©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Direct Activation of Protein Kinase C by 1,25-Dihydroxyvitamin D(*)

(Received for publication, November 14, 1994; and in revised form, January 19, 1995)

Simon J. Slater (1) Mary Beth Kelly (1) Frank. J. Taddeo (1) Jonathan D. Larkin (1) Mark D. Yeager (1) John A. McLane (2) Cojen Ho (1) Christopher D. Stubbs (1)(§)

From the  (1)Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and (2)Hoffmann-LaRoche, Nutley, New Jersey 07110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The key metabolite of vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3)), induces rapid cellular responses that constitute a so-called ``non-genomic'' response. This effect is distinguished from its ``classic'' genomic role in calcium homeostasis involving the nuclear 1,25-D(3) receptor. Evidence is presented that protein kinase C (PKC) is directly activated by 1,25-D(3) at physiological concentrations (EC = 16 ± 1 nM). The effect was demonstrable with single PKC-alpha, -, and - isoform preparations, assayed in a system containing only purified enzyme, substrate, co-factors, and lipid vesicles, from which it is inferred that a direct interaction with the enzyme is involved. The finding that calcium-independent isoform PKC- was also activated by 1,25-D(3) shows that the calcium binding C2 domain is not required. The level of 1,25-D(3)-induced activation, paired with either diacylglycerol or 4beta-12-O-tetradecanoylphorbol-13-acetate, was greater than that achievable by any individual activator alone, each at a saturating concentration, a result that implies two distinct activator sites on the PKC molecule. Phosphatidylethanolamine present in the lipid vesicles potentiated 4beta-12-O-tetradecanoylphorbol-13-acetate- and diacylglycerol-induced PKC activities, whereas 1,25-D(3)-induced activity decreased, consistent with 1,25-D(3)-activated PKC possessing a distinct conformation. The results suggest that PKC is a ``membrane-bound receptor'' for 1,25-D(3) and that it could be important in the control of non-genomic cellular responses to the hormone.


INTRODUCTION

The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3)), (^1)a steroid type hormone, is known for its regulatory role in calcium homeostasis. The hormone, derived either from 7-dehydroxycholesterol by the action of ultraviolet light on the skin or from the diet, facilitates the absorption of calcium from the intestine, its mobilization from bone, and its resorption in the kidney (DeLuca et al., 1990; Burgos-Trinidad et al., 1990; Studzinski et al., 1993). Apart from its role in calcium uptake, 1,25-D(3) is widely acknowledged to be an important regulator of cell growth and differentiation (Nemere et al., 1993; Darwish and DeLuca, 1993; Lowe et al., 1992).

Within target cells, it binds to the vitamin D receptor in the nucleus, which regulates gene expression by interacting with transcription factors. However, recently, it has become clear that many cellular responses to the hormone are too rapid to be mediated by gene expression controlled by the vitamin D receptor. For example, 1,25-D(3) rapidly stimulates the turnover of phosphoinositides and phosphatidylcholines in a wide range of cell types, leading to increases in the levels of inositol triphosphate and diacylglycerol (DAG) (de Boland et al., 1994; Morelli et al., 1993; Civitelli et al., 1990; Bourdeau et al., 1990; Wali et al., 1990). These rapid effects, typical of a membrane receptor-type response, have led to the recognition of the importance of the non-genomic role for 1,25-D(3) in cell regulation.

PKC occupies a central position in signal transduction and controls diverse cellular processes, including growth and differentiation (Nishizuka, 1992; Stabel and Parker, 1991; Bell et al., 1992; Hug and Sarre, 1993). The release of calcium from intracellular stores, induced by inositol triphosphate, along with an increase in extracellular calcium influx promoted by 1,25-D(3) (Morelli et al., 1993) triggers a phospholipid-dependent translocation of calcium-dependent PKC isoforms to the membrane. DAG liberated by phosphoinositide hydrolysis then activates the membrane-bound PKC by inducing a conformational change in the enzyme. A number of studies implicate an involvement of PKC in the non-genomic actions of 1,25-D(3) (for example, see Boland et al.(1991), Simboli-Campbell et al.(1994), de Boland and Norman(1990), Simpson et al.(1989) Lissoos et al.(1993), van Leeuwen et al.(1992)). Evidence for this comes from a similarity in the effects of PKC activators (e.g. phorbol esters) to those of 1,25-D(3) on certain cellular responses and from the reversibility of these responses by PKC inhibitors (Simpson et al., 1989; Khare et al., 1993; de Boland and Norman, 1990; van Leeuwen et al., 1992).

