(Received for publication, June 14, 1995; and in revised form, August 23, 1995)
From the
A variety of approaches have been employed to demonstrate that
the interaction of protein kinase C II with phorbol
ester-containing membranes is reversible, is not accompanied by
significant insertion of the protein into the hydrophobic core of the
membrane, and is qualitatively similar to the interaction with
diacylglycerol (DG). First, we show that under conditions when protein
kinase C is bound with equal affinity to membranes containing either DG
or phorbol myristate acetate (PMA), increasing ionic strength causes a
similar reduction in membrane binding. The similar sensitivity to ionic
strength indicates that the forces mediating the binding of protein
kinase C to PMA are not significantly different from those mediating
the binding to DG. At sufficiently high concentrations of PMA and
relatively low concentrations of phosphatidylserine, the binding of
protein kinase C to membranes became markedly less sensitive to ionic
strength, suggesting that under these conditions direct
non-electrostatic interactions with PMA dominate over electrostatic
interactions with the lipid headgroups. Importantly, regardless of the
strength of the interaction with PMA, protein kinase C exchanges
between vesicle surfaces: protein kinase C bound first to phorbol
ester-containing multilamellar vesicles exchanged to large unilamellar
vesicles upon addition of an excess surface area of the latter. Lastly,
the enzyme's intrinsic tryptophan fluorescence was not quenched
by bromines located at various positions in the hydrophobic core of the
membrane. In contrast, the enzyme's tryptophan fluorescence was
significantly quenched by probes positioned at the membrane surface. In
summary, our results are consistent with protein kinase C binding
reversibly to PMA- or DG-containing membranes primarily via
interactions at the membrane interface.
Generation of diacylglycerol (DG) ()in the plasma
membrane is the key signal in activating most members of the
lipid-regulated family of protein kinase C
isozymes(1, 2) . This lipid second messenger has
recently been shown to regulate protein kinase Cs function by
dramatically increasing the enzyme's affinity for
phosphatidylserine (PS)-containing
membranes(3, 4, 5) . The resulting high
affinity membrane interaction leads to a conformational change that
activates the enzyme: specifically, the autoinhibitory pseudosubstrate
domain of the molecule (6) is removed from the active site,
thus allowing substrate binding and catalysis(7, 8) .
Phorbol esters are functional analogues of DG: they compete for the
same binding site on the molecule as DG(9) , and they can
replace DG in activating the enzyme(10, 11) . Unlike
DGs, which are metabolized within minutes(12, 13) ,
phorbol esters are long lived in the cell. As a result, these molecules
have proved invaluable in activating protein kinase C in situ,
and a plethora of studies over the past decade have described the
phorbol ester-dependent translocation of protein kinase C to cellular
membranes(11, 14) . However, several reports have
questioned whether phorbol esters regulate protein kinase C by the same
mechanism as DGs. Notably, it has been proposed that phorbol esters
``convert'' protein kinase C into a constitutively active
form that is irreversibly inserted into the
membrane(15, 16, 17, 18) . Evidence
for this derives from observations that the association of protein
kinase C with membranes containing phorbol esters, both in vivo and in vitro, is only partially reversed by chelation of
Ca(15, 16, 17, 18, 19) ,
contrasting with the quantitative dissociation of protein kinase C from
vesicles containing DG(19) . Direct evidence for the
penetration of protein kinase C into the hydrocarbon core of the
membranes has also been reported, but this interaction was not unique
to phorbol esters (20, 21) .
In this contribution, we address the nature of the interaction of protein kinase C with phorbol ester-containing membranes. Using a variety of biophysical and biochemical approaches, we show that the interaction of protein kinase C with phorbol ester-containing membranes occurs primarily via interactions at the membrane interface, in the absence of significant interaction with the hydrocarbon core of the membrane. Furthermore, we show that this interaction is reversible and that the apparent irreversibility noted in the literature results from the remarkably high affinity association with phorbol esters. Thus, DGs and phorbol esters regulate protein kinase C by the same mechanism, with observed differences reflecting primarily differences in the strength of the interaction.
