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Molecular Endocrinology, doi:10.1210/me.2006-0559
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Molecular Endocrinology 21 (11): 2637-2650
Copyright © 2007 by The Endocrine Society

Direct Binding and Activation of Protein Kinase C Isoforms by Aldosterone and 17ß-Estradiol

Rodrigo Alzamora, Laura R. Brown and Brian J. Harvey

Department of Molecular Medicine, Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland

Address all correspondence and requests for reprints to: Rodrigo Alzamora, Molecular Medicine, Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, P.O. Box 9063, Dublin 9, Ireland. E-mail: ralzamora{at}rcsi.ie.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Protein kinase C (PKC) is a signal transduction protein that has been proposed to mediate rapid responses to steroid hormones. Previously, we have shown aldosterone directly activates PKC{alpha} whereas 17ß-estradiol activates PKC{alpha} and PKC{delta}; however, neither the binding to PKCs nor the mechanism of action has been established. To determine the domains of PKC{alpha} and PKC{delta} involved in binding of aldosterone and 17ß-estradiol, glutathione S-transferase fusion recombinant PKC{alpha} and PKC{delta} mutants were used to perform in vitro binding assays with [3H]aldosterone and [3H]17ß-estradiol. 17ß-Estradiol bound both PKC{alpha} and PKC{delta} but failed to bind PKC mutants lacking a C2 domain. Similarly, aldosterone bound only PKC{alpha} and mutants containing C2 domains. Thus, the C2 domain is critical for binding of these hormones. Binding affinities for aldosterone and 17ß-estradiol were between 0.5–1.0 nM. Aldosterone and 17ß-estradiol competed for binding to PKC{alpha}, suggesting they share the same binding site. Phorbol 12,13-dybutyrate did not compete with hormone binding; furthermore, they have an additive effect on PKC activity. EC50 for activation of PKC{alpha} and PKC{delta} by aldosterone and 17ß-estradiol was approximately 0.5 nM. Immunoblot analysis using a phospho-PKC antibody revealed that upon binding, PKC{alpha} and PKC{delta} undergo autophosphorylation with an EC50 in the 0.5–1.0 nM range. 17ß-Estradiol activated PKC{alpha} and PKC{delta} in estrogen receptor-positive and -negative breast cancer cells (MCF-7 and HCC-38, respectively), suggesting estrogen receptor expression is not required for 17ß-estradiol-induced PKC activation. The present results provide first evidence for direct binding and activation of PKC{alpha} and PKC{delta} by steroid hormones and the molecular mechanisms involved.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PROTEIN KINASE C (PKC) belongs to a gene family of serine/threonine protein kinases that are key regulatory enzymes in signal transduction. The PKC family consists of several subfamilies (conventional: {alpha}, ßI, ßII, {gamma}; novel: {delta}, {epsilon}, {theta}, {eta}; atypical: {lambda}, {zeta}, {iota}) classified according to their requirements for Ca2+, phospholipids (phosphatidylserine) and diacylglycerol (DAG), or phorbol esters for activation (1, 2, 3, 4). The conventional and novel PKC isoforms contain an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain (4). The regulatory domain of conventional PKC isoforms contains two common regions, C1 and C2. The C1 domain mediates DAG and phorbol ester binding through distinct low- and high-affinity binding sites (5), whereas the C2 domain mediates Ca2+ and phosphatidylserine binding and contains the receptor for activated C kinase binding site (6, 7). PKC requires acidic phospholipids for its activity, and, in the presence of activators, the enzyme has the highest binding affinity for membranes containing phosphatidylserine. Upon activation, PKC isoforms are translocated to distinct subcellular compartments (membranes) and cell structures to phosphorylate their respective substrates (4, 8). This compartmentalization is required for the phosphorylation of specific substrates and the regulation of different physiological functions (4, 9). Preventing PKC translocation from the cytoplasm to membranes inhibits PKC function and the subsequent phosphorylation of specific substrates (7, 9).

PKC isoforms regulate gene expression and a variety of cellular functions, including growth, differentiation, tumor promotion, aging, and apoptosis (1, 2, 5); however, the biological significance of the heterogeneity in the PKC family is not clear. The distinct subcellular distribution, the presence of several isoforms in the same cell, and differential activation or inhibition by different stimuli suggest that each isoform is involved in the regulation of different functions and has a unique role in the cell (2, 4, 9, 10).

Steroid hormones regulate cellular processes by binding to intracellular receptors that, in turn, interact with discrete nucleotide sequences to alter gene expression; this process typically takes at least 30–60 min (11, 12). In contrast, other regulatory actions of steroid hormones are manifested within seconds to a few minutes (13, 14). These time periods are far too rapid to be due to changes at the genomic level and are therefore termed "nongenomic," to distinguish them from the classical steroid hormone action of regulation of gene expression. The rapid effects of steroid hormones are manifold, ranging from activation of MAPK, adenylyl cyclase, PKC, and G proteins (13). In some cases, these rapid actions of steroids are mediated through the classical steroid receptor that can also function as a ligand-activated transcription factor, whereas in other instances the evidence suggests that these rapid actions do not involve the classical steroid receptors and are mediated by a putative membrane receptor (13, 14, 15, 16, 17, 18).

