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

Transcription

1 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 of Biochemistry, Indiana University School of Medicine, Indianapolis, Indium Protein phosphatase C was purified 1500-fold from rat liver by a six-step procedure including a fractionation step with 80% ethanol at room temperature and two successive chromatographic separations on DEAE- Sephadex. This preparation restored hydrox~ethylglutaryl-coa (HMG-CoA) reductase activity in liver microsomes pretreated with MgATP and also inactivated HMG-CoA reductase kinase. The relative rates of activation of HMG-CoA reductase, inactivation of reductase kinase, and dephosphorylation 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-cellulose. 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, ) 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 phosphorylation. 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 microsomes. The activities of HMG-CoA reductase kinase and HMG-CoA reductase kinase kinase were not influenced by CAMP or the specific heat-stable inhibitor of CAMP-dependent protein kinase. Similarly, high concentrations 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 discussed in the light of the recent observation (Ingebritsen, T. S., Geelen, M. J. H., Parker, R. A., Evenson, K. J., and Gibson, D. M. (1979) J. Biol. Chern. 254, ) 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 Institutes of Health (AM19199 and AM21278), the American Heart Association, 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. $. Present address, Department of Biochemistry, University of Dundee, DDl4HN, Scotland, United Kingdom Cholesterol biosynthesis in mammalian liver is regulated principally through the microsomal enzyme hydroxymethylglutaryl-coa reductase (NADPH, EC ), which catalyzes 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 microsomal preparation of reductase kinase became inactive at 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 dephosphorylation 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 enzymes 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 at 0900 (ie. at the peak of the reductase diurnal cycle). Preparation of Different Forms of HMG-CoA Reductase, Reductase Kinase, and Reductase Kinase Kinase Active ~icrosoma~ H M ~ ~ Reducstase ~ o A 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 at 37'C to inactivate reductase kinase (6-8) and then centrifuged at 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.

2 HMG-CoA Reductase Kinase, 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 pro~ein/m~~. suspension The was incubated with 4 mm MgCL and 2 mm ATP for 20 min at 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 resuspended in Buffer A (5 to 10 mg of protein/ml). The inactive microsomal 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 supernatant (cytosol) was frozen in liquid nitrogen and stored at -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 resuspended 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 microsomal HMG-CoA reductase. The two extracts were comhined, concentrated 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 studies. 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 glycogen.protein complex has been described elsewhere (1, 10). Other Protein Preparations Protein Kinase Inhibitor-The specific heat-stable protein inhibitor 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 phosphorylase 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 at 37OC. Subcellular Fractionation of Reductase Kinase and 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 Kinase Kinase, and Phosphatase Enzymes 1139 TABLE I Co-purification of phosphorylase phosphatase, reductase phosphatase, and 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 phosphatase was attained. The indicated ratios are either reductase phospha- tase divided by phosphorylase phosphatase or reductase kinase phosphatase divided by phosphorylase phosphatase. Reductase phosphatase and reductase kinase phosphatase activities could not he reliably quantitated in the crude hoinogexlat,e because of the presence of factors which severely inhibit HMG-CoA reductase (14. 15). Definition of units is found under Materials and Methods. ki- Phosphorylase Reductase nase phosphaphosphatase phosphatase tase Fraction.- ~- - Total IJnits/ IJnits/ (!nits/ unltq me IllZ me Homogenate 15, Supe~atant, ph , Ammonium sulfate 4, VB pellet, Ethanol precipita- 7, tion Ammonium sulfate 4, pellet, 40-75%!. DEAE-Sephadex, 1, Column I DEAE-Sephadex, 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 percentage of the activity in the 12,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 ~ ~ tase ~ ki- ~ kinase ~ ki- ~ i. nase nase nase 12,OOO X g Supernatant Cytosol Microsomes Combined microsomal extracts I Extracted microsomes ~. 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 enzyme has previously been described in detail (6, IO). The assay was started by addition of 0.05 ml of a co-factor-substrate mixture containing 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 at 37 C. HMG-CoA Reductase Kinase Assay-In this assay, kinase-deficient 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 reductase) (Fig. ia). The apparent K,#, for ATP was found to be 0.2 mm in

