[CANCER RESEARCH
45,1958-1963,
May 1985]
Modulation of Protein Kinase C Activity and [3H]Phorbol 12,13-Dibutyrate
Binding by Various Tumor Promoters in Mouse Brain Cytosol
Karen L. Leach1 and Peter M. Blumberg
Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20205
Subcellular localization studies have demonstrated [3H]PDBu
ABSTRACT
Using protein kinase C partially purified from mouse brain
cytosol, we examined the effect of a number of phorbol ester
and nonphorbol tumor promoters on protein kinase C enzymatic
activity and [3H]phorbol 12,13-dibutyrate binding. Mezerein and
phorbol 12-retinoate 13-acetate, second stage tumor promoters,
as well as the weak tumor promoter 4-O-methylphorbol 12myristate 13-acetate stimulated kinase activity to the same ex
tent as did the complete tumor promoter phorbol 12-myristate
13-acetate. In contrast, the nonphorbol ester tumor promoters
anthralin, cantharidin, benzoyl peroxide, and 7-bromomethylbenz(a)anthracene did not affect kinase activity. The unsaturated
fatty acids palmitoleic, oleic, linoleic, linolenic, and arachidonic
acids, some of which have been reported to be weak tumor
promoters, stimulated protein kinase C activity in the presence
of phospholipids, as well as causing some activation in the
absence of phospholipids. The saturated fatty acids butyric,
lauric, myristic, and palmitic acids had relatively little effect. The
fatty acids showed generally similar structure-activity relation
ships for inhibition of [20-3H]phorbol 12,13-dibutyrate binding as
for stimulation of kinase activity. The unsaturated fatty acids
typically decreased binding levels for the reconstituted aporeceptor, while the saturated fatty acids did not. The nature of this
inhibition was explored in the case of arachidonic acid. Scatchard
analysis demonstrated decreases in both the maximum number
of binding sites as well as the apparent binding affinity, indicative
of a complex mechanism. As expected for a lipophilic ligand, the
effect of the arachidonic acid was reduced in the presence of
elevated levels of phospholipid. Our results suggest that fatty
acids are capable of modulating the phorbol 12,13-dibutyrate
receptorprotein
kinase C.
INTRODUCTION
The phorbol esters are the most potent class of tumor pro
moters for mouse skin (11, 12). In addition, these compounds
induce a wide variety of responses in many biological systems.
This laboratory and others (7, 9, 15, 37, 42, 44) have used
[3H]PDBu2 to demonstrate specific high affinity receptors for
phorbol esters in many different intact cells and tissue prepara
tions. The structure-activity requirements for binding are con
sistent with the receptors mediating a number of the biological
responses to the phorbol esters, including their tumor-promoting
activity.
1Present address: The Upjohn Company, Department of Cell Biology, Kalama-
binding activity in both the paniculate and cytoplasmic fractions
(2, 27, 38). The distribution is dependent upon the conditions of
cell lysis; lysis in the presence of chelators increases the [3H]
PDBu binding activity recovered in the 100,000 x g supernatant
fraction (17, 33). The cytoplasmic receptor requires the addition
of phospholipids to reconstitute binding activity, and this receptor
copurifies with PKC (2, 27, 38).
PKC requires calcium and phospholipids for enzymatic activity
(see Ref. 45 for review). In vitro, diacylglycerols stimulate PKC
activity by shifting the calcium concentration dependency curve
to lower calcium concentrations. Castagna ef al. (3) demon
strated that PMA stimulates PKC activity in a manner similar to
diacylglycerols, and Sharkey ef al. (41) have shown that diacyl
glycerols competitively inhibit phorbol ester binding. Further, the
structure activity requirements of phorbol diesters for biological
activity in mouse skin are similar to those for PKC activation (3).
In addition to the phorbol esters, a number of non-phorbol
ester compounds including anthralin, benzoyl peroxide, and fatty
acids act as complete tumor promoters in mouse skin (see Ref.
5 for review). These structurally unrelated tumor promoters,
however, are typically 10,000 to 500,000 times less potent than
PMA (6). Previous studies (6) have demonstrated that the non
phorbol tumor promoters do not in general induce similar biolog
ical effects to the phorbol esters in chick embryo fibroblasts and
do not inhibit [3H]PDBu binding.