Several studies have described direct activation of different PKC isoforms by steroid hormones. Our laboratory has shown direct activation of PKC{alpha} by aldosterone and 17ß-estradiol whereas PKC{delta} was activated by 17ß-estradiol only (19). Direct activation of conventional ({alpha}, ß, and {gamma}) and PKC{epsilon} isoforms was observed with 1,25-(OH)2-vitamin D3 at physiological concentrations (20). The adrenal androgen, dehydroepiandrosterone, has been shown to activate conventional and PKC{zeta} isoforms (21, 22). Similar results have been observed with glucocorticoids (22, 23). Therefore, it has been postulated that steroid hormones exert some of their nongenomic effects via direct interaction with PKC isoforms. However, the binding sites and mechanism by which they rapidly regulate PKC activity are not known.

In this work, we have begun the characterization of the biochemical events underlying this regulation. We have previously described direct activation of PKC{alpha} and PKC{delta} by aldosterone and 17ß-estradiol. Here, we have used glutathione S-transferase (GST) fusion PKC mutants to demonstrate the existence and localization of binding sites for these hormones on PKC{alpha} and PKC{delta} isoforms. Using PKC activity assays, we have shown that preincubation of PKC with nanomolar concentrations of aldosterone and 17ß-estradiol increases PKC activity and the level of autophosphorylation.

Taken together, this is the first evidence for a direct, high-affinity binding of aldosterone and 17ß-estradiol to PKC isoforms and the resulting increase in PKC activity. Given the significance of these in vitro results, we hypothesize that aldosterone and 17ß-estradiol might also be involved in directly regulating PKC activity in vivo.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of PKC Isoforms by Aldosterone and 17ß-Estradiol
Using an in vitro assay system, in which phosphatidylserine, calcium, and substrate concentrations corresponded to those yielding maximal activation, we examine the effect of aldosterone and 17ß-estradiol on human PKC{alpha} and PKC{delta} activity. Aldosterone significantly activated PKC{alpha} in a concentration-dependent manner between 0.1 and 10 nM (Fig. 1AGo). The EC50 for PKC{alpha} activation was 0.45 ± 0.05 nM, indicating that this hormone is a potent activator of PKC{alpha} activity. However, aldosterone had no effect on PKC{delta} activity even at concentrations as high as 10 nM (Fig. 1AGo). Similar effects were obtained with other mineralocorticoids such as fludrocortisone and deoxycorticosterone acetate. The relative potency for these hormones to activate PKC{alpha} was aldosterone = fludrocortisone > deoxycorticosterone acetate >> hydrocortisone (Table 1Go).


Figure 1
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  		Fig. 1. Dose-Response Curves for the Activation of PKC Isoforms by Aldosterone and 17ß-Estradiol

A, Activation of PKC{alpha} ({circ}) and PKC{delta} (bullet) by aldosterone. B, Activation of PKC{alpha} ({circ}) and PKC{delta} (bullet) by 17ß-estradiol. Values are mean ± SEM of four experiments done in triplicate. *, P < 0.05; **, P < 0.01.

 

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  		Table 1. Modulation of PKC Activity by Steroid Hormones

 
Similarly, 17ß-estradiol had a stimulatory effect on PKC{alpha} activity (Fig. 1BGo). This effect was concentration dependent within 0.1 and 10 nM with an EC50 of 0.33 ± 0.03 nM. In contrast to aldosterone, 17ß-estradiol did activate PKC{delta} activity in a concentration-dependent manner within the same concentration range (Fig. 1BGo). The EC50 for PKC{delta} activation by 17ß-estradiol was 0.41 ± 0.04 nM. Similar effects were obtained for other estrogens such as estriol and estrone. The relative potency for these hormones to activate PKC{alpha} and PKC{delta} was 17ß-estradiol > estriol >> estrone. The biologically inactive isomer 17{alpha}-estradiol had no effect on PKC activity (Table 1Go).

Activation of PKC Isoforms by Aldosterone and 17ß-Estradiol in the Presence of 4ß-Phorbol 12,13-Dibutyrate Ester (PDBu)
To characterize aldosterone and 17ß-estradiol interactions with PKC, we examined the effect of these hormones on kinase activity induced by phorbol esters such as PDBu. Figure 2Go shows the effect of a maximally stimulating concentration of aldosterone (5 nM) and 17ß-estradiol (5 nM) on the dose-response curve for PKC{alpha} and PKC{delta} activation by PDBu. The results show an additive effect of both hormones on the PDBu-induced activation of either PKC{alpha} or PKC{delta}. The effect was independent of the concentration of PDBu. These results indicate a minimal level of allosteric interaction between these hormones and PDBu. The increased activation shown here with PDBu paired with either aldosterone or 17ß-estradiol suggests a mechanism involving a two-site mechanism of action.


Figure 2
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  		Fig. 2. Effect of Maximally Activating Concentrations of Aldosterone and 17ß-Estradiol on the Dose-Response Curve for PKC Activation by PDBu

A, Activation of PKC{alpha} by PDBu in the presence of aldosterone (5 nM) or 17ß-estradiol (5 nM) compared with a methanol vehicle control (0.01%). B, Activation of PKC{delta} by PDBu in the presence of 17ß-estradiol (10 nM) compared with a methanol vehicle control (0.01%). Values are mean ± SEM of four experiments done in triplicate.