1,25-D(3) also induces an increase in the level of PKC expression, independent from increases in DAG levels (Obeid et al., 1990); however, this would again be too slow to explain the relatively fast cellular responses to the hormone. The rapid nature of the non-genomic responses, along with the differing specificities for structural derivatives of 1,25-D(3), compared with those involving the vitamin D receptor (Norman et al., 1993), has led to a proposal that there may be a cell membrane-bound receptor(s) for 1,25-D(3) (Lieberherr et al., 1989; Nemere et al., 1993). A recent report has provided evidence for the existence of such a receptor (Nemere et al., 1994); however, molecular details of the membrane-bound receptor or receptors, and the mechanism by which the signal provided by 1,25-D(3) binding to this receptor might be transduced, remains to be resolved.

The similar effects of 1,25-D(3) on PKC activation in vivo to those of DAG and phorbol esters suggested to us the possibility of a direct activation of PKC and that the enzyme itself might act as a membrane-associated 1,25-D(3) receptor. To test this hypothesis, we compared the effects of 1,25-D(3) with those of DAG and the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA), on the activity of PKC using a cell-free assay system with purified PKC. The results reveal that PKC is directly and potently activated by 1,25-D(3) at physiological concentrations in a manner similar to that by DAG.


EXPERIMENTAL PROCEDURES

Materials

Myelin basic protein (MBP) was obtained from LC Services (Woburn, MA). The PKC- pseudosubstrate peptide was custom synthesized by Biosynthesis, Inc. (Lewisville, TX). Lipids were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). TPA, vitamin D(3), and vitamin D(2) were from Sigma. 1,25-D(3) was kindly provided by Dr. M. Uskokovic (Hoffmann-LaRoche). Recombinant PKC- (rat) was from Upstate Biotechnology, Inc. ATP was from Boehringer Mannheim, and [-P]ATP was from DuPont NEN. All other chemicals were of analytical grade and were obtained from Fisher.

PKC Expression and Purification

PKC-I (calcium-dependent isoform mixture) was purified from rat brain as previously described (Slater et al., 1994a). PKC-alpha (bovine) was prepared using the baculovirus-insect cell expression system as previously described (Burns et al., 1990; Stabel et al., 1991). PKC- (rat) was prepared by subcloning the cDNA, obtained from a rat brain cDNA library, into a baculovirus expression vector. (^2)The infection of Sf9 insect cells and purification of the PKC followed previously described procedures (Burns et al., 1990; Stabel et al., 1991).

PKC Activity

Activity was determined, based on a previously described method (Kitano et al., 1986), by measuring the incorporation of P from [-P]ATP (DuPont NEN) into a peptide substrate, exactly as previously detailed (Slater et al., 1994a). For PKC-I, -alpha, and - preparations, a peptide corresponding to the phosphorylation site of MBP (50 µM) was used. For PKC- activity (calcium-independent), the assay mixture contained 0.1 mM EGTA in place of calcium and an ``-peptide'' in place of MBP, based on the pseudosubstrate sequence of PKC--peptide (Ohno et al., 1988; House and Kemp, 1987), in which the single alanine residue was replaced by a serine (Saido et al., 1992). In experiments where calcium concentrations were varied, calcium-EGTA buffers were used (Fabiato and Fabiato, 1979). The conditions of the experiment were adjusted so that less than 10% of the available ATP was hydrolyzed in the assay. Peptide substrates were present at saturating concentrations, and the linearity of the assay was previously confirmed (Slater et al., 1994a). A ``basal'' activity, i.e. unstimulated calcium-dependent activity, was determined for PKC-I, -alpha, and -; for PKC-, there was no calcium-dependent basal activity. For PKC-, activities were calculated after subtraction of phospholipid vesicle-dependent activity. PKC activities, being obtained using different enzyme preparations, accordingly varied slightly between different experiments.