where A and A
are the
measured activities of the bottom and top fractions, respectively. The
fraction of sedimented vesicles,
, was calculated from the
distribution of
H-labeled PC, which was included in trace
amounts in all lipid mixtures. The fraction of kinase activity found in
the supernatant in the absence of lipid,
, was equal, within the
limits of experimental error, to the value expected for a
non-sedimenting protein (i.e. 0.73 under the experimental
conditions used). All experiments were performed in a standard solution
composed of 100 mM KCl, 20 mM Tris, pH 7.0, and 0.3
mg ml
BSA. Concentrations of additions that varied
depending on experiment are given with appropriate results. The
apparent membrane affinity of the enzyme with membranes was defined as
the ratio of membrane-bound to free enzyme divided by the total lipid
concentration.
Figure 4:
Kinetics of the association of protein
kinase C with vesicles. A concentrated solution of protein kinase C
(1.5 µM) was injected into a rapidly stirring suspension
of large unilamellar vesicles (8 µM lipid) to yield a
final concentration of 60 nM. The vesicles were suspended in a
solution containing 100 mM KCl, 10 mM Tris, pH 7.0,
0.5 mM EGTA, and either 0.45 mM or 0.7 mM
CaCl. The final concentration of free Ca
was 1 µM (dashed line) or 200
µM (thin and thick solid lines).
Vesicles were composed of 25 mol % POPS, 74 mol % egg PC, and either 1
mol % PMA (thin solid and dashed lines) or 1 mol %
DODG (thick solid line). Samples were excited at 280 nm and
emission was recorded at 520 nm. Excitation and emission slits were set
at 4 nm.
The primary evidence supporting the hypothesis that phorbol
esters cause an irreversible association of protein kinase C with
membranes derives from multiple observations that the
Ca-dependent binding of protein kinase C to
PMA-containing membranes cannot be completely reversed by chelation of
Ca
(15, 16, 17, 18, 19) .
The possibility that this chelator-dependent binding is reversible was
explored in Fig. 1Fig. 2Fig. 3.
Figure 1:
Effect of
ionic strength on the diacylglycerol or phorbol ester-induced
association of protein kinase C with membranes. The binding of protein
kinase C (1 nM) to POPC vesicles (0.5 mM lipid)
containing 50 mol % (closed symbols) or 25 mol % (open
symbols) POPS, and either 2 mol % DODG (), 0.01 mol % PMA
(
), or 1 mol % PMA (
) was measured in the presence of 0.5
mM EGTA. Results are expressed as the apparent membrane
affinity determined from the amount of membrane-bound and free enzyme,
as described under ``Experimental Procedures.'' Data
represent the average of at least two independent experiments; error bars indicate the range or S.D. Points for 100 and 200
mM KCl were offset slightly to the right and left of the
actual concentration to improve their
visibility.
Figure 2:
Dissociation of protein kinase C from
membranes by chelation of Ca. A, protein
kinase C (60 nM) was incubated with large unilamellar vesicles
(5 µM lipid) composed of 25 mol % DNS-PE, 74 mol % POPC,
and 1 mol % PMA for 45 min, 22 °C, in a solution containing 0.2
mM CaCl
, 100 mM KCl, and 20 mM Tris, pH 7.0. A 200-fold concentrated solution of EGTA was
injected into a continuously stirred enzyme-lipid mixture at the 15th
second of the time scan (thick line) to yield a final
concentration of 1 mM. The thin line represents
injection of a concentrated suspension of protein kinase C, at 15 s,
into a cuvette containing 1 mM EGTA and vesicles with 1 mol %
PMA. Except for Ca
, the final composition of the
suspension in which protein kinase C was present before chelation of
Ca
(i.e. thick line) was identical to that
when protein kinase C was added to a solution with chelator and no
Ca
(i.e. thin line). Excitation and emission
wavelengths were 280 and 520 nm, respectively; both slits were set at 4
nm. B, protein kinase C was incubated as in A except
that vesicles contained 1 mol % DODG instead of PMA. At 15 s, EGTA was
injected into a continuously stirred enzyme-lipid mixture to yield a
final concentration of 1 mM (thick line). Thin
line represents the emission in the absence of protein kinase C
but otherwise identical conditions.