 
Aldosterone- and 17ß-Estradiol-Induced Autophosphorylation of PKC Isoforms
To determine some of the molecular events required for steroid hormone-induced PKC activation, we examined changes in the level of PKC autophosphorylation. The autophosphorylation of PKC{alpha} and PKC{delta} was measured using an in vitro assay with purified human PKC stimulated for 15 min with either aldosterone or 17ß-estradiol. Autophosphorylation of PKC is a step required for activation of these kinases and is often used as an index of PKC activation. Figure 3Go shows the effect of increasing concentrations (0.01–10 nM) of aldosterone and 17ß-estradiol on PKC{alpha} and PKC{delta} autophosphorylation levels. Consequent with the kinase activity assays, these hormones induced a sharp increase in the phosphorylation of both kinases. This effect was concentration dependent between 0.1 and 10 nM. The EC50 for hormone-induced autophosphorylation of PKC{alpha} by aldosterone was 1.07 ± 0.31 nM and 0.53 ± 0.22 nM for 17ß-estradiol. Autophosphorylation of PKC{delta} induced by 17ß-estradiol had an EC50 of 0.65 ± 35 nM. These results show that aldosterone and 17ß-estradiol induce autophosphorylation of PKC{alpha} and PKC{delta} isoforms with a potency similar to that observed in the activity assays. This suggests that hormone-induced activation of PKC{alpha} and PKC{delta} is mediated by autophosphorylation of PKC.


Figure 3
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  		Fig. 3. Autophosphorylation of PKC Isoforms Induced by Aldosterone and 17ß-Estradiol

PKC{alpha} and PKC{delta} were exposed to several concentrations of hormones and blotted using specific phospho-PKC antibodies; membranes were then stripped and blotted with regular PKC antibodies. Effect of aldosterone on PKC{alpha} (A), and effect of 17ß-estradiol on PKC{alpha} (B) and PKC{delta} (C). Upper blot shows autophosphorylation of PKC isoforms; lower blot shows total PKC isoforms; graph shows dose-response curves for the effect. Values in graphs are mean ± SEM of four experiments. *, P < 0.05; **, P < 0.01.

 
Binding of Aldosterone and 17ß-Estradiol to PKC Isoforms
To test our hypothesis of a direct interaction between aldosterone and 17ß-estradiol with PKC{alpha} and PKC{delta} and to determine which PKC domains contain the binding site for these hormones, different truncation and deletion mutants of PKC{alpha} and PKC{delta} were expressed as GST fusion proteins (Fig. 4Go). Proteins were produced using an in vitro expression system. This expression system is coupled with a redox system for formation of disulfide bonds and supplemented with a chaperone mixture for correct folding of proteins. Additionally, the reaction mixture was supplemented with 10 µM ZnCl2 for correct folding of zinc fingers contained in the C1 domain.


Figure 4
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  		Fig. 4. Expression of PKC{alpha} and PKC{delta} Mutants as GST Fusion Proteins

GST fusion proteins with PKC{alpha} (A) and PKC{delta} (B) domains were generated by PCR using the indicated combination of sense (F) and antisense (R) primers, followed by ligation into pIVEX-GST plasmid, and subsequent in vitro expression. Structure of GST-PKC mutants are illustrated schematically: C1A and C1B, cysteine-rich regions; C2, calcium binding domain; C3 and C4, catalytic domains; V1–5, variable domains. For mutants containing an internal deletion the domains were amplified using the first two pairs of primers. PCR products were then fused by overlap extension PCR using the second pair of primers. Predicted molecular weights of the respective GST-PKC mutants are shown.

 
Full-length PKC{alpha}-GST fusion protein showed aldosterone binding in a concentration-dependent manner with a binding affinity of 0.56 ± 0.31 nM and a stoichiometry of 0.58 ± 0.08 mol/mol of protein (Fig. 5Go). All fusion PKC{alpha} mutants containing the C2 domain readily bound aldosterone with affinities and stoichiometry similar to that of the full-length kinase. Consequently, a C2-PKC{alpha} fusion protein also showed high-affinity aldosterone binding. Fusion proteins lacking the C2 domain failed to bind aldosterone at any concentration. Control GST showed no aldosterone binding. Figure 5Go shows saturation curves for full-length PKC{alpha} (A), a C1A deletion mutant PKC{alpha}-{Delta}C1A (B), a C2 deletion mutant PKC{alpha}-{Delta}C2 (C), and a single C2 domain C2-PKC{alpha} (D) and their respective Scatchard plots (E, F, G, and H). Analysis of binding data for all PKC{alpha}-GST fusion mutants is shown in Table 2Go.


Figure 5
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  		Fig. 5. Comparison of Aldosterone Binding to GST Fusion Proteins

PKC{alpha} (A), PKC{alpha}-{Delta}C1A (B), PKC{alpha}-{Delta}C2 (C), and C2-PKC{alpha} (D). [3H]Aldosterone binding depicted as a function of total aldosterone concentration present or the corresponding Scatchard plots (E, F, G, and H) are shown. Binding assays were performed three times in triplicate.

 

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  		Table 2. Binding Affinities and Stoichiometry for Hormone Binding to GST Fusion PKC Mutants

 
Similarly, full-length PKC{alpha}-GST fusion protein showed 17ß-estradiol binding in a concentration-dependent manner with a binding affinity of 1.03 ± 0.15 nM and stoichiometry of 0.55 ± 0.02 mol/mol of protein (Fig. 6Go). All fusion PKC{alpha} mutants containing the C2 domain showed high specific 17ß-estradiol binding. Moreover, the C2-PKC{alpha} fusion protein also showed 17ß-estradiol binding with affinity constant and stoichiometry similar to that of the full-length PKC{alpha}. Fusion proteins lacking the C2 domain failed to bind 17ß-estradiol. Figure 6Go shows saturation curves for full-length PKC{alpha} (A), a C1A deletion mutant PKC{alpha}-{Delta}C1A (B), a C2 deletion mutant PKC{alpha}-{Delta}C2 (C), and a single C2 domain C2-PKC{alpha} (D) and their respective Scatchard plots (E, F, G, and H). Analysis of 17ß-estradiol binding data for all PKC{alpha}-GST fusion mutants is shown in Table 2Go.


