Protein Kinases C Translocation Responses to Low Concentrations
of Arachidonic Acid*
Joseph T.
O'Flaherty
,
Brad A.
Chadwell,
Mary W.
Kearns§,
Susan
Sergeant§, and
Larry W.
Daniel
From the Departments of Internal Medicine and
§ Biochemistry, Wake Forest University School of Medicine,
Winston-Salem, North Carolina 27157
Received for publication, February 5, 2001, and in revised form, April 17, 2001
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ABSTRACT |
Arachidonic acid (AA) directly activates protein
kinases C (PKC) and may thereby serve as a regulatory signal during
cell stimulation. The effect, however, requires a
20
µM concentration of the fatty acid. We find
that human polymorphonuclear neutrophils (PMN) equilibrated with
a ligand for the diacylglycerol receptor on PKC,
[3H]phorbol dibutyrate (PDB), increased binding of
[3H]PDB within 15 s of exposure to
10-30
nM AA. Other unsaturated fatty acids, but not a saturated
fatty acid, likewise stimulated PDB binding. These responses, similar
to those caused by chemotactic factors, resulted from a rise in the
number of diacylglycerol receptors that were plasma membrane-associated
and therefore accessible to PDB. Unlike chemotactic factors, however,
AA was fully active on cells overloaded with Ca2+
chelators. The major metabolites of AA made by PMN, leukotriene B4 and 5-hydroxyicosatetraenoate, did not mimic AA, and an
AA antimetabolite did not block responses to AA. AA also induced PMN to
translocate cytosolic PKC
,
II, and
to membranes.
This response paralleled PDB binding with respect to dose requirements, time, Ca2+-independence, resistance to an AA
antimetabolite, and induction by another unsaturated fatty acid but not
by a saturated fatty acid. Finally, HEK 293 cells transfected with
vectors encoding PKC
I or PKC
fused to the
reporter enhanced green fluorescent protein (EGFP) were studied.
AA caused EGFP-PKC
translocation from cytosol to plasma membrane at
0.5 µM, and EGFP-PKC
translocation from cytosol to
nuclear and, to a lesser extent, plasma membrane at as little as 30 nM. We conclude that AA induces PKC translocations to
specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they
occur in PMN, do not require changes in cell Ca2+ or
oxygenation of the fatty acid. AA seems more suited for
signaling the movement than activation of PKC.
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INTRODUCTION |
Members of the AGC superfamily of kinases move about cells
phosphorylating key proteins on serine and threonine. The execution of
this movement, or translocation, from one cell compartment to another
is crucial to the function of these kinases. Typically, signal pathways
entrained by cell stimulation convert resident lipids to products that
help guide these kinases to the substrates and milieus appropriate for
their phosphorylating activity. One family of AGC kinases, the protein
kinases C (PKC),1 affects
virtually all aspects of cell physiology. The family was first studied
as a phosphorylating activity that developed during the rise in
cytosolic Ca2+ ([Ca2+]i) and
formation of diacylglycerol (DG) attending cell stimulation. High
[Ca2+]i, it was found, caused PKC to move from a
latent state in cytosol to plasma membranes. Upon attaching to
plasmalemmal phospholipids, particularly phosphatidylserine, the
translocated PKC engaged membranous DG and thereby became active. It is
now known that human PKC exist as four subfamilies of 13 isoforms (1-4). Conventional PKC
,
I,
II, and
are sensitive to Ca2+ and DG; novel PKC
,
,
,
and
are sensitive only to DG; and atypical PKC
and
are
sensitive to neither signal. A more distantly related group of µ,
, and PKD-2 isoforms binds DG but is Ca2+-insensitive
and may or may not translocate depending on cell type (5-7).
Many factors besides Ca2+, DG, and phospholipids influence
the disposition of PKC. Various organelle-associated modulator,
scaffold, or substrate proteins bind and thereby recompartmentalize and assist in activating PKC, often in an isoform-specific manner (1-4,
8-18). The phosphorylation of regulatory residues is prerequisite for
PKC activity but also may alter their location (1-4, 6, 15, 19, 20).
Finally, unsaturated fatty acids (UFAs) share with DG the
ability to activate PKC (21). Unlike DG, they target most PKC isoforms
and can operate in the absence of phospholipid or Ca2+
cofactors (reviewed in Ref. 22). Because UFAs likewise activate PKC in
whole cells (22-32), arachidonic acid (AA) has been proposed to
participate in signaling for activation of the enzymes. AA is the major
UFA released during cell stimulation and among UFAs has high efficacy
in activating PKC. It nonetheless achieves this activation only at
20-100 µM, levels unlikely to occur physiologically. On the other hand, UFAs also cause cells to translocate PKC. Observed mainly at UFA concentrations that activate PKC, this effect has been either ignored or viewed as consequential and secondary in importance to PKC activation (21-33). We show here that AA induces PKC
translocation in polymorphonuclear neutrophils (PMN) and HEK 293 cells
at concentrations 2-4 orders of magnitude below those reported to
activate the enzymes. Based on the criterion of potency, AA is
better suited as a translocating than activating signal for
PKC.