Preparation of Vesicles

Chloroform solutions of the required lipids (750 µM) were mixed with 1,25-D(3), vitamin D(3), or vitamin D(2) (in ethanol), as appropriate, the solvents were removed under a stream of nitrogen, and the mixture was co-dispersed by vortexing in 50 mM Tris/HCl buffer, pH 7.4, to form multilamellar lipid vesicles. Large unilamellar vesicles (LUV) were made by the extrusion technique, using an Avestin Liposofast Extruder (MM Developments, Ottawa, Canada) as previously described (MacDonald et al., 1991). Vesicle preparations were kept in the dark, under nitrogen, and used within 1 h. When DAG was added, the concentration of the phosphatidylcholine was reduced by the same mole percentage accordingly. Vesicles of a similar composition have been previously shown to be stable under the PKC assay conditions used here (Boni and Rando, 1985).


RESULTS

The hypothesis that PKC might be directly activated by 1,25-D(3), and therefore constitute a membrane-associated receptor for the hormone, was first tested by comparing its effects on PKC activity with that of DAG and TPA. Using an in vitro assay system, in which phosphatidylserine, calcium, and substrate concentrations corresponded to those yielding maximal stimulation, calcium-dependent PKC-I (rat brain, alpha, beta, isoform mixture) was activated by 1,25-D(3) in a dose-dependent manner, as shown in Fig. 1. The concentration required for half-maximal activation (EC) was 16 ± 1 nM, close to that for TPA (calculated from the data of Fig. 1and Fig. 3, respectively), indicating that the compound is a potent activator of PKC. Fig. 2shows that PKC activation by the metabolic precursor, vitamin D(3), which lacks the 1- and 25-hydroxyl moieties, was negligible compared with an equimolar concentration of 1,25-D(3) (150 nM), as was activation by the same concentration of vitamin D(2), which also differs in the steroid side chain structure. Therefore, 1- and/or 25-hydroxylation appear to be required for PKC recognition.


Figure 1: Dose response curve for the activation of PKC by 1,25-D(3). 1,25-D(3) was incorporated into BPS/POPC LUV (1:4, molar) at the required concentration, and PKC-I activity was determined as described under ``Experimental Procedures.'' Data are the average of triplicate determinations (±S.D.).




Figure 3: The effect of a maximally activating concentration of 1,25-D(3) on the dose response curve for PKC activation by TPA. 1,25-D(3) was incorporated into vesicles consisting of BPS/POPC LUV (1:4, molar) at a concentration of 150 nM, and PKC-I activity was determined as described under ``Experimental Procedures.'' Filled and hollowcircles, with and without 1,25-D(3), respectively. Data are the average of triplicate determinations (±S.D.).




Figure 2: Effect of vitamin D(3)-related metabolites on the activity of PKC. Activity was measured in the presence of equimolar concentrations (150 nM) of 1,25-D(3), vitamin D(3), vitamin D(2), or TPA at a concentration of 0.5 µM, each incorporated into BPS/POPC LUV (1:4, molar), and PKC-I activity was determined as described under ``Experimental Procedures.'' Data are the average of triplicate determinations (±S.D.).



The possibility that the effect of 1,25-D(3) on PKC activity was mediated by an alteration in membrane bilayer physical properties, such as membrane fluidity or lipid order, was discounted due to the negligible effect that 1,25-D(3) had on the anisotropy of 1,6-diphenyl-1,3,5-hexatriene, used as a measure of membrane fluidity (Slater et al., 1994a), even at the high level of 1 µM (results not shown).

Activation by 1,25-D in the Presence of TPA or DAG

Fig. 3shows the effect of a maximally stimulating concentration of 1,25-D(3) (150 nM) on the dose response curve for PKC-I activation by TPA. The results show an increase in the level of stimulation independent of the TPA concentration, indicating a negligible level of allosteric interaction between 1,25-D(3) and TPA activation. Fig. 4A shows that an increased level of PKC-I activation could also be observed in the presence of DAG together with 1,25-D(3) over that obtained with either activator alone, a result also obtained for PKC-alpha, -, and - (Fig. 4, B-D). In a previous study, evidence was provided supporting the hypothesis that there are two discrete activator binding sites on PKC with differing affinities for diacylglycerols and phorbol esters (Slater et al., 1994b). This conclusion was drawn from the observation that the level of activity of PKC achieved by a combination of DAG together with TPA, both being at maximally stimulating concentrations of each compound, was greater than that achievable by either activator alone. The increased activation shown here with 1,25-D(3) paired with DAG or TPA similarly suggests a mechanism involving a two-site mechanism of interaction.


