- Email: [email protected]
Receptors for tumor-promoting phorbol esters in rat ventral prostate
Cancer Letters, 68 (1993)
143
143 - 147
Elsevier Scientific Publishers Ireland Ltd
Receptors for tumor-promoting M.J. Carmena, Departamento
(Received (Accepted
de
phorbol esters in rat ventral prostate
M.P. Garcia-Paramio Bioquimica
y Biologia
and J.C. Prieto
Molecular,
Universidad
Alcal6 de Henares
(Spain)
30 March 1992) 24 November 1992)
Summary The presence of tumor-promoting phorboi ester receptors in rat prostate was investigated by studying fhe binding of phorbol die&et 12,13-dibutyrate (PDBu) in both soluble and particulaate subcellular fractions. Binding of [3H]PDBu to the soluble fraction was optimal
afier the addition of phosphatidylserine
(0.1
mg/ml) and Ca 2+ (1 mM). Both subcellular fractions exhibited a single class of PDBu receptor (Kd between 97 and 128 nM) as shown by saturation binding experiments. Phorbol esters with tumor-promoting activity
showed a higher affinity for these receptors than did endogenous ligands such as diacyglycerols whereas phorbol esters without tumor-promoting activity were ineffectioe euen at concentraHons as high as 1 PM. These properties are highly representatiue of protein kinase C activity.
Keywords:
tumor-promoting phorbol ester binding; protein kinase C; signal transduction Introduction
Tumor-promoting Correspondence
to: J.C.
y Biologfa Molecular, Henares,
de Alcalo’, E-28871
phorbol Prieto, Departamento
0
1992
Printed and Published in Ireland
are
de Bioquimica
Universidad de Alcalfi, E-28871
Spain.
0304-3835/92/$05.00
esters
Alcal
de
lipophylic agents that, circumventing the phosphoinositide pathway by binding directly to protein kinase C (PKC) at the cell membrane, may activate this phospholipid- and Ca*+-dependent enzyme [ 11. Because of their structural similarity, both diacylglycerol (DAG, the endogenous activator of PKC) and tumor-promoting phorbol esters mimic each other in binding to the same receptor (PKC) as a result of which the enzyme is redistributed and translocated away from the cytosol to the plasma membrane where it finds the necessary phosphatidylserine [Z] . Like other processes related to prostatic growth and function, PKC activity in the prostate gland appears to be under androgenic control [3]. Results from our laboratory have shown that the treatment of isolated rat prostatic epithelial cells with tumor-promoting phorbol esters desensitized the stimulation of cyclic AMP accumulation by fl-adrenergic agonists [4] or by vasoactive intestinal peptide (VIP) [5], a finding that suggests interaction between the cyclic AMP and phospholipid metabolizing pathways. However, a better understanding of PKC as a transducer of DAG and Ca*+ second messengers in prostatic tissue requires the characterization of the phorbol ester receptors. The objective of this paper is to investigate the characteristics of phorbol ester receptors in the rat ventral prostate using [3H]phorbol diester 12,13_dibutyrate (PDBu) and different subcellular preparations.
Elsevier Scientific Publishers Ireland Ltd.
144
Materials and Methods
Results
Chemicals 4cr-phorbol 12,13_didecanoate PDBu, (PDD), phorbol 12-myristate, 13-acetate (PMA) , phosphatidylserine and 1,2-dioleoylglycerol (DOG) were from Sigma (St. Louis, MO). [3H]PDBu (20 Ci/mmol) was from New England Nuclear (Boston, MA). All other chemicals were of reagent grade.
