Phosphorylation of native 97-kDa 3-hydroxy-3-methylglutaryl ...

Vol 264. No.9,Issue of March 25, pp. 4811-4887 1989 Printed ind.S A .

T H EJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular B~ology.lnc.

Phosphorylation of Native 97-kDa 3-Hydroxy-3-methylglutarylcoenzyme A Reductase from Rat Liver IMPACT ON ACTIVITY AND DEGRADATION OF THE ENZYME* (Received for publication, January 7,

1988)

Rex A. Parker$, Steven J. Miller, and David M. Gibson From the Department of Biochemistry, Indiana UniversitySchool of Medicine, Indianapolis, Indiana46223 and the $. Department of Cardiouascular Biochemistry, Pharmaceutical Researchand Development Diuision, Bristol-Myers Company, Wallingford, Connecticut06492

Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [r-s2P]ATP in the presence of reductase kinase, revealeda major 97-kDa “P band which disappeared upon competition with pure unlabeled 63-kDa HMG-CoA reductase. A linear correlation between the expressed/totalHMG-CoA reductase activity ratio(E/T) and the fraction of ‘‘P released from the 97-kDaenzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state inisolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T)followed by an enhanced rate of decay of totalreductaseactivity which was proportional to theloss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonateinduced reductase degradation inhepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to &foldgreater yields of s o h ble 52-66-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase wasphosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated “Plabeled reductase resolved a 62-60-kDa doublet which contained “P solely in the upper band. Thew data suggest that a major phosphorylation site of HMG-CoA reductase lies within the “linker”segment joining the membrane spanning and cytoplasmic domains of the native 97-kDa protein.

The native, monomeric form of microsomal 3-hydroxyl-3methylglutaryl-coenzyme A (HMG-CoA)’ reductase is an in-

* This work was supported by grants from Bristol-Myers and the Grace M. Showalter Foundation. This work was presented in part at the meetings of the IVth Symposium on HMG-CoA Reductase, August 24,1985,Breukelen, The Netherlands, the 13th International Congress of Biochemistry, August 25-30, 1985, Amsterdam, The Netherlands, andthe 78th Meeting of the American Society of Philadelphia, PA. The costs of Biological Chemists, June 7-11, 1987, publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18U.S.C. Section 1734solely to indicate this fact. I The abbreviations used are: HMG-CoA, 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (E.C. 1.1.1.34),referred to as reductase in the text. Reductase kinase is the protein kinase that catalyzes the phosphorylation of reductase (4, 5). MVL, d,l-mevalonolactone; MOPS, 3-(N-morpholino)propanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitri1o)tetraacetic acid; DTT, dithiothreitol; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

trinsic 97-kDa protein of the endoplasmic reticulum which includes a long cytosolic extension (1-3; for review see Refs. 4 and 5). Treatment of isolated microsomes by freezing and thawing releases a “53”-kDa (52-56 kDa) enzymatically active, soluble fragment(s) through the action of endogenous leupeptin-sensitive proteases (6), indicating that thecytosolic domain harbors the active site. One modality of control of HMG-CoA reductase in vivo and in vitro is reversible phosphorylation of the enzyme (4, 5, 712). Incubation of freshly isolated microsomes with ATP ( M e ) in the presence of reductase kinase severely impairs catalytic activity (diminished Vmer).Fluoride-sensitive proteinphosphatases restore reductase to full activity. Early evidence that the enzyme is reversibly phosphorylated under these circumstances relied on experiments carried out by pretreatment of native microsomal reductase with [~-~‘Ppl ATP ( M P ) followed by proteolytic release of the 32P-labeled 52-56-kDa-soluble fragment (13-15). The catalytkactivity of the homogeneous, soluble 52-56-kDaenzyme is inversely proportional to thedegree of [3ZP]phosphorylation.The phosphorylated soluble reductase is reactivated concomitant with dephosphorylation by protein phosphatases(16). While phosphorylation of the native 97-kDa microsomal reductase by liver reductase kinase has been demonstrated (17), the implicit inverse relationship between the expressed activity of native reductase and its degree of phosphorylation has been reported only recently (18). This is a crucial consideration since virtually all intracellular changes in native reductase attributedto reversible phosphorylation are measured in terms of the catalytic activity of isolated microsomes (4,5,9). The usual experimental format involves the separation of microsomes in thepresence of fluoride and EDTA to capture the “expressed” activity of reductase, and with the same microsomes (washed free of fluoride and treated with added protein phosphatase) to obtain “total” activity (8, 9). The latter is taken to be equivalent to themass of the completely dephosphorylated, fully active enzyme. The ratioof expressed to total activity (E/T) of isolated, microsomal reductase thus gives a measure of the percent of enzyme in the dephosphorylated mode (8, 9). Variation in the E/T value of reductase in microsomes isolated from intact cells and tissues has been observed in response to endocrine signals and to metabolic products of mevalonate (4,5,9,11,12). Recently, we reported that proteolytic release of soluble reductase from native microsomes using a calcium-dependent, neutral protease (calpain-2) (19) was enhanced when reductase was phosphorylated (20). The present study examines the impact of phosphorylation of native reductase on its catalytic activity and on its sensitivity to degradation.

