- Email: [email protected]
Profiling of inhibitors of protein kinase C
PROFILING OF PROTEIN LAWRENCE
INHIBITORS KINASE C
P. WENNOGLE, HALYNA ARC0 Y. JENG
E. WYSOWSKYJ
Research Department, Pharmaceuticals Division, CIBA-GEIGY Summit, NJ 07901
OF and Corp.,
INTRODUCTION
It is well established that protein kinase C (ATP: protein transphosphorylase, protein kinase first EC 2.7.1.37), a Ca2+- and phospholipid-dependent identified by Nishizuka and co-workers (l), participates in the message transduction for a large class of hormones and neurotransmitters which generate diacylglycerols by stimulating phosphatidylinositol turnover (2). Recent studies have shown that protein kinase C is the major receptor of the phorbol ester tumor promoters (3-5). In view of the involvement of protein kinase C in tumor promotion, oncogene activation, protein phosphorylation, feedback mechanisms in signal transduction, and cellular responses to growth factors (for reviews, see Refs 6-8), significant efforts have focused on searching for inhibitors of this enzyme (9-12). We have broadened this concept by designing a simplified protocol for screening compounds capable of modulating the activity of protein kinase C at different activation states. In this article, three classes of inhibitors are identified. The results suggest that our approach may provide a rapid screening method and yield useful information in the understanding of inhibitory mechanisms. MATERIALS
AND
METHODS
Marerials. Preswollen diethylaminoethyl
cellulose (DE52) and cellulose phosphate P8 1 papers were obtained from Whatman (Clifton, NJ). ATP was a product of Calbiochem (La Jolla, CA). 1,2-Diolein, histone III-S (H-5505), phosphatidylserine, trifluoperazine, apomorphine, and Phenyl-Sepharose CL4B were purchased from Sigma (St. Louis, MO). [r-32P]ATP was from New England Nuclear (Boston, MA). N-14-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenyl j- 1H-tetrazol-5-carboxamide (LY 170198), a LTD, antagonist, was synthesized by Dr. A. Sallmann of CIBA-GEIGY, Basel. Partial purification of protein kinase C. The cytosol from 70 mouse brains was prepared and fractionated by DE52 column chromatography as described (13). Fractions containing high protein kinase activity were pooled, 287
288
L. P. WENNOGLE,
et al.
concentrated, and desalted with Amicon stirred cells (Danvers, MA) using PM30 membranes. The final protein concentration was about 20 mg ml-’ in a buffer containing 0.5 mM EGTA, 0.5 mM EDTA, 1 mM DTT, and 20 mM Tris-HCl, pH 7.4 (buffer A). The concentrated DE52 peak material was further fractionated by a column of Phenyl-Sepharose (5 g of PhenylSepharose per 1 ml of the DE52 pool) equilibrated with 1.5 M NaCl in buffer A. Proteins were eluted with a linear 1.5 to 0 M NaCl gradient in buffer A (40 ml of total gradient vol per 1 ml of the DE52 pool). Protein kinase C was eluted at the end of the gradient and was frozen until use in liquid nitrogen in the presence of 0.01% Triton X- 100 (13). About 60% of the enzymatic activity was recovered from the Phenyl-Sepharose column chromatography. The protein kinase specific activity was about 1 pmol min-’ mg-’ of protein. Protein kinase C at this stage was about 50% pure and was used as the enzyme source in this study. Biochemical assays. Protein kinase C assays were carried out in a 250 /*l reaction mixture containing 0.2 pg partially purified protein kinase C, 5 pM [y-32P]ATP, 100 pg ml-’ histone III-S, 20 mM Tris-HCl, pH 7.4 and, if used, various concentrations of Ca2+ and phosphatidylserine, as described in the legends of the Figures. The reaction mixture was incubated at 30°C for 10 min followed by chilling at 0°C. EDTA (25 r-11)was added to each tube to a final concentration of 5 mM and duplicate, 40-~1 aliquots were analyzed for 32P incorporation into histone III-S using phosphocellulose paper (14). Proteins were determined by the method of Bradford (15) using bovine y-globulins as a standard.
