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Gonadotropin release and redistribution of calcium-activated, phospholipid-dependent protein kinase in phorbol-stimulated rat pituitary cells
ARCHIVES
Vol.
OF BIOCHEMISTRY
249, No. 2, September,
AND
BIOPHYSICS
pp. 557-562,1986
Gonadotropin Release and Redistribution of Calcium-Activated, Phospholipid-Dependent Protein Kinase in Phorbol-Stimulated Rat Pituitary Cells KENJI
HIROTA,’
Endocrinology
TAKAKO
HIROTA:
GRETI
AGUILERA,
AND
and Reproduction Research Branch, National Institute of Child Develqmwnt, National Institutes of Health, Bethesda, Maryland Received
May
KEVIN Health 20205
J. CATF
and Human
12,1986
The effect of phorbol esters on calcium-activated, phospholipid-dependent kinase (protein kinase C) and luteinizing hormone (LH) secretion was examined in cultured rat anterior pituitary cells. The potent tumor promoter 12-O-tetra-decanoylphorbol-13acetate (TPA) stimulated LH secretion and activated pituitary protein kinase C in the presence of calcium and phosphatidylserine. The enzyme activity present in cytosol and particulate fractions was eluted at about 0.05 M NaCl during DE52-cellulose chromatography. Preincubation of pituitary cells with TPA markedly decreased cytosolic protein kinase C activity and increased enzyme activity in the particulate fraction. The maximal TPA-induced change in enzyme activity, with a 76% decrease in cytosol and a 4.3-fold increase in the particulate fraction, occurred within 10 min. The dose-dependent changes in protein kinase C redistribution in TPA-treated cells were correlated with the stimulation of LH release by the phorbol ester. These results suggest that activation of protein kinase C by TPA is associated with intracellular redistribution of the enzyme and is related to the process of secretory granule release from gonadotrophs. o 1986 Academic
Press. Inc.
Phorbol esters are the most potent noncarcinogenic tumor promoters, and enhance tumor growth in mouse skin and other tissues (l-3). In addition to their action as tumor promoters, phorbol esters induce a variety of biological responses such as induction of ornithine decarboxylase, phospholipid synthesis, prostaglandin production, and peptide hormone release (4-7). These biological response to phorbol esters appear to be mediated by a phorbol ester receptor, which has been
1 Present address: Department of Obstetrics & Gynecology, Medical School, Osaka University, Fukushima, Fukushimaku, Osaka, Japan. ‘Present address: Department of Nutrition & Physiology, Medical School, Osaka University, Nakanoshima, Kitaku, Osaka, Japan. a To whom correspondence should be addressed.
identified in mouse skin and other tissues (g-10). Recently, several reports have provided evidence that calcium-activated, phospholipid-dependent protein kinase (protein kinase C) is a receptor for phorbol esters. Thus, TPA4-binding protein in brain tissues copurifies with protein kinase C (ll), and the distribution pattern of phorbol ester binding sites among tissues parallels that for protein kinase C activity (12). Calcium and phosphatidylserine, factors required for the activation of protein kinase C, enhance phorbol ester binding. Phorbol esters directly activate protein ki’ Abbreviations used: TPA, 12-O-tetra-deeanoylphorbol-13-acetate; LH, luteinizing hormone; GnRH, gonadotropin releasing hormone; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; BSA, bovine serum albumin; PS, phosphatidylserine. 55 ‘7
0003-9861/86 Copyright All rights
$3.00
0 1986 by Academic Press. Inc. of reproduction in any form reserved.
558
HIROTA
nase C after becoming intercalated into the plasma membrane (13,14), and phorbol ester binding can be inhibited by diacylglycerol (15), the major endogenous activator of protein kinase C (16). Phorbol esters such as TPA have been previously shown to increase LH release from pituitary cells in a dose-dependent manner (‘7), indicating that protein kinase C could be involved in the mechanism of action of the hypothalamic GnRH. The present report describes the effects of phorbol esters on protein kinase C in rat pituitary cells, and examines the relationship between LH secretion and activation of protein kinase C. MATERIALS
AND
METHODS
Materials. [T-~‘P]ATP (20-30 Ci/mmol) was puchased from ICN and NEN. DEAE-cellulose (DE52) was obtained from Whatman, phosphatidylserine, 1,2diolein, trypsin, and TPA from Sigma Chemical Company, and histone Hl from Boehringer-Mannheim or Sigma Chemical Company (histone 111s). Dispersion and culture of putuitary cells. Anterior pituitary glands of adult female Sprague-Dawley rats (200-250 g) killed at random stages of the estrous cycle were used for the preparation of primary cultures of pituitary cells. Pituitary cells were dispersed by trypsin treatment as described previously (17,18) and were cultured at 106 cells per well in 35-mm multiwell plates (Falcon). Cell cultures were maintained at 37°C under 5% Cot/air in Medium 199 containing 10% horse serum and antibiotics (100 units penicillin and 100 pg streptomycin/ml). After 2 or 3 days in culture, pituitary cells were washed and incubated in Medium 199 containing 25 mM Hepes and 0.1% BSA. For stimulation of LH release, phorbol esters were made up in ethanol and added to cell cultures in 1-~1 aliquots to a total incubation volume of 1 ml. Preparation of q&sol and particulate fractions. After incubation, the cells were released from each well with a rubber policeman and transferred to small plastic tubes. The culture wells were rinsed with icecold Medium 199 and the combined cell suspension was centrifuged for 10 min at 100s. Each cell pellet was resuspended and dispersed in 200 ~1 buffer A (20 mM Tris * HCI, ph 7.5,50 mM 2-mercaptoethanol, and 2 mM EDTA) in a small glass homogenizer, and the homogenate was centrifuged at 100,OOOg for 1 h at 4°C. The supernatant was saved for assay of cytosolic protein kinase C, and the pellet was rehomogenized in 200 ~1 of buffer A containing 1% Triton X-100 and at 0-5°C for 30 min. The suspension was then centrifuged at 100,OOOg for 60 min and the supernatant was assayed for membrane-associated protein kinase C
ET
AL.
