Activation of hormone-sensitive lipase and phosphorylase kinase

Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 4843-4847, November 1977 Biochemistry Activation of hormone-sensitive lipase and phosphorylase kinase by purified cyclic GMP-dependent protein kinase (cyclic AMP-dependent protein kinase/protein kinase inhibitor/cholesterol esterase) JOHN C. KHOO, PAMELA J. SPERRY, GORDON N. GILL, AND DANIEL STEINBERG Division of Metabolic Disease and Division of Endocrinology, Department of Medicine, University of California, San Diego, La Jolla, California 92093 Communicated by Nathan 0. Kaplan, August 12,1977 ABSTRACT Cyclic GMP-dependent protein kinase, purified to homogeneity from bovine lung, was shown to activate hormone-sensitive lipase partially purified from chicken adipose tissue. The degree of activation was the same as that effected by cyclic AMP-dependent protein kinase although higher concentrations of the cyclic GMP-dependent enzyme were required (relative activities expressed in terms of histone H2b phosphorylation units). Activation by cyclic AMP-dependent protein kinase was completely blocked by the heat-stable protein kinase inhibitor protein from skeletal muscle but activation by the cyclic GMP enzyme was not inhibited. Lipase fully activated by cyclic AMP-dependent protein kinase showed no further change in activity when treated with cyclic GMP-dependent protein kinase. Lipase activated by cyclic GMP-dependent protein kinase was reversibly deactivated by purified phosphorylase phosphatase (from bovine heart); full activity was restored by reincubation with cyclic GMP and cyclic GMPdependent protein kinase. Cholesterol esterase activity in the chicken adipose tissue fraction, previously shown to be activated along with the triglyceride lipase by cyclic AMP-dependent protein kinase, was also activated by cyclic GMP-dependent protein kinase. Crude preparations of hormone-sensitive triglyceride lipase from human or rat adipose tissue and cholesterol esterase from rat adrenal were also activated by cyclic GMP-dependent protein kinase. Purified hosphorylase kinase (rabbit skeletal muscle) was also shown to be activated by cyclic GMP-dependent protein kinase. The present results, together with those of other workers on histone phosphorylation, suggest that the substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinase may be similar. This is discussed in the light of a model recently proposed with regard to the relationship between the subunit structures of the two kinases. The physiologic significance of the findings remains to be established. Changes in intracellular concentrations of cyclic GMP (cgmp) have been observed in association with a wide variety of metabolic and hormonal perturbations (reviewed in refs. 1 and 2). In many circumstances the cellular levels of cgmp change reciprocally with those of cyclic AMP (camp), leading to the suggestion that these two cyclic nucleotides regulate metabolic processes in opposite directions-the yin-yang hypothesis (2). Under certain conditions, parallel changes in the concentrations of the two cyclic nucleotides are observed (3, 4). It has been established in several mammalian systems that camp regulates enzymic activity by phosphorylations catalyzed by campdependent protein kinase (5) and it has been suggested that cgmp may work in an analogous fashion through cgmpdependent protein kinase (6). The latter enzyme has now been demonstrated in many tissues (cf. ref. 7) including adipose tissue (8). Thus far, however, covalent enzyme modification with changes in enzyme activity catalyzed by cgmp-dependent kinase has not been demonstrated. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adertisement" in accordance with 18 U. S. C. 1734 solely to indicate this fact. 4843 The role of camp-dependent protein kinase in the activation of hormone-sensitive lipase from adipose tissue (9) and of phosphorylase kinase from skeletal muscle (10) is well established. The availability of a highly purified preparation of cgmp-dependent protein kinase (7) led- us to test the possibility that this kinase might modify the activity of these interconvertible enzymes. MATERIALS AND METHODS Materials. ['4C]Triolein and cholesterol [1-14CJoleate were purchased from Dhom Products, Ltd. Phosphorylase b (rabbit skeletal muscle), phosphoglucomutase, glucose-6-p dehydrogenase, histone H2b, camp, cgmp, and ATP were obtained from Sigma Chemical Co. ['y-32p]atp was prepared by the