3 1140 HMG-GOA Reductase Kinase, Kinase Kinase, and Phosphatase Enzymes A C 0 2 V RK Ius1 PHOSPnORYLASE PHOSPNATASE Imul PHOSPHATASE (mu1 FIG. 1. Dose-response curves for the assay of HMG-CoA tivity) after a 10-min incubation in the standard reductase phosphareductase kinase (RK), HMG-GOA reductase phosphatase, and tase assay. C, net decrease in reductase kinase activity (milliunits per HMG-CoA reductase kinase phosphatase. A, reductase kinase tube) due to added protein phosphatase C (expressed as phosphorylactivit,y in microsomal extracts was assayed using a 5-min incubation ase phosphatase activity) after a 10-min incubation in the standard in the standard reductase kinase assay. The ordinate shows the reductase kinase phosphatase assay. Active reductase kinase exdecrease in HMG-CoA reductase activity (milliunits per tube) due to tracted from the microsomes (see under "Materials and Methods") added reductase kinase (expressed as micrograms of protein). Control was used as substrate. In the control incubations without added reductase activity (no reductase kinase) was millunits per tube. protein phosphatase C total reductase kinase activity at IO min was B, increase in reductase activity (milliunits per tube) due to added miiliunits per tube. 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, reductase phosphatase, and reductase kinase phosphatase assays were all carried out at 37 C using similar incubation conditions. In the phosphorylase 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, pli 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 ml of substrate: phosphorylase a (1 mg/ml) in Buffer C (1 nm EDTA, 5 mm dithiothreitol, and 50 mm imidazole, ph 7.4), inactive microsomal 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 phosphorylase in a activity was estimated (9). The reductase phosphatase assay was terminated by addition of 0.05 mi of the reductase co-factor-substrate mixture to start the reductase assay. Since NADPH and HMG-CoA inhibit reductase activation (2, 13), no further increase in activity was observed 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 reductase 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 at 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).

4 HMG-CoA Reductase Kinase, Kinase Kinase, and Phosphatase Enzymes 1141 Materials Reagents were obtained from the following sources. 3-Hydroxy-3- [3-"C]methylglutaryl-CoA (50 Ci/mol) was from New England Nuclear, ~~-[2-~'H]mevalonic acid and adenosine 5'-[y-"'P]triphosphate (3 Ci/mmol) were from Amersham, Sephadex (3-25 and DEAE-Sephadex 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 , twice crystallized), 28 uni~/mg 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 previous communication (8), we showed that a partially purified preparation of protein phosphatase C was capable of catalyzing both the reactivation ofhmg-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, therefore, 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 dephosphorylation is associated with both the reactivation of reductase and the inactivation of reductase kinase. Reactivation of Reductase Kinase-Inactive reductase kinase, extracted from the microsomes (see under "Materials and Methods"), wasslowly 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 reactivation 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 NaF ImHl FIG. 2. Parallel inhibition of reductase phosphatase, reductase 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 concentration of NaF. / I i CYTOSOL fmgl FIG. 3. Dose-responee curve for reactivation of reductase kinase by cytosolic reductase kinase kinase. The indicated quantity 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 preparation 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 between reductase kinase activity and reductase kinase activation (i.e. reductase kinase kinase activity) using assays in which the inactivation of HMG-CoA reductase was the ultimate 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. Reductase 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 preparations. This second peak may be due either to the binding of C