In the present study, we have examined the effect of a number
of tumor-promoting agents on PKC activity.3 The mode of action
of the various non-phorbol ester tumor promoters is unknown,
and modulation of PKC activity represents one mechanism by
which some of these compounds may promote tumor growth.
For compounds which this laboratory had shown previously not
to inhibit phorbol ester binding to the membrane receptor, we
wished to determine whether they might activate PKC directly
through interaction at a different site. In addition, in the case of
the highly lipophilic fatty acids, their activity to inhibit PDBu
binding had not been examined previously because of potential
problems of enhanced nonspecific binding. Using the partially
purified reconstituted aporeceptor, this technical problem can
now be minimized.
A third issue investigated by these experiments was the
concentration dependency relationship of PKC activation by
PMA. This topic was of interest because of several literature
reports demonstrating bell-shaped dose-response curves for
biological effects of PMA. These observations include the induc
tion of sister chromatid exchange in mouse 10T1/2cells (32), the
zoo. Ml 49001. To whom requests for reprints should be addressed.
2 The abbreviations used are: [3H]PDBu, [20-3H]phorbol 12,13-dibutyrate; PKC,
calcium phospholipid-dependent protein kinase C; PMA, phorbol 12-myristate 13acetate; EGTA, ethyleneglycol bis(/3-aminoethyl ether)-/V,A/,W',A/'-tetraacetic acid;
enhancement of colony formation in transformed human leuko
cytes (49), and the reduction in fibroblast cloning efficiency (25).
We wished to determine whether a biphasic concentration de-
PS, phosphatidylserine; PC, phosphatidylcholine; PRA, phorbol 12-retinoate 13acetate; DPS, 12-deoxyphorbol 13-isobutyrate; BSA, bovine serum albumin.
Received 8/6/84; revised 12/6/84; accepted 1/18/85.
of the American Association for Cancer Research (26).
CANCER RESEARCH
3 A preliminary account of these findings was presented at the annual meeting
VOL. 45 MAY 1985
1958
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PROTEIN KINASE C AND PHORBOL
pendency curve of PMA activation of PKC could account for
these observations.
MATERIALS AND METHODS
Sources of materials were as follows: 13HjPDBu (specific activity, 12.2
to 13.4 Ci/mmol) and fr-^PJATP (specific activity, 1000 Ci/mmol), New
England Nuclear (Boston, MA); nonradioactive phorbol esters, Chemicals
for Cancer Research (Eden Prairie, MN); PC, Supelco (Bellefonte, PA);
DEAE-cellulose (DE52) and phosphocellulose paper, Whatman (Clifton,
NJ); anthralin, Ruger Chemical Co. (Irvington, NJ); benzoyl peroxide,
Chemical Dynamics Corp. (South Plainfield, NJ); and PRÄ, LC Services
Corp. (Wobum, MA). DPB was prepared by Dr. Joseph Dunn (8). 7Bromomethylbenz(a)anthracene
was a gift from Dr. Anthony Dipple,
Frederick Cancer Research Facility, Frederick, MD. All other materials
were from Sigma Chemical Co. (St. Louis, MO).
Tissue Preparation. Whole brains from 6- to 8-week-old female CD1 mice (Charles River Breeding Laboratories) were homogenized with a
Potter-Elvehjem homogenizer at 4 °Cin 1 volume of 20 HIM Tris-CI (pH
7.4) which contained 2 rtiM EDTA, 10 mw EGTA, and 0.25 M sucrose.
The homogenate was centrifuged at 100,000 x g for 60 min at 4 °C.
Column Chromatography. The 100,000 x g supernatant was applied
to a DE52 column (2.5 x 15 cm) equilibrated with 20 DIM Tris-CI (pH 7.4)
containing 2 HIM EDTA, 5 mM EGTA, and 5 rriM dithtothreitol (Buffer B),
and the column was washed with 450 ml Buffer B. The enzyme was
eluted from the column with a 300-ml linear concentration gradient of
NaCI (0 to 0.3 M) in Buffer B, and fractions of 10 ml were collected. Each
fraction was assayed for protein kinase C activity in the presence of 1
mM Caci? and PS (160 fig/ml). Peak fractions were pooled and stored
at -20 °Cin the presence of 50% glycerol.