4877

4878

Phosphorylation of 97-kDa HMG-CoA Reductase EXPERIMENTAL PROCEDURES~ RESULTS

Correlation of 97-kDaReductase Dephosphorylation with Reactivation and E/T Ratio-The effects of protein phosphatase were compared using as substrate both 32P-labeledand unlabeled inactive 97-kDa microsomal reductase. Incubation of equal aliquots of microsomes for 15 min at 37 “C with increasing concentrations of partially purified, broad specificity protein phosphatase produced a graded loss of 32Plabel in 97-kDa reductase immunoprecipitates. Under parallel conditions, the rise in expressed reductase activity in unlabeled phosphorylated microsomes mirrored the loss of immunoprecipitable [32P]phosphate(Fig. 3A), as determined by scintillation counting of radioactivity in the excised 97-kDa bands. Both effects of phosphatase were time-dependent and were fully blocked by inclusion of 50 mM NaF during incubations (not shown). The relationship between activity and degree of phosphorylation is described (Fig. 3B) by the linear equation, E/T = 0.80 (l-P/Po)+ 0.14, in which E/T is the ratio of expressed/total reductase activity and P/Po is the ratio of remaining/initial [32P]phosphate.The intercept at 0.14 indicates that in this experiment the fully phosphorylated enzyme retains 14%of full activity. The dataof Fig. 3 fit this equation with r = 0.993 (linear regression by least squares). Therefore, the degree of phosphorylation of native 97-kDa reductase is linearly proportional to thepercentage of catalytically latent enzyme. These results provide cogent evidence that theratio of expressed to total reductase activity (E/Tratio) for a given microsomal preparation is a valid index of the phosphorylation state of native 97-kDa HMG-CoA reductase in isolated microsomes (see “Discussion”). Expressed and Total Reductase Activity Are Modulated by Mevalonute-productFeedback-Supplementing cells with exogenous mevalonolactone (MVL) provides a model in which the naturalfeedback regulatory signals derived from mevalonate may be experimentally simulated. Previous reports established that administration of MVL to rats (40, 41) or to isolated hepatocytes (35) results in an initial rapid decrease in expressed reductase activity (phosphorylation) which is succeeded by an irreversible loss of total reductase activity (see “Discussion”). In the present studywith hepatocytes, the loss in total reductase activity following the initial drop in expressed activity (phosphorylation) is correlated with diminution in reductase mass by immunoblotting. Microsomes were isolated from treated hepatocytes under conditions which preserve the in situ phosphorylation state and the intact97-kDa reductase protein (“Experimental Procedures”). As previously observed in hepatocytes incubated for 15 min in the presence of 5 mM MVL (35), expressed reductase activity fell to less than half of the control value, while no change occurred in total reductase activity at this time point. This was reflected in a significantly lower reductase E/T ratio for MVL-treated cells (22%) compared with controls (48%), indicating a net increase in the degree of phosphorylation of microsomal reductase within 15 min of addition of MVL. In longer time courses of hepat yte incubations, total reductase activity declined in contro s. To determine if this represented conversion to an inac ’ve state which could not be restored by dephosphorylati n, fixed amounts of microsomal protein were titrated with increasing

\

I

Portions of this paper (including “Experimental ProJedures,” Figs. 1 and 2, and Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of astandard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.

0

5

15

10

20

pl ProteinPhosphatase

1B

O

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.I

r

l

r

,

.2 .3 .4 .5

l

l

l

l

l

,

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( 1 -P/Po)

FIG.3. Concomitant reactivation and removal of [S*P]phosphate from 97-kDa microsomal reductase. Aliquots (180pg of protein) of 32P-phosphorylatedmicrosomal reductase were incubated (15min a t 37 “ C ) in 200 pl of Buffer B containing 20 pl of a dilution series of partially purified rabbit liver protein phosphatase (“Experimental Procedures”). The incubations were terminated with 100 mM NaF, and immunoprecipitation was conducted as given in Fig. 1. Phosphorylated microsomal reductase, prepared using unlabeled ATP from a portion of the microsomes ,used to obtain the 3ZP-labeled enzyme, was treated with protein phosphatase in parallel. The expressed HMG-CoA reductase activity was then assayed in duplicate aliquots. The “P radioactivity remaining in the corresponding 97kDa reductase bands excised from the gel was determined by liquid scintillation counting after rehydration as described under ”Experimental Procedures.” Panel A, the increase in HMG-CoA reductase catalytic activity (open symbols) mirrors the decrease in immunoprecipitable ”P in the 97-kDa reductase band (closed symbols) as the amount of protein phosphataseincreases (given as pl added/sample). Panel B, the plot shows that the expressed/total reductase activity ratio (E/T) bears a linear relationship to the fraction of dephosphorylated enzyme (1 - P/Po),where P/Po is the ratio of remaining/ initial 32Pin the 97-kDa reductase band. The linear equation, (E/T) = 0.80 (1P/Po)+ 0.14,fits these data with r = 0.993.