RESULTS
AND
DISCUSSION
The Assay System Protein kinase C is a complex enzyme which has been shown to contain multiple sites for interaction with substrates and activators. The enzyme can exist in various states of activation. In addition to Ca2+ and phosphatidylserine, protein kinase C can be further activated by 1,Zdiolein (16) which competes with phorbol esters for a binding site in the regulatory domain of the enzyme (17, 18). We designed a protocol for rapid initial screening of inhibitors or activators of protein kinase C (Fig. la). In the presence of 10 pg ml-’ each of phosphatidylserine and 1,2-diolein, protein kinase C was maximally stimulated (Fig. la, D). Under these conditions, the enzyme showed lo- to 15-fold stimulation, compared with that in the absence of either activator (Fig. la, A). Phosphatidylserine alone at 10 pg ml-’ gave about 20% of the maximal activity (Fig. la, B), while the inclusion of additional 2 pg ml-’ diolein yielded about 75% maximal activity (Fig. la, C).
INHIBITORS OF PROTEIN KINASE C
100 -
289
a
A
B
C
D
16
b ?
10
P i0 6
0
FIG. 1. Protein kinase C assays. (a) The assay conditions were the same as described in Materials and Methods except that the concentrations of phosphatidylserine and diolein in the different assays were: A, 0 and 0; B, 10 and 0; C, 10 and 2; D, 10 and 10 /.~gml-‘, respectively. The maximally activated protein kinase C in the D assay was set at 100% and the ercent activities were expressed as mean + SEM (n = 10). The error in the D assay was 4%. (b) Ca R dependence of the assays. The Ca2+ concentrations in the A, B and C assays were: open bars, 0; hatched bars, 10; shaded bars, 100 FM. Filled bars, 200 pM EDTA.
The assays shown in Figure la were referred to as the A, B, C, and D assays, respectively. Ca2+ was not added to these assays, as the residual Ca2+ in the house distilled water (about 10 PM) sufficed. Inclusion of exogenous CaZt up to 100 PM did not give significant additional stimulations in the B and C assays, whereas the addition of 200 @M EDTA abolished the stimulation (Fig. lb). These results indicate that the partially purified protein kinase C employed in this study required both Ca2+and phosphatidylserine for activity and was stimulated by diolein.
290
L. P. WENNOGLE,
et al.
Active Compounds
As expected, analogs of phorbol esters and diolein were found to be activators in the B and C assays (results not shown). Numerous inhibitors were also detected. Since phospholipid-interfering agents may inhibit protein kinase C non-specifically, the active inhibitors were subsequently tested in the C assay supplemented with excess phosphatidylserine (200 pg ml-‘, referred to as the CP assay) to overcome the perturbation of phospholipids by these agents. For compounds which exerted their inhibitory activities via nonspecific interaction with phospholipid vesicles, the inhibitory potencies should be significantly reduced in the CP assay. Three classes of inhibitors were identified, and the structures of representative compounds from each class are shown in Figure 2. These three compounds illustrated different characteristics in the B, the C, and the CP assays (Fig. 3). In the presence of excess phosphatidylserine, the inhibition by
Tr if luoperazine
HO
A pomorphine
LY 170198 FIG. 2. The structures
of three classes of inhibitors
of protein
kinase C
291
INHIBITORS OF PROTEIN KINASE C 100
80
E
‘S
3
00
E E
E 8
40
& n 20
0
J
Apomorphine Trifluoperazine
FIG. 3. Effects of high concentrations of phosphatidylserine on the inhibition ofprotein kinase C by selected compounds. Protein kinase C activities were measured in the presence of 20 phi apomorphine, 50 PM trifluoperazine, or 12.5 pM LY 170198 under different concentrations of phosphatidylserine and diolein: open bars, 10 and 0; filled bars, 10 and 2; shaded bars, 200 and 2 c(g ml-‘, respectively. Error bars, SEM from 6-8 experiments.