For subsequent assays, an aliquot of the DE52-purified preparation
was desalted by Chromatography on 2 PO-10 columns immediately
before use in order to remove chelators. The enzyme preparation (2.5
ml) was applied to a PD-10 column equilibrated with 20 mw Tris-CI (pH
7.4) at 4 °C. The column was washed with 0.5 ml Tris-CI, and the
enzyme was eluted with 2.5 ml Tris-CI. The 2.5-ml-column eluate was
applied to a second PD-10 column equilibrated with Tris-CI and eluted
with 3.0 ml Tris-CI.
Binding Assay. [3H]PDBu binding was determined in a 250-fil incu
bation mixture containing [3H]PDBu (3 to 100 nw), 50 mw Tris-CI (pH
7.4), 1 mM CaCI2, 7.5 mM magnesium acetate, bovine 7-globulin at 4
mg/ml, PS:PC (1:2) at 60 or 600 fig/ml. 2 to 10 fig DE52-purified enzyme,
and other ligands as specified. Fatty acids were dissolved in methanol
and diluted with 50 mM Tris-CI (pH 7.4) prior to addition to the assay.
Dilutions of [3H]PDBu and PDBu were made in 50 mM Tris-CI containing
bovine -j-globulin at a concentration of 10 mg/ml. Aliquots of PS and PC
ESTER BINDING
ing phospholipids, PS:PC mixtures (1:2, w/w) were added at a final
concentration of 60 fig/ml. Phospholipids were prepared by sonication
according to the procedure described above. Fatty acids were dissolved
in methanol and diluted with 20 mM Tris-CI (pH 7.4) prior to addition to
the assay. The dilutions of phorbol esters were made in 20 mM Tris-CI
(pH 7.4) containing BSA at a concentration of 1 mg/ml. Anthralin and
cantharidin were dissolved in dimethyl sulfoxide and diluted with 20 mM
Tris-CI (pH 7.4) prior to addition to the assay. Benzoyl peroxide (dissolved
in the acetone, then diluted in 20 mM Tris-CI) was preincubated with the
phospholipid mixture for 30 min at 37 °Cbefore addition to the kinase
assay. 7-Bromomethylbenz(a)anthracene
was dissolved in dry benzene
and mixed with aliquots of PS and PC (in chloroform). The solvent was
evaporated under nitrogen gas, and the mixture was sonicated as
described above. Incubations were carried out in 1.5-ml tubes for 5 min
at 30 °C.Immediately after incubation, the tubes were placed on ice,
aliquots (25 fil) were spotted onto 2- x 2-cm squares of phosphocellulose
paper, and the paper was washed 3 times in water, rinsed in acetone
and then in petroleum ether, and dried (48). Radioactivity was assayed
in 4 ml of Aquasol (New England Nuclear). Enzymatic activity is ex
pressed as pmol Å“P incorporated per 5 min.
RESULTS
Mouse brain cytosol partially purified by DE52 Chromatography
was used to assay PKC activity. PMA increased PKC activity
with maximal stimulation at approximately 100 HM PMA (Chart
1). Although PMA stimulation of PKC activity has been reported
previously by several laboratories, to our knowledge, no group
has examined the effect of PMA at concentrations greater than
200 nu. Biphasic curves for biological effects or PMA have been
demonstrated in several literature reports (25, 32, 49). In order
to determine whether these effects could be explained by a
biphasic concentration dependency curve of PMA activation of
PKC, we examined much higher concentrations of PMA than
have been reported previously. We found that PKC activity did
not change further with increasing concentrations of up to 10 ¡M
PMA.
The weakly promoting phorbol ester, 4-O-methyl-PMA, stim
ulated PKC activity as did the second-stage promoters, mezerein
and PRA (Table 1). DPB is an inflammatory but relatively nonpromoting phorbol ester that binds to a subclass of the |3H|PDBu binding sites in mouse skin membrane preparations (8).
(in chloroform) were evaporated under nitrogen gas and sonicated in 50
mw Tris-CI for two 30-s bursts at 35 watts with a Cell Disruptor sonifier
(Heat Systems/Ultrasonics,
Plainview, NY) before addition to the incu
bation mixture.
Incubations were carried out in 1.5-ml tubes for 30 min at 37 °C.
Immediately after incubation, the tubes were placed on ice, and 187 n\
of 35% polyethylene glycol (w/v) were added to precipitate proteins. The
remainder of the binding assay was carried out as described previously
(27). All binding and kinase experiments were repeated at least 2 times.