-

4879

Phosphorylation of 97-kDa HMG-GOAReductase amounts of antireductase IgG. In a typical case, while total reductase activity decreased 55% over 3 h in controls, the immunotitration titerremained constant. Therefore,the basal loss of reductase total activity in hepatocytes represents a reduction in number of reductase molecules in the microsomes and implies that conversion of reductase to an immunoreactive but catalytically inert form does not occur under basal conditions. In a typical hepatocyte 2-h time course (Fig. 4),the steady decrease in microsomal reductase total activity in controls was accelerated by incubation of cells with MVL or with 25hydroxycholesterol. At the earliest times examined (6 and 12 min), the reductase expressed/total activity (E/T) ratio decreased more than %fold in MVL-treated cells and toa lesser extent in 25-hydroxycholesterol-treated cells. It should be emphasized that these changes in E/T preceded the increased rate of total enzyme decay. In order todemonstrate that microsomal reductase total activity accurately reflected cellular reductase protein mass under these conditions, immunoblotting was conducted using 7000 x g supernatant fractions of hepatocytes. This crude fraction was employed to account for the possibility that decreases in microsomal total reductase activity could represent redistribution of the enzyme to membranes excluded by the standard microsome isolation procedure (see “Experimental Procedures”). In immunoblots of samples from the experiment shown in Fig. 4, the 97-kDa species of reductase was essentially the only band detected (not shown). The ratio of microsomal total reductase activity/97-kDa reductase immunoblot mass remained approximately constant for all incubation conditions (Table XI). This indicates a concordance between the microsomal total reductase activity and the concentration of intact reductase molecules in intracellular membranes during the processes of basal and MVL-product-stimulated decay of reductase. Effects of Several Inhibitors of Cellular Protein Turnover on Basal and Stimulated Reductase Degradation-In whole rat studies the diurnal downswing in rat liver reductase total activity results from a diminishing level of reductase mRNA and a decrease in relative rate of reductase synthesis at times beyond mid-dark (42). Therefore, hepatocytes were isolated SO that the zero time of cell incubation corresponded to the

0 6 12

I

I

60

120

Minutes

FIG. 4. A decrease in reductase E/Tratio precedes the enhanced rate of decline of total reductase activity in hepatocytes treated with MVL or 26-hydroxycholestero1.Hepatocytes from cholestyramine-fed rats were prepared and incubated under standard conditions (“Experimental Procedures”) with the following additions made at zero time: control, basal conditions .(e, 0);MVL, 1.0 mM (A,A); 25-hydroxycholesterol, 25 p~ (m,0);propylamine, 15 mM (#); MVL + propylamine (4);25-hydroxycholesterol + propylamine (@). At the indicated times, aliquots of cells were removed, microsomes were isolated, and HMG-CoA reductase expressed and total activities were determined (“Experimental Procedures”). Total HMG-CoA reductase activity (closed symbok) is given in units of microsomal reductase activity/g of cellwetweight; the expressed reductase activity (open symbols) is given as the ratio of expressed/ total activity (E/T), shown as percent.

TABLEI1 Hepatocyte HMG-CoA reductase total activity and 97-kDa immunoblot mass decay at similar rates underbasal and stimulated degradation conditiom Immunoblot analysis with antireductase IgG was conducted after urea-SDS-acrylamide gel electrophoresis of aliquots of 7000 X g supernatants, prepared, as described under “Experimental Procedures,” from hepatocytes in the experiment described in Fig. 4. The 97-kDa band of the immunoblot autoradiograph was densitometrically scanned and integrated (AU-mm). These data are compared with the total reductase activity in microsomes (units/g), along with the ratio between the two values. 25-Hydroxycholestero1is 25-OH, (d,l) mevalonolactone is MVL. Condition

Control Control MVL Control MVL 25-OH Control MVL %-OH Propylamine MVL + Propylamine 25-OH + Promlamine

Min

0 6

60 120

HMG-CoA reductase 97-kDa AIJ-mm

Activity units/tr

79 76 10.4 75 10.4 60 8.73 39 9.35 23 40 8.82 24 12 57 62 9.54 9.82 22

7.86 7.28 7.22 6.88 4.17 2.40 4.69 2.72 1.27 5.16 6.50 2.24

Ratio

10.0

9.58 8.53

9.44 11.0

9th hour of a 12-h dark cycle for the donor rats. With a low replacement synthesis rate, changes over time of the cellular concentration of an enzyme reflect alterations inthe enzyme’s degradation rateand provide reasonable estimates of the apparent first-order degradative rate constant ( k F p ) if the time course is extended for one or more enzyme half-lives. As described under “Experimental Procedures,” we employed this approach to determine kypfor basal reductase degradation in hepatocytes. The mean value ( N = 29 independent experiments) for basal kPP= 0.333 2 0.047 h-’, corresponding to t%!= 2.08 & 0.26 h. In thepresence of 10 KM cycloheximide, these values were essentially unchanged ( N = 8), with kyp= 0.343 f 0.088 h-’, and tlp = 2.02 h, suggesting that the assumption of low de novo synthesis of reductase under the conditions employed is valid. Also,this value for reductase tlp under basal conditions is in agreement with values obtained by others using different methods (43). Several agents known to perturb intracellular protein degradation were examined for effects on total reductase decay in 2-3 h time courses in hepatocytes (Fig. 5). The lysosomotropic agents propylamine and methylamine significantly decreased reductase kFp to 57 and 63% of control values, respectively. The monovalent carboxylic ionophore monensin decreased basal k p to 49% of control, yet the potassium ionophore nigericin had no effect. Amino acids at 5 X normal concentration also suppressed basal k p , suggestive of inhibition of autophagic vacuole formation as reported by Seglen et al. (44). Insulin decreased basal reductase kppto 52% of control (Fig. 5) in accord with our previous observations that insulin both increased reductase E/T and spared totalactivity (9). Overall, the results indicate an involvement of acidic subcompartments of the hepatocyte in basal reductase degradation. The data are also consistent with the report of Tanaka etal. (45) that NH&l decreased the basal degradation rate of reductase in a temperature-sensitive mutant mouse mammary cell line. In the hepatocyte 2-h degradation model, incubation with 1.0 m M (R,S)-MVL increased reductase kyp 2.0-foldover control (Fig. 6). Incubation with 25-hydroxycholesterol at 12 p M caused a similar 2.1-fold increase in k P . These effects