method of Glynn and Chappell (11). camp-dependent protein kinase (specific activity, 45 nmol of 32p incorporated per mg of histone H2b per mg of protein per min) was purified from rabbit skeletal muscle according to the method of Wastila et al. (12) through the first DEAE-cellulose chromatography step. Protein kinase inhibitor was also purified from rabbit skeletal muscle through the DEAE-cellulose chromatography step by the method of Walsh et al. (13). Phosphorylase kinase was purified from rabbit skeletal muscle by the method of Cohen (14). Phosphorylase phosphatase, a generous gift of E. Y. C. Lee, was purified from bovine heart to homogeneity by-the method of Brandt et al. (15). The specific activity was 7890 units/mg of protein. Preparation of cgmp-dependent Protein Kinase and Hormone-Sensitive Lipase. cgmp-dependent protein kinase was purified from bovine lung by affinity chromatography on 8-NH2(CH2)2NH-cAMP-Sepharose (7, 16). The homogeneous enzyme prepared by elution with 0.1 mm cgmp in 2% Ampholine and 10% glycerol was concentrated to 1 mg/ml. This purified enzyme had a specific activity of 1375 nmol of 32p incorporated per mg of histone H2b/mg of protein per min. Because cgmp-dependent protein kinase was purified by competitive elution with cgmp, removal of the nucleotide by chromatography on Sephadex G-200 or Sephadex G-50 at 300 was required when effects of added cyclic nucleotide were to be examined. Hormone-sensitive lipase from chicken adipose tissue was prepared by the method of Khoo and Steinberg (17) to the ph 5.2 precipitate step. The specific activity ranged from 13 to 45 nmol of oleic acid released per mg of protein per hr. Endogenous camp-dependent protein kinase was inactivated by incubating the ph 5.2 precipitate fraction (5.2 P fraction) at 500 for 20 min in the presence of 10,uM camp. This heat treatment caused a loss of 40% of the lipase activity. The camp was then removed by dialysis and chromatography on Sephadex G-50. Abbreviations: cgmp, cyclic GMP; camp, cyclic AMP; EGTA, ethylene glycol-bis(,b-aminoethyl ether)-n,n'-tetraacetic acid.

4844 Biochemistry: Khoo et al. 0 10 20 30 40 50 Protein kinase, units/ml FIG. 1. Activation of hormone-sensitive lipase as a function of the concentration of cgmp-dependent protein kinase (0) and camp-dependent protein kinase (A). The chicken adipose tissue 5.2 P lipase fraction (75,g) was first freed of endogenous camp-dependent protein kinase by heat treatment in the presence of camp and then activated at 300 for 10 min under the conditions described in Materials and Methods. In some cases, protein kinase inhibitor (78 Mug/ml) was added to the incubation mixture (@, A). This heat treatment also inactivated endogenous phosphoprotein phosphatase. Enzyme Assay. The conditions for activation, deactivation, and assay of hormone-sensitive lipase or cholesterol esterase were as described (18, 19). The activation mixture (0.1 ml) contained 5 mm magnesium acetate, 0.5 mm ATP, 10,uM cgmp or camp, cgmp- or camp-dependent protein kinase as indicated, 50-150 Mug of 5.2 P fraction, and 20% glycerol/i mm EDTA/25 mm Tris, ph 7.4. After incubation at 300 for 10 min, triglyceride lipase activity was assayed by adding 0.7 ml of an emulsion containing 0.1 mm [14C]triolein, bovine serum albumin at 5 mg/ml, 2 mm EDTA, and 5 mm sodium phosphate (ph 7.0) and incubating for 30 min at 300. Free [I4C]oleic acid was extracted with chloroform/methanol/ benzene/water at ph 11.5 (19). Cholesterol esterase was assayed by using cholesterol [1-14C]oleate added in ethanol as described (18). Activation of phosphorylase kinase was carried out in a reaction mixture of 50 Mul containing 10 mm magnesium acetate, 0.3 mm ATP, 0.5 mm EGTA, 10MM cgmp or camp, cgmpor camp-dependent protein kinase at the indicated concentrations, purified phosphorylase kinase at 0.25 mg/ml, 25 mm f3-glycerophosphate, and 15 mm 2-mercaptoethanol, ph 6.8. After 20 min at 300, the reaction was terminated by addition of 0.5 ml of ice-cold buffer (25 mm,b-glycerophosphate/15 mm 2-mercaptoethanol, ph 6.8). Phosphorylase kinase was assayed by a modification (20) of the method of Krebs et al. (21). Phosphorylase a formed was assayed in the direction of glucose 1-P formation (22). One unit of phosphorylase is defined as the enzyme activity yielding 1,umol of glucose 1-P per min. cgmp-dependent protein kinase and camp-dependent protein kinase were assayed with histone H2b substrate as described by Gill et al. (7). One unit of