trifluoperazine was greatly relieved, indicating the phospholipid-interfering nature of this compound. Trifluoperazine seemed to be able to interact with phospholipids readily, since sonication of trifluoperazine and phosphatidylserine simultaneously did not have greater effects (results not shown). The inhibition of protein kinase C by apomorphine or by LY 170198 was not reversed by phosphatidylserine. LY 170198 was equally potent in the inhibition of protein kinase C either in its partially activated (low phosphatidylserine, B assay) or in the diolein-stimulated state (C assay). Apomorphine, on the other hand, was not as active in the B assay (Fig. 3). The difference in the inhibitory potencies measured in B and C assays probably reflects a variation in the affinities of these inhibitors for protein kinase C at different states of activation. Consistent with this hypothesis are the observations that the IC,O values for LY 170198 measured in the B and C assays were similar, whereas the I&, value for apomorphine was 3-fold greater in the absence of diolein (Table 1). Isoquinolinesulfonamides have the same properties as those of the apomorphine (results not shown) and have been shown to compete for ATP binding (9). A substantial difference in the slopes of the dose-response curves was also observed (Fig. 4). While apomorphine gave a dose-response curve typified by that of a competitive displacer, trifluoperazine and LY 170198 showed steep displacement curves, indicating a Hill coefficient of greater than one. The reason for the apparent cooperativity in inhibition is not currently understood.
292
L. P. WENNOGLE,
er al.
TABLE 1. VARlATION OF INHIBITORY POTENCIES UNDER DIFFERENT ASSAY CONDITIONS
Compound
B assay
C assay
28 ND 2.8
9 40 3.1
Apomorphine Trifluoperazine LY 170198
The concentrations of phosphatidylserine diolein were: B assay, 10 and 0; C assay, 2 ~18ml-‘, respectively. ND, not determined.
and 10 and
20
0 1
100
1000
IlnhibitIrI, PM FIG. 4. Dose-dependent inhibition of protein kinase C by compounds shown in Figure 2. The reaction mixtures contained 10 pg ml-’ phosphatidylserine, 2 pg ml-’ diolein, and various concentrations of trifluoperazine (o), apomorphine (e), and LY 170198 (A). Each data point represents the average of quadruplicate determinations.
SUMMARY
A rapid screen assay for protein kinase C was described which allowed the detection of inhibitors and activators of the enzyme at different states of activation. Upon secondary evaluation of the active inhibitors, three classes of compounds were identified. The inhibition of protein kinase C by one class of compounds, exemplified by trifluoperazine, could be reversed in the presence of excess phosphatidylserine, indicating the phospholipid-interfering nature of these compounds. While the other two classes of compounds, represented by apomorphine and LY 170198, respectively, did not exert their inhibition by interfering with phospholipids, their inhibitory potencies differed depending on the state of activation ofprotein kinase C. LY 170198 was equally potent in the inhibition of protein kinase C either in its partially activated state in the
INHIBITORS OF PROTEIN KINASE C
293
presence of low concentrations of phosphatidylserine or in the dioleinstimulated state. Apomorphine, on the other hand, was less active against protein kinase C in the partially activated state. Isoquinoiinesulfonamides have the same properties as those of the apomorphine and have been shown to compete for ATP binding. The mechanism of inhibition of protein kinase C by LY 170198 needs to be further investigated.
5.
6. 7. 8.
9.
10.
11.
12. 13. 14. 15.
REFERENCES Y. NISHIZUKA,Studiesandperspectives ofprotein kinase C,Science233,305-312(1986). A. A. ABDEL-LATIF, Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers, Pharmacol. Rev. 38,227-272 (1986). J. E. NIEDEL, L. J. KUHN and G. R. VANDENBARK, Phorbol diester receptor copurifies with protein kinase C, Proc. Nor/. Acad. Sci. V.S.A. 80,36-40 (1983). U. KIKKAWA, Y. TAKAI, Y. TANAKA, R. MIYAKE and Y. NISHIZUKA, Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters, J. Biol. Chem. 258, 11442-I 1445 (1983). G. PASTI, J.-C. LACAL, B. S. WARREN, S. A. AARONSON and P. M. BLUMBERG, Loss of mouse Iibroblast cell response to phorbol esters restored by microinjected protein kinase C, Nature 324,375-377 (1986). L. DIAMOND, Tumor promoters and cell transformation, Phormac. Ther. 26, 89-145 (1984). G. S. MARTIN, The erbB gene and EGF receptor, Cuncer Surveys 5, 199-219 (1986).