activity as described below. Except where specified, protein kinase C assays were performed directly on cytosols and detergent extracts of the particulate fractions from six wells each containing lo6 cells. Aliquots of cytosols and membrane extracts from three wells were assayed in the presence of Ca”, and those from three wells were assayed in the absence of Ca*+, to give triplicate estimates of protein kinase C activity for each data point. Protein determinations were performed by the method of Lowry et al. (19), with bovine serum albumin as standard. DEAE-cellulose chromatography. The supernatant (cytosol) or detergent-extracted membrane fractions from 7 X lo7 pituitary cells were applied to small DE52 columns (0.7 X 4 cm) previously equilibrated with buffer A. After a column wash with buffer A, elution was performed with a linear NaCl(0 to 400 mM) gradient in buffer A and an aliquot of each fraction was assayed for protein kinase activity. Protein kinase assay. Protein kinase C was assayed by measuring the calciumand phospholipid-dependent phosphorylation of Hl histone in the presence of diolein or TPA (14). The assay mixture (0.25 ml) contained 20 mM TriseHCl buffer, pH 7.5, 1.25 pmol of magnesium nitrate, 50 pg of histone, 0.2 pg of 1,2diolein, 5 pg of phosphatidylserine, 0.125 ymol of CaC12, 2.5 nmol of [y-32P]ATP (5-10 X lo5 cpm/nmol); and 30-50 ~1 of sample. Identical assay mixtures were prepared in the absence of Ca*+ to estimate basal phosphokinase activity in aliquots from sets of 3 three matching wells, or from column fractions. After incubation for 4 min at 3O”C, the phosphorylated histones were precipitated by addition of 25% trichloroacetic acid, collected, and washed on membrane filters (pore size 0.45 pm, Millipore Corp.), and analyzed by fl-spectrometry. Protein kinase C activity was calculated by subtraction of radioactivity incorporated in the absence of Ca”. Radioimmunoassay of LH. At selected times as specified for each experiment, culture medium was aspirated, diluted 1:5, and stored at -20°C. LH concentrations were measured by radioimmunoassay as previously described (20) with the RP-2 rat LH preparation provided by the National Pituitary Agency, Baltimore, Md., as standard. RESULTS
Protein Kinase C in Cytosol of Cultured Pituitary Cells The properties of protein kinase C in cytosol of cultured, trypsin-dispersed cells from the rat anterior pituitary glands are shown in Fig. 1. The enzymatic activity of protein kinase C in rat pituitary cytosol was maximum in the simultaneous presence of Ca2+ , PS, and the unsaturated dia-
GONADOTROPIN
RELEASE
AND
I
lJ --+ I-
-
c
+
+
+ -
-
-
+
-
+
+ + -
+ +
+ +
-
PROTEIN
KINASE
REDISTRIBUTION
559
whether the decrease in protein kinase following TPA treatment represented a real loss of enzyme activity, or was due to association of the enzyme with other subcellular fractions. As shown in Fig. 3, protein kinase C was increased in the particulate fraction when pituitary cells were incubated with 1 PM TPA for 60 min. In this experiment, LH release was 3.5 f 0.2 ng per lo5 control cells and 16.0 + 0.3 ng per lo5 in TPA-treated cells. The dose-dependent nature of the change in enzyme location is evident in Fig. 4, which shows the effects of increasing TPA concentration on protein kinase C activity in cytosol and particulate fractions. The maximum decrease in cytosolic enzyme activity, and the maximum increase in membrane-associated protein kinase C, were observed af-
FIG. 1. Activation of protein kinase C in cytosol of anterior pituitary by Ca*+, PS, 1,2-diolein (Di), and TPA. Protein kinase C was assayed by measuring the incorporation of =P into Hl histone from [Y-~P]ATP. The reaction mixture contained 20 mM Tris-HCl (pH 7.5). 5 mM Mg nitrate, 50 pg Hl histone, and 10 pM ATP (1-2 X lo6 cpm). Also, PS (5 pg), 1,2-diolein (0.2 Gg), Caa+ (0.5 mM), and TPA (20 ng) were added as indicated. Data are the means (*SD) of triplicate assays, and the results are representative of four similar experiments.
cylglycerol, 1,2-diolein. When any one of these activators was omitted, protein kinase activity was substantially reduced. TPA was as effective as diolein in stimulating protein kinase C activity, and caused maximal enzyme activation in the presence of Ca2+ and PS. Anion exchange chromatography of the 100,OOOg supernatant of untreated pituitary cells on DE52-cellulose revealed a peak of protein kinase C activity eluting at about 0.05 M NaCl. Following exposure of pituitary cells to 1 PM TPA for 60 min, there was a pronounced decrease in protein kinase activity of the cytosol fraction (Fig. 2). Effect of TPA on Protein Kinase Llistrihtion
The amount of protein kinase C activity associated with the particulate fraction of pituitary cells was measured to determine
FRACTION
NUMBER
FIG. 2. Effect of TPA on protein kinase C activity in cytosol of rat pituitary cells. Pituitary cells (7 X lo7 cells) were treated for 60 min at 37°C with 0.1% ethanol (A) or with 1 pM TPA in 0.1% ethanol (B). After treatment the medium was removed for assay of LH and the cells were collected, disrupted by homogenization, and centrifuged at 100,000~ for 1 h. The supernatants were applied to DE52-cellulose columns (0.7 X 4 cm). After sample addition the column was washed with buffer A (20 ml), then eluted with a linear (O-O.4 M) NaCl gradient. Fractions were collected and 50-~1 aliquots of each fraction were assayed for protein kinase activity in the presence (0) or absence (0) of 0.5 mM Caa’ 9 PS I and diolein. Data are the means of duplicate assays, and the results are representative of three similar experiments.