Figure 6
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  		Fig. 6. Comparison of 17ß-Estradiol Binding to GST Fusion Proteins

PKC{alpha} (A), PKC{alpha}-{Delta}C1A (B), PKC{alpha}-{Delta}C2 (C), and C2-PKC{alpha} (D). [3H]17ß-estradiol binding depicted as a function of total 17ß-estradiol concentration present or the corresponding Scatchard plots (E, F, G, and H) are shown. Binding assays were performed three times in triplicate.

 
Using the PKC{delta}-GST fusion proteins we also tested binding of 17ß-estradiol. Full-length PKC{delta}-GST fusion protein showed 17ß-estradiol binding in a concentration-dependent manner with a binding affinity of 0.85 ± 22 nM and stoichiometry of 0.64 ± 0.04 mol/mol of protein (Fig. 7Go). Similar to aldosterone binding, all fusion PKC{delta} mutants containing the C2 domain showed high specific 17ß-estradiol binding. The C2-PKC{delta} fusion protein also showed 17ß-estradiol binding with similar affinity constant whereas fusion proteins lacking this domain failed to bind 17ß-estradiol. Figure 7Go shows saturation curves for full-length PKC{delta} (A), a C1 deletion mutant PKC{delta}-{Delta}C1 (B), a C2 deletion mutant PKC{delta}-{Delta}C2 (C), and a single C2 domain C2-PKC{delta} (D) and their respective Scatchard plots (E, F, G, and H). Analysis of 17ß-estradiol binding data for all PKC{delta}-GST fusion mutants is shown in Table 2Go. As expected, full-length PKC{delta} and its mutants failed to bind aldosterone (Table 2Go).


Figure 7
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  		Fig. 7. Comparison of 17ß-Estradiol Binding to GST Fusion Proteins

PKC{delta} (A), PKC{delta}-{Delta}C1 (B), PKC{delta}-{Delta}C2 (C), and C2-PKC{delta} (D). [3H]17ß-estradiol binding depicted as a function of total 17ß-estradiol concentration present or the corresponding Scatchard plots (E, F, G, and H) are shown. Binding assays were performed three times in triplicate.

 
Competition assays performed on full-length PKC{alpha} showed that [3H]aldosterone binding was displaced by aldosterone [inhibition constant (Ki), 0.41 ± 0.14 nM] and 17ß-estradiol (Ki, 1.33 ± 0.98 nM), indicating that both hormones share the same binding site on PKC{alpha} (Fig. 8AGo). In contrast, PDBu did not displace aldosterone binding to PKC{alpha}, suggesting a two-site binding site model (Fig. 8AGo). Similarly, [3H]17ß-estradiol binding to PKC{alpha} was displaced by aldosterone (Ki, 0.40 ± 0.15 nM) and 17ß-estradiol (Ki 1.38 ± 0.73 nM) but not by PDBu (Fig. 8BGo). In contrast, binding of [3H]17ß-estradiol to full-length PKC{delta} was only displaced by 17ß-estradiol (Ki, 0.83 ± 0.57 nM), suggesting a specific site for estrogens on this PKC isoform (Fig. 8CGo). To control the binding properties of PKC{alpha} and PKC{delta}, competition assays for PDBu were carried out. [3H]PDBu binding to both PKC isoforms was displaced by nonradioactive PDB with a Ki of 22.2 ± 2.1 nM for PKC{alpha} and Ki 54.7 ± 10.4 nM for PKC{delta} (Fig. 8DGo).


Figure 8
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  		Fig. 8. Competition with Specific Binding of [3H]Aldosterone or [3H]17ß-Estradiol to PKC{alpha} and PKC{delta}

Radiolabeled hormones (present at 10 nM throughout) were competed with by increasing concentrations of nonradioactive aldosterone ({circ}), 17ß-estradiol (bullet), and the phorbol ester PDBu ({square}). A, Competition with [3H]aldosterone binding to PKC{alpha}. B, Competition with [3H]17ß-estradiol binding to PKC{alpha}. C, Competition with [3H]17ß-estradiol binding to PKC{delta}. D, Competition with [3H]-PDBu binding to PKC{alpha} ({blacktriangleup}) and PKC{delta} ({Delta}) by nonradioactive PDBu. Values are mean ± SEM of four experiments performed in triplicate.

 
Binding of Aldosterone and 17ß-Estradiol to the C2 Domain of PKC{alpha} and PKC{delta} Is Required for Hormone-Induced PKC Activation
Recombinant PKC isoforms expressed in vitro retain the binding properties of PKC expressed in eukaryotic cells but they are not catalytically active. Therefore, we examined the effect of aldosterone and 17ß-estradiol on human PKC{alpha} and PKC{delta} expressed in Sf9 insect cells, which are known to have kinase activity. Hormone-induced PKC activity was measured in the presence of increasing concentrations of PKC{alpha} and PKC{delta} C2 domains. Aldosterone- and 17ß-estradiol-induced PKC{alpha} activity was inhibited by coincubation with C2-PKC{alpha} in a concentration-dependent manner suggesting that sequestration of these hormones by C2-PKC{alpha} reduces binding and activation of full-length PKC{alpha} (Fig. 9AGo). Similarly, 17ß-estradiol-induced PKC{delta} activity was inhibited by coincubation with C2-PKC{delta} in a concentration-dependent manner, suggesting sequestration of 17ß-estradiol by C2-PKC{delta} reduces binding and activation of full-length PKC{delta} (Fig. 9BGo). In both cases, PDBu-induced PKC activity was not affected by coincubation with the C2 domain (Fig. 9Go, A and B). In contrast, coincubation with C1-domains did not affect the hormone-induced PKC activity but reduced PDBu-induced PKC activity due to sequestration of PDBu, thus preventing binding to C1 domains (Fig. 9Go, C and D). These data suggest binding of aldosterone and 17ß-estradiol to the C2 domains of PKC{alpha} and PKC{delta} is a prerequisite for kinase activation.