A. Y. JENG and P. M. BLUMBERG, Biochemical mechanisms of action of the phorbol ester class of tumor promoters, in The Parhobiology of Neoplasia (A. E. SIRICA, ed.), Plenum, New York (1987), in press. H. HIDAKA, M. INAGAKI, S. KAWAMOTO and Y. SASAKI, Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C, Biochemisrry 23,5036-5041 (1984). R. S. TURNER and J. F. KUO, Phospholipid-sensitive Ca*+-dependent protein kinase (protein kinase C): The enzyme, substrates, and regulation, pp. 75-110 in Phospholipidsund Cellular Regularion, Vol. 2 (J. F. KUO, ed.), CRC Press, Boca Raton, Florida (1985). Y. A. HANNUN, C. R. LOOMIS, A. H. MERRILL, JR. and R. M. BELL, Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets, J. Biof. Chem. 261, 12604-12609 (1986). T. TAMAOKI, H. NOMOTO, I. TAKAHASHI, Y. KATO, M. MORIMOTO and F. TOMITA, Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase, Biochem. BioDhvs. Res. Commun. 135. 397-402 (3986). A. Y. JENG, N. A.‘SHARKEY and P. M. BLUMBE‘RG, Purification of stable protein kinase C from mouse brain cytosol by specific ligand elution using fast protein liquid chromatography, Cancer Res. 46, 1966-1971(1986). J. J. WITI’ and R. ROSKOSKI, JR., Rapid protein kinase assay using phosphocellulosepaper absorption, Anal. Biochem. 66,253-258 (1975). M. M. BRADFORD, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248-254 (1976).
16. M. CASTAGNA, Y. TAKAI, K. KAIBUCHI, K. SANO, U. KIKKAWA and Y. NISHIZUKA, Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters, J. Biol. Chem. 257,7847-7851 (1982). 17. N. A. SHARKEY, K. L. LEACH and P. M. BLUMBERG, Competitive inhibition by diacylglycerol of specific phorbol ester binding, Proc. Nat/. Acud. Sci. U.S.A. 81,607-610 (1984).
18. M.-H. LEE and R. M. BELL, The lipid binding, regulatory domain of protein kinase C. A 32-kDa fragment contains the calcium and phosphatidylserine-dependent phorbol diester binding activity, J. Biol. Chem. 261, 14867-14870 (1986).
INHIBITORS KINASE C
P. WENNOGLE, HALYNA ARC0 Y. JENG
E. WYSOWSKYJ
Research Department, Pharmaceuticals Division, CIBA-GEIGY Summit, NJ 07901
OF and Corp.,
INTRODUCTION
It is well established that protein kinase C (ATP: protein transphosphorylase, protein kinase first EC 2.7.1.37), a Ca2+- and phospholipid-dependent identified by Nishizuka and co-workers (l), participates in the message transduction for a large class of hormones and neurotransmitters which generate diacylglycerols by stimulating phosphatidylinositol turnover (2). Recent studies have shown that protein kinase C is the major receptor of the phorbol ester tumor promoters (3-5). In view of the involvement of protein kinase C in tumor promotion, oncogene activation, protein phosphorylation, feedback mechanisms in signal transduction, and cellular responses to growth factors (for reviews, see Refs 6-8), significant efforts have focused on searching for inhibitors of this enzyme (9-12). We have broadened this concept by designing a simplified protocol for screening compounds capable of modulating the activity of protein kinase C at different activation states. In this article, three classes of inhibitors are identified. The results suggest that our approach may provide a rapid screening method and yield useful information in the understanding of inhibitory mechanisms. MATERIALS
AND
METHODS
Marerials. Preswollen diethylaminoethyl
cellulose (DE52) and cellulose phosphate P8 1 papers were obtained from Whatman (Clifton, NJ). ATP was a product of Calbiochem (La Jolla, CA). 1,2-Diolein, histone III-S (H-5505), phosphatidylserine, trifluoperazine, apomorphine, and Phenyl-Sepharose CL4B were purchased from Sigma (St. Louis, MO). [r-32P]ATP was from New England Nuclear (Boston, MA). N-14-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenyl j- 1H-tetrazol-5-carboxamide (LY 170198), a LTD, antagonist, was synthesized by Dr. A. Sallmann of CIBA-GEIGY, Basel. Partial purification of protein kinase C. The cytosol from 70 mouse brains was prepared and fractionated by DE52 column chromatography as described (13). Fractions containing high protein kinase activity were pooled, 287
288
L. P. WENNOGLE,
et al.