5 1142 HMG-CoA Reductase Kinase, Kinase Kinase, and Phosphatase Enzymes - D E 3 E. - > z t v < L Y 2oo I ADD Mg ATP +RKK +NaF I TIME (MIN.) FIG. 4. inactivation and reactivation of cytosolic reductase kinase (RK). Kat liver cytosol was equilibrated with huffer containing 5 mm EDTA, 5 mm dithiothreitol, and 50 mm imidazole, ph 7.4. by desalting over Sephadex G-25. This active reductase kinase preparation was then preincubated without dilution at 37 C for 30 min. NaF was added at either the beginning (0) or the end of the preincubation (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 (KKK)(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. I AHLE 111 some reductase kinase to reductase kinase kinase or to an Effect of CAMP nnd the hmt-stnhleprotein inhihitor-c~f(,ami - artifact resulting from incomplete inhibit,ion of reductase ki- dependent protein kinase (FKf) on reductase frinttsr, reductase nase kinase ir; the reductase kinase assay. These studies kinnse kincrse. and histone hinnsc rrctit ity in c?tostrl crncl microsonml extracts indicate that reductase kinase and reductase kinase kinase are Iieductase kinase and reductase kinase kinase assays were perdifferent enzymes. formed using the standard methods. Histone kinase activity was Reductase Kinnse and Reductase Kinase Kinase are Dis- assayed using reductase kinase assay conditions (Method 2) or retiuctinct front ~A~P-de~enden~ Protein Kinase-The effects of tase kinase kinase assay conditions (Method 3). Where indicated, CAMP and the specific, heat-stable protein inhibitor of CAMP- CAMP (0.01 RIM) or the protein kinase inhibitor (PKL 0.1:XJ mg of dependent protein kinase on reductase kinase and reductase protein/nll, final concentration) were included the assays. Activities kinase kinase activities are shown in Table 111. CAMP (0.01 are expressed as units/mg of protein. Reductase kinase kinase activity mm) failed to stimulate either activity when cytosol or microin microsomal extracts was estimated from the initial rate of reactivation of inactive reductase kinase in the extract. 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). experiments These 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 to FRACTION FIG. 5. ~hrom~to~aphy of reductase kinase (RK) and reductase kinase kinase (RKK) in cytosol (A) and microsomal extracts (B) on DEAE-cellulose. A, 0.7 ml of cytosol was eyuilibrated 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 DKAIScellulose 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 ml were collected at 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. Cytosol Control + PKI + CAMP + CAMP ~ 1 Control PKI CAMP + CAMP + Microsomal extracts 1.20 I.2f 0. I indicated reductase that kinase and PKI -~ z

6 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 immediately governed by the relative activities of two modulating enzymes: reductase kinase, which phosphorylates and from rabbit skeletal muscle or the type I1 protein kinase from inactivates HMG-CoA reductase, and a broad specificity probovine heart in the presence of CAMP and MgATP. No tein phosphatase termed protein phosphatase C (9, 17, 181, significant changes in either reductase or reductase kinase which restores reductase activity. Reductase kinase activity activity were observed even after incubation for up to 60 min itself is activated through phosphorylation by a second protein with 100 to 200 times the (histone) kinase activity normally present when cytosol or microsomal extracts were assayed for kinase termed reductase protein phosphatase C. kinase kinase and inactivated by reductase kinase or reductase kinase kinase activity as in In the present communicat.ion, we have established the Table 111. following points regarding the bicyclic reductase system. 1) Subcellular Distribution of the Reductase Kinase and Re- Protein phosphatase C (and not traces of a contaminating ductase Kinase Kinase Actiuities-Previous studies indicated enzyme) catalyzes both the activation of HMG-CoA reductase that reductase kinase and reductase kinase kinase are present in both the cytosolic and microsomal fractions obtained from and the inactivation of reductase kinase. This result indicates that protein dephosphorylat,ion accompanies each reaction. 2) 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 Reductase kinase and reduct,ase kinase kinase are distinct, 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 centrifuging a rat liver homogenate at 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 experiments indicate that, both reductase kinase and reductase kinase kinase are cytosolic enzymes, in contrast to HMG-CoA reductase which is firmly bound to thendoplasmic 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 REDUCTASE KINASE KINASE ATP (Mg*+) PHOSPHATASE PHOSPHATASE FIG. 6. Bicyclic model for regulation of HMG-CoA reductase. cytosolic enzymes. 3) Neither kinase is identical with CAMPdependent 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 incorporated into the HMG-CoA reductase (19, 21) and the reductase 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 kinases. These authors have pointed out the need to use both CAMP and the specific heat-stable inhibitor of CAMP-dependent 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 was it not, determined whether th 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 phosphorylase (23-25) and of myosin light chains (26, 27). In the skeletal muscle phosphorylase system a single broad specificity 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~sphatases.