Figure 4: Activation of PKC-I, -alpha, -, and - preparation by 1,25-D(3) in combination with DAG or TPA. Activities of PKC-I, -alpha, and -, stimulated by TPA and DAG, were measured in the presence (filledbars) and absence (hollowbars) of 1,25-D(3). A, PKC-I; B, PKC-alpha; C, PKC-; D, PKC-. Data are the average of triplicate determinations (±S.D.). Other details are as described under ``Experimental Procedures.''



Effect of PE

The activity of PKC depends on an optimum interaction of the regulatory domain with the membrane lipids. Apart from phosphatidylserine, other lipids also influence activity, especially PE (Slater et al., 1994a; Kaibuchi et al., 1981; Bazzi et al., 1992; Orr and Newton, 1992). Fig. 5shows that the presence of PE attenuated the 1,25-D(3)-mediated activation of PKC-I, opposite to the potentiating effect PE has on DAG or TPA-induced activation, indicating that DAG and TPA-activated PKC forms differ from the 1,25-D(3)-activated form of the enzyme.


Figure 5: Effect of PE on PKC activated by 1,25-D(3). Activation by 1,25-D(3) (150 nM), TPA (0.5 µM), and DAG (4 mol % of the total phospholipid concentration) was measured with (filledbars) and without (hollowbars) 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE). The vesicle compositions were BPS/POPC LUV (1:4, molar) or BPS/POPC/POPE LUV (1:2:2, molar), containing 150 nM 1,25-D(3) where added. Data are the average of triplicate determinations (±S.D.). Other details are as described under ``Experimental Procedures.''



Calcium Dependence of 1,25-D(3)Activation

The interdependence between DAG and calcium in the activation of calcium-dependent PKC isozymes is well documented, the presence of DAG leading to a decrease in the calcium requirement for maximal activation (Nelsestuen and Bazzi, 1991; Kishimoto et al., 1980; Mosior and Epand, 1993; Hannun et al., 1986; Wolf et al., 1985; Hannun and Bell, 1990) and an increase in PKC translocation to the membrane. The effect of 1,25-D(3) on the calcium-induced PKC-I activation, shown in Fig. 6, is similar to that of DAG, both activators reducing the concentration of calcium required for half-maximal activation by 2-3 orders of magnitude. This indicates that activation by 1,25-D(3) and DAG have a similar requirement for calcium. The Ca-independent isoform PKC- was also stimulated by 1,25-D(3) (see Fig. 4D). This shows that the Ca binding C2 domain is not essential for the 1,25-D(3) action on PKC.


Figure 6: Effects of 1,25-D(3) and DAG on the calcium requirement for PKC activation. PKC-I activity was determined as a function of calcium concentration using calcium-EGTA buffers. The 1,25-D(3) concentration was 150 nM, and DAG was 4 mol % of the total phospholipid concentration. Hollowcircles, basal; filledcircles, 1,25-D(3); filledtriangles, DAG. Data are the average of triplicate determinations (±S.D.). Other details are described under ``Experimental Procedures.''




DISCUSSION

The principal result of this study is the finding that PKC is potently and directly activated by 1,25-D(3). This suggests that PKC serves as a membrane-bound receptor for the hormone, providing a rapid mechanism for transducing a 1,25-D(3)-initiated signal, additional to the well known activation of the enzyme initiated by hormone receptor-activated phospholipase C-induced phosphoinositide breakdown. The assay system used contained only PKC, phospholipid vesicles, substrate, and necessary cofactors so that a direct interaction of 1,25-D(3) involving some specific site on the enzyme itself is indicated. This is supported by the finding that the metabolic precursor vitamin D(3) and also vitamin D(2), which lack the 1- and 25-hydroxyl moieties of 1,25-D(3), were without significant effect on the enzyme activity, indicating a degree of structural specificity.

