc- tase kinase and reductase kinase kinase are both present in rat liver cytosol. However, the observation that reductase kinase kinase activity was blocked in the high ionic strength buffer used in the reductase kinase assay suggested that the two enzymes were distinct. In order to confirm this idea, rat liver cytosol was chro~atographed on DEAE-cellulose (Fig. 5Af in an attempt to separate the two enzymes. Under the conditions used, each activity was initially retained on the column. The two activities were then separated by eluting the column with a linear NaCl gradient from 0 to 0.6 M NaCl. Reductase kinase kinase was eluted as a single peak at 0.25 M NaCl. Reductase kinase was eluted as a major peak at 0.15 M NaCl and a minor peak at 0.25 M NaCl which co-eluted with the peak of reductase kinase kinase. The amount of this second peak was variable and was absent in some prepara- tions. This second peak may be due either to the binding of

1142 HMG-CoA Reductase Kinase, Kinase Kinase, and Phosphatase Enzymes

2oo I 150

- D E

E

>

. 3 -

100 z v t < L Y

50

4 ADD Mg A T P

+RKK +NaF

I 0 30 60

TIME (MIN.) FIG. 4. inactivation and reactivation of cytosolic reductase

kinase (RK) . Kat liver cytosol was equilibrated with huffer contain- ing 5 mM EDTA, 5 mM dithiothreitol, and 50 mM imidazole, pH 7.4. by desalting over Sephadex G-25. This active reductase kinase prep- aration was then preincubated without dilution at 37°C for 30 min. NaF was added at either the beginning ( 0 ) or the end o f the prein- cubation (0) to give a final concentration of 50 mM. After the preincubation, aliquots (20 pl) of the incubation mixtures containing either active (0) or inactivated (0) reductase kinase were further incubated with a particulate preparation of reductase kinase kinase ( K K K ) (see under “Materials and Methods”) for up to 40 min under the conditions used in the standard reductase kinase kinase assay. Control incubations were carried out in the absence of reductase kinase to correct for the small amount of reductase kinase activity in the reductase kinase kinase preparation. Reductase kinase activity is expressed as milliunits per mg of cytosolic protein.

some reductase kinase to reductase kinase kinase or to an artifact resulting from incomplete inhibit,ion of reductase ki- nase kinase ir; the reductase kinase assay. These studies indicate that reductase kinase and reductase kinase kinase are different enzymes.

Reductase Kinnse and Reductase Kinase Kinase are Dis- tinct front ~ A ~ P - d e ~ e n d e n ~ Protein Kinase-The effects of CAMP and the specific, heat-stable protein inhibitor of CAMP- dependent protein kinase on reductase kinase and reductase kinase kinase activities are shown in Table 111. CAMP (0.01 mM) failed to stimulate either activity when cytosol o r micro- somal extracts were assayed. On the other hand, histone kinase activity in these fractions was stimulated by 0.01 mM CAMP when assays were performed ~rnder the conditions used in either the reductase kinase or reductase kinase kinase assay. When higher levels of CAMP (up to 1 mM) or of cGMP, cIMP, or cCMP (0.01 to 1 mm) were added to the reductase kinase assay, no increase in activity was observed (data not shown). Finally, addition of a high level of the specific protein kinase inhibitor (which completely blocked histone kinase activity in the cytosol or microsomal extracts) failed to inhibit either reductase kinase or reductase kinase kinase activity in these fractions (Table 111).

reductase kinase kinase are distinct from CAMP-dependent protein kinase. The possibility that CAMP-dependent protein kinase may act as a second reductase kinase or reductase kinase kinase was ruled out by the following experiment.

n

0 0 z

- .6

- .3

- 0 - t o 20 30

FRACTION

FIG. 5. ~ h r o m ~ t o ~ a p h y of reductase kinase ( R K ) and re- ductase kinase kinase (RKK) in cytosol ( A ) and microsomal extracts (B) on DEAE-cellulose. A, 0.7 ml of cytosol was eyuili- brated with Buffer C containing 50 ~ I M NaF hy gel filtration on Sephadex G-25 and then applied to a column (0.7 X 9 cm) of DKAIS- cellulose equilibrated with the same buffer. The column was washed with the buffer until the A,,, was less than 0.01 and then eluted with a linear gradient (total volume. 70 ml) from 0 to 0.f hl NaCl in the buffer. Fractions of 2 m l were collected a t a flow rate of 20 ml/h and then assayed for reductase kinase (0) and reductase kinase kinase (0) activity. Fractions assayed for reductase kinase kinase activity were desalted on columns of Sephadex G-25 equilibrated with Buffer C plus 50 mM NaF to remove NaCl which inhibited the activity. H , separation of reductase kinase in 0.7 ml of microsomal extract by the same chromatographic system.