Figure 3:
Reversibility of protein kinase C's
association with membranes containing diacylglycerol or phorbol esters.
Protein kinase C (1 nM) was incubated with multilamellar
vesicles composed of 2 mol % DODG, 50 mol % POPS, and 48 mol % POPC (filled columns; 2.2 mM total lipid) or 1 mol % PMA,
25 mol % POPS, and 74 mol % POPC (blank columns; 5.5 mM total lipid) in the solution described in the legend to Fig. 1. After 15 min of incubation at 22 °C, large
unilamellar vesicles of identical composition to that of the
multilamellar vesicles were added to the enzyme-lipid mixture to yield
a 2-fold increase in the total amount of lipid. Buffer was added to the
control samples. After incubation for another 15 min, uni- and
multilamellar vesicles were separated by centrifugation at 10,000
g for 6 min at 22 °C. The percentage of protein
kinase C activity associated with the sedimented multilamellar vesicles
was calculated as described under ``Experimental
Procedures.''
Fig. 1compares the binding of protein kinase C II to
membranes containing either DG or PMA in the presence of excess
chelator (0.5 mM EGTA, no added Ca
). Note
that the same amount of membrane association can be achieved for
membranes containing either activator when the difference in affinity
of protein kinase C for each activator is taken into account.
Specifically, protein kinase C bound with equal affinity to membranes
containing the same amount of PS (50 mol %) and either 2 mol % DG (filled circles) or 0.01 mol % PMA (filled squares),
in the absence of Ca
; the difference in activator
concentrations is consistent with the approximately 250-fold increase
in protein kinase C's affinity for PMA compared with DG reported
recently(27) . To test whether the chelator-resistant
interaction differed for PMA compared with DG, the ability of
increasing ionic strength to reduce the strength of the membrane
interaction was compared for both activators. Increasing ionic strength
caused a marked reduction in the affinity of protein kinase C for PS
membranes containing either 2 mol % DG (filled circles) or
0.01 mol % PMA (filled squares). The magnitude of this
reduction was similar for membranes containing both activators. The
similar effect of ionic strength on both interactions suggests that any
difference in hydrophobic contributions in the association of protein
kinase C with DG- versus PMA-containing membranes is small.
The sensitivity of protein kinase C's membrane interaction to ionic strength depended on the membrane content of PS and PMA. When the PMA concentration was increased to 1 mol % and the PS concentration decreased concomitantly from 50 to 25 mol %, protein kinase C's membrane interaction became markedly less sensitive to ionic strength. This ionic strength insensitivity at high concentrations of phorbol esters is consistent with the finding that 1 mol % PMA is sufficient to cause significant association of protein kinase C with membranes in the absence of PS ((27); also see Fig. 7). Thus, the direct association with PMA that can be observed at high concentrations of this ligand likely does not involve the salt-sensitive electrostatic interactions between protein kinase C and PS headgroups that dominate when the PS concentration is relatively high and the DG or PMA concentration relatively low (filled symbols).
Figure 7:
Association of protein kinase C with
phorbol ester-phosphatidylcholine membranes in liquid crystalline or
gel phase. Protein kinase C (1 nM) was incubated with vesicles
(5 mM lipid) composed of 2 mol % PMA and either 98 mol % POPC
() or 98 mol % DPPC (
) for the indicated times at 22
°C; the incubation medium contained 0.5 mM EGTA, 0.3 mg
ml
BSA, 100 mM KCl, and 20 mM Tris, pH 7.0. Membrane-bound enzyme was separated from free enzyme
by centrifugation, as described under ``Experimental
Procedures.'' The indicated times include the 19-min separation
procedure. Data represent the mean and range of results from two
independent experiments.
Fig. 2examines the effect of chelation of Ca by EGTA on the association of protein kinase C with membranes
containing the same concentration of PMA or DG and 25 mol % DNS-PE,
which can substitute for PS in activating protein kinase C(4) .