Figure 1 shows the phosphatidylserine dependence of [3H]PDBu binding to the soluble fraction from rat ventral prostate as representative of the phospholipid need for PKC activation. Binding was practically nil in the absence of phosphatidylserine and increased quickly up to the approximately 0.2 - 0.3 mg/ml which was required for maximal responses, The regulatory role of Ca*+ on [3H]PDBu binding was examined in both soluble and particulate cell fractions (Fig. 2) as an index of the requirement for this cation in PKC activation. Increasing concentrations of Ca*+ increased the binding of [3H]PDBu in the two subcellular preparations but some difference in the
Preparation of subcellular fractions The ventral lobes of the prostate from male Wistar rats (250 -300 g) were excised and homogenized into 3 volumes of ice-cold 25 mM Tris-HCI (pH 7.5) containing 0.25 M sucrose, 2.5 mM magnesium acetate, 2.5 mM EGTA and 2.5 mM ditiothreitol. Tissue homogenate was centrifuged at 100 000 x g for 1 h and the supernatant was referred to as the soluble fraction. The corresponding pellet was resuspended in homogenization buffer and considered to be the particulate fraction. All subcellular fractions were stored at - 85OC. Protein concentration was determined [6] using bovine serum albumin as a standard. Study of [3mPDBu binding Phorbol binding to subcellular fractions of rat ventral prostate was studied essentially as described elsewhere [7]. Standard binding assays were carried out in 0.16 ml of 50 mM Tris - HCI (pH 7.4) containing 0.1 mg of protein sample, 31.25 nM [3H]PDBu, 1 mM CaCls, 0.78 magnesium acetate, 0.2 mg/ml phosphatidylserine and 0.78 mM EGTA. The incubation was allowed to proceed at 37OC for 15 min. The nonspecific binding was defined in the presence of an excess of unlabelled PDBu (5 PM). Separation. of bound from free [3H]PDBu was achieved by centrifugation at 13 000 X g for 15 min (particulate fraction) or precipitation in polyethylene glycol followed by centrifugation (soluble fraction). The pellets were then washed and counted by liquid scintillation.
1.5
[Phosphatidylserinel, bg/ml Fig. 1. Effect of phosphatidylserine on [3H]PDBu binding in soluble subcellular fraction. The assay was carried out as described in Methods, with 0.1 mg of protein sample and the indicated concentrations of the phospholipid. Each point is the mean of triplicates in a representative
experiment.
145
1.8
”
0
0.5
I’
I B/F 0.020.011
0, 0
ah,
2
t.0
[Ca2+l,mM Fig. 2. Modification of [3H]PDBu binding to soluble (left panel) and particulate (right panel) subcellular fractions by addition of Ca’+. The assay was performed at increasing concentrations of added divalent cation. Each point is the mean of triplicates in a representative experiment.
extent of the Ca2+ requirement could be appreciated since maximal binding values were reached at either 0.5 mM Ca2+ in particulate cell fraction and 1 .O mM Ca2+ in soluble cell fractions. Figure 3 shows the PDBu binding activities in the two subcellular fractions studied. Saturation experiments using [3H]PDBu and increasing concentrations of the unlabelled tumor-promoting phorbol ester gave results that were interpreted by Scatchard analysis [8] in terms of a single-site model. The dissociation constant (I&) was calculated to be 97 * 16 nM (SE) with a binding capacity of 2.1 f 0.3 pmol PDBu bound per mg protein in the soluble fraction. The corresponding values in the particulate fraction were 128 f 12 nM and 2.8 f 0.3 pmol PDBu bound per mg protein. The binding affinity of the receptor for other phorbol esters and DOG was estimated by dose-dependent inhibition of [3H]PDBu binding in the soluble cell fraction (Fig. 4). When comparing the I&, values, PMA and the
0
Sal [Total PDBul, MI
Fig. 3. Saturation binding analysis of PDBu binding to soluble (top) and particulate (bottom) subcellular fractions. The assay was carried out with a fixed (31.25 nM) concentration of [3H]PDBu and increasing doses of the unlabelled phorbol ester. Each point is the mean f SE, n = 6 triplicate determinations. The insets correspond to the Scatchard analysis of the mean binding data.