‘I’AHLE 111 Effect of CAMP nnd the hmt-stnhleprotein inhihitor-c~f(,AMI”- dependent protein kinase (FKf) on reductase frinttsr, reductase

kinnse kincrse. and histone hinnsc rrctit’ity in c?tostrl crncl microsonml extracts

Iieductase kinase and reductase kinase kinase assays were per- formed using the standard methods. Histone kinase activity was assayed using reductase kinase assay conditions (Method 2 ) or retiuc- tase kinase kinase assay conditions (Method 3 ) . Where indicated, CAMP (0.01 RIM) or the protein kinase inhibitor (PKL 0.1:XJ mg of protein/nll, final concentration) were included in the assays. Activities are expressed as units/mg of protein. Reductase kinase kinase activity in microsomal extracts was estimated from the initial rate o f reacti- vation of inactive reductase kinase in the extract.

Cytosol

Microsomal extracts

Control + PKI + CAMP + CAMP +

1 3 ~ 1

Control + PKI + CAMP + CAMP +

P K I

0.40 0.51 0.47 0.63

1.20 I .2 f 1 . 0 6 1.11

0.084 0.088 0.079 0.079

0 . I44 0.149 0.144 0.161

These experiments indicated that reductase kinase and -~_____

HMG-CoA Reductase Kin.ase, Kinase Kinase, and Phosphatase Enzym.es 1143

Active reductase or inactive reductase kinase were incubated with high concentrations of either the type I protein kinase from rabbit skeletal muscle or the type I1 protein kinase from bovine heart in the presence of CAMP and MgATP. No significant changes in either reductase or reductase kinase activity were observed even after incubation for up to 60 min with 100 to 200 times the (histone) kinase activity normally present when cytosol or microsomal extracts were assayed for reductase kinase or reductase kinase kinase activity as in Table 111.

Subcellular Distribution of the Reductase Kinase and Re- ductase Kinase Kinase Actiuities-Previous studies indicated that reductase kinase and reductase kinase kinase are present in both the cytosolic and microsomal fractions obtained from rat liver (5,6,8). However, the quantitative distribution of the two enzymes in these fractions was not determined.

As shown in Table 11, the subcellular distribution of both reductase kinase and reductase kinase kinase was very similar to that of CAMP-dependent protein kinase which has been designated a cytosolic marker protein (16). The bulk of each activity was recovered in the supernatant obtained after cen- trifuging a rat liver homogenate at 12.000 X g for 15 min. Further centrifugation of the fraction at 100,000 X g for 1 h revealed that 80 to 90% of each activity was in the supernatant (cytosol), while 20%) was recovered in the pellet (microsomes). After extracting the microsomes with neutral buffer, 75 to 100% of the reductase kinase, reductase kinase kinase, and CAMP-dependent protein kinase activities in this fraction were recovered in the soluble extracts, leaving only 1% of the activity initially present in the 12,000 X g supernatant in the membrane fraction. HMG-CoA reductase was not removed from the microsomes under these conditions.

When microsomal extracts were chromatographed on DEAE-cellulose (Fig. 5B), reductase kinase was eluted as a single peak in the same position as the major peak of reductase kinase in the cytosolic fraction. In this preparation of the microsomal extract reductase kinase kinase was too low for measurement in the dilute column effluent. These experi- ments indicate that, both reductase kinase and reductase kinase kinase are cytosolic enzymes, in contrast to HMG-CoA reductase which is firmly bound to the endoplasmic reticulum.

DISCUSSION

The results presented demonstrate that liver HMG-CoA reductase is regulated by the bicyclic system depicted in Fig. 6 (8). In this system, the catalytic efficiency of reductase is

ATP (Mg*+)

REDUCTASE KINASE KINASE

PHOSPHATASE

PHOSPHATASE

FIG. 6. Bicyclic model for regulation of HMG-CoA reductase.

immediately governed by the relative activities of two modu- lating enzymes: reductase kinase, which phosphorylates and inactivates HMG-CoA reductase, and a broad specificity pro- tein phosphatase termed protein phosphatase C (9, 17, 181, which restores reductase activity. Reductase kinase activity itself is activated through phosphorylation by a second protein kinase termed reductase kinase kinase and inactivated by protein phosphatase C.

In the present communicat.ion, we have established the following points regarding the bicyclic reductase system. 1) Protein phosphatase C (and not traces of a contaminating enzyme) catalyzes both the activation of HMG-CoA reductase and the inactivation of reductase kinase. This result indicates that protein dephosphorylat,ion accompanies each reaction. 2) Reductase kinase and reduct,ase kinase kinase are distinct, cytosolic enzymes. 3 ) Neither kinase is identical with CAMP- dependent protein kinase. 4) The latter enzyme does not catalyze the inactivation of reductase nor the reactivation of reductase kinase.