Membrane binding was assessed by resonance energy
transfer(24, 28) . Protein kinase C was incubated for
45 min with vesicles containing 25 mol % DNS-PE and 1 mol % of either
PMA or DG in the presence of 200 µM Ca
. Fig. 2shows that chelation of Ca
resulted in a
rapid dissociation of protein kinase C from membranes, as assessed by a
decrease in fluorescence energy transfer from tryptophans to dansyl-PE.
The half-time for dissociation was approximately 3 s for protein kinase
C bound to vesicles containing either PMA (Fig. 2A) or
DODG (Fig. 2B). Dissociation was complete for
DG-containing vesicles as evidenced by the decrease in light emission
to the level observed for vesicles in the absence of enzyme (Fig. 2B, thin line) after compensation for
the dilution caused by addition of the EGTA. The same level of emission
was observed for vesicles in the presence or absence of EGTA, as well
as in the presence of protein kinase C and EGTA (data not shown). For
PMA-containing membranes, approximately 10% of the enzyme remained
membrane-bound (Fig. 2A, thick line). However,
a comparable level of association was measured within minutes after
protein kinase C was added to vesicles of the same composition
suspended in a solution containing 0.5 mM EGTA and no
exogenous Ca
(Fig. 5A, thin
line). Thus, the fraction of protein kinase C that remained
associated with membranes upon chelation of Ca
represented the fraction of protein kinase C that associates with
such membranes in the absence of Ca
, rather than
enzyme that had become irreversibly bound as a result of the initial
Ca
-dependent interaction.
Figure 5:
Quenching of the intrinsic tryptophan
fluorescence of protein kinase C by dansyl fluorophores localized at
the water-lipid interface. Protein kinase C (60 nM) and large
unilamellar vesicles (150 µm lipid) composed of 74 mol % POPC and
either 25 mol % POPS and 1 mol % DODG (dashed line), 25 mol %
DNS-PE and 1 mol % DODG (thick solid line), or 25 mol % DNS-PE
and 1 mol % PMA (thin solid line) were incubated in a solution
composed of 0.2 mM CaCl, 100 mM KCl, and
20 mM Tris, pH 7.0. Samples were excited at 280 nm and the
emission from 300 to 360 nm recorded. Excitation and emission slits
were set at 4 nm; spectra were corrected for inner filter
effect.
At high concentrations
of PMA and high lipid/protein ratios, the majority of protein kinase C
molecules are vesicle associated, even in the presence of chelator (Fig. 1). To address the reversibility of this
Ca-independent membrane association (i.e. the binding to PMA-containing membranes in the absence of
Ca
), we asked whether membrane-bound enzyme could
translocate to enzyme-free vesicles. In the experiment in Fig. 3, protein kinase C was bound to multilamellar vesicles
containing 1 mol % of either DG or PMA, and then incubated with an
equal concentration of lipid in the form of large unilamellar vesicles
of the same composition. Separation of the multilamellar vesicles from
the unilamellar vesicles by centrifugation revealed that approximately
90% of the protein kinase C bound to the DG-containing multilamellar
vesicles had now partitioned with the large unilamellar vesicles;
control experiments indicated that the unilamellar vesicles had about
10 times more surface area. This result is consistent with complete
equilibration of the enzyme between the two types of vesicles. For
PMA-containing membranes, protein kinase C did not equilibrate
completely between the two types of vesicles: 17 ± 6% remained
bound to multilamellar vesicles 15 min after addition of the large
unilamellar vesicles. Nonetheless, the majority of the enzyme did
exchange from the multilamellar vesicles to the unilamellar vesicles
within the course of the assay.