Protein Kinase C Assay. Protein kinase C activity was assayed by
measuring the incorporation of Å“P into historie H1 from [-r-^PJATP. The
reaction mixture contained, in a total volume of 50 pi, 20 mM Tris-CI (pH
7.4), 7.5 HIM magnesium acetate, 25 MM [r-^PJATP (1 to 2 x 102 cpm/
pmol), 750 fig histone H1, 12.5 fig BSA, 0.5 mM EGTA, 0.6 mM CaCI2,
0.5 HIM dithtothreitol, 1 to 2 pg DE52-purified enzyme, and other com
pounds as specified in the chart legends. The presence of BSA in the
assay did not affect the fatty acid results. The Ca2+:EGTA buffer system
was used to control free calcium levels in the assay. Free calcium
concentrations were calculated as described (36). In the assays contain
CANCER RESEARCH
01
10.000
Chart 1. PMA stimulation of PKC activity. PMA in dimethyl sulfoxide was diluted
with 20 mM Tris-CI containing BSA (1 mg/ml) and added directly to the PKC assay
containing PS:PC. PKC activity was measured as described in 'Materials and
Methods." Points, mean of 4 determinations within a single experiment; bare, SE.
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PROTEIN
KINASE C AND PHORBOL
ESTER BINDING
Table 1
EHectof phorbol ester and nonphorbol tumor promoters on PKCactivity
The effect of each of the compounds on PKC activity was measured in the
presence of the PS:PC mixture, as described in "Materials and Methods." Control
refers to PKC activity in the presence of PS:PC plus 200 nw PDBu.
CompoundPMA(100nw)
activity (% of
control)100
±3a
102 ±1
Mezerein(3000 nM)
PRA (3000 nM)
95 ±6
96 ±2
DPB (3000 nM)
4-O-Methyl-PMA(3000 nM)
116± 4
1 ±5
Anthralin (10 >JM)
12± 5
Cantharidin(IO^M)
Benzoyl peroxide (100 jig/ml)
0.2 ±.9
7-Bromomethylbenz(a)anthracene(100 >»g/ml)
0.2 ±.9
230 ±6
Oleic acid (350 UM)PKC
8 Average ±rangeof duplicatedeterminationswithin a singleexperiment. Similar
results were obtained in 2 additional experiments.
This phorbol ester also increased PKC levels. All 4 compounds
increased enzyme levels to the same maximal degree as did 200
nM PDBu, although concentrations of up to 3000 nM were
required for maximal stimulation.
A number of non-phorbol ester tumor promoters were tested
for their ability to stimulate PKC activity in the presence of
phospholipids. Anthralin, cantharidin, benzoyl peroxide, and 7bromomethylbenz(a)anthracene all were unable to stimulate PKC
activity (Table 1). Fatty acids, such as oleic acid, have weak
tumor-promoting activity. When added to enzyme preparations
at 350 iiM, oleic acid markedly stimulated PKC activity. The level
of stimulation by oleic acid was over 2-fold that caused by 200
nM PDBu.
Various fatty acids differed in their ability to stimulate PKC
activity. The concentration dependency curves for representative
fatty acids, palmitic, palmitoleic, and arachidonic acids, are
shown in Chart 2A. The activation concentration dependency
curves for the other saturated and unsaturated fatty acids ex
amined (see Table 2) corresponded closely to those of palmitic
and palmitoleic acids, respectively, and thus have not been
included in Chart 2. The saturated fatty acids, such as palmitic
acid, had relatively little effect on PKC activity, either in the
presence or absence of phospholipids.
Table 2 is a summary of the effects of a number of fatty acids
on both PKC and [3H]PDBu binding activity. Since maximal
effects on PKC activity occurred at approximately 300 tiM (100
Mg/ml) for the fatty acids, as shown in Chart 2A, the effect of
this concentration was used in compiling Table 2. In the absence
of phospholipids, the saturated fatty acid butyric, like palmitic
acid, was ineffective at stimulating PKC activity (Table 2). Both
lauric and myristic acids increased PKC activity somewhat in the
presence of phospholipids to 57 and 42 pmol/5 min, respectively.
As with palmitoleic acid, the unsaturated fatty acids oleic, linotele,
linolenic, and arachidonic acids all increased PKC levels more
than did the saturated fatty acids, in both the presence and
absence of PS:PC mixture. Fatty acids increased PKC activity in
the presence of 200 nM PDBu as well; the unsaturated fatty
acids and lauric acid increased PKC activity to over twice the
level as did 200 nM PDBu alone, while myristic and palmitic acids
increased PKC activity between 40 and 70%.