Figure 9
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  		Fig. 9. Effect of C2 Domain Competition for [3H]Aldosterone or [3H]17ß-Estradiol Binding to PKC{alpha} and PKC{delta} on Kinase Activity

The effect of coincubation with increasing concentrations (expressed as molar ratios) of C1 and C2 domains on PKC{alpha} and PKC{delta} activity stimulated by 5 nM aldosterone ({circ}), 5 nM 17ß-estradiol (bullet), and 30 nM PDBu ({square}). A, Effect of C2-PKC{alpha} domain on stimulated PKC{alpha} activity. B, Effect of C2-PKC{delta} domain on stimulated PKC{delta} activity. C, Effect of C1-PKC{alpha} domain on stimulated PKC{alpha} activity. D, Effect of C1-PKC{delta} domain on stimulated PKC{delta} activity. Values are mean ± SEM of four experiments performed in triplicate.

 
Hormone-Induced PKC Activation Is Independent of Steroid Hormone Receptor Expression
To determine the role of steroid hormone receptors on the aldosterone- and 17ß-estradiol-induced activation of PKC{alpha} and PKC{delta} and the properties of this effect in cells, we examine the effect of 17ß-estradiol on PKC{alpha} and PKC{delta} activity in estrogen receptor positive (ER+) MCF-7 cells and the estrogen receptor negative (ER–) HCC-38 cells. Kinase activity for each isoform was measured from total lysates, using specific PKC isoform inhibitors, such as HBDDE-sensitive for PKC{alpha} and rottlerin-sensitive for PKC{delta}. In HCC-38 cells a 10-min treatment with 17ß-estradiol induced a concentration-dependent increase in PKC{alpha} and PKC{delta} activity with an EC50 of 1.34 ± 0.64 nM and 0.54 ± 0.08 nM, respectively (Fig. 10AGo). Similarly, in MCF-7 cells a 10-min treatment with 17ß-estradiol induced a concentration-dependent increase in PKC{alpha} and PKC{delta} activity with an EC50 of 0.74 ± 0.16 nM and 0.51 ± 0.04 nM, respectively (Fig. 10BGo). Pretreatment of both cell lines with the pure antiestrogen ICI 182,780 failed to prevent the stimulatory effect of 17ß-estradiol on PKC activity even at concentrations as high as 10 µM (data not shown). These data indicate that in both cell lines 17ß-estradiol caused a dose-dependent increase in both PKC{alpha} and PKC{delta} activity, indicating ER expression is not required for the 17ß-estradiol-mediated increase in PKC activity. In order to evaluate in vivo the additive effect of steroid hormones with classic activators of PKC such as DAG and phorbol esters, we tested the effect of 17ß-estradiol on bombesin-induced PKC activity in HCC-38 cells. Bombesin is a peptide hormone that activates DAG and inositol 1,4,5-triphosphate synthesis, and intracellular Ca2+ mobilization leading to PKC activation. In HCC-38 cells, bombesin increased PKC{delta} activity in a concentration-dependent manner with a maximal response at 100 nM bombesin. Pretreatment with 0.1 or 1 nM 17ß-estradiol for 10 min significantly increased the bombesin-induced PKC activation even at concentrations in which bombesin effect was maximal (Fig. 10CGo). Aldosterone had no effect on either basal or bombesin-induced PKC{delta} activity (data not shown). These results suggest that 17ß-estradiol-induced PKC activation has an additive effect on other regulators of PKC, which acts through the DAG-dependent signaling pathway.


Figure 10
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  		Fig. 10. Effect of 17ß-Estradiol on PKC Activity in ER-Positive and -Negative Cells

The effect of 17ß-estradiol on PKC{alpha} and PKC{delta} activity was measured in MCF-7 (ER+) and HCC-38 cells (ER–) as HBDDE-sensitive (PKC{alpha}) and rottlerin-sensitive (PKC{delta}) kinase activity. A, Effect of 17ß-estradiol on PKC{alpha} (bullet) and PKC{delta} ({circ}) activity in HCC-38 cells. B, Effect of 17ß-estradiol on PKC{alpha} (bullet) and PKC{delta} ({circ}) activity in MCF-7 cells. C, Effect of 17ß-estradiol on bombesin-induced PKC{delta} activity in HCC-38 cells. Concentration-response curves represent bombesin ({circ}), bombesin + 0.1 nM 17ß-estradiol (bullet), bombesin + 1 nM 17ß-estradiol ({square}). Values are mean ± SEM of four experiments performed in triplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The main findings in the present study are the direct binding of PKC{alpha} and PKC{delta} by aldosterone and 17ß-estradiol through interaction with their C2 domain, leading to autophosphorylation and activation of these kinases. These results suggests that PKC{alpha} and PKC{delta} may serve as receptors for aldosterone and 17ß-estradiol, providing a new rapid mechanism for transducing the rapid effects of these hormones, additional to the well-known activation of PKC by membrane-receptor-mediated activation of phospholipase C that leads to increases in DAG synthesis (3, 5, 8). Because kinase activity was observed in an in vitro system containing only PKC, phospholipids, vesicles, substrates and the necessary cofactors, it can be assumed that aldosterone and 17ß-estradiol directly interact with specific binding sites on PKC{alpha} and PKC{delta}. This hypothesis is supported by the finding that other mineralocorticoids and estrogens show different potency on activating PKC{alpha} and that the biologically inactive 17{alpha}-estradiol had no effect on the enzyme activity, indicating a degree of structural specificity rather than a nonspecific effect. Furthermore, this specificity is shown by the fact that PKC{delta} was activated only by biologically active estrogens such as 17ß-estradiol and estriol but insensitive to 17{alpha}-estradiol and aldosterone.