Partial reversibility of protein
kinase C's association with membranes containing PMA suggested
that the dissociation rate of the enzyme from such membranes could have
been a limiting factor over the time scale of the experiment. However,
because the association of protein kinase C with vesicles containing
more than one molecule of PMA increases with time, ()prolonging the time of incubation for the exchange
experiment described above was unlikely to provide an unambiguous
answer. Unfortunately, technical limitations prevented the use of
resonance energy transfer to measure the dissociation of protein kinase
C from membranes at low occupancy of PMA molecules by the protein; this
technique requires substantial occupancy of the membrane surface by
protein. As an alternative approach to determining dissociation rates,
we compared the rates of initial association of protein kinase C with
membranes containing DG or PMA using resonance energy transfer from
tryptophans to dansyl-PE. Fig. 4shows that protein kinase C
associated rapidly with DG-containing vesicles (thick solid
line); the apparent association constant was 0.26 ± 0.04
s
. Despite the greater than 200-fold higher affinity
of protein kinase C for vesicles containing 1 mol % PMA compared with 1
mol % DG, the apparent association constant was 0.16 ± 0.02
s
for the PMA-containing membranes (thin solid
line), approximately half that for association with the
DG-containing membranes. Thus, the dissociation of protein kinase C
from vesicles containing PMA is likely to be at least 2 orders of
magnitude slower than that from vesicles containing an equal
concentration of DG. To measure the association rate under conditions
resulting in comparable membrane association of protein kinase C, the
Ca
concentration was reduced 200-fold for the
incubation with PMA-containing vesicles. In the presence of 1
µM Ca
, the fluorescence emission in the
presence of vesicles containing 1 mol % PMA (dashed line)
reached the level obtained in the presence of vesicles containing 1 mol
% DG and 200 µM Ca
within 10 min, and
did not change significantly afterward (data not shown). However, the
apparent association constant of protein kinase C under low
Ca
conditions decreased only by a factor of 6 to
0.026 ± 0.001 s
, despite the 200-fold lower
membrane affinity. This observation suggests that the association of
protein kinase C with phorbol esters may be rate limiting in the
binding of enzyme to membranes containing phorbol esters. In summary,
under conditions when the apparent association constants were identical
for vesicles containing the same mol % DG or PMA, the association of
the enzyme with the latter proceeded at a significantly slower rate. In
the presence of 0.2 mM Ca
and 1 mol% of
either PMA or DG in the membrane, biphasic kinetics were observed in
the association of protein kinase C with membranes. The apparent rate
constant for the slower component was the same (0.023 ± 0.002
s
) for both PMA- and DG-containing vesicles,
suggesting that it did not reflect an irreversible membrane insertion
which is thought to require phorbol esters and not DG(19) . The
slower component in Fig. 4likely reflects steric saturation of
the membrane surface because of the high ratio of enzyme/lipid.
The
sensitivity of protein kinase C's intrinsic tryptophan
fluorescence to either surface-positioned (Fig. 5) or
membrane-inserted quenchers (Fig. 6) was used as an independent
and direct method to address the degree of penetration of the enzyme
into the hydrophobic core of the membrane. Protein kinase C II has
9 tryptophans(29, 30, 31) ; 5 of these are in
the membrane-interacting regulatory domain. Fig. 5shows that
the presence of dansyl-labeled PE in PC vesicles caused a 40% decrease
in the intrinsic emission of the enzyme (solid lines) relative
to vesicles that did not have the probe but had equimolar PS (dashed line). No quenching was observed when neither DG nor
PMA was included in vesicles, in the presence of 0.5 mM EGTA (i.e. under conditions where little protein kinase C was
vesicle-associated) (not shown). Under this latter condition (i.e. free protein kinase C but vesicles present), or in the presence of
only protein kinase C (no vesicles), the fluorescence emission at 300
nm was approximately 15% lower than in the presence of vesicles with
bound protein kinase C but without the quencher (dashed line).
For these experiments, an excess of lipid was used so that >99% of
the protein kinase C was membrane-bound. An almost identical degree of
quenching was observed for vesicles containing 1 mol % of either DG (thick solid line) or PMA (thin solid line) and 25
mol % DNS-PE. Despite the 200-fold difference in protein kinase
C's affinity for membranes containing 1 mol % PMA compared with 1
mol % DG, the spectra for both membranes had similar shapes indicating
that protein kinase C's tryptophans are in similar environments
whether bound to DG- or PMA-containing membranes. Both the quenching by
DNS-PE (Fig. 5) and the resonance energy transfer from
tryptophan to the dansyl moiety (Fig. 2) indicate that at least
some of the tryptophans of protein kinase C are proximal to the
interface.