560
HIROTA A
CONTROL
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I I’
DISCUSSION - 0.2
- 0.1
-0 TPA
FRACTION
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,’
,’
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These studies have demonstrated that TPA directly activates pituitary protein kinase C in the presence of calcium and phosphatidylserine, and induces a prominent decrease in cytosolic protein kinase C and an increase in membrane-associated enzyme activity. Protein kinase C in rat pituitary cells was activated by diolein and TPA in the presence of phosphatidylserine and calcium, and was eluted at about 0.05 M NaCl during DE52-cellulose chromatog-
NUMBER
FIG 3. Effect of TPA on protein kinase C activity in the membrane fraction. Pituitary cells (7 X lo7 cells) were treated for 60 min at 37°C with 0.1% ethanol (A) or with 1 pM TPA in 0.1% ethanol (B). After treatment the medium was saved for assay of LH. Cells were homogenized and centrifuged at 100,OOOg for 1 h. After solubilization of the pellet with 1% Triton X100 in buffer A, the mixture was centrifuged at 100,000g for 1 h. The detergent-solubilized supernatant fraction (1 ml) was applied to DE52-cellulose columns, which were washed with buffer A and eluted with an O-O.4 M NaCl gradient. Fractions were assayed for protein kinase C with (0) or without (0) 0.5 mM Ca*+, PS, and diolein. Data are the means of duplicate assays, and the results are representative of three similar experiments.
ter treatment with 1 PM TPA, a concentration that maximally stimulated LH release from pituitary cells. To determine the time course of TPAstimulated LH release and protein kinase C activation in cytosol and membrane fractions, pituitary cells were incubated with 1 PM TPA at 37°C for various periods up to 60 min (Fig. 5). After 10 min of TPA treatment, little protein kinase C activity was present in the cytosolic fraction, and enzyme activity remained low throughout the incubation period. In contrast, protein kinase C activity in the particulate fraction increased from 10 to 60 min. At 10 min, cytosolic enzyme activity decreased to 22% from its initial 90% of total activity, and particulate enzyme activity increased to 43% from its initial 10% of total activity.
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’
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TPA (MI
FIG. 4. Concentration dependence of TPA-stimulated LH release (above) and redistribution of protein kinase C activity in cytosol and membrane fractions (below). Cultured pituitary cells (106) were treated in triplicate with the indicated concentrations of TPA for 1 h at 3’7°C. Aliquots of the culture medium were assayed for LH content and cells were analyzed for protein kinase activity in the cytosol (0) and membrane (0) fractions. Data are the means (*SD) of triplicate assays, and the results are representative of three similar experiments.
GONADOTROPIN
RELEASE
AND
TIME kninI
FIG. 5. Time course of LH release and protein kinase activity in cytosol and membrane fractions following stimulation by TPA. Pituitary cells (106) were treated with 1 pM TPA for the times indicated, aliquots of the culture medium were removed for assay of LH concentration, and cells were collected for assay of protein kinase activity in the cytosol (0) and membrane (0) fractions. Data are the means (*SD) of triplicate assays, and the results are representative of two similar experiments.
raphy. These findings are consistent with the properties of the enzyme in other tissues (21-23). TPA did not fully activate protein kinase C in the absence of calcium. However, TPA increases the affinity of the enzyme for calcium, activates protein kinase C in the presence of low concentrations of calcium (13,14), and can elicit certain cellular responses without a change in cytosolic calcium concentration (24). In pituitary cells, TPA might also activate protein kinase C without calcium influx or mobilization, since the phorbol ester has no detectable effect on intracellular calcium measurements performed with Quin2 (E. McCoy and K. J. Catt, unpublished data). During TPA treatment of pituitary cells, the loss of cytosolic protein kinase C was not accompanied by an equivalent increase of membrane-associated enzyme activity, which accounted for about 60% of the concomitant decrease in cytosolic protein kinase C (Fig. 1B). Membrane-associated protein kinase C may be inactivated during extraction by Triton X-100, or TPA treat-
PROTEIN
KINASE
REDISTRIBUTION
561
ment may favor protease generation and inactivation of protein kinase C (25). In the rat brain, phorbol ester binding capacity is higher in the membrane fraction after homogenization in the presence of Ca2+, but is higher in the cytosol when homogenization is performed in the presence of calcium chelators (11). Also, a shift of phorbol ester binding between cytosol and membranes as a function of free Ca’+ has been observed (26,27). Recent studies have suggested that protein kinase C and phorbol ester binding activities are located within a single bifunctional protein (11). Such a proposal is consistent with the occurrence of a decrease in cytosolic protein kinase C and an increase in membrane-associated activity during TPA treatment. Previously, treatment with phorbol ester was shown to cause a decrease of protein kinase C in the cytosol in EL4 mouse thymoma cells (28) and an increase in membranes of yolk sac cells (29). Also, incubation of human platelets with TPA caused a redistribution of protein kinase C from cytosol to membrane (30). Other examples of protein kinase C redistribution in target cells exposed to TPA or stimulatory ligands include the effects of GnRH and TPA in pituitary gonadotrophs (31), of isoprenaline and TPA in pinealocytes (32), and of interleukins upon CTG cells and FDC-PI cells (33, 34). Thus, translocation of protein kinase C appears to accompany activation of the enzyme by TPA and hormonal ligands in several tissues. Some of the endogenous substrates of protein kinase C include receptors for insulin (35) and EGF (36-38), an insulin granule membrane protein (39), and a 40kDa protein in platelets (40, 41). Each of these proteins has been shown to be directly or indirectly phosphorylated by protein kinase C. In pituitary gonadotrophs, it is possible that protein kinase C phosphorylates plasma-membrane components including the GnRH receptor (42) after undergoing translocation from cytosol to membrane-associated sites. In this report, we have characterized the protein kinase C in rat pituitary cells, and have shown that redistribution of protein
562
HIROTA
kinase C by TPA is associated with stimulation of LH release from pituitary gonadotrophs. With the recent demonstration that GnRH causes a similar change in protein kinase C activity during stimulation of gonadotropin release from purified gonadotrophs (31), these findings provide further evidence to support the intermediate role of protein kinase C in the mechanism of pituitary gonadotropin secretion.