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EXPERIMENTAL PROCEDURES |
Reagents and Buffers--
The following were purchased:
[3H]phorbol dibutyrate ([3H]PDB) (19.1 Ci/mmol) and polyvinylidene difluoride membranes (PerkinElmer Life Sciences); PDB, phorbol 12-myristate 13-acetate,
dioctanoylglycerol, N-formyl-methionyl-leucyl-phenylalanine (FMLP),
nordihydroguaiaretic acid, sphinganine, 0.01%
poly-L-lysine plates, and fatty acid-free BSA (Sigma);
intracellular Ca2+ chelators (Molecular Probes);
fatty acids (NuChek Prep, Elysean, MN); PKC isoform standards and
rabbit antibodies to these isoforms (PanVera, Madison WI); horseradish
peroxidase-linked rabbit anti-IgG (Transduction Laboratories);
pPKC
-EGFP (CLONTECH); rabbit antibody to
enhanced green fluorescent protein (EGFP) (Santa Cruz Biotechnology); Supersignal chemiluminescence kits (Pierce); silicone oil (Versilube F50; General Electric Corp., Westview NY); DH5
-competent cells, LipofectAMINE kits, Maxi-prep DNA isolation kits (Promega), and LB medium (Life Technologies, Inc.); and HEK 293 cells (ATCC). Leukotriene (LT)B4 and
5-(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoate (5-HETE)
were prepared, and protease and phosphatase inhibitors were purchased
(33-35). pPKC
-EGFP was a gift from Prof. N. Saito (Biosignal
Research Center, Kobe University, Kobe, Japan). Human PMN were
suspended in a modified Hanks' balanced solution (33); HEK 293 cells
were grown in Dulbecco's modified essential medium (DMEM) with
streptomycin (100 µg/ml) and penicillin (100 units/ml), ± 10% fetal
calf serum. Where indicated, Hanks' buffer and DMEM were made 100 µM in ascorbic acid plus sufficient NaOH to maintain a
constant pH.
[3H]PDB Binding--
PMN (5 × 106) isolated from human donor blood (33) were incubated in
1 ml of Hanks' buffer at 37 °C for 15 min, equilibrated with 125 pM [3H]PDB for 5 min, challenged with 1 µl
of ethanol or a UFA dissolved in ethanol, and centrifuged through 400 µl of silicone oil. Cell pellets and supernatant fluids were counted
for radioactivity. Results are expressed as fmol of total recovered
radiolabel that are cell-associated. [3H]PDB binding
attains equilibrium within 1 min regardless of PDB concentration (125 pM-10 µM) or PMN calcium status (34, 35).
PKC Immunodetection--
PMN (2.5 × 108) in 5 ml of Hanks' buffer were incubated at 37 °C for 20 min; challenged
with 5 µl of ethanol or UFAs in ethanol; diluted in 40 ml of 4 °C
Hanks' buffer; and centrifuged (400 g; 5 min; 4 °C). Cells were
suspended in 5 ml of isotonic saline, incubated for 5 min at 4 °C
with 2 mM diisopropylfluorophosphate, and washed twice in
Hanks' buffer. Cells (2 × 108/ml) were suspended in
Hanks' buffer containing 2 mM EGTA, 1 mM PMSF,
1 µg/ml leupeptin, 10 µM benzamidine, 10 µM pepstatin, and 0.2 µg/ml aprotinin. Suspensions were
sonicated (4 °C; 5 strokes (3 s) of a Heat Systems sonicator,
setting of 2) and centrifuged (800 × g for 10 min at
4 °C) to remove unbroken cells, nuclei, and other debris.
Supernatant fluid from the centrifugation was layered onto sucrose
gradients and centrifuged (150,000 × g for 30 min at
4 °C) to obtain fractions enriched in cytosol, plasma membrane, and
granule markers (36, 37). Fractions were assayed for protein (Bio-Rad
protein assay; BSA as standard) and stored in 10% glycerol
(-70 °C). Samples of defined protein mass, PKC standards, and
prestained Mr markers were resolved on 7%
SDS-polyacrylamide gels and electrotransferred to polyvinylidene
difluoride membranes. The membranes were washed three times in TBS-T
(10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween, pH
7.5), blocked for 1 h with 5% Carnation nonfat dry milk in
TBS-T, and washed three times in TBS-T. After a 2-h incubation with
primary antibody (1/200 in 3% BSA/TBS-T, 0.02% sodium azide),
membranes were washed six times in 300 ml of TBS-T, incubated for
1 h with secondary antibody (1/2000 in 5% milk/TBS-T), and washed
three times in TBS-T. Blots were developed with enhanced
chemiluminescence kits as instructed by the manufacturer. Individual
bands were quantified with ImageQuant (Molecular Dynamics, Sunnyvale CA).
Ca2+ Studies--
PMN (1 × 107/ml)
were incubated (37 °C) for 1 h with fura-2 AM or Quin2 AM in
Hanks' buffer (1 µM EGTA, no Ca2+), washed
twice in this buffer, and incubated (37 °C) for 20 min in Hanks'
buffer ± CaCl2. Cells loaded with 1 µM
fura-2 or Quin2 and incubated with 0 or 1.4 mM
Ca2+ are termed Ca2+-depleted and
Ca2+-repleted, respectively; cells incubated with a 30 µM concentration of a probe and no Ca2+ are
termed Ca2+-chelated. To assay
[Ca2+]i, PMN loaded with fura-2 were incubated at
1 × 107/ml in Hanks' buffer ± 1.4 mM CaCl2 at 37 °C, excited alternately at
340 and 380 nm, and monitored at 510 nm with an Aminco-Bowman spectrofluorometer. To obtain [Ca2+]i, the ratio
of emission intensities at the exciting wavelengths were compared with
those of a buffer containing 1 µM fura-2 pentapotassium
salt and CaCl2 clamped at 0-800 nM free Ca2+ with EDTA (34). Results are given in nM
[Ca2+]i.