Since the unsaturated fatty acids markedly stimulated PKC
activity, both in the presence of phospholipids alone and with
phospholipids plus PDBu, we asked whether these compounds
3000
FATTY ACID, MM
Chart 2. Effect of fatty acids on PKC and [3H]PDBubinding activity. A, effect
on PKC activity. PKC was assayed in presence of palmitic acid (O), palmitic acid
plus PS:PC (•),
palmitoleic acid (D), palmitoleic acid plus PS:PC (•),
or 200 nM
PDBu plus PS:PC(A). B, effect on [3H]PDBubinding activity. Binding activity was
measuredas described in 'Materials and Methods" in the presenceof palmitic acid
(O), palmitoleicacid (•),
or arachidonicacid (A). Points, average; bare, range.
Table 2
Effect of fatty acids on PKCand pHJPDBu binding activity
The effect of 100 ^g (approximately 300 I¡M)
of each of the fatty acids per ml
on PKC and |3H]PDBu binding activity was measured in both the presence and
absence of the PS:PC mixture, or in the presence of the PS:PC mixture plus 200
RMPDBu, as described in "Materials and Methods." Values are the average of
duplicate determinations within a single experiment. Similar results were obtained
in 2 additionalexperiments.
bound
min)CompoundBuffer PKC activity (pmol/5
(pmol/mg)-PS:PC1.61.712.86.26
aloneButyricLauricMyristicPalmiticPalmitoleicOleicUnoleicLinolenicArachidonicPDBu-P
might affect [3H]PDBu binding activity as well. The fatty acids
showed generally similar but not identical structure-activity rela
tionships for inhibiting [3H]PDBu binding in the presence of
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PROTEIN KINASE C AND PHORBOL
PS:PC as for stimulating kinase activity. Saturated fatty acids,
such as palmitic acid, had no effect on [3H]PDBu binding activity
(Chart 2B). Unsaturated fatty acids, however, inhibited binding
levels in a concentration-dependent manner. The K, for inhibition
by palmitoleic, linoleic, and arachidonic acids, determined from
the 50% inhibition dose, was approximately 50 /IM.
The summary of the fatty acid effects on binding activity is
shown in Table 2. The percentage of inhibition of binding by the
unsaturated fatty acids ranged from a low of 10% with oleic acid
to over 90% with linolenic acid. In the absence of phospholipids,
[3H]PDBu binding activity was less than 2 pmol/mg, but the
addition of several of the fatty acids, both saturated and unsat
urated, significantly increased binding levels.
In order to explore the mechanism of binding inhibition by the
fatty acids, arachidonic acid was chosen as a representative
unsaturated fatty acid, and Scatchard analysis was carried out
(Chart 3). Arachidonic acid decreased both the affinity of
[3H]PDBu as well as the maximal extent of binding at saturation.
ESTER BINDING
We reasoned that the highly lipophilic fatty acids will interact
with phospholipids, and thus, their effects may depend on not
only absolute concentrations but also their proportion relative to
the phospholipids. Therefore, we also carried out Scatchard
analysis in the presence of a higher concentration of PS:PC
(Chart 4). As predicted, we found that increasing the PS:PC
concentration 10-fold in the presence of 260 tit* arachidonic acid
(80 ng/m\) decreased the extent of inhibition. The total binding
sites increased to 194 ±8 pmol/mg (n = 2), which was similar
to control levels (210 ± 18, n = 2), and the binding affinity
increased somewhat as well, to 1.75 ±0.6 nw (n = 2), although
it still did not reach the control value (0.64 ±0.06 nw, n = 2).
DISCUSSION
In these studies, we used the phospholipid mixture PS:PC
(1:2) to reconstitute enzymatic activity. We chose these phos
Total binding sites decreased from the control value of 221 ±6 pholipids, rather than PS alone, in order to mimic more closely
the in vivo phospholipid environment (13, 47). This mixture
(SE) pmol/mg (n = 6) to 154 ±1 pmol/mg (n = 3) in the presence
of 260 fiM arachidonic acid (80 /¿g/ml)while the apparent Ka (Ka')
results in lower basal PKC activity, compared to PS alone (19),
shifted approximately 10-fold, from 0.58 ±0.07 nw to 5.22 ± thus enhancing the degree of stimulation by PDBu and 1,2diolein. Reconstitution with the PS:PC mixture in place of PS
0.33 nw.
had little effect on the binding affinity of [3H]PDBu for the
cytosolic receptor (data not shown).