The simplest interpretation of the present data is that the changes observed in reductase and reductase kinase act,ivity result from the reversible phosphorylation of the two enzymes (8). A second possibility, namely that proteins which regulate reductase and reductase kinase activity are the targets for the reversible phosphorylation, appears unlikely in view of the recent experiments of Beg et a!. (19,201 and Keith et al. (21). These workers reported that ,I2P from [y-”’PIATP was incor- porated into the HMG-CoA reductase (19, 21) and the reduc- tase kinase (20) proteins following incubation with reductase kinase and reductase kinase kinase, respectively. In the case of reductase (19, 211, phosphorylation was accompanied by inactivation of the enzyme; however, in the case of reductase kinase (ZO), the effects of phosphorylation on the activity of the enzyme were not reported. The precise stoichiometry of the two phosphorylation reactions remains to be determined.

In the present studies we have used the criteria suggested by Traugh et al. (22) in classifying both reductase kinase and reduct,ase kinase kinase as CAMP-independent protein ki- nases. These authors have pointed out the need to use both CAMP and the specific heat-stable inhibitor of CAMP-depend- ent protein kinase in order to determine whether an unknown kinase is identical with either the free catalytic subunit or the holoenzyme form of CAMP-dependent protein kinase. Our results are not in agreement with the work of Beg et al. (20), who recently reported that CAMP stimulated reductase kinase activity in microsomal extracts. In the latter studies it was not, determined whether the effects of CAMP could be blocked by the prot,ein kinase inhibitor nor was it ascertained whether it was possible to mimic the effect of CAMP using the catalytic subunit of cAM1’-dependent protein kinase. Consequently, the mechanism of action of CAMP in this in uitro system is not certain.

The reductase system is the third example of a bicyclic phosphorylation system in mammalian tissues. The other two systems are the reversible phosphorylation of glycogen phos- phorylase (23-25) and of myosin light chains (26, 27). In the skeletal muscle phosphorylase system a single broad specific- ity protein phosphatase, termed protein phosphatase-1, has been shown to dephosphorylate both phosphorylase a and the B subunit of phosphorylase kinase, thereby inactivating both enzymes (25 ) . An analogous situation exists in the reductase system where protein phosphatase C has been shown to dephosphorylate bot,h HMG-CoA reductase and reductase kinase producing coordinate changes in the two activities. Therefore, the activity of phosphorylase and of HMG-CoA reductase should be exquisitely sensitive to regulatory signals which determine the activity of these protein ~ h ~ s p h a t a s e s .

1144 HMG-CoA Reductase Kinase, Kinase Kinase, and Phosphatase Enzymes

The observation that CAMP-dependent protein kinase is not an integral component of the bicyclic reductase system was unexpected in view of our recent observation (28) that addition of glucagon or dibutyryl CAMP to isolated hepato- cytes caused both an inactivation of HMG-CoA reductase and an increase in reductase kinase activity in these cells. Since other actions of gIucagon in the liver are known to be exerted through CAMP-dependent protein kinase (24), it seems likely that this kinase must regulate one of the components of the bicyclic reductase system.

fn the phosphorylase system CAMP-dependent protein ki- nase increases the activity of the enzyme by two distinct mechanisms. The fist is through the phosphorylation of the psubunit of phosphorylase kinase which activates the enzyme, and the second is through the inactivation of protein phos- phatase-1. This latter effect is achieved by the phosphoryla- tion and activation of a regulatory protein, inhibitsor-1, which inhibits protein phosphatase-1 specifically. Since protein phosphatase-1 and protein phosphatase C appeared to be quite similar enzymes, we have previously suggested (1, 2 ) that the phosphorylation of inhibitor-1 may also explain the regulation of the reductase system by glucagon and CAMP. In order to further investigate this idea, experiments have re- cently been carried out to determine whether protein phos- phatase C and protein phosphatase-1 are the same enzyme. These studies (29) yielded the surprising result that “protein phosphatase C” of liver is a mixture of two distinct broad specificity protein phosphatases (M, = 33,000 to 34,000). One enzyme is very similar to protein phosphatase-1 while the second appears to be reiated to an enzyme, termed protein phosphatase-2, which has also been implicated in the control of glycogen metabolism in mammalian skeletal muscle (25). The two low molecular weight phosphatases have very similar physical properties but can be distinguished both by their substrate specificity and by the fact that protein phosphatase- 1 but not protein phosphatase-2 is inhibited by inhibitor-1 and a second heat-stable protein termed inhibitor-2. Both phosphatases are capable of dephosphorylating HMG-CoA reductase and reductase kinase.,” Studies are in progress to delineate the relative importance of these two types of phos- phatase in the hormonal regulation of the bicyclic reductase system by insulin and glucagon.

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