In Situ PKC Translocation--
Our two vectors encoded for human
PKC
I and PKC
fused to EGFP. pPKC
-EGFP was
transformed in DH5
-competent cells and grown on LB agar (kanamycin,
30 µg/ml). Colonies were picked and expanded in LB medium
(kanamycin, 30 µg/ml). After isolation with Maxi-prep DNA isolation
kits, DNA was digested with KpnI and BamI to
yield a fragment running with the appropriate size (2027 base pairs) on
1% agarose gels. pPKC
-EGFP was transformed in DH5
-competent cells and plated on LB agar (ampicillin, 50 µg/ml). Colonies were selected and expanded in LB medium (ampicillin, 50 µg/ml). On reaching an absorbance of 0.4 AU (400 nm), cultures
were treated with 170 µg/ml chloramphenicol and grown overnight.
Digestion of isolated DNA with HindIII gave appropriately
sized fragments of 1400 and 5600 base pairs on agarose gels. HEK-293
cells (2 × 105 cells on 35-mm
poly-L-lysine-coated dishes) were transfected with 2 µg
of a vector using the Life Technologies, Inc. LipofectAMINE kit and
protocol. Cells were grown for 10-12 days in DMEM, 10% fetal calf
serum with 700 µg/ml gentamycin. Viable cells were diluted to single
cells and expanded in DMEM, 10% fetal calf serum, 400 µg/ml
gentamycin. Final colonies, which expressed appropriate molecular
weight proteins that reacted with antibody to EGFP and either
PKC
I or PKC
on Western blots, were maintained in this medium and transferred to serum-free DMEM supplemented with insulin, transferrin, and selenium for 18 h. Cells were challenged while being monitored by fluorescent (excitation at 488 nm, emission at 510 nm) and Nomarsky optics with a Zeiss LSM-510 confocal laser scanning system.
Cell Toxicity--
As little as a 20 µM
concentration of a UFA caused PMN to leak preloaded fura-2 or Quin2,
leak cytosolic LDH, and take up trypan blue (assayed as in Ref. 34). At
30 µM, AA induced leakage of LDH and the two chromophores
but only several minutes after the peak in PKC translocation responses;
at
50 µM, it caused leakage by 2 min of PMN challenge.
5,8,11,14,17-Eicosapentaenoic acid had these respective
immediate and delayed effects at 20 and 30 µM;
8,11,14-eicosatrienoic acid and 11,14,17-eicosatrienoic acid at 30 and
50 µM; and 8,11-eicosadienoic acid at
100
µM and
100 µM. Eicosanoic acid lacked
these effects at 100 µM. We restricted most PMN analyses
to UFA concentrations
10-fold below the level in which each fatty
acid caused delayed cytolysis. HEK-293 cells did not leak LDH or
EGFP-PKC isoforms nor alter uptake of trypan blue in the presence of
100 µM AA. We did note that AA, at
1-10 µM, caused HEK 293 cells to round, shrink, and, mostly in
EGFP-PKC
-expressing transfectants, form blebs. Because resting cells
had blebs that were only faintly fluorescent and therefore hard to
capture in our reproductions, AA, by causing cytosolic EGFP-PKC
to
attach to plasmalemma, may have highlighted preexisting blebs more than caused new bleb formation. In any case, the data indicate that AA
produces morphology changes that are not due to overt alterations in
plasma membrane integrity.
 |
RESULTS |
PDB Binding--
[3H]PDB has been used to track PKC
in astrocytoma cells (38), macrophage tumor cells (29), platelets (39),
and PMN (34, 40). The label binds to DG receptors. In
consequence, its association with cells rises as these receptors move
from the cell interior to surface membrane (34, 38). The top
panels of Fig. 1 give the PDB
binding of PMN exposed to a 100 nM concentration of the leukocyte chemotactic factor (CF) FMLP. Cells were loaded with 1 µM Quin2 and incubated with 1 µM EGTA plus
0 (Ca2+-fixed) or 1.4 mM
(Ca2+-repleted) CaCl2 or, alternatively, loaded
with 30 µM Quin2 and incubated with 1 µM
EGTA and 0 µM Ca2+
(Ca2+-chelated). On stimulation, Ca2+-repleted
PMN rapidly increased binding of [3H]PDB.
Ca2+-fixed PMN had slower changes, and
Ca2+-chelated PMN had no such changes. Only
Ca2+-repleted PMN mounted Ca2+ transients (Fig.
1, bottom panels). The refractoriness of
Ca2+-chelated PMN was not due to the side effects of
Ca2+ deprivation or chelators; after 5 min of incubation
with 1.4 mM CaCl2, Ca2+-chelated
PMN responded fully to FMLP (data not shown). These results, which
agree with those found in PMN challenged by other CFs, imply PDB
binding responses to CFs consist of an early component associated with
[Ca2+]i rises and a late component that is
independent of these rises but still requires some minimal level of
cell Ca2+ (34, 35).

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Fig. 1.