kinase activity is defined as that amount of enzyme transferring 1 nmol of 32p from [y-32p]atp to recovered histone H2b per min. RESULTS Activation of Hormone-Sensitive Lipase from Chicken Adipose Tissue. The partially purified fraction of chicken hormone-sensitive lipase (5.2 P fraction) used in the first part of these studies still contained significant levels of endogenous camp-dependent protein kinase. Thus, as shown in Table 1, activation was observed with addition of MgATP and camp Table 1. Proc. Nati. Acad. Sci. USA 74 (1977) Activation of hormone-sensitive lipase from chicken adipose tissue* Lipase activity, Ratio: act. nmol oleic acid/ with additions/ mg protein act. with Additionst per hr MgATP alone MgATP alone 33 1.0 +camp 747 22.6 +camp and protein kinase inhibitor 41 1.2 +cgmp 30 0.9 +cgmp-dependent protein kinase 72 2.2 +cgmp and cgmpdependent protein kinase 414 12.5 +cgmp, cgmp-dependent protein kinase, and protein kinase inhibitor 515 15.6 * 5.2 P fraction (75 Mg) of chicken adipose tissue hormone-sensitive lipase was incubated with the indicated cofactors for 10 min at 300; [14C]triolein emulsion was then added and incubation was continued for 30 min at 300. Lipase activity was determined from release of [14C]oleic acid. t The concentrations of the cofactors were: Mg(OAc)2, 5 mm; ATP, 0.5 mm; cgmp or camp, 10,uM; protein kinase inhibitor, 78 Mug/ml; and cgmp-dependent protein kinase, 5.3 units/ml. alone. Addition of protein kinase inhibitor completely blocked this, indicating that the activation was due to endogenous camp-dependent protein kinase. Addition of MgATP and cgmp alone caused no activation, indicating that the endogenous protein kinase of this adipose tissue fraction was not readily activated by cgmp under these conditions. This is in agreement with earlier studies in rat adipose tissue showing that, although the Ka for camp-stimulated activation of hormone-sensitive lipase was 1.1 X 10-7 M, significant cgmp-stimulated activation was seen only at 1 X 10-4 M (23). Addition of MgATP and cgmp-dependent protein kinase yielded a 2-fold increase in triglyceride lipase activity; with addition of both cgmp-dependent protein kinase and cgmp a 12-fold increase was obtained (Table 1). This approximately 6-fold enhancement by cgmp is comparable to the previously reported (7) enhancement by cgmp of cgmp-dependent protein kinase activity in histone phosphorylation. Protein kinase inhibitor did not block activation due to cgmp-dependent protein kinase plus cgmp. On the contrary, it caused a slight increase (24%) in lipase activation, compatible with previous reports that it enhances cgmp-dependent protein kinase activation (24). When both camp and cgmp (10-5 M) were added with cgmp-dependent protein kinase, there was no further increase in lipase activation above that seen with camp alone (data not shown). In the course of these studies it was found that the endogenous protein kinase associated with adipose tissue lipase could be selectively and almost totally inactivated by heating in the presence of camp, making activation dependent on addition of camp-dependent protein kinase. Activation of the lipase as a function of added camp-dependent protein kinase is shown in Fig. 1. Enzyme activity was increased more than 20-fold. Addition of protein kinase inhibitor completely blocked this activation. Substitution of purified cgmp-dependent protein kinase and cgmp under the same conditions yielded the same maximal degree of activation. Expressed in units based on histone phosphorylation, cgmp-dependent protein kinase was less effective than camp-dependent protein kinase under the

Biochemistry: Khoo et al. Proc. Natl. Acad. Sci. USA 74 (1977) 4845 200 1I80 // = 80 E~~~~~~~~~~~~ r 60 80 10 20 3 'Eli08 o7 v 1061 Cyclic nucleotide, M FIG. 2. Effect of varying concentrations of cgmp (0) and camp (^)on the activation of hormone-sensitive lipase by cgmp-dependent protein kinase. Conditions for activation were as described in Materials and Methods except that 2 mm theophylline was included in the incubation mixtures and 100 jig of chicken adipose tissue 5.2 P was used. The cgmp-dependent protein kinase preparation used wvas purified by affinity chromatography, and cgmp was removed by dialysis and chromatography on Sephadex G-200. The amount of cgmp-dependent protein kinase added was 7.8 units/ml. Basal lipase activity before the addition of cyclic nucleotides and cgmp-dependent protein kinase was 20 nmol of free fatty