The finding that the level of activation achieved by a maximally stimulating concentration of these hormones in combination with the phorbol ester PDBu was higher than that achieved with any activator alone implies that aldosterone and 17ß-estradiol are not merely competing with PDBu for its binding site in the C1 domain of PKC (24, 25, 26, 27, 28, 29) but rather interacting with a different binding site. To confirm this hypothesis, we performed competition assays between PDBu and aldosterone or 17ß-estradiol. We found that PDBu does not compete with either aldosterone or 17ß-estradiol binding to PKC{alpha} and PKC{delta}, confirming that these hormones do not bind to the same site for phorbol esters. These data suggest that these hormones can act both as effectors and promoters of PKC activity. This concept is supported by the in-cell studies showing an additive effect of 17ß-estradiol and bombesin, a well-known PKC agonist, on PKC activity in HCC-38 cells.

In the study presented here, we have used GST-PKC fusion proteins to determine the binding site for aldosterone and 17ß-estradiol to human PKC{alpha} and PKC{delta}. Recombinant GST-PKC fusion proteins have been widely used to study binding of activators such as DAG and phorbol esters to PKC (25, 26, 27, 28, 29, 30). In the vesicle assay, high-affinity hormone binding was seen with any fusion protein containing the C2 domain, but not in those lacking this domain. Furthermore, no significant differences in either hormone binding or stoichiometry were observed between any of the C2-containing mutant PKCs. Thus, the presence or absence of other PKC domains did not affect binding of these hormones to C2-containing mutant PKCs. These results clearly indicate the C2 domain is essential and sufficient for binding of aldosterone and 17ß-estradiol to a common binding site in the C2 domain of PKC{alpha} whereas PKC{delta} exclusively binds 17ß-estradiol.

Aldosterone and 17ß-estradiol-binding stoichiometries range between 0.5 and 1.0 mol of ligand per mol of protein, suggesting a stoichiometry of 1:1 for hormone binding to PKC{alpha} and PKC{delta}. A possible concern regarding these experiments is that, in many cases, hormone-binding stoichiometries are significantly lower than 1, suggesting that, although soluble, GST fusion proteins might not necessarily be functional. However, binding stoichiometries for PKC described in this study are higher, on average, than those reported for other ligands known to bind PKC with a 1:1 stoichiometry (30), indicating the majority of soluble, purified C2-containing fusion proteins do represent functional aldosterone and 17ß-estradiol binding sites.

In recent years several studies have focused on the direct binding and activation of PKC isoforms by compounds that bind the C2 domain. The C2 domain of conventional PKC isoforms mediates calcium and phosphatidylserine binding and contains the binding site for activated C kinase receptor (31, 32, 33). Novel PKC isoforms lack calcium-coordinating acidic residue side chains; hence novel PKCs are maximally activated by DAG and phorbol esters, without requiring calcium. Recent studies have shown that retinoic acid directly activates PKC{alpha} and PKC{delta} upon binding of the C2 domain in two locations coincident with the two binding sites previously reported for acidic phospholipids (34, 35, 36, 37). These observations were supported by [3H]retinoic acid binding, site-directed mutagenesis, and crystallographic studies.

The traditional model of PKC activation focuses on allosteric modulation by calcium and DAG. However, more recent studies have identified a series of sequential priming phosphorylations at highly conserved serine/threonine phosphorylation motifs in all PKC isoforms that lock the enzyme in a closed, stabilized, catalytically competent and protease/phosphatase-resistant conformation (38, 39, 40, 41). Upon activation, PKCs are believed to undergo autophosphorylation, on a conserved proline-flanked turn motif and a hydrophobic motif, which is important for reversible PKC isoform stimulation by physiological agonists (42). These phosphorylations on conventional PKC isoforms generally have been characterized as intramolecular autophosphorylation events (43). In this study both aldosterone and 17ß-estradiol induced autophosphorylation of PKC{alpha} and PKC{delta} on the hydrophobic motif. Because this event was observed in an in vitro assay the phosphorylations are likely to occur through an intramolecular mechanism.