The finding that the level of activation achieved by 1,25-D(3), in combination with either a maximally stimulating concentration of TPA or DAG, was greater than that achievable by any activator alone suggests 1,25-D(3) is not simply competing with the other activators at a single activator binding site. A similar increased activity was also obtained in the presence of DAG added together with TPA (Slater et al., 1994b). A trivial explanation that the individual isoforms comprising the PKC-I preparation (each had differing affinities for each activator) is ruled out since the effect was demonstrable with single PKC-alpha, -, and - isozymes.

Previous studies indicate that PKC has two activator binding sites, one on each of the two C1 domain-zinc finger regions, which act as high and low affinity phorbol ester binding sites (Burns and Bell, 1991; Kazanietz et al., 1992). The nuclear 1,25-D(3) receptor also contains the zinc finger motif; therefore, it is possible that 1,25-D(3) binds at the region of the two C1-zinc finger domains contained within PKC in a similar manner. The results of the present study are inconsistent with 1,25-D(3), TPA, and DAG activators binding with equal affinities at each site, since equal competition by paired activators for binding and activation at each site would not have led to a greater activity than that obtainable with a single activator. Thus, the activator pairs, 1,25-D(3) and TPA or 1,25-D(3) and DAG, most likely bind with reversed affinities to the two sites, as previously proposed for TPA and DAG (Slater et al., 1994b). Thus, for TPA and DAG added together, for example, TPA would bind and/or activate more strongly to the so-called high affinity phorbol ester site while DAG would bind more strongly and/or activate more strongly at the second low affinity phorbol ester site.

An alternative explanation is that occupation of only one of the two activator binding sites is responsible for activation, while the other ``effector'' site acts to promote or amplify activation. This is consistent with the observation that addition of a second activator increases the level of activation beyond the overall level of stimulation achievable by the first activator alone. With this model, a single activator alone would promote its own stimulation by interacting at both sites, or it could promote the effect of a second activator, provided the second activator bound more strongly to the effector site. 1,25-D(3) both activates PKC and augments the level of TPA and DAG stimulation, suggesting that 1,25-D(3) can also act both as an effector and promoter. This model implies an order of binding affinities for activator pairs opposite for the two sites. While binding studies should answer this question, this is not trivial. Usually in the study of the binding of phorbol esters, the short chain phorbol-12,13-dibutyrate is used since it has low nonspecific binding to the membrane. However, this would not be adequate to test the above model since phorbol-12,13-dibutyrate has a very different activating potency from TPA. Further, TPA and 1,25-D(3) both bind nonspecifically to the membrane so that separation of bound from free activator is experimentally difficult. The idea of a promoter site raises the intriguing and testable hypothesis that there may be a class of compounds that act solely as promoters while alone being unable to activate the enzyme.

Evidence supporting the idea that the 1,25-D(3)-activated PKC conformation differs from the TPA or DAG-activated forms, comes from the opposite effects of PE on 1,25-D(3) activation. A number of studies suggest that PE promotes an optimal PKC-lipid bilayer interaction, thereby amplifying the level of activation (Slater et al., 1994a; Kaibuchi et al., 1981; Bazzi et al., 1992; Orr and Newton, 1992). However, the effect is dependent on the experimental conditions, and we have shown PE can have an apparent opposite inhibitory effect (Slater et al., 1994a). This was explained on the basis of the level of PKC activity being a biphasic function of the PE level or of the physical effect of PE, which is to induce a stress at the head group region (Slater et al., 1994a). This is caused by small head group compared to acyl chain volume of PE leading to a molecular cone shape, by contrast to the cylindrical shape of phosphatidylcholine. Fitting cone-shaped PE into an essentially planar bilayer surface induces surface stress since optimal packing of PE molecules induces a concave surface (Israelachvili et al., 1980; Kirk et al., 1984; Gruner, 1985; Hui and Sen, 1989; Seddon, 1990). The other half of the bilayer, tending to curve in the opposite direction, and other lipids such as phosphatidylcholine etc., frustrates the expression of this curvature stress and maintains the bilayer form, although the system retains an elastic curvature stress energy. PKC insertion into the bilayer, together with the known conformational change (Bazzi and Nelsestuen, 1988; Brumfeld and Lester, 1990; Snoek et al., 1988; Bosca and Moran, 1993; Slater et al., 1994a), is energetically favorable since it acts to reduce the curvature stress. Thus, there is a level of curvature stress that leads to adoption of an optimal conformation of PKC yielding maximal activity (Slater et al., 1994a). The finding that PE addition leads to amplified activity when DAG or TPA stimulated but reduced activity with 1,25-D(3) suggests that there are distinct activator-dependent PKC conformations, with different levels of curvature stress for optimal interaction/activity.