acid released per mg of protein/hr. conditions used. Addition of protein kinase inhibitor had no effect on the activation due to cgmp-dependent protein kinase, ruling out participation of camp-dependent protein kinase in the observed activation. Lipase activation by cgmp-dependent protein kinase as a function of cyclic nucleotide concentration is shown in Fig. 2. Half-maximal activation was obtained at X 10-7 M cgmp or 1.2 X 10-6 M camp, thus confirming the relative cyclic nucleotide specificity of the cgmp-dependent protein kinase. However, the Ka for cgmp and camp were about 10 times greater than those reported for histone phosphorylation, and the ratio camp/cgmp (i.e., 4:1) needed for half-maximal activation was much lower than that reported for histone phosphorylation (ratio, 50:1) (7). Evidence that the activation by cgmp-dependent protein kinase, like that by camp-dependent protein kinase, reflects phosphorylation of the lipase was obtained by demonstrating reversible deactivation by a purified protein phosphatase (from bovine heart). As shown in Fig. 3, lipase previously activated by cgmp-dependent protein kinase showed a progressive fall in activity during incubation with the phosphatase. At 20 mn. an aliquot was removed and again incubated (10 min) with cgmp-dependent protein kinase and cgmp. This restored lipase activity to that of the fully activated preparation. Activation of Other Acyl Hydrolases. Previous studies have shown that hydrolase activities against cholesterol esters and lower glycerides in chicken adipose tissue are closely associated with hormone-sensitive triglyceride lipase and, like it, are activated by camp-dependent protein kinase although to different degrees (18). In the present studies the cholesterol esterase and diglyceride hydrolase activities of the 5.2 P fractions were increased 5- to 6fold by cgmp-dependent protein kinase (data not shown). Hormone-sensitive triglyceride lipase in rat (23) and human (25) adipose tissue has been previously shown to be activated by camp-dependent protein kinase. As shown in Table 2, both are also activated by cgmp-dependent protein kinase. Activation with cgmp-dependent protein kinase was carried out in the presence of protein kinase inhibitor to prevent partici- 0 10 20 30 Time, min FIG. 3. Reversible deactivation of hormone-sensitive lipase. Chicken adipose tissue 5.2 P (750,g/ml) was fully activated with cgmp-dependent protein kinase. The activated enzyme preparation was immediately passed through a Sephadex G-50 column to remove ATP, cgmp, and Mg2+. The enzyme eluted in the void volume was supplemented with 5 mm Mg2+ and 0.35 unit of purified bovine heart phosphorylase phosphatase, and incubation was carried out at 300 (0). Reactivation of the deactivated lipase was effected at 20 min by ATP, 10 MM cgmp, and cgmp-dependent protein kinase at 5 units/ml and incubating for 10 min at 300 (t). The basal lipase activity prior to activation was 13 nmol of free fatty acid released per mg of protein/hr. pation by endogenous camp-dependent protein kinase. The degree of activation was comparable to that observed with camp-dependent protein kinase. Cholesterol esterase from rat adrenal has been previously shown to be activated, although to a limited extent (40 + 16%), by camp-dependent protein kinase (26, 27). As shown in Table 2, it was also activated by cgmp-dependent protein kinase. Activation of Phosphorylase Kinase. Protein kinase-dependent activation of phosphorylase kinase (rabbit muscle) is shown in Fig. 4. Activation by camp-dependent protein kinase plus camp was almost completely inhibited by added protein kinase inhibitor. Full activation was also obtained with Table 2. Activation of hormone-sensitive lipase from human and rat adipose tissue and of cholesterol esterase from rat adrenal* Enzyme activity, nmol free fatty acid/mg protein per hr Human Rat Rat adipose adipose adrenal tissue tissue cholesterol Additions lipase lipase esterase Mg2+ 118 44 30 MgATP, cgmp 102 43 32 MgATP, cgmp, cgmp-dependent protein kinase, protein kinase inhibitor 289 70 42 MgATP, camp, camp-dependent protein kinase 241 80 46 * The 5.2 P fraction of human omental adipose tissue was used; in the case of the rat tissues, the 100,000 X g supernatant fraction was used. The conditions for activation and the concentrations of the additions were as described under Materials and Methods and in Table 1. The concentration of camp-dependent protein kinase used was 10 units/ml.