Phosphorylation of 97-kDa HMG-CoA Reductase

4880

propylamine and monensin each only partially prevented the stimulation of kPPproduced by the oxysterol (Fig. 6). These data suggest that functional acidic compartments in the cell 1 are required for MVL product feedback stimulation of reduc2 tase degradation. One possible explanation of the differential blockade of the effects of 25-hydroxycholesterol compared 0.30 a with mevalonate, produced by propylamine or monensin, is P that the active feedback effector derived from mevalonate is D 3 $ distinct from this oxysterol and from those endogenous me40 0.20 tabolites which mayincrease after treatmentwith the oxyste"\N CD 4 rol. This is consistent with the proposal (46) that in addition 2 3 to oxysterols non-sterol isoprenoids may act as reductase 5 W feedback effectors. Additionally, the rapid decrease in reduc0.10, tase E/T (increased phosphorylation) caused by MVL treatIO ment is of greater magnitude than that seen with 25-hydroxycholesterol (Fig. 4), suggesting that 25-hydroxycholesterol 20 does not mediate these effects of MVL. 0. Reductase Phosphorylation Enhances Its Proteolysis in ViFIG. 5. Basal degradation of reductase in hepatocytes is tro-An in vitro model was developed in order to study problocked by several inhibitors of lysosome function. The firstorder apparent degradative rate constant for HMG-CoA reductase teolytic degradation of microsomal reductase in defined phos( k P P )was estimated by total activity decay in hepatocyte incubations phorylation states. In thisapproach, a useful protease was the as described in detail under "Experimental Procedures," and dis- Ca2+-dependentneutral thiol protease calpain-2 (33) purified cussed further in the text. Reductase kFp (h-', left ordinate) and t L from rat liver by the method of DeMartino (32). A similar (h, right ordinate) are shown for each experimental condition (open preparation of calpain wasused by Liscum et al. (19) in bars) compared with simultaneously conducted controls (crosshatched bars). Data represent mean +- S.E. for N independent deter- mapping the domains of UT-1 cell reductase. In a preliminary minations of kFp. The confidence intervals for significance of differ- study using this approach, we reported (20) that the soluble ence were determined by paired t tests; **p < 0.002; *p < 0.05. fraction of calpain digests of phosphorylated microsomes conConcentrations of agents in cell incubations were: propylamine, 15 tained up to 6 times more cleaved 52-56-kDa reductase than mM; methylamine, 15 mM; monensin, 50 pM; nigercin, 50 pM; amino digests of dephosphorylated preparations. acids, 5 X Eagle's minimum essential medium levels; insulin, 50 nM. In the present studies, the incipient stages of limited calpain-2 proteolysis of microsomal reductase were examined in N= 16** 15*' 6 ' 7** order to clarify the differences that phosphorylation state of 1 reductase conferred. The soluble and membrane-bound cleav0.8C age products of reductase generated by three calpain concentrations at three time points were analyzed by immunoblotting (Fig. 7). Only 97-kDa reductase was seen in control lanes ,- 0.6C (panels A and E ) . As the concentration of protease increased (each pair of lanes), for each time point (panels B-D), the '$ release to the supernatant phase of soluble 52-56-kDa reductase was enhanced 4- to &fold for phosphorylated (evenn i' 0.40 numbered lanes) compared with dephosphorylated reductase (odd-numbered lanes). At the lowest protease concentration, #! a fragment of reductase which adhered to themembrane was 0.20 generated while only very low levels of the soluble enzyme were produced. The relative levels of reductase total activity (assayed following dephosphorylation), in both the membranes and supernatants separated after 1 min of proteolysis, 0 closely reflected the yields assessed by densitometry of the FIG. 6. Propylamine and monensin fully suppress the increased reductase degradation produced by MVL but only immunoblot (Fig. 8). A greater loss of membrane-bound total partially block the effect of 25-hydroxycholesterol. Each reductase activity and native97-kDa mass, and ahigher yield paired set of conditions shows the effect of selected agents on reduc- of soluble activity and 52-56-kDa reductase (5-fold at a caltase k,F' (h-', left ordinate)and t'n (h, right ordinate) in hepatocytes, pain concentration of 12 pg/mg) was observed for the phosas determined by the method given in Fig. 5. The concentrations were: MVL, 1.0 mM; 25-hydroxycholestero1, 12 pM; propylamine, 15 phorylated compared to dephosphorylated reductase (Fig. 8). Densitometric absorbances from the immunoblots of both mM; and monensin, 50 pM. The data represent mean 2 S.E. for N native 97-kDa and calpain-2-solubilized 52-56-kDa reductase independent determinations of kyp;p < 0.002; *p < 0.05. were linearly proportional to the quantity of enzyme loaded on the gels (data notshown). When normalized to the amount were concentration-dependent for bothagents(datanot shown). It should be noted that the levels employed (Fig. 6) of total activity (dephosphorylated before assay), the yields were selected to provide an approximate doubling of kPP in of reductase in the immunoblots for both 97- and 52-56-kDa order to study inhibition of the enhanced degradation. In forms were independent of phosphorylation state. This indicontrast to their partialeffects on basal kBP,propylamine and cates that theepitopes recognized by the polyclonal antibody monensin each completely blocked the increase in k P p caused under the immunoblot conditions do not differ significantly byMVL. In experiments not shown, the inclusion of pro- for the two states of phosphorylation, consistent with the pylamine in cell incubations with MVL did not prevent the immunotitration results cited above. Reductase Phosphorylation Site-In order to examine the MVL-induced rapid decrease in microsomal reductase E/T ratio. When tested in the presence of 25-hydroxycholesterol, distribution of the phosphorylation site among reductase N = IO'*