endogenous ligand DOG showed a four times higher and a seven times lower respective affinity than PDBu for the receptors, whereas PDD was ineffective at concentrations up to at least 1 PM. Discussion Present data provide the first demonstration of the presence of specific receptors for tumorpromoting phorbol esters in the prostate and serve as a further characterization of PKC activity in this important gland of the male genitourinary tract. Specific t3H]PDBu binding activity was detected in both soluble and particulate subcellular fractions. However,
-+’ 00
tllnlabelled compound), -log M Fig. 4. Dose-dependent inhibition of [3H]PDBu binding to soluble cellular fraction by various phorbol esters and DOG. The unlabelled compounds tested were PMA ( q, PDBu (O), DOG (A) and PDD (0). Data are means of triplicates in a representative experiment.
some differences could be established since the soluble binding sites showed total dependency on both phosphatidylserine and Ca*+ whereas those in the particulate fraction only needed a modest exogenous supplement of the divalent cation to express maximal activity. This feature is well established in other systems characterizing phorbol ester binding and/or PKC activation [9 - 121 and it suggests that PKC itself could be the prostatic phorbol ester receptor. However, the existence of other prostatic phorbol ester receptors in addition to PKC remains to be defined. Present results on the soluble fraction of prostatic tissue confirm that the environment of the phorbol ester receptors in this compartment does not allow the corresponding binding unless a membranelike environment is mimicked. In this respect, it might be stressed that under physiological
conditions PKC is active when bound to membranes [13] and that membrane insertion needs Ca*+ and is favoured by phorbol esters 1141. The binding of PDBu in rat prostate was shown to represent a single class of receptors in both soluble and particulate subcellular fractions, Kd values being 97 and 128 nM respectively. These values are near those reported in systems such as mouse brain [15] and C6 rat glioma cells [16] with apparent Kd values between 40 and 90 nM. However, other systems that also show homogeneous phorbol ester binding have been reported to possess Kd values that fall within the 1 - 10 nM range [ 10,17,18]. Factors such as differences in lipid environment and even in the protein portions of the receptors could explain, at least in part, these apparent discrepancies. The lack of statistical significance suggests that the same receptor is present in the two preparations and that some mechanism of subcellular translocation is likely to exist as happens in other PKC activation or phorbol ester binding systems [ 131. A single class of phorbol ester receptor is the common observation in the majority of tissues studied [ 191. It is important to note that multiple (three or four) isozymes of Ca*+ /phospholipid-dependent PKC have been detected in the rat ventral prostate [3]. Thus, the corresponding functional moieties which interact with phorbol esters must be very similar if only a single class of receptors can be detected as shown in the present report. From the point of view of specificity, tumorpromoting phorbol esters like PMA and PDBu showed higher binding affinity than the endogenous ligand DOG. This agrees with observations in other systems and is likely to be related to the prolonged activation of PKC that is elicited by those phorbol compounds [9]. The observation that PDD did not displace [3H]PDBu binding again reinforces the hypothesis that the receptor studied in the prostate is PKC since PDD is a phorbol ester which is practically devoid of tumor-promoting ability and does not activate PKC [20]. In summary, tumor-promoting
prostatic tissue possesses phorbol ester receptors that
147
could presumably be PKC. This finding should allow the use of measurements of phorbol ester binding activity as an easy method to quantify PKC activity. Since PKC is under androgenic regulation in the prostate, as also are the general growth and function of this gland [3], the study of prostatic phorbol ester receptors during postnatal development and aging or in the autophagic response after castration as well as in pathological conditions including hyperplastic and neoplastic prostate would be of interest for the further specification of the corresponding molecular events. The exact biological responses mediated through these sites deserve further studies in prostatic tissue. In this context, previous results from our laboratory have already shown that phorbol esters used at concentrations similar to those in the present study desensitized fl-adrenergic [4] and VIP [5] stimulated cyclic AMP accumulation, which is a further indication of the interrelationship between the two main routes of signal transduction and again supports the possible identity between PKC and the phorbol ester receptor in the prostate gland.
in 5
6
7
8 9
10
11
12
13
Acknowledgments
This work was supported by grants from the FISS (90/74) and the University of Alcal6. M.P.G.P. was supported by a fellowship from the Diputacion de Guadalajara. We thank C.F. Warren from the I.C.E. of the U.A.H. for editorial assistance.