concentrated, and desalted with Amicon stirred cells (Danvers, MA) using PM30 membranes. The final protein concentration was about 20 mg ml-’ in a buffer containing 0.5 mM EGTA, 0.5 mM EDTA, 1 mM DTT, and 20 mM Tris-HCl, pH 7.4 (buffer A). The concentrated DE52 peak material was further fractionated by a column of Phenyl-Sepharose (5 g of PhenylSepharose per 1 ml of the DE52 pool) equilibrated with 1.5 M NaCl in buffer A. Proteins were eluted with a linear 1.5 to 0 M NaCl gradient in buffer A (40 ml of total gradient vol per 1 ml of the DE52 pool). Protein kinase C was eluted at the end of the gradient and was frozen until use in liquid nitrogen in the presence of 0.01% Triton X- 100 (13). About 60% of the enzymatic activity was recovered from the Phenyl-Sepharose column chromatography. The protein kinase specific activity was about 1 pmol min-’ mg-’ of protein. Protein kinase C at this stage was about 50% pure and was used as the enzyme source in this study. Biochemical assays. Protein kinase C assays were carried out in a 250 /*l reaction mixture containing 0.2 pg partially purified protein kinase C, 5 pM [y-32P]ATP, 100 pg ml-’ histone III-S, 20 mM Tris-HCl, pH 7.4 and, if used, various concentrations of Ca2+ and phosphatidylserine, as described in the legends of the Figures. The reaction mixture was incubated at 30°C for 10 min followed by chilling at 0°C. EDTA (25 r-11)was added to each tube to a final concentration of 5 mM and duplicate, 40-~1 aliquots were analyzed for 32P incorporation into histone III-S using phosphocellulose paper (14). Proteins were determined by the method of Bradford (15) using bovine y-globulins as a standard.
RESULTS
AND
DISCUSSION
The Assay System Protein kinase C is a complex enzyme which has been shown to contain multiple sites for interaction with substrates and activators. The enzyme can exist in various states of activation. In addition to Ca2+ and phosphatidylserine, protein kinase C can be further activated by 1,Zdiolein (16) which competes with phorbol esters for a binding site in the regulatory domain of the enzyme (17, 18). We designed a protocol for rapid initial screening of inhibitors or activators of protein kinase C (Fig. la). In the presence of 10 pg ml-’ each of phosphatidylserine and 1,2-diolein, protein kinase C was maximally stimulated (Fig. la, D). Under these conditions, the enzyme showed lo- to 15-fold stimulation, compared with that in the absence of either activator (Fig. la, A). Phosphatidylserine alone at 10 pg ml-’ gave about 20% of the maximal activity (Fig. la, B), while the inclusion of additional 2 pg ml-’ diolein yielded about 75% maximal activity (Fig. la, C).
INHIBITORS OF PROTEIN KINASE C
100 -
289
a
A
B
C
D
16
b ?
10
P i0 6
0
FIG. 1. Protein kinase C assays. (a) The assay conditions were the same as described in Materials and Methods except that the concentrations of phosphatidylserine and diolein in the different assays were: A, 0 and 0; B, 10 and 0; C, 10 and 2; D, 10 and 10 /.~gml-‘, respectively. The maximally activated protein kinase C in the D assay was set at 100% and the ercent activities were expressed as mean + SEM (n = 10). The error in the D assay was 4%. (b) Ca R dependence of the assays. The Ca2+ concentrations in the A, B and C assays were: open bars, 0; hatched bars, 10; shaded bars, 100 FM. Filled bars, 200 pM EDTA.
The assays shown in Figure la were referred to as the A, B, C, and D assays, respectively. Ca2+ was not added to these assays, as the residual Ca2+ in the house distilled water (about 10 PM) sufficed. Inclusion of exogenous CaZt up to 100 PM did not give significant additional stimulations in the B and C assays, whereas the addition of 200 @M EDTA abolished the stimulation (Fig. lb). These results indicate that the partially purified protein kinase C employed in this study required both Ca2+and phosphatidylserine for activity and was stimulated by diolein.
290
L. P. WENNOGLE,
et al.