5*

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4

5

5*

0.40

L

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4881

cleavage products, parallel proteolytic digests of both 32Plabeled and unlabeled phosphorylated 97-kDa reductase were carried out and analyzed by immunoprecipitation and immunoblotting, respectively. Electrophoresis of unfractionated digests was conducted on replicate urea-SDS-polyacrylamide gels, and the radioactivity in excised bands was determined directly (Fig. 9). The results indicate quantitative conversion of reductase mass from the 97-kDa form to products in the 52-56-kDa range. However, tracking the phosphate label under these conditions showed that the 32Pinitially present in 97-kDa reductase was recovered in the 52-56-kDa fragments at lower levelsof proteolysis but was lost at higher concentra1 2 3 4 5 6 1 2 3 4 5 6 - m tions of calpain-2. This suggested that the phosphorylation I D I E site of native reductase resides within the fragment formed under limited proteolysis and isremoved with more extensive FIG. 7. Initial stagesof calpain-2 proteolysis of microsomal 97-kDa reductase in dephosphorylated uersus phosphorylated digestion, while the immunoreactive enzyme mass in the 52states. Aliquots (330 pg of protein) of microsomes prepared in the 56-kDa band remained essentially constant. two extremes of reductase phosphorylation state (asdescribed under The possibility that a phosphatase activity present in the "Experimental Procedures") were incubated with the indicated levels calpain preparation explains the results of Fig. 9 is unlikely of calpain-2 plus 2 mM CaC12 in 150 pl of Buffer C. Reactions were due to the high purity of the calpain in relation to the levels terminated with leupeptin (0.2mM) and EGTA (25 mM). After ultracentrifugation (100,000 X g a t 20 "C for 6 min in a Beckman of phosphatase required for efficient dephosphorylation of Airfuge), the membrane fractions (top panel) and soluble superna- reductase (see Fig. 3). Incubation of microsomes with calpain tants (bottom panel) from each sample were analyzed by Western in the presence of leupeptin, which inhibits calpain proteolytic immunoblotting ("Experimental Procedures") using urea-SDS gels activity, did not alter the E/T ratio of phosphorylated reducand antireductase IgG. The autoradiograph exposure time for the tase (data not shown). Furthermore, it is unlikely that a latent bottom panel was triple that of the top panel. Odd-numbered lanes phosphatase activity is revealed in the microsomal preparacontained dephosphorylated reductase; even-numbered lanes contained phosphorylated reductase. Part A shows controls, no protease tions during proteolysis since the reductase E/T activity ratio and no Ca2+.Parts B-D contained Ca2+plus calpain, 1 pg (lanes 1 assayed in digests remained essentially unchanged during and 2 ) ; 2 pg (lanes 3 and 4 ) ; or 4 pg (lanes 5 and 6).Part E shows initial stages of 52-56-kDa fragment generation (data not controls, no protease but with Ca2+(lanes 1 and 2 ) ; with Ca2+and 2 shown). pg of calpain plus 20 pg of calpastatin (lanes 3 and 4 ) ; or with Ca2+ With different batches of microsomes and of calpain-2 and 2 pg of calpain plus 0.20 mM leupeptin (lanes 5 and 6).Incubation unfractionated digests were analyzed as described for Fig. 9 times at 37 "C were: 0 min (part A ) , 1 min (part B), 2 min (part C ) , except that electrophoresis employed replicate SDS-polyand 4 min (parts D and E ) . acrylamide gradient gels for improved resolution. Immunoblots of digests of unlabeled phosphorylated reductase (Fig.

52-56 kDa lmmunoblot

2

0 K I-

z

80

70

0 0 LL

0

aQ

30

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20

5

2.5

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20

30

40

CALPAlN rglmg " " "

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FIG. 8. Effects of phosphorylation on distribution of reductase activity and immunoreactive products after brief proteolysis. The proteolytic fragments generated from phosphorylated compared to dephosphorylated native reductase by incubation with the indicated levels of calpain were quantified by densitometry of the Westernblot shown in Fig. 7 (part B). Total reductase activity (dephosphorylated before assay) was also determined in aliquots of the centrifuged digests. The data shown represent yields, in terms of percent of control 97-kDa band or totalreductase activity. Densitometry data were corrected for film exposure time differences. Symbols used open symbols, phosphorylated reductase; closed symbols, dephosphorylated reductase. A = total activity in membranes; V = total activity in supernatants; 0 = 97-kDa band in membranes; 0 = 5256-kDa band in membranes; 0 = 52-56-kDa band in supernatants.