Noteworthy in this study is the fact that all the events observed (activity, autophosphorylation, and binding) are induced by nanomolar concentrations of aldosterone and 17ß-estradiol, which are consistent with the physiological concentrations of free circulating hormones (~0.1–0.5 nM). Therefore, it is expected that direct binding and activation of PKC{alpha} and PKC{delta} play a role in the physiological functions of both hormones, especially on their rapid nongenomic effects. Several studies have shown that both aldosterone and 17ß-estradiol exert rapid effects in several tissues, including PKC and MAPK activation, increase of intracellular Ca2+ concentration, and regulation of ion channel activity (13, 14, 15). In most cases, activation of PKC is critical for the activation of other rapid responses. Hence, several groups have postulated that these kinases can act as receptors for steroid hormones (44) or that hormone-induced PKC activity may act as a coregulator of a membrane receptor-mediated response (20). PKC{alpha} is almost ubiquitously distributed in tissues within the body. However, the rapid responses to aldosterone and estrogen are tissue specific. Thus, other factors must be involved to confer cellular and tissue specificity for PKC{alpha}-mediated responses. These factors include intracellular calcium, other kinases including MAPK, PDK1, and the expression levels of each PKC isoform. The latter is particularly relevant for PKC{delta}, which we have shown to be differentially expressed at mRNA and protein levels in male and female colonic crypts (45). Because activation of conventional PKC isoforms depends on calcium binding, the aldosterone and 17ß-estradiol direct activation of PKC{alpha} must operate in concert with an increase in intracellular calcium. Several studies have shown that these hormones can increase the turnover of DAG and inositol triphosphate, leading to an increase in intracellular calcium and ultimately to PKC activation (13, 14, 15, 19). Therefore, direct activation of PKC{alpha} by aldosterone and 17ß-estradiol may represent a coactivation mechanism in which they will enhance the response induced by DAG and calcium similar to the manner in which they enhance the effect of PDBu. Similarly, 17ß-estradiol could also enhance the effect of DAG-induced PKC{delta} activation. However, because this PKC isoform does not require calcium, the 17ß-estradiol-induced activation of PKC{delta} could take place independent of membrane receptor-mediated increase in DAG. Thus, PKC{delta} can act as a receptor for 17ß-estradiol.

In conclusion, we have demonstrated for the first time a direct interaction between 17ß-estradiol and aldosterone with PKC{alpha} and of 17ß-estradiol with PKC{delta} and have located the binding site for these hormones as being within the C2 domain. These new data support the hypothesis that discrete steroid hormone-binding sites exist in PKC isoforms with affinities for specific steroid hormones. The binding affinities are well within the physiological concentration for these hormones. Therefore, these PKC isoforms can act as receptors for steroid hormones and mediate cross talk with membrane receptor-mediated signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
[1,2,6,7-3H]Aldosterone (80 Ci/mmol), [2,4,6,7-3H]17ß-estradiol (95 Ci/mmol), and [{gamma}-32P]ATP (30 Ci/mmol) were from PerkinElmer (Buckinghamshire, UK). PDBu, human recombinant PKC{alpha} and PKC{delta}, histone-1, and 2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl ether (HBDDE) were from Calbiochem (Nottingham, UK). Pfu-Ultra Hot-Start DNA polymerase was from Stratagene (Cambridge, UK). Restriction enzymes, ligase, and phosphatase were from New England Biolabs (Hertfordshire, UK). Antibodies against Ser(643)-phospho-PKC{delta}, PKC{alpha}, and PKC{delta} were from Cell Signaling Technologies (Hertfordshire, UK), Ser(657)-phospho-PKC{alpha} was from Upstate (Dublin, Ireland). Protease inhibitor cocktail, horseradish peroxidase-conjugated anti-His6 antibody, and pIVEX-GST plasmid were from Roche Applied Science (Burgess Hill, UK). All other reagents were from Sigma-Aldrich (Dublin, Ireland).

PKC Assay
PKC activity was assayed with Triton X-100 mixed micelles, using the phosphorylation of histone-1, as described by Hannun and Bell (46). Micelles were prepared by dissolving phosphatidylserine (2.8 mg) in chloroform in a glass tube. After evaporation of chloroform under a stream of nitrogen, 1 ml of 3% Triton X-100 (prepared fresh) was added and the mixture was sonicated. Unless stated otherwise, the reaction mixture contained 20 mM Tris (pH 7.5), 20 mM MgCl2, 1.0 mM CaCl2, 0.25 mM EGTA, 0.25 mM EDTA, 1 mM ß-mercaptoethanol, 0.28 mg/ml phosphatidylserine, 0.3% Triton X-100, 50 µM histone-1, 20 µM [{gamma}-32P]ATP, and 150 mU of PKC in a final volume of 25 µl. Samples were incubated for 15 min at 37 C, and reactions were terminated by spotting an aliquot of reaction mixture on phosphocellulose disks (P-81, 2.3 cm, Whatman, Clifton, NJ). After washing the disks with 1% phosphoric acid, {gamma}-32P incorporation into histone-1 was determined by liquid scintillation spectrometry. Non-PKC-specific activity was determined in samples incubated in the absence of the enzyme.

Cell Culture
MCF-7 and HCC-38 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were grown in phenol red-free DMEM with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.01 mg/ml insulin at 37 C in an atmosphere containing 5% CO2 and 100% humidity. When confluent, cells were subcultured into six-well culture plates and grown using the same conditions as above. At confluence, cells were serum starved for 24 h and then treated with PDBu, aldosterone, or 17ß-estradiol for the required times.

Protein Extraction and Western Blotting
Cells were lysed by hypotonic shock on ice for 45 min [Lysis buffer: 20 mM Tris, pH 7.4; 0.5% Nonidet P-40; 250 mM NaCl; 3 mM EDTA; 3 mM EGTA; leupeptin, 1 µg/µl; 500 mM dithiothreitol; 5 mM phenylmethylsulfonylfluoride; and complete mini EDTA-free protease inhibitor cocktail tablets (one tablet/7 ml of lysis buffer; Roche Applied Science)]. After incubation the samples were clarified at 12,000 rpm for 10 min. The cleared supernatant was collected, and the protein content was quantified by the Bradford method. Protein samples (50 µg) were combined with 2x sample buffer, boiled at 95 C for 5 min, and spun at 12,000 rpm for 2 min. Western blot analysis was carried out as previously described (45).