Active Compounds
As expected, analogs of phorbol esters and diolein were found to be activators in the B and C assays (results not shown). Numerous inhibitors were also detected. Since phospholipid-interfering agents may inhibit protein kinase C non-specifically, the active inhibitors were subsequently tested in the C assay supplemented with excess phosphatidylserine (200 pg ml-‘, referred to as the CP assay) to overcome the perturbation of phospholipids by these agents. For compounds which exerted their inhibitory activities via nonspecific interaction with phospholipid vesicles, the inhibitory potencies should be significantly reduced in the CP assay. Three classes of inhibitors were identified, and the structures of representative compounds from each class are shown in Figure 2. These three compounds illustrated different characteristics in the B, the C, and the CP assays (Fig. 3). In the presence of excess phosphatidylserine, the inhibition by
Tr if luoperazine
HO
A pomorphine
LY 170198 FIG. 2. The structures
of three classes of inhibitors
of protein
kinase C
291
INHIBITORS OF PROTEIN KINASE C 100
80
E
‘S
3
00
E E
E 8
40
& n 20
0
J
Apomorphine Trifluoperazine
FIG. 3. Effects of high concentrations of phosphatidylserine on the inhibition ofprotein kinase C by selected compounds. Protein kinase C activities were measured in the presence of 20 phi apomorphine, 50 PM trifluoperazine, or 12.5 pM LY 170198 under different concentrations of phosphatidylserine and diolein: open bars, 10 and 0; filled bars, 10 and 2; shaded bars, 200 and 2 c(g ml-‘, respectively. Error bars, SEM from 6-8 experiments.
trifluoperazine was greatly relieved, indicating the phospholipid-interfering nature of this compound. Trifluoperazine seemed to be able to interact with phospholipids readily, since sonication of trifluoperazine and phosphatidylserine simultaneously did not have greater effects (results not shown). The inhibition of protein kinase C by apomorphine or by LY 170198 was not reversed by phosphatidylserine. LY 170198 was equally potent in the inhibition of protein kinase C either in its partially activated (low phosphatidylserine, B assay) or in the diolein-stimulated state (C assay). Apomorphine, on the other hand, was not as active in the B assay (Fig. 3). The difference in the inhibitory potencies measured in B and C assays probably reflects a variation in the affinities of these inhibitors for protein kinase C at different states of activation. Consistent with this hypothesis are the observations that the IC,O values for LY 170198 measured in the B and C assays were similar, whereas the I&, value for apomorphine was 3-fold greater in the absence of diolein (Table 1). Isoquinolinesulfonamides have the same properties as those of the apomorphine (results not shown) and have been shown to compete for ATP binding (9). A substantial difference in the slopes of the dose-response curves was also observed (Fig. 4). While apomorphine gave a dose-response curve typified by that of a competitive displacer, trifluoperazine and LY 170198 showed steep displacement curves, indicating a Hill coefficient of greater than one. The reason for the apparent cooperativity in inhibition is not currently understood.
292
L. P. WENNOGLE,
er al.
TABLE 1. VARlATION OF INHIBITORY POTENCIES UNDER DIFFERENT ASSAY CONDITIONS
Compound
B assay
C assay
28 ND 2.8
9 40 3.1
Apomorphine Trifluoperazine LY 170198
The concentrations of phosphatidylserine diolein were: B assay, 10 and 0; C assay, 2 ~18ml-‘, respectively. ND, not determined.
and 10 and
20
0 1
100
1000
IlnhibitIrI, PM FIG. 4. Dose-dependent inhibition of protein kinase C by compounds shown in Figure 2. The reaction mixtures contained 10 pg ml-’ phosphatidylserine, 2 pg ml-’ diolein, and various concentrations of trifluoperazine (o), apomorphine (e), and LY 170198 (A). Each data point represents the average of quadruplicate determinations.
