Proc. Nati. Acad. Sci. USA Vol. 80, pp. 2477-2480, May 1983

Regulation of three key in cholesterol metabolism by /dephosphorylation (3-hydroxy-3-methylglutaryl-coenzyme A reductase/acyl-coenzyme A:cholesterol acyltransferase/cholesterol 7a-hydroxylase/homeostasis) TERENCE J. SCALLEN* AND AJIT SANGHVIt *Department of Biochemistry, School of Medicine, University of New Mexico, Albuquerque, New Mexico 87131; and tDepartment of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Communicated by N. E. Bradbury, January 17, 1983 ABSTRACT Our laboratories have investigated the role of phosphorylation and activated by dephosphorylation (10-19) (Fig phosphorylation/dephosphorylation in the regulation of three key 2). Recently, the in vivo significance of the phosphorylation of enzymes in cholesterol metabolism. 3-Hydroxy-3-methylglutaryl- HMG-CoA reductase was demonstrated, both for rats receiving coenzyme A (HMG-CoA ) reductase (EC 1.1.1.34), the major reg- a single dose of mevalonic acid by intragastric tube (20) and for ulatory in cholesterol , is inhibited by phos- rats receiving a single meal containing 2% cholesterol (21). Re- phorylation. Acyl-CoA:cholesterol O-acyltransferase (ACATase; cent studies from several other laboratories also support the EC 2.3.1.26) and cholesterol 7a-hydroxylase (EC 1.14.13.7), key biological significance of phosphorylation/dephosphorylation regulatory enzymes in the utilization of cholesterol, are activated of by phosphorylation. In view of these results, we propose that short- HMG-CoA reductase as a short-term regulatory mechanism term regulation of the concentration of intracellular unesterified (22-27). cholesterol is achieved by a coordinate phosphorylation/dephos- Acyl-CoACholesterol Acyltransferase ACATase (EC2.3.1.26). phorylation of these three enzymes. For example, if cholesterol ACATase is a membrane-bound microsomal enzyme that cata- enters the liver cell, HMG-CoA reductase would be inhibited by lyzes the formation of long-chain fatty-acyl cholesterol esters in phosphorylation and biosynthesis of cholesterol would be re- rat liver and other tissues (Fig. 1). This enzyme is important in duced; however, reactions utilizing cholesterol would be acti- regulating the concentration of unesterified cholesterol within vated, due to the phosphorylation of ACATase and cholesterol 7a- the cell, and thus it provides a mechanism for the removal of hydroxylase. Thus, the phosphorylation/dephosphorylation of these a potentially harmful excess of unesterified cholesterol by con- three enzymes provides an elegant short-term mechanism for the version to a form that can be stored intracellularly without del- homeostasis of intracellular unesterified cholesterol. eterious effects to the cell (1). In a preceding article (28), evidence is presented that ACAT- As has been noted by Spector et aL (1) and also by Kandutsch ase is capable of being regulated by phosphorylation/dephos- et al. (2), it is crucial that the concentration of unesterified cho- phorylation. It is significant that, whereas HMG-CoA reduc- lesterol within the cell be regulated within narrow limits. Unes- tase is inactivated by phosphorylation (Fig. 2), ACATase is terified cholesterol, present in cell membranes, functions to activated by phosphorylation (Fig. 3A). ACATase is therefore modulate the fluidity of the phospholipid bilayer (3). It has been regulated by phosphorylation