Autophosphorylation Assay
To determine autophosphorylation of PKC isoforms induced by aldosterone or 17ß-estradiol, recombinant human PKC isoforms (1 ng) were incubated in PKC assay mixture with nonradioactive ATP in a final volume of 25 µl. Samples were treated with several concentrations of aldosterone or 17ß-estradiol (0.01–10 nM) for 15 min at 37 C. Reaction was stopped by addition of Laemli sample buffer, and samples were subjected to standard Western blot (45) with phospho-PKC{alpha} and phospho-PKC{delta} antibodies.

Expression of GST-PKC Fusion Proteins
To generate large quantities of protein for characterization of binding, several deletion and truncation mutants of human PKC{alpha} and PKC{delta} were expressed as amino-terminal fusion proteins with hexa-histidine (His6) and GST. Briefly, specific regions of human PKC{alpha} and PKC{delta} were amplified by PCR using the pBlueBac/PKC{alpha} and pBlueBac/PKC{delta} clones (ATCC) containing human PKC cDNA as a template. The restriction site of all forward primers was AflIII. The restriction site of all reverse primers was EcoRV. For mutants with internal deletions, fragments of the PKC domains to be recombined were generated in separate PCRs and the amplified products were then fused by overlap extension PCR as described by Horton et al. (47). Table 3Go shows all primers used for amplification of PKC mutants. DNA amplification was carried out for 25 cycles, each at 95 C for 1 min, average primer pair melting temperature minus 5 C for 1 min, and 72 C for 2 min. The PCR products were double digested with AflIII and EcoRV, analyzed in 2% agarose gels, and isolated using Gel Extraction Kit (QIAGEN, Crawley, UK) following manufacturer’s protocol. The digested PCR products were ligated into NcoI/SmaI double-digested GST-pIVEX plasmid. After ligation, DH5{alpha} Escherichia coli competent cells (Invitrogen, Paisley, Scotland, UK) were transformed with the plasmids. Positive clones were initially selected by restriction analysis and subsequently confirmed by DNA sequencing (MWG Biotech, Ebersberg, Germany).


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  		Table 3. Primers Used for Amplification of PKC Mutants

 
In Vitro Protein Synthesis and Purification
Protein was synthesized in vitro using the rapid-translation system RTS100 E. coli Disulfide Kit (Roche Applied Science), according to the manufacturer’s instructions using 200 ng of plasmid DNA per reaction. Reactions were incubated at 30 C for 12 h in an RTS ProteoMaster Instrument (Roche Applied Science). To act as a control, GST was synthesized from an empty pIVEX-GST plasmid. Proteins were purified with nickel nitrilotriacetic acid agarose spin columns (QIAGEN) according to the manufacturer’s instructions. The mixture from the in vitro protein synthesis was loaded into a nickel nitrilotriacetic acid spin column and washed twice with a four-bed volume of wash buffer A (100 mM NaH2PO4; 10 mM Tris-Cl, pH 6.3). The recombinant protein was eluted with a one-bed volume of elution buffer B (100 mM NaH2PO4; 10 mM Tris-Cl, pH 5.9), followed by another elution with a one-bed volume of buffer C (100 mM NaH2PO4; 10 mM Tris-Cl, pH 4.5). All buffers contained a protease inhibitor mixture. The eluted His6-tagged different constructs of PKC proteins were examined using standard Western blot analysis with horseradish peroxidase-conjugated anti-His6 antibody. Purified proteins were stored at –70 C.

Binding Assays
[3H]Aldosterone and [3H]17ß-estradiol binding to PKC mutants was measured using the polyethylene glycol precipitation assay developed by Kazanietz et al. (48), with minor modifications. For determination of the dissociation constants (Kd) and the number of sites (Bmax) for the different PKC preparations, typical saturation curves with increasing concentrations of the radioactive ligand (between 0.01 and 10 nM) were obtained in triplicate. The assay mixture (250 µl) contained 0.2 nmol GST fusion protein, 50 mM Tris-HCl (pH 7.4), 100 µg/ml phospholipids, 4 mg/ml bovine IgG, 100 µM CaCl2, and variable concentrations of ligand. The assay was carried out at 37 C for 15 min. For competition assays a fixed concentration of radiolabeled aldosterone or 17ß-estradiol (5 nM) was incubated against five to seven increasing concentrations of the competing nonradioactive PDBu and hormones.

Data Analysis
Densitometric analysis of Western blots was performed using Genetools software (Syngene, Cambridge, UK). Statistical analysis of the data was obtained using paired t test for analysis between two groups. One-way ANOVA and Tukey’s post hoc test were used for multiple analyses of more than two groups. Nonlinear regression analysis of binding data was performed using Igor-Pro 3.0 software (Wavemetrics, Inc., Lake Oswego, OR). P values ≤ 0.05 were considered to be significant. Data are expressed as means ± SEM unless specifically stated to be from a representative experiment.


   FOOTNOTES
 
This work was supported by The Wellcome Trust Grant 06089/Z/00/Z and Higher Education Authority of Ireland PRTLI Grants cycle 1 and 3. R.A. was recipient of a Wellcome Trust Studentship Prize 06379/Z/01/Z. L.R.B. was recipient of a Science and Technology Work Experience Program for Secondary School Summer Studentship.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 31, 2007

Abbreviations: DAG, Diacylglycerol; ER, estrogen receptor; GST, glutathione S-transferase; PDBu, 4ß-phorbol 12,13-dibutyrate; PKC, protein kinase C.

Received for publication December 29, 2006. Accepted for publication July 25, 2007.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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