SUMMARY
A rapid screen assay for protein kinase C was described which allowed the detection of inhibitors and activators of the enzyme at different states of activation. Upon secondary evaluation of the active inhibitors, three classes of compounds were identified. The inhibition of protein kinase C by one class of compounds, exemplified by trifluoperazine, could be reversed in the presence of excess phosphatidylserine, indicating the phospholipid-interfering nature of these compounds. While the other two classes of compounds, represented by apomorphine and LY 170198, respectively, did not exert their inhibition by interfering with phospholipids, their inhibitory potencies differed depending on the state of activation ofprotein kinase C. LY 170198 was equally potent in the inhibition of protein kinase C either in its partially activated state in the
INHIBITORS OF PROTEIN KINASE C
293
presence of low concentrations of phosphatidylserine or in the dioleinstimulated state. Apomorphine, on the other hand, was less active against protein kinase C in the partially activated state. Isoquinoiinesulfonamides have the same properties as those of the apomorphine and have been shown to compete for ATP binding. The mechanism of inhibition of protein kinase C by LY 170198 needs to be further investigated.
5.
6. 7. 8.
9.
10.
11.
12. 13. 14. 15.
REFERENCES Y. NISHIZUKA,Studiesandperspectives ofprotein kinase C,Science233,305-312(1986). A. A. ABDEL-LATIF, Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers, Pharmacol. Rev. 38,227-272 (1986). J. E. NIEDEL, L. J. KUHN and G. R. VANDENBARK, Phorbol diester receptor copurifies with protein kinase C, Proc. Nor/. Acad. Sci. V.S.A. 80,36-40 (1983). U. KIKKAWA, Y. TAKAI, Y. TANAKA, R. MIYAKE and Y. NISHIZUKA, Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters, J. Biol. Chem. 258, 11442-I 1445 (1983). G. PASTI, J.-C. LACAL, B. S. WARREN, S. A. AARONSON and P. M. BLUMBERG, Loss of mouse Iibroblast cell response to phorbol esters restored by microinjected protein kinase C, Nature 324,375-377 (1986). L. DIAMOND, Tumor promoters and cell transformation, Phormac. Ther. 26, 89-145 (1984). G. S. MARTIN, The erbB gene and EGF receptor, Cuncer Surveys 5, 199-219 (1986).
A. Y. JENG and P. M. BLUMBERG, Biochemical mechanisms of action of the phorbol ester class of tumor promoters, in The Parhobiology of Neoplasia (A. E. SIRICA, ed.), Plenum, New York (1987), in press. H. HIDAKA, M. INAGAKI, S. KAWAMOTO and Y. SASAKI, Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C, Biochemisrry 23,5036-5041 (1984). R. S. TURNER and J. F. KUO, Phospholipid-sensitive Ca*+-dependent protein kinase (protein kinase C): The enzyme, substrates, and regulation, pp. 75-110 in Phospholipidsund Cellular Regularion, Vol. 2 (J. F. KUO, ed.), CRC Press, Boca Raton, Florida (1985). Y. A. HANNUN, C. R. LOOMIS, A. H. MERRILL, JR. and R. M. BELL, Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets, J. Biof. Chem. 261, 12604-12609 (1986). T. TAMAOKI, H. NOMOTO, I. TAKAHASHI, Y. KATO, M. MORIMOTO and F. TOMITA, Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase, Biochem. BioDhvs. Res. Commun. 135. 397-402 (3986). A. Y. JENG, N. A.‘SHARKEY and P. M. BLUMBE‘RG, Purification of stable protein kinase C from mouse brain cytosol by specific ligand elution using fast protein liquid chromatography, Cancer Res. 46, 1966-1971(1986). J. J. WITI’ and R. ROSKOSKI, JR., Rapid protein kinase assay using phosphocellulosepaper absorption, Anal. Biochem. 66,253-258 (1975). M. M. BRADFORD, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248-254 (1976).
16. M. CASTAGNA, Y. TAKAI, K. KAIBUCHI, K. SANO, U. KIKKAWA and Y. NISHIZUKA, Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters, J. Biol. Chem. 257,7847-7851 (1982). 17. N. A. SHARKEY, K. L. LEACH and P. M. BLUMBERG, Competitive inhibition by diacylglycerol of specific phorbol ester binding, Proc. Nat/. Acud. Sci. U.S.A. 81,607-610 (1984).
18. M.-H. LEE and R. M. BELL, The lipid binding, regulatory domain of protein kinase C. A 32-kDa fragment contains the calcium and phosphatidylserine-dependent phorbol diester binding activity, J. Biol. Chem. 261, 14867-14870 (1986).