4846 Biochemistry: Khoo et- al. 0 2 4 6 8 10 Protein kinase, units/ml FIG. 4. Activation of phosphorylase kinase as a function of protein' kinase concentration. The conditions for the activation of phosphorylase kinase from rabbit skeletal muscle (12.5,tg) were as described under Materials and Methods. Activation by camp-dependent protein kinase plus camp was carried out either in the absence (M) or in the presence (78,g/ml) of protein kinase inhibitor (A); activation by cgmp-dependent protein kinase plus cgmp was carried out in the presence (78,ug/ml) of protein kinase inhibitor (0). The activation was terminated by 1:10 dilution with ice-cold buffer, and phosphorylase kinase activity was immediately assayed as described. cgmp-dependent protein kinase but this required a somewhat higher concentration of kinase (expressed in histone phosphorylation units). This activation depended absolutely on the presence of ATP. Activation by cgmp-dependent protein kinase was not inhibited by protein kinase inhibitor. In fact, the data shown for cgmp-dependent protein kinase activation were obtained in the presence of the same inhibitor concentration that fully inhibited camp-dependent protein kinase activation. DISCUSSION Both camp- and cgmp-dependent protein kinases have been purified to homogeneity (7, 28, 29). Although many physical properties of the two enzymes are similar, the subunit structure and response to cycle nucleotides differ. camp-dependent protein kinase is a tetramer, R2C2, which in the presence of camp exists as R2-(cAMP)2 + 2C (28-30). cgmp-dependent protein kinase is a dimer, (RC)2, which in the presence of cgmp exists as (RC)2-(cGMP)2 (7, 16). The present studies demonstrate the ability of purified cgmp-dependent protein kinase to act on two well-characterized, interconvertible enzyme systems that have previously been shown to be activated by camp-dependent protein kinase (10, 23). Hormone-sensitive lipase activated by cgmp-dependent protein kinase was deactivated by a purified protein phosphatase and then fully reactivated, supporting the interpretation that cgmp-dependent protein kinase catalyzes the same phosphorylation as that catalyzed by camp-dependent protein kinase (23). However, this remains to be demonstrated directly. Concurrently with the present studies, it was found (D. K. Blumental, J. T. Stull, and G. N. Gill, unpublished data) that cgmp-dependent protein kinase also catalyzes the phosphorylation of cardiac troponin. In these three cases, the reactions parallel those catalyzed in the same systems by campdependent protein kinase. Studies by Hashimoto et al. (31) have shown that the two kinases act on the same phosphorylation sites in histone. Taken together, the results suggest that the substrate specificities of the two kinases are similar. Nishiyama et al. (32), Takai et al. (33), and Inoue et al. (34) Proc. Natl. Acad. Sci. USA 74 (1977) did not observe activation of phosphorylase kinase by cgmp-dependent protein kinase. Although these authors used enzyme preparations isolated from other sources (silkworms or bovine cerebellum), it is unlikely that this explains the different results because the cgmp-dependent protein kinases they studied phosphorylate histone at the same sites phosphorylated by camp-dependent protein kinase (31). Kuo et al. (35) also failed to obtain phosphorylase kinase activation with a 150- fold-purified cgmp-dependent protein kinase from fetal guinea pig lung, which would be expected to be very similar to the bovine lung enzyme used in the present studies. The possibility that other substances in partially purified preparations modify not only the level of activity but also substrate specificity was considered. However, a partially purified preparation of cgmp-dependent protein kinase, purified to the DEAE-cellulose chromatography step prior to affinity chromatography (7), catalyzed similar activation of hormone-sensitive lipase and phosphorylase