in a manner exactly opposite to demonstrated that changes in the membrane cholesterol con- that of HMG-CoA reductase. In addition to being regulated by tent, by altering fluidity, can affect permeability, as well as the phosphorylation/dephosphorylation, the rate of cholesterol es- properties of membrane-bound enzymes and transport systems ter formation in rat liver microsomes is also affected by the (4-7). The presence of a storage pool (cholesterol ester) and, in availability of endogenous cholesterol substrate (29, 30), pre- the case of liver cells, the utilization of cholesterol via bile acid sumably delivered to ACATase by sterol carrier protein2 (SCP2) formation provide a means not only for the storage and utili- (31, 32). zation of cholesterol, but also for the control of the amount of Cholesterol 7a-Hydroxylase (Cholesterol 7a-Monooxygen- unesterified cholesterol present within the cell. ase, EC 1.14.13.7). The conversion of cholesterol to 7a-hy- In this article, we briefly review evidence consistent with droxycholesterol (Fig. 1), a reaction catalyzed by cholesterol 7a- the concept that regulation of three key enzymes in cholesterol hydroxylase, is the major regulatory step in bile acid biosyn- metabolism by phosphorylation/dephosphorylation provides a thesis (33, 34). Recent studies (35) have indicated that short-term short-term mechanism for intracellular unesterified cholesterol rapid changes in the activity of this enzyme may involve phos- homeostasis. The evidence presented supports our proposal that phorylation/dephosphorylation, with the active enzyme being enzymes involved in the regulation of cholesterol synthesis- phosphorylated (Fig. 3B). It was observed that incubation of rat e.g., 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) re- liver microsomes with Escherichia coli alkaline ductase-are inactivated by phosphorylation, whereas en- produced a loss of cholesterol 7a-hydroxylase activity in pro- zymes involved in cholesterol utilization or storage are acti- portion to the amount of phosphatase added (35). Much of this vated by phosphorylation. loss was recovered by removal of the phosphatase, followed by HMG-CoA Reductase (EC 1.1.1.34). HMG-CoA reductase the addition of ATP, Mg2+, and a cytosolic protein fraction. It catalyzes the conversion of HMG-CoA to mevalonic acid (Fig. was shown recently (36) that cholesterol 7a-hydroxylase activity 1). This enzyme is the major regulatory enzyme in cholesterol is lost when rat liver microsomes are incubated in the presence biosynthesis (8, 9). Initially, evidence from several laboratories of various divalent cations e.g., Mg2 . This inactivation was demonstrated that HMG-CoA reductase activity is inhibited by Abbreviations: HMG-CoA reductase, 3-hydroxy-3-methylglutaryl- The publication costs ofthis article were defrayed in part by page charge coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acyl- payment. This article must therefore be hereby marked "advertise- ating), EC 1.1.1.34]; ACATase, acyl-coenzyme A:cholesterol O-acyl- ment" in accordance with 18 U.S.C. ยง1734 solely to indicate this fact. transferase (EC 2.3. 1.26). 2477 Downloaded by guest on September 28, 2021 2478 Biochemistry: Scallen and Sanghvi Proc. Nad. Acad. Sci. USA 80 (1983) Acetyl-CoA CHOLLESTEROL1 SYNMTHESIS 4 HMG-CoA I HMG-CoA v Reductase Mevalonate *f INHIBITS O -j------| CHOLESTEROL 1-~----|PHOSPHORYLATION|\