14
15
16
References Jeng, A.Y. and Blumberg, P.M. (1989) Biochemical mechanisms of action of the phorbol ester class of tumor promoters. In: The Pathobiology of Neoplasia, pp. 371-383. Editor: A.E. Sirica. Plenum Publishing Corp., New York. Hannun, Y.A. and Bel, R.M. (1986) Phorbol ester binding and activation of protein kinase C on Triton X-100 mixed micelles containing phosphatidylserine. J. Biol. Chem., 261, 9341-9347. Goueli, S.A. (1990) Differential regulation of prostatic protein kinase C isozymes by androgens. FEBS Leit., 264, 53-55. Carmena, M.J., Escudero, F. and Prieto, J.C. (1988) Inhibitory action of tumor-promoting phorbol esters upon fladrenergic agonist stimulation of cyclic AMP accumulation
17
18
19
20
rat
prostatic
epithelial
cells.
Biochem.
Int.,
17,
427 - 432. Escudero, F., Carmena, M.J. and Prieto, J.C. (1987) Tumor-promoting phorbol esters interfere with the vasoactive intestinal peptide receptor/effecter system in rat prostatic epithelial cells. Biochem. Biophys. Res. Commun., 149, 221- 226. Lowry, O.H., Rosebrough, N.J., Fan, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent, J. Biol. Chem., 193, 265-275. Jaken, S. (1987) Mesurement of phorbol ester receptors in intact cells and subcellular fractions. Methods Enzymol., 141, 275-287. Scatchard, G. (1949) The attractions of protein for small molecules and ions. Ann. N.Y. Acad. Sci., 51,660 - 672. Kerr, D.E., Kissinger, L.F., Gentry, L.E., Purchio, A.F. and Shoyab, M. (1987) Structural requirements of diacyl glycerols for binding and activating phospholipid-dependent, Ca2+-sensitive protein kinase. Biochem. Biophys. Res. Commun., 148, 776-782. Dowd, J.P., Alila, H.W. and Hansel, W. (1990) Phorbol ester receptors in bovine luteal cells: realationship to protein kinase C. Mol. Cell. Endocrinol., 69, 199-206. Dougherty, R. W. and Niedel, J. (1986) Cytosolic calcium regulates phorbol diester binding affinity in intact phagocytes. J. Biol. Chem., 261, 4097-4100. Lee, M.H. and Bell, R.M. (1989) Phospholipid functional groups involved in protein kinase C activation, phorbol ester binding to mixed micelles. J. Biol. Chem., 264, 14797 - 14805. Epand, R.M. and Lester, D.S. (1990) The role of membrane biophysical properties in the regulation of protein kinase C activity. Trends Pharmacol. Sci., 11, 317 -320. Bazzi, M.D. and Nelsestuen, G.L. (1988) Constitutive activity of membrane-inserted protein kinase C. Biochem. Biophys. Res. Commun., 152, 336-343. Sharkey, N.A., Leach, K.L. and Blumberg, P.M. (1984) Competitive inhibition by diacylglycerol of specific phorbol ester binding. Proc. Natl. Acad. Sci. U.S.A., 81, 607 - 710. Leach, K.L., Frost, M.M., Blumberg, P.M. and Bressler, J.P. (1987) Second stage tumor promoters: differences in biological potency and phorbol ester receptor affinity in C6 cells. Cancer Lett., 36, 139- 147. Ashendel, CL. (1989) Protein kinase C: a mediator and regulator of receptor-linked signal transduction. In: Receptor Phosphorylation, pp. 163- 175. Editor: V.K. Mougdil. CRC Press, Boca Raton, Florida. Konig, B, di Nitto, P.A. and Blumberg, P.M. (1985) Stoichiometric binding of diacylglycerol to the phorbol ester receptor. J. Biol. Chem., 27, 255 -265. Leach, K.L., Powers, E.A., Ruff, V.A., Jaken, S. and Kaufman, S. (1989) Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells. J. Cell Biol., 109, 685-695. Thomopoulos, P. (1985) Kinase C, ester du phorbol et recepteurs hormonaux membranaires. Ann. Endocrinol., 46. 307 - 312.