PDB binding and Ca2+ transient
responses to FMLP. For PDB binding (top panels), PMN
were Ca2+-repleted, Ca2+-fixed, or
Ca2+-chelated with Quin2 (fura-2 gave similar results);
equilibrated with 125 pM [3H]PDB ± 5 µM PDB; exposed to 100 nM FMLP; and counted
for radioactivity. Results are fmol (mean ± S.E. for 5-7
experiments) of label specifically bound (bound by cells incubated with
[3H]PDB minus that bound by cells incubated with
[3H]PDB plus PDB) after correction for the specific
binding of PMN stimulated by the vehicle for FMLP, 62.5 µg of BSA.
For Ca2+ transients (bottom panels), PMN were
Ca2+-manipulated with fura-2 and challenged with 100 nM FMLP. Results are mean ± S.E.
[Ca2+]i (nM) for 4-6 experiments. By
itself, BSA did not stimulate PDB binding or
[Ca2+]i rises.
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Similar to CFs,
10 nM AA stimulated
Ca2+-repleted PMN to bind [3H]PDB (Fig.
2). Unlike CFs, however, AA was equally
active on Ca2+-fixed and Ca2+-chelated cells
(Fig. 3, top panels). AA
provoked [Ca2+]i rises only in
Ca2+-repleted PMN (Fig. 3, bottom panels),
although these rises were slow-paced and, in view of the response to AA
executed by Ca2+-fixed and Ca2+-chelated PMN,
contributed little to the effect of the fatty acid on PDB binding.
Neither LTB4 nor 5-HETE, the major metabolites of AA in
PMN, mediated the action of AA. 5-HETE (5 µM) did not stimulate PDB binding (data not shown), and LTB4, although
it elicited the response in Ca2+-repleted and
Ca2+-fixed PMN, differed from AA in having no effect on
Ca2+-chelated PMN (34). Moreover, PMN pretreated with
LTB4 had reduced or down-regulated PDB binding responses to
a second LTB4 challenge yet responded fully to AA (data not
shown) and an inhibitor of AA oxygenases, nordihydroguaiaretic acid,
failed to reduce responses to AA (Table
I). Finally, UFAs that are not substrates
for lipoxygenases or cyclooxygenases stimulated PDB binding. Their
potencies were inversely related to saturation level, with the
saturated fatty acid, arachidic acid, being devoid of activity
(Fig. 4).

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Fig. 2.
PDB binding responses to AA. PMN were
Ca2+-repleted with Quin2 (fura-2 gave similar results),
exposed to AA, and analyzed for fmol of [3H]PDB-specific
binding (mean ± S.E. for 5-7 experiments) as in Fig. 1 except
that data were corrected for the action of ethanol (1 µl/ml), which
by itself did not stimulate PDB binding or
[Ca2+]i rises.
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Fig. 3.
PDB binding and Ca2+ transient
responses to AA. PMN were Ca2+-manipulated with Quin2
(fura-2 gave similar results), exposed to 1 µM AA, and
assayed for fmol of [3H]PDB specifically bound (top
panels) or nM [Ca2+]i
(bottom panels) as in Fig. 1. Data are mean ± S.E. for
5-8 experiments.
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Table I
PDB binding responses in variably treated PMN
PMN were Ca2+-repleted with Quin2, equilibrated with 125 pM [3H]PDB for 4 min, pretreated with an agent
for 2 min, filtered, and counted for radioactivity. Alternatively, PMN
were treated with nordihydroguaiaretic acid, PDB, PMA,
dioctanoyl-glycerol8, or sphinganine for 30 min; equilibrated
with 125 pM [3H]PDB for 5 min; and stimulated with AA for 2 min before filtering and assaying PDB binding.
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Fig. 4.
PDB binding responses to fatty acids.
PMN were Ca2+-repleted with Quin2, exposed to an indicated
fatty acid for 2 min, and assayed for fmol of PDB specifically bound as
in Fig. 1. Data are mean ± S.E. for 3-8 experiments. Unsaturated
fatty acids had 2-5 cis double bounds at the indicated
carbon positions.
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PMN pretreated with a DG receptor agonist, either PDB, PMA, or
dioctanoylglycerol, bound PDB minimally and did not raise this binding
in response to UFAs (Table I). The same agents, when added 2 min after
UFAs, reversed the rise in PDB binding by
95% within 0.5 min (data
not shown). Sphinganine, which blocks PKC attachment to membranes, also
inhibited responses to UFAs (Table I). Thus, baseline as well as
stimulated PDB binding is reversible and sensitive to a drug disrupting
PKC-membrane interactions. We also observed that
Ca2+-repleated PMN treated with 100 µM
ascorbic acid for 10 min increased the binding of [3H]PDB
by 10.5 ± 2 fmol (mean ± S.E. for three experiments) 2 min after challenge with 1 µM AA. This value was 9.9 ± 0.8 fmol for cells not treated with the antioxidant. Finally,
UFAs increased the number of sites bound by PDB in whole cells but did
not alter the Kd for this binding or the number of
sites detected in sonicated PMN (Table
II). We conclude that AA and other UFAs induce PMN to raise the number of receptors available to PDB. In
achieving the effect, they do not require processing by oxygenases, [Ca2+]i rises, the generation of reactive oxygen
species, or, because dioctanoylglycerol blocked but never
increased PDB binding (Table I), the endogenous formation of DG.