FIG.9. Proteolysis of 97-kDa phosphorylated HMG-CoA reductase leads to a 52-56-kDa form with diminishing '*P content. Aliquots (160 pg of protein) of phosphorylated microsomal reductase prepared with 32P-labeledor unlabeled ATP (as described in Fig. 3) were incubatedin parallel with the indicated levels of calpain-2 plus 2 mM CaC12 for 4 min a t 37 "C in 75 pl of Buffer C. Reactions were terminated with leupeptin (0.2 mM) and EGTA (20 mM), and the unfractionated digests were analyzed by Western immunoblot of the unlabeled reductase or by immunoprecipitation of the 32P-labeledreductase, using replicate urea-SDS-acrylamide gels run simuntaneously. Both analysesemployed the same antireductase IgG preparation. The lZ5Iand 32Pbands of the nitrocellulose and gel, respectively, were located by autoradiography, excised, and quantified by scintillation counting. The data are given as % of control cpm (open symbols, closed symbols, 32P)recovered in the 97-kDa (circles) and the 52-56-kDa (triangles) bands, plotted as a function of the concentration of calpain (pg calpain/mg microsomal protein).


4882

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Origin

1

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33

, 35

,

37

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, 43

33

35

37

39

41

43

SCAN OISTANCE. m m

FIG. 11. Densitometric scans demonstrate loss of the "Pphosphorylated residue from reductase during generation of the mass doublet at 52-66-kDa. Results of laser densitometry

scan of the 52-56-kDa region of lane 6 (panel A ) and lane 7 (panel B ) of the autoradiograph of Fig. 10 are shown. Panel B corresponds to twice the concentration of protease compared to panel A. The plot gives absorbance units ( A U ) versus mm electrophoretic migration distance from the bromphenol front (distance from front to origin is equal to 120 mm) for both the 32P-containingfragment and for the

-

n

fragments revealed by immunoblotting.

P 0

c

i E

immunoblot doublet formed. In a similar manner, the "Plabeled aggregate above 100 kDa ultimately diminished in a intensity in parallel with the 56-kDa band at thehigher levels of proteolysis (lane 7).Densitometric scans of the 52-56-kDa region of autoradiographs of the immunoblot compared with 29 immunoprecipitates clearly depict the loss of the 32P-labelas the lower fragment of the 52-56-kDa reductase doublet formed during proteolysis (Fig. 11). FIG. 10. Loss of a major phosphorylation site of reductase The most direct interpretation of the dataavailable is that correlatea with the generation of a doublet of reductase protein at 62-66-kDa. Aliquots (160 pgof protein) of labeled and one or more major phosphorylation sites of native 97-kDa unlabeled phosphorylated microsomes were proteolyzed and unfrac- HMG-CoA reductase lie between twocalpain-sensitive loci in tionated digests were examined as described in Fig. 9 except that the reductase protein which are separated by a peptide segindependent sources of microsomes and of calpain were used, and ment of up to 4 kDa. The datafurther indicate that phosphoelectrophoresis was conducted on replicate 5-12% gradient acrylam- rylation within this region both inactivates the enzyme and ide SDS gels. Shown are the autoradiographs of the immunoblotted unlabeled phosphorylated reductase (upper panel)and of the immu- enhances the susceptibility of reductase to proteolytic cleavnoprecipitated 32P-labeledreductase (lower panel). Proteolysis con- age by calpain in the vicinity of these sites. H

0

jFmnt

ditions (4 min at 37 "C) for both upper and lower panels were: lane 2,control, no protease; lanes 3-7, Ca" plus calpain at 2.5, 5, 10, 20, and 40 pg/mg microsomal protein, respectively. Lane I, upper panel, is control (duplicateof lane 2);and lane 1, lowerpanel, is a preimmune IgG control.

10,upper panel) were compared with immunoprecipitates of digests of [32P]phosphorylated reductase (Fig. 10, lower panel). As indicated in Fig.2, the apparent aggregates of reductase with M , greater than 100,000 were observed in the 32P-labeledimmunoprecipitates but not in the immunoblot. The observation that aggregates slightly reduced in size (compared with controls) were isolated under conditions in which the 97-kDa 32P-bandwas fully cleaved (Fig. 10, lanes 5-7) suggests that the cysteine residues involved in disulfide formation are located in the cytoplasmic domain of 97-kDa reductase, on the COOH-terminal side of the cleavage points. The results of the comparative proteolytic mapping (Fig. 10)differed in another, more significant way. The lower levels of calpain (lanes 3 and 4 ) generated a distinct 56-kDa species of reductase protein which also contained labeled phosphate. With higher protease concentrations a second band of reductase protein (approximately52 kDa) emerged to form a nearly fully resolved doublet of 52-56-kDa in the immunoblot (Fig. 10, upper panel, lanes 5-7). However, the major 32P-labeled product seen in this M, range in immunoprecipitatesremained as a single band at 56kDawhich greatly diminished in intensity (Fig. 10,lower, lanes 5-7) as the lower band of the

DISCUSSION

The decrease in HMG-CoA reductase activity attending preincubation of rat liver microsomes with ATP(Mg2') and protein kinases, and itsreactivation by protein phosphatases, has been extensively studied by several laboratories (4, 5). The expressed activity of the enzyme vanes by morethan 10fold between the two extremes. In the present study 97-kDa microsomal reductase was both inactivated and phosphorylated in separate incubations with ATP and [y-32P]ATP.Approximately 0.1 mol of 32P04/molof 97-kDa reductase was apparently incorporated during inactivation to 8%of control. The calculation of this stoichiometry is severely limited by uncertainties in the molecular activity of the native enzyme and thespecific radioactivity of the ATP during the reaction. It is probable that isotopic dilution of the T-POI of ATP occurred during the incorporation in unwashed microsomal membranes due to vigorous endogenous ATPase and phosphate exchange reactions (47,48). Thiscatalytically inactive, 32P-labeledreductase was restored toward full activity with variable quantities of protein phosphatase (Fig. 3). In agreement with Beg et al. (18),we have demonstrated that the fraction of total microsomal reductase catalytic activity that is latent is a linear function of the amount of 32Pbound in the 97-kDa band. In other words, the E/T ratio (expressed catalytic activity to total activity) is an accurate representation of the percent of microsomal reductase in the dephos-