ET
AND RANDALL, 20.
21.
22. 23. 24.
40.
12. ASHENDEL, C. L., STALLER, J. M., AND BOUTWELL, R. K. (1983) B&hem. Biophys. Res. Cmnmun 112,340-345. 13. YAMANISHI, J., TAKAI, Y., KAIBUCHI, K., SANO, K., CASTAGNA, M., AND NISHIZUKA, Y. (1983) Biochem Biophys. Res. Ccrmmun. 112,778-786. 14. CASTAGNA, M., TAKAI, Y., KAIBUCHI, K., SANO, K., KIKKAWA, U., AND NISHIZUKA, Y. (1982) J. BioL Chem. 257,7847-7851. 15. SHARKEY, N. A., LEACH, K. L., AND BLUMBERG, P. M. (1984) Proc. NatL Acad. Sci. USA 81,607610. 16. NISHIZUKA, Y. (1984) Nature (London) 309, 693699. 17. DENEF, C., HAUTEKEETE, E., DEWOLF, A., AND VANDERSCHUEREN, B. (1978) Endxrirwlogy 103, 724-735.
18. HYDE, C. L., CHILDS, G., WAHL, L. M., NAOR, Z., AND CAlT, K. J. (1982) En&crirwZogy 111,14211423. 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,
R. J. (1951)
J. Biol.
Chem.
193,
265-275.
REFERENCES 1. BOUTWELL, R. K. (1974) CRC Cd. Rev. TozicoL 2, 419-443. 2. SIVAK, A. (1979) B&hem Biophya Acta 560,6789. 3. GOERTTLER, K., LOHRKE, H., AND HESSE, B. (1980) Curcin0genesi.s 1,707-713. 4. WEINSTEIN, J. B., LEE, L. S., FISHER, P. B., MUFSON, A., AND YAMASAKA, H. (1979) J. SupramoL Stmct. 12,195-208. 5. BLUMBERG, F. M. (1980) CRC &it. Rev. ToxicoL 8, 153-234. 6. OSBORNE, R., AND TASHJIAN, A. H. (1981) EndoCrinolog~ 108,11641170. 7. NAOR, Z., AND CAT-T, K. J. (1981) J. BioL Chem. 256, 2226-2229. 8. DELCLOS, K. B., NAGLE, D. S., AND BLUMBERG, P. M. (1980) Cell 19,1025-1032. 9. LEHRER, R. I., COHEN, L. E., AND KOEFFLER, H. P. (1983) Cancer Res. 43,3563-3566. 10. SHOYAB, M., WARREN, T. C., AND TODARO, G. J. (1981) Carcinogenesis 2,1273-1276. 11. NIEDEL, J. E., KUHN, L. J., AND VANDENBARK, G. R. (1983) Proc. Natl. Acad Sci. USA 80,36-
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25. 26.
27. 28.
29. 30. 31. 32.
LOUMAYE, E., NAOR, Z., AND CATT, K. J. (1982) Endocrinology 111,730-736. KIKKAWA, U., TAKAI, Y., MINAKUCHI, R., INOHARA, S., AND NISHIZUKA, Y. (1982) J. BioL Chem. 257, 13,341-13,348. WISE, B. C., RAYNOR, R. L., AND Kuo, J. F. (1982) J BioL Chem. 257,8481-8488. CLARK, L. C., DAVIS, J. S., AND LEMAIRE, W. L. (1983) J. Clin EndowinoL Metab. 57, 872-874. KISHIMOTO, A., KAJIKAWA, N., SHIOTA, M., AND NISHIZUKA, Y. (1983) J. BioL Chem. 258,11561164. RINK, T. J., SANCHEZ, A., AND HALLAM, T. J. (1983) Nature (Lcdm) 305,317-319. BLUMBERG, P. M., DELCLOS, K. B., DUNN, J. A., JAKEN, S., LEACH, K. L., AND YEH, E. (1983) Ann. N. Y. Acad Sci. 407,303-315. LEACH, K. L., JAMES, M. L., AND BLUMBERG, P. M. (1983) Proc. NatL Acad. Sci. USA 80,4208-4212. KRAFT, A. S., ANDERSON, W. B., COOPER, H. L., AND SANDO, J. J. (1982) J. BioL Chem. 257, 13,193-13,196. KRAFT, A. S., AND ANDERSON, W. B. (1983) Nature (h&m) 301,621-623. TAPLEY, P. M., AND MURRAY, A. W. (1984) Biochem Biophys. Res. Commun. 122,158-164. HIROTA, K., HIROTA, T., AGUILERA, G., AND CATT, K. J. (1985) J. BioL Chem. 260,3243-3246. SUGDEN, D., VANACEK, J., KLEIN, D. C., THOMAS, T. P., AND ANDERSON, W. B. (1985) Nature (London)
314,359-361.