Using the PS:PC system, we demonstrated that the concen
tration dependency curve for PMA stimulation of PKC activity
increased with increasing PMA concentrations, attaining a pla
teau level by 100 nw PMA. Further increases in PMA concentra
tions of up to 10 UM had no further effects. These findings
400 -
300
» 200
OD
u.
m
100
200
PHIPDBu BOUND, pmol/mg
Chart3.
Scatchard
analysis of specific |3H|PDBu binding as a function
100
of
200
[3H|PDBu BOUND, pmol/mg
arachidonic acid concentration. Specific binding was measured ¡nthe presence of
PS:PC (60 «g/ml)and arachidonic acid at 0 (•).50 (O), 130 (A), and 260 P) „M.
Chart4. Scatchard analysis of PS:PC at 600 eg/ml. Specific binding was
measured in the presence of PS:PC (600 ng/mi) with 0 IM arachidonic acid (•),
or
Points, average of triplicate determinations; lines were determined by linear regres
260 ¿IM
arachidonic acid (•).
sion analysis.
CANCER RESEARCH
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PROTEIN
KINASE C AND PHORBOL
suggest that the biphasic curves are not a result of inhibition of
PKC at high PMA concentrations. Instead, the biphasic response
curves may be the result of more than one receptor mediating
the effects of PMA or one receptor in various affinity states or
with different degrees of accessibility; both heterogeneity in
phorbol ester binding (4, 8,15) and modulation of binding affinity
and PKC activity by phospholipids (19, 24) have been demon
strated.
A number of nonphorbol esters have tumor-promoting activity
(40). The mode of action of these tumor promoters is unknown,
and thus, it was of interest to examine the effect of several of
these compounds on PKC activity. Neither anthralin nor cantharidin inhibited [3H]PDBu binding in chick fibroblasts (7), and neither
compound affected PKC activity in our in vitro assays. 7-Bromomethylbenz(a)anthracene is an effective, complete carcinogen
in mouse skin, and it acts as a tumor promoter after initiation
with 7,12-dimethylbenz(a)anthracene as well (39). The promoter
benzoyl peroxide, a free radical-generating compound, induces
several similar biological effects as the phorbol esters, such as
inhibition of metabolic cooperation (43). None of these nonphor
bol promoters activated PKC in the presence of phospholipids.
In addition, the compounds did not affect PDBu stimulation of
the enzyme (data not shown). On the basis of their inability to
inactivate PKC, our results indicate that these promoters need
not act directly on the PDBu receptorPKC. These agents may
affect PKC activity at another step in the pathway, such as
modulating phospholipase C activity and diacylglycerol formation
(18); alternatively, the may act through an entirely different
pathway.
Fatty acids represent another class of compounds with tumorpromoting activity. Undiluted oleic acid as well as oleic and lauric
acids dissolved in chloroform exhibited significant tumor-pro
moting properties when applied to mouse skin 6 times/week.
The saturated fatty acids, stearic and palmitic, did not induce
tumor growth (14). In addition to their tumor-promoting activity,
several of the fatty acids have been shown to induce other similar
biological effects as the phorbol esters. For example, lauric acid
stimulated deoxyglucose transport in chick embryo fibroblasts
(6), and butyric acid induced the expression of Epstein-Barr virus
early antigen in human lymphoblasts (21). In our experiments,
lauric acid affected PKC and [3H]PDBu binding activity more than
the other saturated fatty acids, consistent with its reported
biological effects. Oleic acid was consistently poor at inhibiting
[3H]PDBu binding activity, although it stimulated PKC activity.
Conversely, linoleic acid was a good inhibitor of binding activity
but a weak stimulator of PKC. The reason for these differences
is not clear, although these results were duplicated with several
different preparations of both oleic and linoleic acid.