Although 1,25-D(3) shares with DAG and TPA an ability to translocate PKC to the membrane (for example, see Morelli et al.(1993), Simboli-Campbell et al.(1992, 1994), Wali et al.(1990)) and shows a calcium-dependent activation, as shown here (for calcium-dependent isoforms), some differences remain. For instance, 1,25-D(3) promotes cell differentiation and is anti-proliferative, while phorbol esters promote proliferation (Blumberg, 1988). Interestingly, as with 1,25-D(3), the potential anti-tumor agent bryostatin also activates PKC while promoting differentiation rather than proliferation (Wender et al., 1988; Kraft et al., 1986), and we have recently found that, as with 1,25-D(3), bryostatin in combination with TPA also produces a greater level of activity than that achievable with either activator alone. (^3)The possibility that distinct active PKC conformers induced by TPA and 1,25-D(3) could contribute to the different effects these compounds have on differentiation and proliferation is supported by the results of the present study and would be a worthwhile area for further investigation.

The finding that 1,25-D(3) both induces differentiation and decreases proliferation, in a variety of cell lines, has led to much interest in its potential therapeutic properties. However, it is limited by accompanying deleterious effects on calcium metabolism at the required doses. Thus, much effort has been directed toward separating the structural specificities required for effects on calcium metabolism from the anti-proliferative and differentiation effects of the hormone (Studzinski et al., 1993), mostly involving manipulation of the sterol side chain (Norman et al., 1993). The finding that PKC activation by the metabolic precursor vitamin D(3) was negligible, as was activation by the same concentration of vitamin D(2), indicates that 1- and/or 25-hydroxylation are required for PKC recognition. Therefore, although further studies are required to fully elucidate the structural specificity of PKC activation by 1,25-D(3), comparison with the structures of other known PKC activators and inhibitors may allow a more rational design of vitamin D analogs, optimized for inducing PKC-mediated differentiation and anti-proliferative effects rather than calcium homeostasis.

In summary, we present evidence that 1,25-D(3) directly activates PKC at physiological concentrations. Thus, PKC acts as a membrane-bound receptor for 1,25-D(3) and as such may be responsible for many of the non-genomic cellular responses to the hormone. Recently, a membrane-associated protein was isolated that also binds 1,25-D(3) (Nemere et al., 1994). Although this protein itself could be PKC, it appears unlikely, and it may be that there are several membrane proteins that can act as 1,25-D(3) receptors. The present finding that PKC is directly activated by 1,25-D(3) reinforces the importance of this hormone in signal transduction events.


FOOTNOTES

*
This work was supported by U. S. Public Health Service Grants AA08022, AA07215, AA07186, and AA07465. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology and Cell Biology, Rm. 271 JAH, Thomas Jefferson University, Philadelphia, PA 19107. Tel.: 215-955-5019; stubbsc{at}jeflin.tju.edu.

(^1)
The abbreviations used are: 1,25-D(3), 1alpha,25-dihydroxyvitamin D(3); PKC, protein kinase C; LUV, large unilamellar vesicles; TPA, 4beta-12-O-tetradecanoylphorbol-13-acetate; DAG, diacylglycerol; PE, phosphatidylethanolamine; BPS, brain phosphatidylserine; POPC, palmitoyl-oleoylphosphatidylcholine.

(^2)
M. D. Yeager, F. J. Taddeo, S. J. Slater, and C. D. Stubbs, manuscript in preparation.

(^3)
S. J. Slater, M. B. Kelly, and C. D. Stubbs, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to S. McCoy for technical assistance, Drs. K. Jansen and J. Benovic for helpful discussions, Dr. R. M. Bell for providing a PKC-alpha baculovirus preparation, and Dr. M. Uskokovic for providing 1,25-D(3).


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