33. FARRAR, W. L., AND ANDERSON, W. 8. (1985) Nature (London) 315,233-235. 34. FARRAR, W. L., THOMAS, T. P., AND ANDERSON, W. B. (1985) Nature (London) 315,235-237. 35. JACOBS, S., SAHYOUN, N. E., SALTIEL, A. R., AND CUATRECASAS, P. (1983) Proc. NatL Acad. Sci. USA 80,6211-6213. 36. COCHET, C., GILL, G. N., MEISENHELDER, J., COOPER, J. A., AND HUNTER, T. (1984) J. BioL Chem. 259,2553-2558. 37. IWASHITA, S., AND Fox, F. (1984) J. BioL Chem. 259,2559-2567. 38.
DAVIS, R. J., AND CZECH, M. P. (1984) J. BioL Chem 259,8545-8549.
39. BROCKLEHURST, Biochem
K. W., AND HUTTON,
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40. KAIBUCHI, K., TAKAI, Y., SAWAMURA, M., HOSHIJIMA, M., FUJIKURA, T., AND NISHIZUKA, Y. (1983) J BioL Chem. 258, 6701-6704. 41. SANO, K., TAKAI, Y., YAMANISHI, J., AND NISHIZUKA, Y. (1983) J. BioL Chem. 256,2010-2013. 42. CATT, K. J., LOUMAYE, E., WYNN, P., SUAREZQUIAN, C., KIESEL, L., IWASHITA, M., HIROTA, K., MORGAN, R., AND CHANG, J. (1984) in Endocrinology (Labrie, F., and Proulx, L., eds.), pp. 57-65, Elsevier, Amsterdam.
Vol.
OF BIOCHEMISTRY
249, No. 2, September,
AND
BIOPHYSICS
pp. 557-562,1986
Gonadotropin Release and Redistribution of Calcium-Activated, Phospholipid-Dependent Protein Kinase in Phorbol-Stimulated Rat Pituitary Cells KENJI
HIROTA,’
Endocrinology
TAKAKO
HIROTA:
GRETI
AGUILERA,
AND
and Reproduction Research Branch, National Institute of Child Develqmwnt, National Institutes of Health, Bethesda, Maryland Received
May
KEVIN Health 20205
J. CATF
and Human
12,1986
The effect of phorbol esters on calcium-activated, phospholipid-dependent kinase (protein kinase C) and luteinizing hormone (LH) secretion was examined in cultured rat anterior pituitary cells. The potent tumor promoter 12-O-tetra-decanoylphorbol-13acetate (TPA) stimulated LH secretion and activated pituitary protein kinase C in the presence of calcium and phosphatidylserine. The enzyme activity present in cytosol and particulate fractions was eluted at about 0.05 M NaCl during DE52-cellulose chromatography. Preincubation of pituitary cells with TPA markedly decreased cytosolic protein kinase C activity and increased enzyme activity in the particulate fraction. The maximal TPA-induced change in enzyme activity, with a 76% decrease in cytosol and a 4.3-fold increase in the particulate fraction, occurred within 10 min. The dose-dependent changes in protein kinase C redistribution in TPA-treated cells were correlated with the stimulation of LH release by the phorbol ester. These results suggest that activation of protein kinase C by TPA is associated with intracellular redistribution of the enzyme and is related to the process of secretory granule release from gonadotrophs. o 1986 Academic
Press. Inc.
Phorbol esters are the most potent noncarcinogenic tumor promoters, and enhance tumor growth in mouse skin and other tissues (l-3). In addition to their action as tumor promoters, phorbol esters induce a variety of biological responses such as induction of ornithine decarboxylase, phospholipid synthesis, prostaglandin production, and peptide hormone release (4-7). These biological response to phorbol esters appear to be mediated by a phorbol ester receptor, which has been
1 Present address: Department of Obstetrics & Gynecology, Medical School, Osaka University, Fukushima, Fukushimaku, Osaka, Japan. ‘Present address: Department of Nutrition & Physiology, Medical School, Osaka University, Nakanoshima, Kitaku, Osaka, Japan. a To whom correspondence should be addressed.
identified in mouse skin and other tissues (g-10). Recently, several reports have provided evidence that calcium-activated, phospholipid-dependent protein kinase (protein kinase C) is a receptor for phorbol esters. Thus, TPA4-binding protein in brain tissues copurifies with protein kinase C (ll), and the distribution pattern of phorbol ester binding sites among tissues parallels that for protein kinase C activity (12). Calcium and phosphatidylserine, factors required for the activation of protein kinase C, enhance phorbol ester binding. Phorbol esters directly activate protein ki’ Abbreviations used: TPA, 12-O-tetra-deeanoylphorbol-13-acetate; LH, luteinizing hormone; GnRH, gonadotropin releasing hormone; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; BSA, bovine serum albumin; PS, phosphatidylserine. 55 ‘7
0003-9861/86 Copyright All rights
$3.00
0 1986 by Academic Press. Inc. of reproduction in any form reserved.