Figure 6:
Effect of bromines located at different
membrane depths on the tryptophan fluorescence of cytochrome b and protein kinase C. Protein kinase C (60
nM) (A) or cytochrome b
(0.5
µM) (B) was incubated with large unilamellar
vesicles (150 µM lipid) composed of 25 mol % POPS, 1 mol %
PMA, and 74 mol % of either POPC (thick solid line),
P(6,7-diBr)SPC (thin solid line), or P(11,12-diBr)SPC (dashed line) as described in the legend to Fig. 3. The
excitation wavelength was 280 nm; excitation, and emission slits were
set at 4 nm. Spectra were corrected for inner filter
effect.
In contrast to the significant quenching of protein
kinase C's tryptophans by the surface-positioned dansyl probe,
bromines situated at either the 6 and 7 or 11 and 12 carbons on the
acyl chain had no detectable effect on tryptophan fluorescence. Fig. 6A shows that the tryptophan emission of protein
kinase C was the same whether bound to vesicles containing 25 mol % PS
and 1 mol % PMA and either 74 mol % POPC (thick solid line),
P(6,7-DiBr)SPC (thin solid line), or P(11, 12-DiBr)SPC (dashed line). Identical results were obtained when PMA was
substituted by DG (data not shown). In marked contrast, the tryptophan
fluorescence of cytochrome b, which does insert
into the hydrophobic core of the membrane(32, 33) ,
was quenched by 57% when bound to vesicles with PC brominated at acyl
chain positions 6 and 7, and by 48% when bound to vesicles with PC
brominated at acyl chain positions 11 and 12 (Fig. 6B).
Note that the quenching of cytochrome b
with PC/PS
vesicles observed in Fig. 6B was qualitatively and
quantitatively similar to that reported in assays using pure PC
vesicles(33, 34) . In summary, unlike cytochrome b
, the tryptophan fluorescence of protein kinase C
is insensitive to quenchers at positions 6 or greater on lipid acyl
chains.
Most of the differences in the association of protein kinase
C with vesicles containing either DG or PMA can be accounted for by the
quantitative differences in the affinity of the enzyme for these two
activators and the differences in association rate constants, rather
than by invoking irreversible membrane insertion mediated by phorbol
esters. However, we have found that the association of protein kinase C
with vesicles containing DG reaches equilibrium within minutes whereas
the association with PMA-containing vesicles increases slowly with time (Fig. 7). This slow component might result from
irreversible penetration of protein kinase C into the hydrophobic core
of the membrane. To test this possibility, we took advantage of the
finding that the interaction of protein kinase C with PMA is strong
enough to cause protein kinase C to associate with vesicles containing
only PC and no PS (Fig. 7).
If the slow increase in
membrane association reflects membrane penetration, then a slower
association with lipids in the gel state versus liquid
crystalline state might be expected. Fig. 7shows that protein
kinase C associated with similar kinetics to PMA-containing vesicles in
the gel phase (DPPC; T
= 41.5
°C(35) ; squares) as to vesicles in the liquid
crystalline phase (egg PC; Tm<0 °C; circles).
Curiously, the enzyme bound with approximately three times higher
apparent affinity to the lipids in the gel phase compared with those in
the liquid crystalline phase. Consistent with no significant
differences in the interaction with both membranes, the PMA-dependent
activation of protein kinase C was similar in the presence of DPPC or
egg PC vesicles: PMA (1 mol %) in DPPC membranes activated protein
kinase C to 0.12 nmol µg
min
(10% of V
) and 1.8 nmol
µg
min
(34% of V
) using as substrates a peptide based on the
MARCKS protein and a peptide based on the pseudosubstrate of protein
kinase C
, respectively. For vesicles composed of egg PC,
corresponding values were 0.07 and 2.2 nmol µg
min
, respectively. The lack of significant
sensitivity in the kinetics of membrane association and activation to
the lipid phase suggests that any putative penetration of protein
kinase C into the bilayer does not involve significant interaction with
the acyl chains.