7 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 hepatocytes 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 kinase 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 phosphatase-1. This latter effect is achieved by the phosphorylation 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 recently been carried out to determine whether protein phosphatase 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 phosphatase in the hormonal regulation of the bicyclic reductase system by insulin and glucagon. T. S. Ingebritsen and P. Cohen, unpublished observations. of Cellular Regulation (Cohen, P., ed) Vol. I, pp , Elsevier/North-Holland Biomedical Press, Amsterdam 3. Dugan, R. E., and Porter, J. W. (1977) in Biochemical Actions of Hormones (Litwack, G., ed) VoI. 4, pp , Academic Press, New York 4. Rodwell, V. W., Nordstrom, J. L., and Mitschelen, J. J. (1976) Adv. Lipid Res. 14, Beg, Z. H., Allmann, D. W., and Gibson, D. M. (1973) Biochem. Biophys. Res. Commun. 54, Nordstrom, J. L., Rodwell, V. W., and Mitschelen, J. J ) J. Biol. Chem. 252, Brown, M. S., Brunschede, G. Y., and Goldstein, J. L ) J. Biol. Chem. 250, Ingebritsen, T. S., Lee, H.3, Parker, R. A., and Gibson, D. M. (1978) Biochem. Biophys. Res. Commun. 81, Brandt, H., Capulong, 2. L., and Lee, E. Y. C. (1975) J. Biol. Chem. 250, Ingebritsen, T. S., and Gibson, D. M. (1981) Methods Enzymol. 7 1, Nimmo, G. A., and Cohen, P. (1978) Eur. J. Biochem. 87, Corhin, J. D., and Reimann, E. M. (1974) Methods Enzymol. 38, Saucier, S. E., and Kandutsch, A.A. (1979) Biochim. Biophys. Acta 572, Nordstrorn, J. L. (1976) Ph.D. thesis, Purdue University 15. Ness. G. C.. and Moffler, M. H. (19781 Arch. Biochem. Biophys. 189, Chen. L.-J.. and Walsh. D. A. (1971) Biochernistrs Lee, E. Y. C., Mellgren, R. L., Killilea, S. D., and Ayiward, J. J. (1977) Fed. Eur. Biochem. SOC. Symp. 42, Killilea, S. D., Brandt, H., Lee, E, Y. C., and Whelan, W. J. (1976) J. Biol. Chem. 251, Beg, Z. H., Stonik, J. A., and Brewer, H. B. (1978) Proc. Natl. Acrid. Sci. U. S. A. 75, Beg, 2. H., Stonik, J. A,, and Brewer, I. B. (1979) Prof. Natl. Acad. Sei. U. S. A. 76, Keith, M. L., Rodwell, V. W., Rogers, D. H.. and Rudne.y, H. (1979) Biochem. Biophys. Res. Commun. 90, Traueh. J. A,, Ashbv. C. D., and Walsh, D. A. (1974) ~ethods Enzymol. 38, Nimmo. N. G.. and Cohen. P. (1977) Adv. Cyclic Nucleotide Res. 8, Krebs, E. G., and Beavo, J. A. (1979) Annu. Rev. Biochem. 48, Cohen, P. (1978) Curr. Top. Cell. Regul. 14, Adelstein, R. S., and Hathaway, D. R. (1979) An. J. Cardiol. 44, Adelstein, R. S., Conti, M. A., Hathaway, D. R., and Klee, C. B. (1978) J. Biol. Chem. 253, REFERENCES 28. Ingebritsen, T. S., Geelen, M. J. H., Parker, R. A,, Evenson, K. J., 1. Gibson, D. M., and Ingebritsen, T. S. (1978) Life Sci. 23, and Gibson, D. M. (1979) J. Bid Chem. 254, Ingebritsen, T. S., Foulkes, J. G., and Cohen, P. (1980) FERS 2. Ingebritsen, T. S., and Gibson, a. M. (1980) in ~olecular Aspec~s Lett. 119, Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, Bradford, M. M. (1976) Anal. Biochem. 72,