558
HIROTA
nase C after becoming intercalated into the plasma membrane (13,14), and phorbol ester binding can be inhibited by diacylglycerol (15), the major endogenous activator of protein kinase C (16). Phorbol esters such as TPA have been previously shown to increase LH release from pituitary cells in a dose-dependent manner (‘7), indicating that protein kinase C could be involved in the mechanism of action of the hypothalamic GnRH. The present report describes the effects of phorbol esters on protein kinase C in rat pituitary cells, and examines the relationship between LH secretion and activation of protein kinase C. MATERIALS
AND
METHODS
Materials. [T-~‘P]ATP (20-30 Ci/mmol) was puchased from ICN and NEN. DEAE-cellulose (DE52) was obtained from Whatman, phosphatidylserine, 1,2diolein, trypsin, and TPA from Sigma Chemical Company, and histone Hl from Boehringer-Mannheim or Sigma Chemical Company (histone 111s). Dispersion and culture of putuitary cells. Anterior pituitary glands of adult female Sprague-Dawley rats (200-250 g) killed at random stages of the estrous cycle were used for the preparation of primary cultures of pituitary cells. Pituitary cells were dispersed by trypsin treatment as described previously (17,18) and were cultured at 106 cells per well in 35-mm multiwell plates (Falcon). Cell cultures were maintained at 37°C under 5% Cot/air in Medium 199 containing 10% horse serum and antibiotics (100 units penicillin and 100 pg streptomycin/ml). After 2 or 3 days in culture, pituitary cells were washed and incubated in Medium 199 containing 25 mM Hepes and 0.1% BSA. For stimulation of LH release, phorbol esters were made up in ethanol and added to cell cultures in 1-~1 aliquots to a total incubation volume of 1 ml. Preparation of q&sol and particulate fractions. After incubation, the cells were released from each well with a rubber policeman and transferred to small plastic tubes. The culture wells were rinsed with icecold Medium 199 and the combined cell suspension was centrifuged for 10 min at 100s. Each cell pellet was resuspended and dispersed in 200 ~1 buffer A (20 mM Tris * HCI, ph 7.5,50 mM 2-mercaptoethanol, and 2 mM EDTA) in a small glass homogenizer, and the homogenate was centrifuged at 100,OOOg for 1 h at 4°C. The supernatant was saved for assay of cytosolic protein kinase C, and the pellet was rehomogenized in 200 ~1 of buffer A containing 1% Triton X-100 and at 0-5°C for 30 min. The suspension was then centrifuged at 100,OOOg for 60 min and the supernatant was assayed for membrane-associated protein kinase C
ET
AL.
activity as described below. Except where specified, protein kinase C assays were performed directly on cytosols and detergent extracts of the particulate fractions from six wells each containing lo6 cells. Aliquots of cytosols and membrane extracts from three wells were assayed in the presence of Ca”, and those from three wells were assayed in the absence of Ca*+, to give triplicate estimates of protein kinase C activity for each data point. Protein determinations were performed by the method of Lowry et al. (19), with bovine serum albumin as standard. DEAE-cellulose chromatography. The supernatant (cytosol) or detergent-extracted membrane fractions from 7 X lo7 pituitary cells were applied to small DE52 columns (0.7 X 4 cm) previously equilibrated with buffer A. After a column wash with buffer A, elution was performed with a linear NaCl(0 to 400 mM) gradient in buffer A and an aliquot of each fraction was assayed for protein kinase activity. Protein kinase assay. Protein kinase C was assayed by measuring the calciumand phospholipid-dependent phosphorylation of Hl histone in the presence of diolein or TPA (14). The assay mixture (0.25 ml) contained 20 mM TriseHCl buffer, pH 7.5, 1.25 pmol of magnesium nitrate, 50 pg of histone, 0.2 pg of 1,2diolein, 5 pg of phosphatidylserine, 0.125 ymol of CaC12, 2.5 nmol of [y-32P]ATP (5-10 X lo5 cpm/nmol); and 30-50 ~1 of sample. Identical assay mixtures were prepared in the absence of Ca*+ to estimate basal phosphokinase activity in aliquots from sets of 3 three matching wells, or from column fractions. After incubation for 4 min at 3O”C, the phosphorylated histones were precipitated by addition of 25% trichloroacetic acid, collected, and washed on membrane filters (pore size 0.45 pm, Millipore Corp.), and analyzed by fl-spectrometry. Protein kinase C activity was calculated by subtraction of radioactivity incorporated in the absence of Ca”. Radioimmunoassay of LH. At selected times as specified for each experiment, culture medium was aspirated, diluted 1:5, and stored at -20°C. LH concentrations were measured by radioimmunoassay as previously described (20) with the RP-2 rat LH preparation provided by the National Pituitary Agency, Baltimore, Md., as standard. RESULTS
Protein Kinase C in Cytosol of Cultured Pituitary Cells The properties of protein kinase C in cytosol of cultured, trypsin-dispersed cells from the rat anterior pituitary glands are shown in Fig. 1. The enzymatic activity of protein kinase C in rat pituitary cytosol was maximum in the simultaneous presence of Ca2+ , PS, and the unsaturated dia-
GONADOTROPIN
RELEASE
AND
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PROTEIN
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559
whether the decrease in protein kinase following TPA treatment represented a real loss of enzyme activity, or was due to association of the enzyme with other subcellular fractions. As shown in Fig. 3, protein kinase C was increased in the particulate fraction when pituitary cells were incubated with 1 PM TPA for 60 min. In this experiment, LH release was 3.5 f 0.2 ng per lo5 control cells and 16.0 + 0.3 ng per lo5 in TPA-treated cells. The dose-dependent nature of the change in enzyme location is evident in Fig. 4, which shows the effects of increasing TPA concentration on protein kinase C activity in cytosol and particulate fractions. The maximum decrease in cytosolic enzyme activity, and the maximum increase in membrane-associated protein kinase C, were observed af-
FIG. 1. Activation of protein kinase C in cytosol of anterior pituitary by Ca*+, PS, 1,2-diolein (Di), and TPA. Protein kinase C was assayed by measuring the incorporation of =P into Hl histone from [Y-~P]ATP. The reaction mixture contained 20 mM Tris-HCl (pH 7.5). 5 mM Mg nitrate, 50 pg Hl histone, and 10 pM ATP (1-2 X lo6 cpm). Also, PS (5 pg), 1,2-diolein (0.2 Gg), Caa+ (0.5 mM), and TPA (20 ng) were added as indicated. Data are the means (*SD) of triplicate assays, and the results are representative of four similar experiments.