The foregoing results indicate that protein kinase C's high affinity membrane interaction induced by phorbol esters is reversible and does not involve significant penetration of the enzyme in the hydrophobic core of the membrane. Several lines of evidence are consistent with protein kinase C-binding membranes via an electrostatic interaction that dominates at relatively low concentrations of phorbol esters, and a non-ionic interaction that dominates at relatively high concentrations of PMA. Each type of interaction is sufficient for membrane binding: high concentrations of acidic lipids result in membrane association in the absence of molecules that bind the phorbol ester domain, and high concentrations of PMA result in protein kinase C binding to neutral lipids. Importantly, both interactions are reversible.
An alternative possibility to account for the lack of effects of brominated PCs on protein kinase C's fluorescence is that the enzyme preferentially binds PS, thus decreasing the local concentration of brominated PC to levels too low to allow interaction with membrane-inserted tryptophans. However, other proteins that preferentially bind anionic lipids are sensitive to brominated PCs, indicating that the highly dynamic behavior of acyl chains precludes this possibility. For example, the tryptophan fluorescence of SecA (39) and the putative membrane-binding domain of cytidylyltransferase (40) were quenched by up to 60% by brominated PCs even though bilayers contained 50 mol % anionic lipids (twice the concentration used in the above experiments); the latter example is particularly relevant because binding of this peptide to anionic lipids displays sigmoidal kinetics consistent with enrichment of the local environment of the peptide with anionic lipids(40) .
Quenching of the intrinsic fluorescence
emission of protein kinase C by spin and fluorescently labeled fatty
acids has been reported(20) . However, the degree of the
reported quenching by 1:1 PC/PS vesicles was identical in the presence
and absence of Ca. Because the association of protein
kinase C with such vesicles in the absence of Ca
is
too weak to be detected(4, 41) , the observed changes
in the intrinsic fluorescence of the enzyme under those conditions are
not likely to reflect membrane association and hence, even less likely,
membrane penetration by the enzyme. Labeling of protein kinase C with a
photoactivable probe residing in the hydrocarbon core of the membrane
has also been reported(21) . However, this labeling was
observed at very low mol % PS, under conditions where no significant
association of protein kinase C with vesicles
occurs(4, 41) , was decreased with increasing
Ca
concentration, and was weaker in the presence of
phorbol esters compared with DG. A second study examining whether
protein kinase C in chromafin granule membranes is labeled with a
photoactivatable probe revealed no significant labeling of protein
kinase C, whether binding was Ca
or PMA mediated. (
)This latter result with natural membranes is consistent
with our lack of evidence for penetration of protein kinase C into
model membranes.
The phorbol ester-induced activity of protein kinase C also displayed little dependence on the state of the membrane. This finding is inconsistent with models of activation of protein kinase C that invoke membrane penetration in order to render the enzyme catalytically competent(17, 45, 46) .
The preceding model accounts for the effects of
phorbol esters on protein kinase C in situ. In particular, the
well-documented increase in the amount of protein kinase C recovered in
membrane fractions of cells stimulated with phorbol esters (e.g. 15, 47, 48) reflects the several orders of magnitude increase in
membrane affinity caused by phorbol esters. This binding is resistant
to chelation because it is of such high affinity. In addition, the
nature of the high affinity interaction indicates that there may be
many cases in vivo where PMA or DG is sufficient to activate
Ca -dependent protein kinase Cs in the absence of an
increase in the intracellular Ca
level. Either high
mol % PS or high lipid concentrations would favor the partitioning of
protein kinase C with membranes. A striking example is provided by rod
outer segments, where the lipid concentrations are so high (0.2 M(49) ) that the interaction of a
Ca
-regulated protein kinase C with PS and DG would be
sufficient for activation of the enzyme at Ca
levels
below the dissociation constant of Ca
from
membrane-bound protein kinase C(24) . In summary, DG and PMA
have similar effects on protein kinase C: they induce an
extraordinarily high affinity interaction of protein kinase C with
membranes that is reversible, but can be of such high affinity that it
occurs in the absence of Ca
, or, in the case of PMA,
absence of PS.