. I I I -Arl%-ATCQ-tIAX I IVAI Lb-

7a-Hydroxylase ACAT CHOLESTEROL UTILIZATION

V 7a-hydroxycholesterol Cholesterol esters

Bile acids FIG. 1. Diagram of cholesterol synthesis and utilization in rat liver. blocked when the incubation was conducted in the presence of may regulate the amount of unesterified cholesterol present in sodium fluoride. These results are consistent with the presence the cell. All three enzymes-i.e., HMG-CoA reductase, ACAT- of an endogenous microsomal , activated ase, and cholesterol 7a-hydroxylase-have a common intra- by divalent cations, that in turn inactivates cholesterol 7a-hy- cellular location, namely, the endoplasmic reticulum. HMG-CoA droxylase by dephosphorylation (Fig. 3B). The recent studies reductase is the major enzyme regulating cholesterol biosyn- by Goodwin et al. (37) confirm the observation (36) that cho- thesis (Fig. 1). In liver cells, cholesterol utilization is regulated lesterol 7a-hydroxylase is capable of being regulated by phos- phorylation/dephosphorylation. A It is significant to note that cholesterol 7a-hydroxylase is ac- tivated by phosphorylation (Fig. 3B), whereas HMG-CoA re- ductase is inactivated by phosphorylation (Fig. 2). Cholesterol 7a-hydroxylase is therefore regulated by phosphorylation in a ACTIVE manner exactly opposite to that of HMG-CoA reductase. In ad- Protein Protein dition to being regulated by phosphorylation/dephosphoryla- Phosphatase ATP, M92+ tion, the rate of 7a-hydroxycholesterol formation in rat liver Mg2+ microsomes is also affected by the availability of endogenous cholesterol substrate (29). Short-Term Intracellular Cholesterol Homeostasis by Phos- phorylation/Dephosphorylation of Three Key Enzymes. The INACTIVE above findings provide a basis for proposing a mechanism that

HMG - CoA Reductase B | 76-Hydroxylase I OPO3- OPO3 INACTIVE ACTIVE Protein j Protein Kinase Protein Reductase Kinase Phosphatase ATP, Mg2+ Phosphatase ATP, Mg2+ Mg2+ |HMG-CoA Reductase |7cx-Hydroxylase| OH OH ACTIVE INACTIVE FIG. 2. Diagram showing the phosphorylation/dephosphorylation FIG. 3. Diagram showing the phosphorylation/dephosphorylation of rat liver HMG-CoA reductase, the major regulatory enzyme in cho- of enzymes in rat liver involved in the regulation of cholesterol utili- lesterol biosynthesis. zation. (A) ACATase (ACAT); (B) cholesterol 7a-hydroxylase. Downloaded by guest on September 28, 2021 Biochemistry: Scallen and Sanghvi Proc. Natl. Acad. Sci. USA 80 (1983) 2479 A. CHOLESTEROL EXCESS was demonstrated that sterol ester was activated by phosphorylation, which would cause increased amounts of Phosphorylation Produces: unesterified cholesterol to be formed and then utilized for ste- 1. HMG-CoA Reductase activity roid hormone production. 2. + ACAT activity The evidence reviewed here is consistent with our proposal 3. 7o-Hydroxylase activity that enzymes involved in the regulation of cholesterol synthe- sis-e.g., HMG-CoA reductase-are inactivated by phospho- rylation, whereas enzymes involved in cholesterol utilization are activated by phosphorylation (Fig. 1). This regulation of key B. CHOLESTEROL DEPRIVATION enzymes in cholesterol metabolism by coordinate phosphoryla- tion/dephosphorylation provides an elegant short-term mech- Dephosphorylation Produces: anism for intracellular unesterified cholesterol homeostasis. I. + HMG-CoA Reductase activity 2. ACAT activity We thank Dr. Philip Hooper (Department of Medicine, University 3. 