cylglycerol, 1,2-diolein. When any one of these activators was omitted, protein kinase activity was substantially reduced. TPA was as effective as diolein in stimulating protein kinase C activity, and caused maximal enzyme activation in the presence of Ca2+ and PS. Anion exchange chromatography of the 100,OOOg supernatant of untreated pituitary cells on DE52-cellulose revealed a peak of protein kinase C activity eluting at about 0.05 M NaCl. Following exposure of pituitary cells to 1 PM TPA for 60 min, there was a pronounced decrease in protein kinase activity of the cytosol fraction (Fig. 2). Effect of TPA on Protein Kinase Llistrihtion
The amount of protein kinase C activity associated with the particulate fraction of pituitary cells was measured to determine
FRACTION
NUMBER
FIG. 2. Effect of TPA on protein kinase C activity in cytosol of rat pituitary cells. Pituitary cells (7 X lo7 cells) were treated for 60 min at 37°C with 0.1% ethanol (A) or with 1 pM TPA in 0.1% ethanol (B). After treatment the medium was removed for assay of LH and the cells were collected, disrupted by homogenization, and centrifuged at 100,000~ for 1 h. The supernatants were applied to DE52-cellulose columns (0.7 X 4 cm). After sample addition the column was washed with buffer A (20 ml), then eluted with a linear (O-O.4 M) NaCl gradient. Fractions were collected and 50-~1 aliquots of each fraction were assayed for protein kinase activity in the presence (0) or absence (0) of 0.5 mM Caa’ 9 PS I and diolein. Data are the means of duplicate assays, and the results are representative of three similar experiments.
560
HIROTA A
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These studies have demonstrated that TPA directly activates pituitary protein kinase C in the presence of calcium and phosphatidylserine, and induces a prominent decrease in cytosolic protein kinase C and an increase in membrane-associated enzyme activity. Protein kinase C in rat pituitary cells was activated by diolein and TPA in the presence of phosphatidylserine and calcium, and was eluted at about 0.05 M NaCl during DE52-cellulose chromatog-
NUMBER
FIG 3. Effect of TPA on protein kinase C activity in the membrane fraction. Pituitary cells (7 X lo7 cells) were treated for 60 min at 37°C with 0.1% ethanol (A) or with 1 pM TPA in 0.1% ethanol (B). After treatment the medium was saved for assay of LH. Cells were homogenized and centrifuged at 100,OOOg for 1 h. After solubilization of the pellet with 1% Triton X100 in buffer A, the mixture was centrifuged at 100,000g for 1 h. The detergent-solubilized supernatant fraction (1 ml) was applied to DE52-cellulose columns, which were washed with buffer A and eluted with an O-O.4 M NaCl gradient. Fractions were assayed for protein kinase C with (0) or without (0) 0.5 mM Ca*+, PS, and diolein. Data are the means of duplicate assays, and the results are representative of three similar experiments.
ter treatment with 1 PM TPA, a concentration that maximally stimulated LH release from pituitary cells. To determine the time course of TPAstimulated LH release and protein kinase C activation in cytosol and membrane fractions, pituitary cells were incubated with 1 PM TPA at 37°C for various periods up to 60 min (Fig. 5). After 10 min of TPA treatment, little protein kinase C activity was present in the cytosolic fraction, and enzyme activity remained low throughout the incubation period. In contrast, protein kinase C activity in the particulate fraction increased from 10 to 60 min. At 10 min, cytosolic enzyme activity decreased to 22% from its initial 90% of total activity, and particulate enzyme activity increased to 43% from its initial 10% of total activity.
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FIG. 4. Concentration dependence of TPA-stimulated LH release (above) and redistribution of protein kinase C activity in cytosol and membrane fractions (below). Cultured pituitary cells (106) were treated in triplicate with the indicated concentrations of TPA for 1 h at 3’7°C. Aliquots of the culture medium were assayed for LH content and cells were analyzed for protein kinase activity in the cytosol (0) and membrane (0) fractions. Data are the means (*SD) of triplicate assays, and the results are representative of three similar experiments.