kinase (data not shown). The use of higher concentrations of cgmp-dependent protein kinase (expressed as units of histone phosphorylating activity) and other differences in assay conditions may partially explain the difference in results. Adipose tissue contains cgmp-dependent protein kinase activity (8) and so changes in cgmp concentration might directly regulate hormone-sensitive lipase. From the present results it appears that regulation at this level would be in the same direction as that exercised by camp. On the other hand, control of lipolysis by cgmp might also be exercised at other levels and in an opposite direction to that exercised by camp. Illiano et al. (36) observed increases in cgmp in response to insulin, and this has been confirmed by Fain and Butcher (37). However, the latter workers found little correlation between insulininduced changes in cgmp levels and suppression of norepinephrine-induced lipolysis. Carbachol also increases cgmp levels but it does not appear to be antilipolytic (37). Furthermore, norepinephrine itself has been reported to increase cgmp levels, a finding difficult to reconcile with a simple push-pull type of regulation. The levels of cgmp and cgmp-dependent protein kinase in rat adipose tissue are apparently low compared to those of camp and camp-dependent protein kinase (8, 36, 37). Thus, it seems unlikely that under ordinary circumstances the hormone-induced changes in cgmp levels would contribute importantly to the lipolytic effect of catecholamines. Exton et al. (38) have reported that cgmp added to liver perfusates increased phosphorylase activity. On the other hand, in recent studies in heart and in liver, changes in cgmp concentration have not been well correlated with changes in phosphorylase activity (4, 39). Thus, it is difficult to assess the possible importance of cgmp-dependent protein kinase in the activation of the lipase and phosphorylase kinase systems in vvo. The lesser potency (based on histone phosphorylation units) of cgmp-dependent protein kinase under the in vitro conditions used does not rule out significant participation in vivo. camp- and cgmp-dependent protein kinases were compared on the basis of histone phosphorylation because most published reports have utilized this substrate. The homogeneous enzymes have similar specific activities: camp-dependent protein kinase, 1660 units/mg with histone Type II A (28); cgmp-dependent protefinklnase, 1375 units/mg with histone H2b (7). Moreover, both protein kinases phosphorylate the same serine hydroxyl groups in histones H1 and H2b (31). Histone phosphorylation units are thus a reasonable but arbitrary standard for comparison. Whatever the physiologic significance, the finding of similar substrate specificities is consonant with the similarity in basic

Biochemistry: Khoo et al. subunit structures in the model proposed by Gill (40). According to this model the two kinases may differ primarily in that the regulatory and catalytic functions of cgmp-dependent protein kinase are present in a single chain while in camp-dependent protein kinase the chain is discontinuous-i.e., regulatory and catalytic subunits are distinct. Note Added in Proof. A recent report by Lincoln and Corbin (41) documented that cgmp-dependent protein kinase catalyzed incorporation of 32P into rat liver pyruvate kinase and fructose-1,6-bisphosphatase, rabbit skeletal muscle glycogen synthase, and phosphorylase kinase, in agreement with the functional changes documented in the present study. We thank Mrs. Mercedes Silvestre for her excellent technical assistance. We are indebted to Dr. Ernest Y. C. Lee, University of Miami, for the highly purified phosphorylase phosphatase, Dr. Steven E. Mayer for the camp-dependent protein kinase, and Dr. Steven R. Gross for the phosphorylase kinase. This project was supported by National Institutes of Health Research Grant HL 12373 from the National Heart, Lung, and Blood Institute and Research Grant BC-209 from the American Cancer Society. G. N.G. is the recipient of Research Career Development Award no. AM70215 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. 