We found that the unsaturated fatty acids both stimulated
PKC activity and inhibited [3H]PDBu activity. Since our first report
(26) of these findings and during the preparation of this paper,
McPhail ef al. (30) also have reported that fatty acids stimulate
PKC activity in neutrophils. In contrast to our results, however,
they found that high concentrations of fatty acids inhibit PKC
activity. Differences in experimental systems may explain the
concentration dependency curves; McPhail ef al. used detergent
extracts from neutrophils, while we used PKC partially purified
from mouse brain, in the absence of any detergents. Although
Kishimoto ef a/. (22) reported that fatty acids do not affect PKC
activity, they may have only examined low concentrations of the
ESTER BINDING
compounds. Our results demonstrate that concentrations
greater than 30 UM are required to stimulate PKC activity.
In the presence of phospholipids, the unsaturated fatty acids
increased PKC levels over 2-fold, compared to PDBu stimulation,
and, except for oleic acid, inhibited [3H]PDBu binding by 50 to
90%, whereas the saturated fatty acids did not. Relatively little
is known about the effects of free fatty acids upon membrane
structure. Fluorescence polarization and conformational binding
studies have shown that free fatty acids readily intercalate into
lipid vesicles, resulting in perturbation of the phospholipid struc
ture (1, 20, 23). C/5-unsaturated fatty acids disorder the lipids to
a much greater extent than do unsaturated fatty acids. As in our
studies, a number of biological activities of fatty acids distinguish
between saturated and unsaturated acids and may reflect the
ability of the fatty acid to perturb lipid structure.
We found from Scatchard analysis that arachidonic acid af
fected both the binding affinity and total number of binding sites,
consistent with the complex perturbation of the interactions
between [3H]PDBu, aporeceptor, and phospholipid. Addition of
higher concentrations of phospholipids reduced the inhibition,
suggesting that the fatty acid:phospholipid ratio is an important
factor in determining its activity. Previously, we had shown that
the activity of diacylglycerols was directly related to their mol
fraction in the phospholipids (41).
The fatty acid modulation of PKC and [3H]PDBu binding activity
occurred in the absence of phospholipids as well as in the
presence. Since both cytosolic activities are phospholipid de
pendent, these results may reflect the ability of the fatty acids to
interact with hydrophobic sites on PKC, allowing activation of
the kinase and binding of |3H|PDBu. We have argued previously
that diacylglycerols are endogenous phorbol ester analogues
(41). Although physical techniques may be required to determine
whether the appropriate fatty acids interact at the phorbol ester
binding site, our evidence strongly argues that this cannot be
their only action, (a) Maximal [3H]PDBu binding as well as binding
affinity is reduced, (b) The fatty acids give greater stimulation of
PKC activity than do the phorbol ester, and the combination of
the 2 ligands gives greater stimulation than either alone, (c) As
described above, certain fatty acids in the absence of phospho
lipids can partially reconstitute the aporeceptor to permit binding.
(d) Variation is found in the relative abilities of the fatty acids to
reconstitute binding and to stimulate PKC. None of these results
would have been expected for a true analogue.
A regulatory role of fatty acids in cellular metabolism has been
documented in detail. The concentrations of fatty acids used
here are in the range found to be active in these other studies.
In vitro, fatty acids have been shown to inhibit glycerol 3phosphate dehydrogenase in rat liver (29), adenylate cyclase in
rat adipose tissue (28), and glycerophosphate acyltransferase in
yeast (31). Unsaturated fatty acids stimulate phosphatidylinositol
phosphodiesterase in rat brain and liver (16, 46), Ca2+-ATPase
in erythrocyte membranes (34), and CTP:phosphocholine cytidylyltransferase in rat lung (10). In vitro, Pelech ef al. (35)
demonstrated
that fatty acids promote translocation
of
CTP:phosphocholine cytidylyltransferase from the cytosol to the
microsomal membrane, where it is then activated by the lipid
environment.
Our results demonstrate that, in vitro, fatty acids modulate
PKC and phorbol ester receptor binding activities; in vivo, these
compounds may also play a role in cellular regulation of PKC.
CANCER RESEARCH VOL. 45 MAY 1985
1962
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PROTEIN KINASE C AND PHORBOL
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26. Leach, K. L., and Blumberg, P. M. Modulation of mouse brain protein kinase
C and [3H]PDBu binding activity. Proc. Am. Assoc. Cancer Res., 25: 147,
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CANCER RESEARCH VOL. 45 MAY 1985
1963
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Modulation of Protein Kinase C Activity and [3H]Phorbol 12,
13-Dibutyrate Binding by Various Tumor Promoters in Mouse
Brain Cytosol
Karen L. Leach and Peter M. Blumberg
Cancer Res 1985;45:1958-1963.
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