GONADOTROPIN
RELEASE
AND
TIME kninI
FIG. 5. Time course of LH release and protein kinase activity in cytosol and membrane fractions following stimulation by TPA. Pituitary cells (106) were treated with 1 pM TPA for the times indicated, aliquots of the culture medium were removed for assay of LH concentration, and cells were collected for assay of protein kinase activity in the cytosol (0) and membrane (0) fractions. Data are the means (*SD) of triplicate assays, and the results are representative of two similar experiments.
raphy. These findings are consistent with the properties of the enzyme in other tissues (21-23). TPA did not fully activate protein kinase C in the absence of calcium. However, TPA increases the affinity of the enzyme for calcium, activates protein kinase C in the presence of low concentrations of calcium (13,14), and can elicit certain cellular responses without a change in cytosolic calcium concentration (24). In pituitary cells, TPA might also activate protein kinase C without calcium influx or mobilization, since the phorbol ester has no detectable effect on intracellular calcium measurements performed with Quin2 (E. McCoy and K. J. Catt, unpublished data). During TPA treatment of pituitary cells, the loss of cytosolic protein kinase C was not accompanied by an equivalent increase of membrane-associated enzyme activity, which accounted for about 60% of the concomitant decrease in cytosolic protein kinase C (Fig. 1B). Membrane-associated protein kinase C may be inactivated during extraction by Triton X-100, or TPA treat-
PROTEIN
KINASE
REDISTRIBUTION
561
ment may favor protease generation and inactivation of protein kinase C (25). In the rat brain, phorbol ester binding capacity is higher in the membrane fraction after homogenization in the presence of Ca2+, but is higher in the cytosol when homogenization is performed in the presence of calcium chelators (11). Also, a shift of phorbol ester binding between cytosol and membranes as a function of free Ca’+ has been observed (26,27). Recent studies have suggested that protein kinase C and phorbol ester binding activities are located within a single bifunctional protein (11). Such a proposal is consistent with the occurrence of a decrease in cytosolic protein kinase C and an increase in membrane-associated activity during TPA treatment. Previously, treatment with phorbol ester was shown to cause a decrease of protein kinase C in the cytosol in EL4 mouse thymoma cells (28) and an increase in membranes of yolk sac cells (29). Also, incubation of human platelets with TPA caused a redistribution of protein kinase C from cytosol to membrane (30). Other examples of protein kinase C redistribution in target cells exposed to TPA or stimulatory ligands include the effects of GnRH and TPA in pituitary gonadotrophs (31), of isoprenaline and TPA in pinealocytes (32), and of interleukins upon CTG cells and FDC-PI cells (33, 34). Thus, translocation of protein kinase C appears to accompany activation of the enzyme by TPA and hormonal ligands in several tissues. Some of the endogenous substrates of protein kinase C include receptors for insulin (35) and EGF (36-38), an insulin granule membrane protein (39), and a 40kDa protein in platelets (40, 41). Each of these proteins has been shown to be directly or indirectly phosphorylated by protein kinase C. In pituitary gonadotrophs, it is possible that protein kinase C phosphorylates plasma-membrane components including the GnRH receptor (42) after undergoing translocation from cytosol to membrane-associated sites. In this report, we have characterized the protein kinase C in rat pituitary cells, and have shown that redistribution of protein
562
HIROTA
kinase C by TPA is associated with stimulation of LH release from pituitary gonadotrophs. With the recent demonstration that GnRH causes a similar change in protein kinase C activity during stimulation of gonadotropin release from purified gonadotrophs (31), these findings provide further evidence to support the intermediate role of protein kinase C in the mechanism of pituitary gonadotropin secretion.
ET
AND RANDALL, 20.
21.
22. 23. 24.
40.
12. ASHENDEL, C. L., STALLER, J. M., AND BOUTWELL, R. K. (1983) B&hem. Biophys. Res. Cmnmun 112,340-345. 13. YAMANISHI, J., TAKAI, Y., KAIBUCHI, K., SANO, K., CASTAGNA, M., AND NISHIZUKA, Y. (1983) Biochem Biophys. Res. Ccrmmun. 112,778-786. 14. CASTAGNA, M., TAKAI, Y., KAIBUCHI, K., SANO, K., KIKKAWA, U., AND NISHIZUKA, Y. (1982) J. BioL Chem. 257,7847-7851. 15. SHARKEY, N. A., LEACH, K. L., AND BLUMBERG, P. M. (1984) Proc. NatL Acad. Sci. USA 81,607610. 16. NISHIZUKA, Y. (1984) Nature (London) 309, 693699. 17. DENEF, C., HAUTEKEETE, E., DEWOLF, A., AND VANDERSCHUEREN, B. (1978) Endxrirwlogy 103, 724-735.
18. HYDE, C. L., CHILDS, G., WAHL, L. M., NAOR, Z., AND CAlT, K. J. (1982) En&crirwZogy 111,14211423. 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,
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40. KAIBUCHI, K., TAKAI, Y., SAWAMURA, M., HOSHIJIMA, M., FUJIKURA, T., AND NISHIZUKA, Y. (1983) J BioL Chem. 258, 6701-6704. 41. SANO, K., TAKAI, Y., YAMANISHI, J., AND NISHIZUKA, Y. (1983) J. BioL Chem. 256,2010-2013. 42. CATT, K. J., LOUMAYE, E., WYNN, P., SUAREZQUIAN, C., KIESEL, L., IWASHITA, M., HIROTA, K., MORGAN, R., AND CHANG, J. (1984) in Endocrinology (Labrie, F., and Proulx, L., eds.), pp. 57-65, Elsevier, Amsterdam.