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Table II
Number of receptors accessed by PDB in variably stimulated intact or
sonicated PMN
5 × 106 PMN were Ca2+-repleted,
Ca2+-fixed, or Ca2+-chelated with Quin2 and sonicated
and assayed for PDB receptors or, alternatively, equilibrated with 125 pM [3H]PDB for 4 min; stimulated for 15 s;
treated with 0.25, 1, 4, 8, 16, 256, 1024, or 10,000 nM PDB
for 1 min; filtered; and counted for radioactivity.
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PKC in PMN--
PMN express
,
I,
II,
, and
but not several other PKC isoforms (25,
29, 30, 43-49). We confirmed that PMN contain the former but lack
,
,
, or
isoforms (36, 37). PKC
,
I,
II, and
localized mostly to cytosol with small
amounts in plasmalemma and traces of PKC
and
in granule
fractions of resting PMN. The antibodies used here had high isoform
specificity except that PKC
I antibody cross-reacted with
PKC
II. Given these findings and the toxicity of UFAs
(see "Cell Toxicity" under "Experimental Procedures"), we
focused on PKC
,
II, and
in experiments on Ca2+-repleted and Ca2+-chelated PMN exposed to
1 µM AA. These restrictions still allow for testing the
effect of AA at low concentrations on the translocation of
Ca2+-sensitive and Ca2+-insensitive PKC
isoforms as a function of cell Ca2+.
Ca2+-repleted and Ca2+-chelated PMN were
challenged with AA and separated into membrane, granule, and cytosol
fractions. Fractions were resolved by SDS-polyacrylamide gel
electrophoresis, transferred to membranes, and probed for PKC. Fig.
5 gives representative Western blots;
Figs. 6 and
7 show response kinetics and dose
dependence, respectively, as judged by densitometric analyses of these
blots. AA stimulated transient (
8 min) falls in cytosol and rises in
membrane PKC
,
II, and
. Granule fraction PKC did
not change (data not shown). PKC
responses showed the least
percentage change and sensitivity to the fatty acid, translocating only
at
100 nM AA. Ca2+-chelated PMN exhibited
some reduction in the speed and extent of PKC
and
II,
but not PKC
, responses. In studies not shown, 11,14,17-eicosatrienoic (3 µM) induced PKC
,
II, and
translocations, arachidic acid (30 µM) lacked this activity, and nordihydroguaiaretic acid
(10 µM), when incubated with PMN for 30 min, did not
alter the response to 1 µM AA. Thus, AA, at 10-100
nM, stimulates PDB binding and the translocation of three
PKC isoforms. The two effects have similar kinetics,
Ca2+-independence, and resistance to an AA antimetabolite.
The data support the idea that PDB binding responses to AA reflect the movements of cytosolic PKC
,
II, and
to
plasmalemma.

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Fig. 5.
PKC ,
II, and in
PMN. Cells were Ca2+-manipulated with Quin2,
challenged with 1 µM AA for 0-4 min (left two
columns) or 0-1000 nM AA for 2 min (right two
columns), and fractionated. Fractions were loaded (20 and 60 µg
of protein for cytosol and membrane fractions, respectively) and
resolved on SDS-polyacrylamide gels, transferred to membranes, reacted
with antibodies, and visualized by enhanced chemiluminescence. All
bands migrated with PKC standards; doublet bands, seen mostly with
PKC , may reflect differences in phosphorylation (44, 48). Ethanol (1 µl/ml) did not alter the distribution of PKC. Each column
of panels gives results from the PMN of one donor and is representative
of at least four experiments.
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Fig. 6.
Kinetics of AA-induced PKC
translocation. PMN were Ca2+-manipulated with Quin2
and processed as in Fig. 5 to obtain Western blots that were quantified
by densitometry. Data are mean ± S.E. (4-7 experiments)
percentage changes in band density, relative to PMN challenged with
ethanol for 0-5 min. Asterisks indicate values
significantly (p < 0.05, Student's paired
t test) above (plasma membrane) or below (cytosol)
ethanol-challenged cells. By itself, ethanol did not alter the location
of any PKC isoform.
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Fig. 7.
PKC translocation at different AA
concentrations. PMN were Ca2+-manipulated with Quin2;
challenged by 0 (1 µl/ml ethanol), 10, 100, or 1000 nM AA
for 2 min; processed; and analyzed as in Fig. 6. Data (mean ± S.E. for 3-6 experiments) are percentage changes in band density
relative to PMN challenged by ethanol. Asterisks indicate
values significantly (p < 0.05, Student's paired
t test) above (plasma membrane (solid lines)) or
below (cytosol (interrupted lines)) those for
ethanol-challenged cells.
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HEK 293 Cells--
HEK 293 cells expressing EGFP-PKC
presented
homogenous fluorescence in the cytosol; nuclei lacked fluorescence. On
exposure to AA, cytosol fluorescence fell, whereas fluorescence at the cell periphery rose (Fig. 8, top
two rows). Nomarsky optical analysis localized the latter
fluorescence to surface membrane (Fig. 9, A-C). These responses occurred at 30, 10, 1, and 0.5 µM AA but not at 0.1 µM AA. Cells exposed
to 30 µM AA exhibited visible changes at
8 min; maximal
changes occurred by 20 min. At 10, 1, and 0.5 µM, just
detectable and maximal effects occurred by ~20 and 30, 30 and 40, or
30 and 45 min, respectively (Fig. 10). In all cases, maximal responses followed detectable responses within 10 min and did not reverse over 60 min. Cells challenged with arachidic
acid (30 µM) had no change in fluorescence pattern over
60 min (data not shown). Finally, addition of an antioxidant, ascorbic
acid (100 µM), to the cell cultures for 10 min did not alter the EGFP-PKC
response to 1 or 10 µM AA (data not
shown).