Phosphorylation of 97-kDa HMG-CoA Reductase phorylated mode. Consequently, measurements of expressed and total reductase activities of microsomes isolated from cells and tissues under various conditions (9, 11, 12) reflect the degree of phosphorylation of this enzyme if care is taken to fix the intracellular state of reductase with fluoride and EDTA. A corollary inthis relationship is thatthe total microsomal reductase activity (the fully dephosphorylated state) isequivalent to the total97-kDa mass by immunoblotting. In the present studies there was no evidence by these methods for an immunoreactive, but catalytically inert, species of 97-kDa reductase that cannot be restored by dephosphorylation. Except for the apparently multimeric forms of reductase (Mrgreater than 100,000) which result from disulfide formation during sample preparation (see Miniprint) no other immunoreactive species were detected. In liver and othertissues mevalonate, the product of HMGCoA reductase, gives rise to various polyisoprenoids and sterols, predominantly cholesterol. Cholesterol, certain oxysterols (e.g.25-hydroxycholestero1) and inseveral systems polyisoprenoid derivatives of mevalonate provide a negative feedback loop that impairs the production of mevalonate (2, 23, 40,41,46,49-54). An acute response to theadministration of mevalonolactone to rats (40,41), toisolated hepatocytes (35, 55), or to fibroblasts (11)results within 15 min in a protein phosphatase-reversible inactivation of HMG-CoA reductase (phosphorylation-engenderedfall in expressed activity). This is followed by an progressive phosphatase-irreversibleloss of total reductase activity. In Fig. 4 and Table 11, we show that the slower fall in total activity is mirrored in the decline of mass of 97-kDa reductase recovered in immunoblots under conditions in which there is no replacement synthesis, i.e. degradation of the native microsomal reductase. As we observed in this (Fig. 4)and earlier studies(35) with suspensions of isolated hepatocytes in simple buffered media supplemented with mevalonolactone, the initial rapid drop in expressed activity (E) precedes a change in total activity (T) so that the E/T ratio falls. On the basis of the microsomal [32P]phosphorylationactivity correlations presented inFig. 3, this would indicate a rise in the degree of phosphorylation of microsomal 97-kDa reductase. Of interest is the apparent reversal of the E/Tratio afterthe totalenzyme level (activity and mass) begins to fall (Fig. 4). This would be expected if the phosphorylated form of the enzyme is preferentially degraded, under these conditions in which new enzyme formation is precluded. These findings have been further elucidated by Marrero et al. (55) who have recently demonstrated that degradation of 32P-labeled97-kDa reductase in hepatocytes is significantly enhanced following treatment of these cells with mevalonate. Leonard and Chen (56) observed that the addition of ATP to permeabilized Chinese hamster ovary cells resulted in phosphatase-irreversibleloss of reductase activity. The data in this and related investigations, both in uiuo and in vitro (11, 20, 35, 40, 41, 55), indicate that increased phosphorylation of reductase may bea prelude to degradation. For example, both expressed and total reductase activity fall in hepatocytes treated with glucagon, whereas expressed activity of reductase rises after insulin and the fall in total activity is blocked (9). Similarly, diurnal variation of liver microsomal reductase depends on covariant changes in expressed and total activities (the latter reflecting the balance between synthesis and degradation of the enzyme) (12). The degradation of several other enzymes and proteins areknown to be affected by phosphorylation (5,5749). In the present study the intracellular route of degradation of microsomal reductase in hepatocytes, under basal conditions or after addition of mevalonolactone, appears to lead