7o-Hydroxylase activity of New Mexico) for a helpful suggestion concerning the arrangement ofFig. 1. This investigation was supported by National Institutes ofHealth FIG. 4. Short-term regulatory adjustments (arrows) for three key Grants HL-16,796 and AM-10,628. enzymes in cholesterol metabolism for cholesterol excess (A) or cho- lesterol deprivation (B). 1. Spector, A. A., Mathur, S. N. & Kaduce, T. L. (1979) Prog. Lipid Res. 18, 31-53. 2. Kandutsch, A. A., Chen, H. W. & Heiniger, H.-J. (1978) Science by ACATase and cholesterol 7a-hydroxylase. Phosphorylation 201, 498-501. of the enzyme that regulates cholesterol synthesis (HMG-CoA 3. Papahadjopoulos, D., Dowden, M. & Kimelberg, H. (1973) Bio- reductase, Fig. 2) inactivates the enzyme, whereas phosphoryl- chim. Biophys. Acta 330, 8-26. ation of the enzymes that regulate cholesterol utilization in rat 4. Deuticke, B. & Ruska, C. (1976) Biochim. Biophys. Acta 433, 638- liver (ACATase and cholesterol 7a-hydroxylase, Fig. 3) acti- 653. vates these enzymes. Therefore, synthesis and utilization are 5. Demel, R. A. & DeKruyff, B. (1976) Biochim. Biophys. Acta 457, 109-132. oppositely regulated by phosphorylation/dephosphorylation (Fig. 6. Szabo, G. (1974) Nature (London) 252, 47-49. 1). 7. Sen, P. C. & Ray, T. K. (1980) Arch. Biochem. Biophys. 202, 8- The regulatory implications of this proposal are shown in Fig. 17. 4. In the instance of cholesterol excess-e.g., dietary choles- 8. Rodwell, V. W., Nordstrom, J. L. & Mitschelen, J. J. (1976) Adv. terol entering the cell-the regulatory adjustments would be as Lipid Res. 14, 1-74. follows (Fig. 4A). Cholesterol synthesis would be inhibited by 9. Dietschy, J. M. & Brown, M. S. (1974)J. Lipid Res. 15, 508-516. 10. Beg, Z. H., Allman, D. W. & Gibson, D. M. (1973) Biochem. phosphorylation of HMG-CoA reductase; however, cholesterol Biophys. Res. Commun. 54, 1362-1369. utilization would be activated by phosphorylation of ACATase 11. Brown, M. S., Brunschede, G. Y. & Goldstein, J. L. (1975)J. Biol. and cholesterol 7a-hydroxylase. The net result would be to Chem. 250, 2502-2509. maintain intracellular unesterified cholesterol within narrow 12. Nordstrom, J. L., Rodwell, V. W. & Mitschelen, J. J. (1977)J. Biol. limits. Chem. 252, 8924-8934. In the instance of cholesterol deprivation-e.g., a choles- 13. Ursini, F., Valenti, M., Ferri, L. & Gregolin, C. (1977) FEBS Lett. 82, 97-101. terol-free diet or a cultured liver cell grown in lipoprotein-de- 14. Ingebritsen, T. S., Lee, H. S., Parker, R. A. & Gibson, D. M. ficient medium-the regulatory adjustments would be as fol- (1978) Biochem. Biophys. Res. Commun. 81, 1268-1277. lows (Fig. 4B). Cholesterol synthesis would be activated by 15. Bove, J. & Hegardt, F. G. (1978) FEBS Lett. 90, 198-202. dephosphorylation of HMG-CoA reductase; however, choles- 16. Goodwin, C. D. & Margolis, S. (1978)J. Lipid Res. 19, 747-756. terol utilization would be significantly decreased by dephos- 17. Beg, Z. H. & Brewer, H. B., Jr. (1978) Proc. Natl. Acad. Sci. USA phorylation of ACATase and cholesterol 7a-hydroxylase. 75, 3678-3682. 18. Beg, Z. H., Stonik, J. A. & Brewer, H. B., Jr. (1979) Proc. Nati. The above discussion relates specifically to the regulation of Acad. Sci. USA 76, 4375-4379. cholesterol metabolism in the liver. However, the same rea- 19. Keith, M. L., Rodwell, V. W., Rodgers, D. H. & Rudney, H. (1979) soning can be applied to various extrahepatic tissues as well. Biochem. Biophys. Res. Commun. 