143
143 - 147
Elsevier Scientific Publishers Ireland Ltd
Receptors for tumor-promoting M.J. Carmena, Departamento
(Received (Accepted
de
phorbol esters in rat ventral prostate
M.P. Garcia-Paramio Bioquimica
y Biologia
and J.C. Prieto
Molecular,
Universidad
Alcal6 de Henares
(Spain)
30 March 1992) 24 November 1992)
Summary The presence of tumor-promoting phorboi ester receptors in rat prostate was investigated by studying fhe binding of phorbol die&et 12,13-dibutyrate (PDBu) in both soluble and particulaate subcellular fractions. Binding of [3H]PDBu to the soluble fraction was optimal
afier the addition of phosphatidylserine
(0.1
mg/ml) and Ca 2+ (1 mM). Both subcellular fractions exhibited a single class of PDBu receptor (Kd between 97 and 128 nM) as shown by saturation binding experiments. Phorbol esters with tumor-promoting activity
showed a higher affinity for these receptors than did endogenous ligands such as diacyglycerols whereas phorbol esters without tumor-promoting activity were ineffectioe euen at concentraHons as high as 1 PM. These properties are highly representatiue of protein kinase C activity.
Keywords:
tumor-promoting phorbol ester binding; protein kinase C; signal transduction Introduction
Tumor-promoting Correspondence
to: J.C.
y Biologfa Molecular, Henares,
de Alcalo’, E-28871
phorbol Prieto, Departamento
0
1992
Printed and Published in Ireland
are
de Bioquimica
Universidad de Alcalfi, E-28871
Spain.
0304-3835/92/$05.00
esters
Alcal
de
lipophylic agents that, circumventing the phosphoinositide pathway by binding directly to protein kinase C (PKC) at the cell membrane, may activate this phospholipid- and Ca*+-dependent enzyme [ 11. Because of their structural similarity, both diacylglycerol (DAG, the endogenous activator of PKC) and tumor-promoting phorbol esters mimic each other in binding to the same receptor (PKC) as a result of which the enzyme is redistributed and translocated away from the cytosol to the plasma membrane where it finds the necessary phosphatidylserine [Z] . Like other processes related to prostatic growth and function, PKC activity in the prostate gland appears to be under androgenic control [3]. Results from our laboratory have shown that the treatment of isolated rat prostatic epithelial cells with tumor-promoting phorbol esters desensitized the stimulation of cyclic AMP accumulation by fl-adrenergic agonists [4] or by vasoactive intestinal peptide (VIP) [5], a finding that suggests interaction between the cyclic AMP and phospholipid metabolizing pathways. However, a better understanding of PKC as a transducer of DAG and Ca*+ second messengers in prostatic tissue requires the characterization of the phorbol ester receptors. The objective of this paper is to investigate the characteristics of phorbol ester receptors in the rat ventral prostate using [3H]phorbol diester 12,13_dibutyrate (PDBu) and different subcellular preparations.
Elsevier Scientific Publishers Ireland Ltd.
144
Materials and Methods
Results
Chemicals 4cr-phorbol 12,13_didecanoate PDBu, (PDD), phorbol 12-myristate, 13-acetate (PMA) , phosphatidylserine and 1,2-dioleoylglycerol (DOG) were from Sigma (St. Louis, MO). [3H]PDBu (20 Ci/mmol) was from New England Nuclear (Boston, MA). All other chemicals were of reagent grade.