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Fig. 8.
EGFP-PKC in HEK 293 cells. Cells
expressing EGFP-PKC (top panels) or (bottom
panels) were exposed to 1 µM AA for 0-39 min and
monitored for fluorescence every 60 s by confocal laser
microscopy. EGFP-PKC fluorescence began to fall in the cytosol and
rise at the cell periphery by 31 min. The change peaked at ~39 min
and did not reverse over the ensuing 21 min. EGFP-PKC fluorescence
began to fall in the cytosol and rise at the nuclear membrane by 2 min.
Changes peaked at 4-8 min and returned to baseline in 12 min. Results
are representative of 10 experiments done on at least two separate
clones of cells.
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Fig. 9.
Location of EGFP-PKCs in HEK 293 cells.
Cells expressing EGFP-PKC were exposed to 10 µM AA for
0 (A) or 31 (B and C) min and examined
by fluorescent (A and B) or Nomarski
(C) optics. Resting cells had homogenous fluorescence
throughout the cytosol; nuclei lacked fluorescence. After 22 min of
challenge, fluorescence in the cytosol shifted to silhouette
plasmalemma. Cells expressing EGFP-PKC were exposed to 10 µM AA for 0 (D) and 5 (E and
F) min or 50 nM AA for 0 (G) and 8 (H and I) min. Cells were examined by fluorescent
(D, E, G, and H) or Nomarski (F and
I) optics. Resting cells had fluorescence throughout the
cytosol and, to a lesser extent, nucleus. Within 2 min of challenge,
fluorescence in the cytosol fell, and that at the nuclear perimeter
rose. Nucleoplasm fluorescence was unaltered. Changes peaked at 5 and
reversed by 12 min. E, F, H, and I show
perinuclear fluorescence-silhouetted nuclear membrane, e.g.
at the indentation of the right nucleus in E and
F. AA also caused irregular rises in plasmalemma
fluorescence, at e.g. 1-3 o'clock in the cell shown in
E, 2-4 o'clock in the upper cell shown in
H, and the contact interface between the two cells in
H.
|
|

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Fig. 10.
Effect of varying AA concentrations on the
location of EGFP-PKC . HEK 293 cells
expressing EGFP-PKC were stimulated and monitored for fluorescence
as in Fig. 8. The two cell clumps in the top row showed
falls in cytosolic and rises in cell periphery fluorescence by 21 min
of challenge with 30 µM AA. Cells in the second
row had similar changes in response to 10 µM AA but
only after 21 min. The two adjacent cells in the third row
had more modest fluorescent changes after 39 min of exposure to 0.5 µM AA, whereas cells in the bottom rows had no
fluorescence change after challenge with the AA vehicle, ethanol (1 µl/ml).
|
|
Cells expressing EGFP-PKC
displayed homogeneous fluorescence in
cytosol and, to a lesser extent, nucleus. AA caused declines in
cytosolic and rises in perinuclear fluorescence but little or no change
in nucleoplasm fluorescence (Fig. 8, bottom two rows). Concurrently with these changes, localized areas of the cell periphery gained fluorescent intensity. Fig. 9, E, F, H, and
I, indicates that rises in the fluorescence intensity
occurred specifically at nuclear and plasma membranes. Cells exposed to
0.1-30 µM AA initiated, maximized, and reversed these
changes by ~2, 8, and 14 min, respectively (Fig.
11). For cells treated with a 30 or 50 nM concentration of the fatty acid, fluorescence increases in plasma and nuclear membrane occurred 2-4 min after challenge (Fig.
9, G-I). Again, 30 µM arachidic acid did not
translocate EGFP-PKC
, and 100 µM ascorbic acid, when
added to cultures for 10 min, did not alter the translocation response
of EGFP-PKC
to 0.1 or 1 µM AA (data not shown).

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[in this window]
[in a new window]
|
Fig. 11.
Effect of varying AA concentrations on the
location of EGFP-PKC . HEK 293 cells
expressing EGFP-PKC were stimulated and monitored as in Fig. 8. The
cell in the top row showed gains in perinuclear
fluorescence. These peaked at 2-4 min and fell to baseline by 14 min
of challenge with 33 µM AA. Similar but progressively
less intense changes occurred with the cell in the second
row challenged with 300 nM AA and the cell in the
third row challenged with 100 nM AA. The two
cells in the fourth row showed subtle changes (better
depicted in Fig. 9, bottom three panels) in response
to 50 nM AA. The two cells in the bottom row had
no change in fluorescence after challenge with 1 µl/ml ethanol.