90, 969-975. The major difference would be that short-term control would 20. Arebalo, R. E., Hardgrave, J. E., Noland, B. J. & Scallen, T. J. be achieved by the opposite regulation of HMG-CoA reductase (1980) Proc. Nati. Acad. Sci. USA 77, 6429-6433. (cholesterol synthesis) and ACATase (cholesterol utilization or 21. Arebalo, R. E., Hardgrave, J. E. & Scallen, T. J. (1981) J. Biol. Chem. 256, 571-574. storage), because cholesterol 7a-hydroxylase would not be 22. Ingebritsen, T. S., Geelen, M. J. H., Parker, R. A., Evenson, K. present to a significant extent in these tissues. Thus the op- J. & Gibson, D. M. (1979) J. Biol. Chem. 254, 9986-9989. posite regulation of HMG-CoA reductase (inactivated by phos- 23. Erickson, S. K., Shrewsbury, M. A., Gould, R. G. & Cooper, A. phorylation) and ACATase (activated by phosphorylation) could, D. (1980) Biochim. Biophys. Acta 620, 70-79. along with cholesterol substrate availability, explain the results 24. Beg, Z. H., Stonik, J. A. & Brewer, H. B., Jr. (1981) Fed. Proc. obtained by Brown et al. with human fibroblasts (38). In those Fed. Am. Soc. Exp. Biol. 40, 1604 (abstr.). 25. Avigan, J. & Beg, Z. H. (1982) Fed. Proc. Fed. Am. Soc. Exp. Biol. studies, cholesterol entering the cell via the low density lipo- 41, 1400 (abstr.). protein receptor produced decreased HMG-CoA reductase ac- 26. Henneberg, R. & Rodwell, V. (1981) Fed. Proc. Fed. Am. Soc. Exp. tivity and increased ACATase activity. Biol. 40, 1604 (abstr.). Another interesting example, consistent with our proposal 27. Parker, R. A., Evenson, K. J. & Gibson, D. M. (1982) Fed. Proc. that enzymes involved in cholesterol utilization are activated by Fed. Am. Soc. Exp. Biol. 41, 881 (abstr.). T. Proc. can be found in the studies of Vahouny and 28. Gavey, K. L., Trujillo, D. L. & Scallen, J. (1983) Nati. phosphorylation, Acad. Sci. USA 80, 2171-2174. colleagues (39) concerning the utilization of esterified choles- 29. Mitropoulos, K. A., Balasubramaniam, S., Venkatesan, S. & terol in the adrenal via sterol ester hydrolase. Esterified cho- Reeves, B. E. A. (1978) Biochim. Biophys. Acta 530, 99-111. lesterol in these cells provides a storage reservoir for substrate 30. Erickson, S. K., Shrewsbury, M. A., Brooks, C. & Meyer, D. J. used in steroid hormone biosynthesis. In these studies (39), it (1980)J. Lipid Res. 21, 930-941. Downloaded by guest on September 28, 2021 2480 Biochemistry: Scallen and Sanghvi Proc. Nati. Acad. Sci. USA 80 (1983)

31. Gavey, K. L., Noland, B. J. & Scallen, T. J. (1981)J. Biol. Chem. 36. Sanghvi, A., Grassi, E. & Diven, W. (1983) Proc. NatI. Acad. Sci. 256, 2993-2999. USA 80, 2175-2178. 32. Poorthuis, B. J. H. M. & Wirtz, K. W. A. (1982) Biochim. Bio- 37. Goodwin, C. D., Cooper, B. W. & Margolis, S. (1982)J. Biol. Chem. phys. Acta 710, 99-105. 257, 4469-4472. 33. Danielsson, H. (1972) Steroids 20, 63-72. 38. Brown, M. S., Dana, S. E. & Goldstein, J. L. (1975)J. Biol. Chem. 34. Myant, N. B. & Mitropoulos, K. A. (1977)J. Lipid Res. 18, 135- 250, 4025-4027. 153. 39. Naghshineh, S., Treadwell, C. R., Gallo, L. L. & Vahouny, G. V. 35. Sanghvi, A., Grassi, E., Warty, V., Diven, W., Wight, C. & Les- (1978)J. Lipid Res. 19, 561-569. ter, R. (1981) Biochem. Biophys. Res. Commun. 103, 886-892. Downloaded by guest on September 28, 2021