Figure 1 shows the phosphatidylserine dependence of [3H]PDBu binding to the soluble fraction from rat ventral prostate as representative of the phospholipid need for PKC activation. Binding was practically nil in the absence of phosphatidylserine and increased quickly up to the approximately 0.2 - 0.3 mg/ml which was required for maximal responses, The regulatory role of Ca*+ on [3H]PDBu binding was examined in both soluble and particulate cell fractions (Fig. 2) as an index of the requirement for this cation in PKC activation. Increasing concentrations of Ca*+ increased the binding of [3H]PDBu in the two subcellular preparations but some difference in the
Preparation of subcellular fractions The ventral lobes of the prostate from male Wistar rats (250 -300 g) were excised and homogenized into 3 volumes of ice-cold 25 mM Tris-HCI (pH 7.5) containing 0.25 M sucrose, 2.5 mM magnesium acetate, 2.5 mM EGTA and 2.5 mM ditiothreitol. Tissue homogenate was centrifuged at 100 000 x g for 1 h and the supernatant was referred to as the soluble fraction. The corresponding pellet was resuspended in homogenization buffer and considered to be the particulate fraction. All subcellular fractions were stored at - 85OC. Protein concentration was determined [6] using bovine serum albumin as a standard. Study of [3mPDBu binding Phorbol binding to subcellular fractions of rat ventral prostate was studied essentially as described elsewhere [7]. Standard binding assays were carried out in 0.16 ml of 50 mM Tris - HCI (pH 7.4) containing 0.1 mg of protein sample, 31.25 nM [3H]PDBu, 1 mM CaCls, 0.78 magnesium acetate, 0.2 mg/ml phosphatidylserine and 0.78 mM EGTA. The incubation was allowed to proceed at 37OC for 15 min. The nonspecific binding was defined in the presence of an excess of unlabelled PDBu (5 PM). Separation. of bound from free [3H]PDBu was achieved by centrifugation at 13 000 X g for 15 min (particulate fraction) or precipitation in polyethylene glycol followed by centrifugation (soluble fraction). The pellets were then washed and counted by liquid scintillation.
1.5
[Phosphatidylserinel, bg/ml Fig. 1. Effect of phosphatidylserine on [3H]PDBu binding in soluble subcellular fraction. The assay was carried out as described in Methods, with 0.1 mg of protein sample and the indicated concentrations of the phospholipid. Each point is the mean of triplicates in a representative
experiment.
145
1.8
”
0
0.5
I’
I B/F 0.020.011
0, 0
ah,
2
t.0
[Ca2+l,mM Fig. 2. Modification of [3H]PDBu binding to soluble (left panel) and particulate (right panel) subcellular fractions by addition of Ca’+. The assay was performed at increasing concentrations of added divalent cation. Each point is the mean of triplicates in a representative experiment.
extent of the Ca2+ requirement could be appreciated since maximal binding values were reached at either 0.5 mM Ca2+ in particulate cell fraction and 1 .O mM Ca2+ in soluble cell fractions. Figure 3 shows the PDBu binding activities in the two subcellular fractions studied. Saturation experiments using [3H]PDBu and increasing concentrations of the unlabelled tumor-promoting phorbol ester gave results that were interpreted by Scatchard analysis [8] in terms of a single-site model. The dissociation constant (I&) was calculated to be 97 * 16 nM (SE) with a binding capacity of 2.1 f 0.3 pmol PDBu bound per mg protein in the soluble fraction. The corresponding values in the particulate fraction were 128 f 12 nM and 2.8 f 0.3 pmol PDBu bound per mg protein. The binding affinity of the receptor for other phorbol esters and DOG was estimated by dose-dependent inhibition of [3H]PDBu binding in the soluble cell fraction (Fig. 4). When comparing the I&, values, PMA and the
0
Sal [Total PDBul, MI
Fig. 3. Saturation binding analysis of PDBu binding to soluble (top) and particulate (bottom) subcellular fractions. The assay was carried out with a fixed (31.25 nM) concentration of [3H]PDBu and increasing doses of the unlabelled phorbol ester. Each point is the mean f SE, n = 6 triplicate determinations. The insets correspond to the Scatchard analysis of the mean binding data.
endogenous ligand DOG showed a four times higher and a seven times lower respective affinity than PDBu for the receptors, whereas PDD was ineffective at concentrations up to at least 1 PM. Discussion Present data provide the first demonstration of the presence of specific receptors for tumorpromoting phorbol esters in the prostate and serve as a further characterization of PKC activity in this important gland of the male genitourinary tract. Specific t3H]PDBu binding activity was detected in both soluble and particulate subcellular fractions. However,
-+’ 00
tllnlabelled compound), -log M Fig. 4. Dose-dependent inhibition of [3H]PDBu binding to soluble cellular fraction by various phorbol esters and DOG. The unlabelled compounds tested were PMA ( q, PDBu (O), DOG (A) and PDD (0). Data are means of triplicates in a representative experiment.