Associated with rises in perinuclear fluorescence, fluorescence in the
cytosol showed partial declines (best seen at the 2 and 4 min time
points for the 30 and 0.3 µM AA challenge) and irregular
gains in fluorescence at the cell periphery (see Fig. 9).
|
|
 |
DISCUSSION |
UFAs directly activate PKC and, when added to hepatocytes (23),
platelets (24, 25), myocytes (26), cultured monocytes (27, 31),
macrophages (27, 28, 31), endothelial cells (30), or PMN (29),
induce PKC to translocate. Although the latter effect may be
consequential to activating these enzymes, the two responses are
separable. For example, a low level of phosphatidylserine or
Ca2+ causes PKC to attach to membranes in vitro
whereas a higher level of either agent also activates the
membrane-attached PKC (1-4, 50, 51). The activating effect of AA on
most PKC isoforms, including
,
II, and
,
reportedly requires at least 20-100 µM. Such extreme
levels of the fatty acid can occur in vivo; microparticles shed by perturbed cells may stimulate PKC and PKC-dependent
functions by delivering up to 100 µM free AA to various
target cells (31, 52). Under most physiological conditions, however, AA
occurs at levels below this. Cells responding to receptor agonists, in particular, are unlikely to propel cytosolic AA to levels engendering PKC activation. We find that
10-100 nM AA causes PMN and
HEK 293 cells to bind [3H]PDB and translocate PKC.
PDB binding responses to AA reflected an increase in the number of
receptors available to the ligand (Tables I and II). In studies not
shown, PMN were challenged for 2 min with 3 µM AA or
arachidic acid, diluted with 9 volumes of 4 °C buffer, disrupted, and fractionated on Percoll gradients. The former but not the latter
fatty acid caused significant falls in cytosol and rises in plasmalemma
PDB receptors but no such change in granule, endoplasmic reticulum, or heavy Golgi fractions. Thus, AA-induced PDB
binding responses involve the movement of PDB receptors from cytosol to plasmalemma. In sucrose gradient experiments, AA, but not arachidic acid, similarly caused PMN to decrease cytosol and increase membrane PKC
,
II, and
(Fig. 5). Although membranes
isolated from the latter gradients contain not only plasmalemma but
also Golgi, endoplasmic reticulum, and other organelles (36),
PKC translocation paralleled [3H]PDB binding. We
accordingly interpret sucrose gradient data as indicating that PKC
moved mainly if not exclusively from cytosol to plasmalemma. We stress,
however, that other PDB receptor-bearing proteins (PKC
I,
chimerin, etc.) may and likely do contribute to PDB binding (41,
42).
PDB binding responses to CFs were slowed in Ca2+-fixed and
absent in Ca2+-chelated PMN (34, 35) (Fig. 1). CFs,
including FMLP, likewise have reduced ability to translocate various
PKC isoforms in PMN deprived of Ca2+ (43). In marked
contrast, AA caused prominent PBD binding and PKC
II,
, and
translocation (Figs. 3 and 5-7) responses regardless of
PMN Ca2+ status. Thus, CFs need a minimal level of cell
Ca2+ to affect PKC, whereas UFAs have far less such a
requirement. Because PKC
and
II, but not PKC
,
exhibited reduced responses to AA in Ca2+-chelated PMN
(Figs. 6 and 7), [Ca2+]i rises likely contribute
to PDB binding (34, 35) (Fig. 1) responses to CFs by enhancing the
movement of Ca2+-sensitive PKC isoforms. On the other hand,
the effect of AA on PDB proved largely indifferent to PMN
Ca2+ status (Fig. 3). The relatively slow-paced
[Ca2+]i rise initiated by AA may explain this
anomaly: Ca2+-actuated PDB binding response to AA overlap
and may be obscured by Ca2+-insenstive PDB binding
response. In any case, studies clearly show that
Ca2+-sensitive PKC isoforms translocate in the absence of
Ca2+ transients in PMN challenged with AA (Figs. 5-7), as
well as CFs (43) or other receptor agonists (32). It will be important to determine whether endogenous AA has a role in these receptor-driven translocations. Relevant to this, agents that block AA-releasing enzymes, phospholipases A2, have recently been found to
inhibit interleukin 2 in stimulating T lymphocytes to translocate PKC activity (53).
AA did not trigger PKC translocation as a result of its oxygenation.
5-HETE and LTB4, the major oxygenation products formed from
AA by PMN, did not mimic the action of AA. Moreover, LTB4 down-regulated PMN to itself but not AA (data not shown),
nordihydroguaiaretic acid did not alter the action of AA (Table I), and
other UFAs stimulated PDB binding (Table II and Fig. 4) and PKC
translocation (data not shown). These results contrast with those found
with many AA activities. For example, AA stimulates PMN to activate cytosolic phospholipase A2. The effect is sensitive to
nordihydroguaiaretic acid and produced by LTB4 and 5-HETE
but not other UFAs. Here, then, PMN convert AA to LTB4 and
5-HETE, which bind respective receptors to initiate signal pathways
activating the enzyme (54, 55). Our data exclude this route for
AA-induced PKC translocation. An alternate metabolic route, however,
could explain PKC translocation responses to UFAs. Cells acylate UFAs
to CoA, transfer the UFA moiety of UFA-CoA to glycerolipids, and
shuttle glycerolipid-bound UFAs to other lipids. PMN conduct these
steps rapidly even if overloaded with Ca2+-chelators (56).