1. Goldberg, N. D., O'Dea, R. F. & Haddox, M. K. (1973) Adv. Cyclic Nucleotide Res. 3, 155-223. 2. Goldberg, N. D., Haddox, M. K., Nicol, S. E., Glass, D. B., Sanford, C. H., Kuehl, F. A. & Estensen, R. (1975) Adv. Cyclic Nucleotide Res. 5, 307-330. 3. Nesbitt, J. A., Anderson, W. B., Miller, Z., Pastan, I., Russell, T. & Gospodarowicz, D. (1976) J. Biol. Chem. 251, 2344-2352. 4. Pointer, R. H., Butcher, F. R. & Fain, J. N. (1976) J. Biol. Chem. 251,2987-2992. 5. Krebs, E. G. (1972) Curr. Top. Cell. Regul. 5,99-133. 6. Kuo, J. F. & Greengard, P. (1970) J. Biol. Chem. 245, 2493-2498. 7. Gill, G. N., Holdy, K. E., Walton, G. M. & Kanstein, C. B. (1976) Proc. Natl. Acad. Sci. USA 73,3918-3922. 8. Lincoln, T. M., Hall, C. L., Park, C. R. & Corbin, J. D. (1976) Proc. Natl. Acad. Sci. USA 73,2559-2563. 9. Steinberg, D. (1976) Adv. Cyclic Nucleotide Res. 7, 157-198. 10. Walsh, D. A., Perkins, J. P. & Krebs, E. G. (1968) J. Biol. Chem. 243,3763-3765. 11. Glynn, I. M. & Chappell, J. B. (1964) Biochem. J. 90, 147-149. 12. Wastila, W. B., Stull, J. T., Mayer, S. E. & Walsh, D. A. (1971) J. Biol. Chem. 246,1996-2003. 13. Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, E. H. & Krebs, E. G. (1971) J. Biol. Chem. 246, 1977-1985. 14. Cohen, P. (1973) Eur. J. Biochem. 34, 1-14. Proc. Natl. Acad. Sci. USA 74 (1977) 4847 15. Brandt, H., Capulong, Z. L. & Lee, E. Y. C. (1975) J. Biol. Chem. 250,8038-8044. 16. Gill, G. N., Walton, G. M. & Sperry, P. J. (1977) J. Biol. Chem. 252,6443-6449. 17. Khoo, J. C. & Steinberg, D. (1974) J. Lipid Res. 15, 602-610. 18. Khoo, J. C., Steinberg, D., Huang, J. J. & Vagelos, P. R. (1976) J. Biol. Chem. 251, 2882-2890. 19. Pittman, R. C., Khoo, J. C. & Steinberg, D. (1975) J. Biol. Chem. 2,50,4505-4511. 20. Khoo, J. C. (1976) Biochim. Biophys. Acta 422, 87-97. 21. Krebs, E. G., Love, D. S., Bratvold, G. E., Trayser, K. A., Meyer, W. L. & Fischer, E. H. (1964) Biochemistry 3,1022-1033. 22. Hardman, J. G., Mayer, S. E. & Clark, B. (1965) J. Pharmacol. Exp. Ther. 150,341-348. 23. Huttunen, J. K. & Steinberg, D. (1971) Biochim. Biophys. Acta 239,411-427. 24. Donnelly, T. E., Jr., Kuo, J. F., Miyamoto, E. & Greengard, P. (1973) J. Biol. Chem. 248, 199-203. 25. Khoo, J. C., Aquino, A. A. & Steinberg, D. (1974) J. Clin. Invest. 53, 1124-1131. 26. Trzeciak, W. H. & Boyd, G. S. (1974) Eur. J. Biochem. 46, 201-207. 27. Pittman, R. C. & Steinberg, D. (1977) Biochim. Biophys. Acta 248,431-444. 28. Hofmann, F., Beavo, J. A., Bechtel, P. J. & Krebs, E. G. (1975) J. Biol. Chem. 250,7795-7801. 29. Rubin, C. S., Erlichman, J. & Rosen, 0. M. (1972) J. Biol. Chem. 247,36-44. 30. Rosen, 0. M. & Erlichman, J. (1975) J. Biol. Chem. 250, 7788-7794. 31. Hashimoto, E., Takeda, M., Nishizuka, Y., Hamana, K. & Iwai, K. (1976) J. Biol. Chem. 251, 6287-6293. 32. Nishiyama, K., Katakami, H., Yamamura, H., Takai, Y., Shimomura, R. & Nishizuka, Y. (1975) J. Biol. Chem. 250, 1297-1300. 33. Takai, Y., Nishiyama, K., Yamamura, H. & Nishizuka, Y. (1975) J. Biol. Chem. 250,4690-4695. 34. Inoue, M., Kishimoto, A., Takai, Y. & Nishizuka, Y. (1976) J. Biol. Chem. 251, 4476-4478. 35. Kuo. J. F., Kuo, W-N., Shoji, M., Davis, C. W., Seery, V. L. & Donnelly, T. E., Jr. (1976) J. Biol. Chem. 251, 1759-1766. 36. Illiano, G., Tell, G. P. E., Siegel, M. I. & Cuatrecasas, P. (1973) Proc. Natl. Acad. Scd. USA 70,2443-2447. 37. Fain, J. N. & Butcher, F. R. (1976) J. Cyclic Nucleotide Res. 2, 71-78. 38. Exton, J. H., Hardman, J. G., Williams, T. F., Sutherland, E. W. & Park, C. R. (1971) J. Biol. Chem. 246,2658-2664. 39. Gardner, R. M. & Allen, D. 0. (1976) J. Pharmacol. Exp. Ther. 198,412-419. 40. Gill, G. N. (1977) J. Cyclic Nucleotide Res. 3, 153-162. 41. Lincoln, T. M. & Corbin, J. D. (1977) Proc. Natl. Acad. Sci. USA 74,3239-3243.