some differences could be established since the soluble binding sites showed total dependency on both phosphatidylserine and Ca*+ whereas those in the particulate fraction only needed a modest exogenous supplement of the divalent cation to express maximal activity. This feature is well established in other systems characterizing phorbol ester binding and/or PKC activation [9 - 121 and it suggests that PKC itself could be the prostatic phorbol ester receptor. However, the existence of other prostatic phorbol ester receptors in addition to PKC remains to be defined. Present results on the soluble fraction of prostatic tissue confirm that the environment of the phorbol ester receptors in this compartment does not allow the corresponding binding unless a membranelike environment is mimicked. In this respect, it might be stressed that under physiological
conditions PKC is active when bound to membranes [13] and that membrane insertion needs Ca*+ and is favoured by phorbol esters 1141. The binding of PDBu in rat prostate was shown to represent a single class of receptors in both soluble and particulate subcellular fractions, Kd values being 97 and 128 nM respectively. These values are near those reported in systems such as mouse brain [15] and C6 rat glioma cells [16] with apparent Kd values between 40 and 90 nM. However, other systems that also show homogeneous phorbol ester binding have been reported to possess Kd values that fall within the 1 - 10 nM range [ 10,17,18]. Factors such as differences in lipid environment and even in the protein portions of the receptors could explain, at least in part, these apparent discrepancies. The lack of statistical significance suggests that the same receptor is present in the two preparations and that some mechanism of subcellular translocation is likely to exist as happens in other PKC activation or phorbol ester binding systems [ 131. A single class of phorbol ester receptor is the common observation in the majority of tissues studied [ 191. It is important to note that multiple (three or four) isozymes of Ca*+ /phospholipid-dependent PKC have been detected in the rat ventral prostate [3]. Thus, the corresponding functional moieties which interact with phorbol esters must be very similar if only a single class of receptors can be detected as shown in the present report. From the point of view of specificity, tumorpromoting phorbol esters like PMA and PDBu showed higher binding affinity than the endogenous ligand DOG. This agrees with observations in other systems and is likely to be related to the prolonged activation of PKC that is elicited by those phorbol compounds [9]. The observation that PDD did not displace [3H]PDBu binding again reinforces the hypothesis that the receptor studied in the prostate is PKC since PDD is a phorbol ester which is practically devoid of tumor-promoting ability and does not activate PKC [20]. In summary, tumor-promoting
prostatic tissue possesses phorbol ester receptors that
147
could presumably be PKC. This finding should allow the use of measurements of phorbol ester binding activity as an easy method to quantify PKC activity. Since PKC is under androgenic regulation in the prostate, as also are the general growth and function of this gland [3], the study of prostatic phorbol ester receptors during postnatal development and aging or in the autophagic response after castration as well as in pathological conditions including hyperplastic and neoplastic prostate would be of interest for the further specification of the corresponding molecular events. The exact biological responses mediated through these sites deserve further studies in prostatic tissue. In this context, previous results from our laboratory have already shown that phorbol esters used at concentrations similar to those in the present study desensitized fl-adrenergic [4] and VIP [5] stimulated cyclic AMP accumulation, which is a further indication of the interrelationship between the two main routes of signal transduction and again supports the possible identity between PKC and the phorbol ester receptor in the prostate gland.
in 5
6
7
8 9
10
11
12
13
Acknowledgments
This work was supported by grants from the FISS (90/74) and the University of Alcal6. M.P.G.P. was supported by a fellowship from the Diputacion de Guadalajara. We thank C.F. Warren from the I.C.E. of the U.A.H. for editorial assistance.