Because UFA-CoA alters PKC activity and because glycerolipids bearing
UFAs have higher affinity for PKC than their saturated fatty
acid-containing counterparts (57-59), UFA-CoA or UFA-glycerolipids may
influence the dispositions of PKC and thereby the translocation
response to UFAs. The same events could operate in cells stimulated to
release endogenous AA. Our studies also do not address other indirect
means for translocating PKC, e.g. by stimulating formation
of phosphatidylinositol phosphates, phosphorylation of PKC, changes in
PKC docking proteins, or issuance of various other signals, although
these events may not occur in Ca2+-chelated cells
challenged by low levels of AA. Studies with pharmacological inhibitors
offer a first step to determine whether AA acts directly, upon
esterification to CoA or glycerolipids, or through signal pathways.
Fluorescent fusion products have been used to monitor the movements of
various PKC isoforms. In CHO cells, EGFP-PKC
localizes to cytosol
and nucleoplasm (60, 61); ATP stimulates the label to shift to
plasmalemma, whereas phorbol esters cause it to move to plasma or
nuclear membranes (61, 62). LLCPK-1 cells localize EGFP-PKC
to plasmalemma and do not translocate it in response to dopamine,
although dopamine translocates other PKC isoforms (63).
EGFP-PKC
II localizes exclusively to cytosol in HEK 293 cells and moves to plasmalemma in less than 1 min of challenge by G
protein-coupled receptor agonists (64). These reports evidence that the
location, as well as the direction and kinetics of EGFP-PKC
and
II movements, varies with cell, isoform, and stimulus
type. Other PKC isoforms show similar variability (60, 65). Most relevant here, however, are reports on UFAs. In CHO cells,
200 µM AA and oleic acid cause rapid, transient shifts in
EFFP-PKC
from cytosol to plasma membrane, whereas 50 µM AA causes a slow, sustained shift in cytosol
EGFP-PKC
to the perinuclear area (66, 67). We are unaware of
reports on the effect of UFAs on other EGFP-PKC isoforms. We show here
that AA caused HEK 293 cells to translocate EGFP-PKC
from cytosol to
the entire nuclear and limited portions of the plasma membrane (Fig. 9,
D-I). Responses occurred and reversed within minutes of
exposure to
30 nM AA (Fig. 8, bottom two rows,
and Fig. 11). AA affected EGFP-PKC
differently, causing it to move
from cytosol to plasma membrane (Fig. 9, A-C). Unlike the
EGFP-PKC
response to AA here or the EGFP-PKC
responses to
200
µM AA in CHO cells (66), EGFP-PKC
movements evolved slowly, did not reverse for 60 min, and occurred at
0.5
µM AA in HEK 293 cells (Fig. 8, top two rows,
and Fig. 10). As found elsewhere (60-68), then, the kinetics and
direction of PKC translocation responses to a given agent are
isoform-specific and may relate to the unique functions of each PKC
isoform (1-4). Interestingly, resting HEK 293 cells localized
EGFP-PKC
to both the cytosol and nucleoplasm but localized
EGFP-PKC
only to the cytosol. This allows for the
possibility that PKC
may have a nuclear membrane-targeting motif that over longer periods aids its penetration into nuclei.
In conclusion, AA causes PMN and HEK 293 cells to translocate PKC
,
I,
II, and
at concentrations 2-4
orders of magnitude below those reportedly activating the enzymes.
Because PKCs must be correctly compartmentalized in order to engage
their cofactors and substrates, AA, as presented to cells by, for
example, microparticles or released during stimulation, may serve to
direct Ca2+-sensitive and Ca2+-insensitive PKC
isoforms to proper membrane targets and thereby assist, modulate, or
(when no Ca2+ transient occurs) replace
[Ca2+]i signals. Indeed, exogenous AA and DG
operate synergistically to stimulate PKC and PKC-dependent
responses in whole cells (1-4) and may similarly cooperate when formed
endogenously. The effect of AA on the translocation of PKC, at least as
it occurs in PMN, proceeds independently of
[Ca2+]i, DG production, and oxygenation to
eicosanoids. The fatty acid may act directly, after acylation to CoA or
glycerolipids, or by stimulating PKC phosphorylation, PKC binding
proteins, or other signal pathways.
 |
ACKNOWLEDGEMENT |
We thank Prof. N. Saito from the Biosignal
Research Center, Kobe University (Kobe, Japan) for his generous gift of
pPKC
-EGFP.
 |
FOOTNOTES |
*
This work was supported by National Institute of Health,
NHLBI, Grant 1 RO1 HL56710.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be 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 Internal
Medicine, Section on Infectious Diseases, Wake Forest University School
of Medicine, Medical Center Blvd., Winston-Salem, NC 27156. Tel.:
336-716-6039; Fax: 336-716-3825; E-mail: joflaher@wfubmc.edu.
Published, JBC Papers in Press, April 27, 2001, DOI 10.1074/jbc.M101093200
 |
ABBREVIATIONS |
The abbreviations used are:
DG, diacylglycerol;
PKC, protein kinases C;
EGFP, enhanced green fluorescent protein;
UFA, unsaturated fatty acid;
AA, arachidonic acid;
PDB, phorbol dibutyrate;
PMA, phorbol 12-myristate 13-acetate;
FMLP, N-formyl-methionyl-leucyl-phenylalanine;
5-HETE, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoate;
LTB4, leukotriene B4;
PMN, polymorphonuclear
neutrophils;
BSA, bovine serum albumin;
DMEM, Dulbecco's modified
essential medium;
CF, chemotactic factor.
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