4883

through acidic subcellular compartments. Although lysosomotropic agents (methylamine, propylamine, and monensin) were effective in blocking the increased rate of degradation of reductase after mevalonolactone (Fig. 61, these agents only partially diminished the enhanced degradation observed after addition of 25-hydroxycholesterol. Tanaka et al. (45) found that NH,Cl impaired basal degradation of reductase in a mutant mammary cell line but had no effect on the accelerated degradation induced by 25-hydroxycholestero1.These authors also showed that inactivation of the ubiquitin degradative pathway (60) did not impair the rate of decay of reductase in these temperature-sensitive cells. The involvement of other proteases that act in thecytosolic space can not be excluded. A limited proteolytic event in the cytosol could initiate a degradative sequence which requires functional acidic compartments for its completion. In order to approach the question of how phosphorylation of microsomal reductase could affect its rate of degradation an in vitro model system employing the Ca2+-dependent, neutral protease, calpain-2 (33), was examined. Liscum et al. (19) had previously shown that thisenzyme cleaved reductase in a manner similar to thefreezing and thawing procedure (6) for release of soluble, catalytically active reductase (52-56kDa). In preliminary studies (20) we showed that cleavage of the native 97-kDa reductase by liver cytosolic calpain-2 was enhanced by pretreatment of microsomes with ATP(MgZ+)in the presence of reductase kinase, compared with protein phosphatase-pretreated microsomes. This was reflected in the recovery of soluble 52-56-kDa reductase (activity and immunoblot mass) in the centrifugal supernatant of the reaction mixture. An immunoreactive cleavage product of similar electrophoretic mobility remained bound to the residual microsomal membranes, much like the 62-kDa membrane-bound fragment of UT-1 cells described by Liscum et al. (19). A more comprehensive kinetic experiment was presented in Figs. 7 and 8. Examination of this system with ATP- and[32P]ATPtreated microsomes (Fig.9) revealed that the32P-labeIedband of 52-56-kDa reductase generated in the whole reaction mixture appeared to diminish at higher calpain levels while the immunoblots of the same product were unchanged. In a second experiment of this kind (Fig. lo), the 52-56-kDa reductase cleavage product was resolved into a doublet by gradient SDS-PAGE immunoblot analysis. The 32P-labeled product remained a single band with a mobility most nearly matching the upper (larger) component of the immunoblot doublet. As the second (lower) band of the doublet increased, a t higher calpain concentrations, the 32P-labeledband became less intense (Figs. 10 and 11).This result indicates that the larger of two major cleavage products of 97-kDa microsomal reductase isphosphorylated while the second with a slightly smaller molecular mass is not. Thus, as proteolysis proceeds with purified calpain-2 the peptide segment with bound 32Pis removed with only a minor decrease in themass of the original fragment. The difference in apparentmolecular masses of the bands generated during loss of the 32Psignal was approximately 4 kDa (fewer than 40-amino acid residues). These data suggest that a site of HMG-CoA reductase phosphorylation, shown to be serine for the ratliver enzyme (38), lies between the two main loci cleaved bypurified calpain-2. Alternatively, the phosphorylation site, and one of the proteolytic clips, may be located near the COOH terminus of reductase. However, Liscum et al. (61) showed that the antibody, raised against a synthetic peptide corresponding to the COOH-terminal 14amino acid sequence of hamster reductase, reacted with both 97-kDa reductase and the soluble 52-56-kDa form of reductase released by a Ca2+-activatedprotease. That is, the 52-

4884


56-kDa-soluble forms of reductase are probably co-extensive with the cytosolic domain of 97-kDa reductase at the COOH terminus. In our experience with electrophoretic mobilities in immunoblots of the reductase fragments released by calpain were indistinguishable from those liberated by the classical freeze-thaw proteolysis procedure used in most purifications of the soluble reductase. Purification of soluble reductase by affinity chromatography often yields a doublet in the 52-56kDa range (2, 24, 26, 27, 29, 62). To obtain fragments the size of the 52-56-kDa-soluble reductase, calpain-2 presumably cleaves the cytosolic domain of the 97-kDa reductase in theso-called “linker” region where the membrane-spanning chain emerges, uiz. between residues 340 and 449. In this region a total of 12 serines are found in the Chinese hamster reductase sequence (63). A comparison of reductase sequences from the threespecies reported to date (human, Chinese hamster, and Syrian hamster) (63-65) reveals that only five of these serines are conserved (residues 346, 349, 356, 384, and 420). It is reasonable to propose that a major site of phosphorylation in our studiesof rat reductase may be among these conserved serines since they also lie in or near so-called PEST sequences (66) which are especially prevalent in proteins that characteristically have short halflives (66,67). (PEST regions are relatively enriched in proline, glutamate, serine, and threonine and delimited with positively charged residues.) Other conserved residues may point to regions that could be cleaved by calpain. Two pairs of arginines (residues 370,371,377, and 378)(62) frame PEST region 1 (66). Also Leu, Lys (350, 351) and Ile, Lys (400,401) are considered to be preferred cleavage sites for calpain (33). The mechanism by which phosphorylation alters catalytic capacity of enzymes probably involves conformational changes in protein structure (68). While inactivation of reductase is a consequence of its phosphorylation, coincident shifts in the protein structure may also influence the accessibility of potentially scissile peptide bonds within the linker region of reductase to endoproteases such as calpain. Phosphoserine residues by enhancing focal calcium binding may promote calcium-dependent protease action at adjacent sites (66). Since cholesterol appears to enhance reductase degradation in cells (52-54, 69), it can be argued that cholesterol enrichment in the endoplasmic reticulum, by facilitating phosphorylation of reductase, could increase its susceptibility to proteolytic attack. The observations of Beg et a2 (70,71) that two Ca2+-signaled protein kinases (protein kinase C and calmodulin-dependent protein kinase) effectively phosphorylate and inactivate HMG-CoA reductase open up the possibility that these kinases could act in concert with Ca’+-dependent calpain (72). Although calpain is potentially useful in localizing the phosphorylation site(s) of native 97-kDa reductase, the apparent predilection of calpain for phosphorylated reductase in vitro serves only as a model for its degradation intracellularly. Hepatocyte proteases that preferentially attack phosphorylated microsomal reductase have yet to be identified. Acknowledgments-We are grateful for the expert technical assistance provided by Karen Evenson McCarthy and Teresa L. Lanier and for meticulous manuscript typing by Margaret Smith. We are indebted to Dr. Charles E. Wilde for his advice on immunoanalysis.

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4886


t

z a.4

--

a l-

I

4887

Phosphorylation of 97-kDa HMG-CoA Reductase 2

3

4

7

TABL8 I