14
15
16
References Jeng, A.Y. and Blumberg, P.M. (1989) Biochemical mechanisms of action of the phorbol ester class of tumor promoters. In: The Pathobiology of Neoplasia, pp. 371-383. Editor: A.E. Sirica. Plenum Publishing Corp., New York. Hannun, Y.A. and Bel, R.M. (1986) Phorbol ester binding and activation of protein kinase C on Triton X-100 mixed micelles containing phosphatidylserine. J. Biol. Chem., 261, 9341-9347. Goueli, S.A. (1990) Differential regulation of prostatic protein kinase C isozymes by androgens. FEBS Leit., 264, 53-55. Carmena, M.J., Escudero, F. and Prieto, J.C. (1988) Inhibitory action of tumor-promoting phorbol esters upon fladrenergic agonist stimulation of cyclic AMP accumulation
17
18
19
20
rat
prostatic
epithelial
cells.
Biochem.
Int.,
17,
427 - 432. Escudero, F., Carmena, M.J. and Prieto, J.C. (1987) Tumor-promoting phorbol esters interfere with the vasoactive intestinal peptide receptor/effecter system in rat prostatic epithelial cells. Biochem. Biophys. Res. Commun., 149, 221- 226. Lowry, O.H., Rosebrough, N.J., Fan, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent, J. Biol. Chem., 193, 265-275. Jaken, S. (1987) Mesurement of phorbol ester receptors in intact cells and subcellular fractions. Methods Enzymol., 141, 275-287. Scatchard, G. (1949) The attractions of protein for small molecules and ions. Ann. N.Y. Acad. Sci., 51,660 - 672. Kerr, D.E., Kissinger, L.F., Gentry, L.E., Purchio, A.F. and Shoyab, M. (1987) Structural requirements of diacyl glycerols for binding and activating phospholipid-dependent, Ca2+-sensitive protein kinase. Biochem. Biophys. Res. Commun., 148, 776-782. Dowd, J.P., Alila, H.W. and Hansel, W. (1990) Phorbol ester receptors in bovine luteal cells: realationship to protein kinase C. Mol. Cell. Endocrinol., 69, 199-206. Dougherty, R. W. and Niedel, J. (1986) Cytosolic calcium regulates phorbol diester binding affinity in intact phagocytes. J. Biol. Chem., 261, 4097-4100. Lee, M.H. and Bell, R.M. (1989) Phospholipid functional groups involved in protein kinase C activation, phorbol ester binding to mixed micelles. J. Biol. Chem., 264, 14797 - 14805. Epand, R.M. and Lester, D.S. (1990) The role of membrane biophysical properties in the regulation of protein kinase C activity. Trends Pharmacol. Sci., 11, 317 -320. Bazzi, M.D. and Nelsestuen, G.L. (1988) Constitutive activity of membrane-inserted protein kinase C. Biochem. Biophys. Res. Commun., 152, 336-343. Sharkey, N.A., Leach, K.L. and Blumberg, P.M. (1984) Competitive inhibition by diacylglycerol of specific phorbol ester binding. Proc. Natl. Acad. Sci. U.S.A., 81, 607 - 710. Leach, K.L., Frost, M.M., Blumberg, P.M. and Bressler, J.P. (1987) Second stage tumor promoters: differences in biological potency and phorbol ester receptor affinity in C6 cells. Cancer Lett., 36, 139- 147. Ashendel, CL. (1989) Protein kinase C: a mediator and regulator of receptor-linked signal transduction. In: Receptor Phosphorylation, pp. 163- 175. Editor: V.K. Mougdil. CRC Press, Boca Raton, Florida. Konig, B, di Nitto, P.A. and Blumberg, P.M. (1985) Stoichiometric binding of diacylglycerol to the phorbol ester receptor. J. Biol. Chem., 27, 255 -265. Leach, K.L., Powers, E.A., Ruff, V.A., Jaken, S. and Kaufman, S. (1989) Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells. J. Cell Biol., 109, 685-695. Thomopoulos, P. (1985) Kinase C, ester du phorbol et recepteurs hormonaux membranaires. Ann. Endocrinol., 46. 307 - 312.