(Received for publication, August 23, 1994; and in revised form, October 24, 1994)
From the
The interaction of protein kinase C (PKC) with lipids was probed by a dual approach. Pyrene-labeled lipid analogues of diacylglycerol, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylcholine (PC) were used both as acceptors of tryptophan excitation energy of PKC and as membrane probes for intra- and intermolecular lipid chain collisions by measuring the ratio of excimer-to-monomer fluorescence intensity (EM). Both in micelles of polyoxyethylene 9-lauryl ether and in dioleoyl-PC vesicles, interaction of PKC with monopyrenyl PS (pyr-PS) in the absence of calcium resulted in a relatively slow decrease of the EM value. This effect on the lipid dynamics was accompanied by quenching of the tryptophan fluorescence of PKC. Addition of calcium resulted in a rapid further decrease of the EM ratio of pyr-PS and in additional quenching of the tryptophan fluorescence. When 4 mol % of pyr-PS was replaced by 0.5 mol % of dipyrenyl-labeled diacylglycerol a decrease of the intramolecular excimer formation rate and tryptophan fluorescence could only be detected in the presence of calcium and PS. Strong binding was also observed with dipyrenyl-labeled PIP (dipyr-PIP), but not with the other dipyrenyl-labeled lipids: PI, PS, or PC. In addition, the EM ratios of dipyr-PIP were not affected by phorbol 12-myristate 13-acetate, indicating that phorbol 12-myristate 13-acetate and dipyr-PIP can bind simultaneously to PKC.
Protein kinase C (PKC) ()is a
serine/threonine-specific kinase involved in cellular
signaling(1) . Interaction with specific lipids is essential in
the activation of this regulatory
kinase(2, 3, 4, 5, 6, 7) .
The components required for activation of the various PKC-isozyme
families (nPKC, cPKC, and
PKC) have now been established by
extensive biochemical, biophysical, and genetic analysis. Essential
factors are anionic lipids(8, 9) , in particular L-phosphatidylserine (PS) (10, 11, 12, 13) and (for cPKC)
calcium. In addition, cPKC and nPKC families require for maximal
activation lipid-like cofactors like sn-1,2-diacylglycerol
(DG) or phorbol esters
(PMA)(14, 15, 16, 17) . Binding to
these cofactors is highly (stereo-)
specific(3, 4, 18, 19) . Specific
structural elements of PMA were shown to interact with cysteine-rich
sequences of PKC (20) and a one-to-one stoichiometry for the
binding of both DG and PMA to PKC has been
reported(4, 15, 21, 22) . Although
the components for PKC activation have been identified now, very little
is known about the mutual dependencies of the interaction of PKC with
these factors. Especially the role of calcium in the PKC-lipid
interaction and/or in the activation of cPKC is unclear. Most initial
investigations of the lipid modulation of PKC activity were performed
with PKC preparations from rat brain which is mainly composed of the
calcium-dependent isozymes(9, 23) . Calcium has long
been considered to be the cofactor that regulates association of this
PKC with anionic lipids(24, 25) . Bell and co-workers (25) have proposed a model for
PKC
Ca
PS
DG complexation. In this
model calcium stabilizes the binding of PKC to PS containing membranes
(see also (2, 3, 4, 6) ). The
interaction of the PKC
Ca
PS complex with
DG induces a release of a pseudo-substrate domain from the active site,
thereby allowing access of
substrates(26, 27, 28, 29) . This
calcium-dependent lipid interaction is essentially reversible (2, 24, 30, 31) and insufficient for
activation of PKC(24) . Furthermore, it was shown that after
initial association, PKC penetrates the membrane which is accompanied
by some additional conformational changes which led to partial calcium
independence of the
binding(24, 31, 32, 33, 34) .
Cofactors like DG and PMA might stabilize this inserted active PKC.
According to this concept calcium modulates the lipid binding of PKC by
inducing conformational changes in the protein. Alternatively, calcium
may bind more aspecifically to PKC or the membrane, thereby affecting
the electrostatic potential at the membrane surface (by neutralization
of repulsive electrostatic interaction forces between PKC and the
membrane (35) and/or the lipid organization in the
bilayer(34, 36) ). Furthermore, calcium is known to
affect hydrophobic interactions (37) and hydration between
phospholipids and membrane-bound protein. These more aspecific effects
of calcium can have dramatic effects on binding of PKC to membranes,
although they do not appear to be of major importance in the
association of PKC with lipids(34, 36) .
In contradiction with earlier observations, Lester and co-workers (31) demonstrated that both association and subsequent penetration of PKC into membranes is independent of divalent cations, but very sensitive to pH. Using circular dichroism, large and specific conformational changes were observed upon binding to phosphatidylserine vesicles in the absence of calcium(31, 38) . Based on these observations it was speculated that the inactive form of PKC may be loosely associated to the membrane. At odds with the concept of calcium modulation of lipid binding, these authors considered phospholipids as modulators for the binding of calcium to PKC(31, 34) . The enzyme is first associated with the lipids and then binds calcium which is needed for activity. Cofactors like DG shift the calcium dependence of PKC activity to lower concentrations of calcium(39, 40) .
In the present report the binding of PKC to PS and DG is re-evaluated by simultaneous observation of quenching of PKC tryptophan fluorescence by resonance energy transfer and of lipid dynamic properties at various conditions. In these studies pyrene-labeled analogues of DG, PS, phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylinositol 4-phosphate (PIP) dispersed in vesicles or mixed micelles served not only as acceptors of tryptophan excitation energy but also as reporters of lipid acyl chain dynamics by measuring the ratio of excimer-to-monomer fluorescence intensity (EM) of the pyrene moiety. Formation of a protein-lipid complex resulted in an increased quenching of tryptophan fluorescence and in a reduced EM ratio of the pyrene lipids. A major advantage of this approach is the simultaneous employment of two independent methods for registration of the same events from both lipid and protein viewpoints. In addition, the approach allowed on-line and specific detection of interaction between PKC and the various lipids, and, combined with the high sensitivity, enabled the use of low concentrations of protein and lipid probes.
Figure 1:
A,
probing of PKC binding to Thesit micelles containing 10 mol % brain PS,
4 mol % pyr-PS (-) or pyr-PC () from the
tryptophan fluorescence quenching of PKC. The upper curve represents the PKC fluorescence signal in presence of PS
containing micelles without pyrene lipids. B, parallel to the
registration of the PKC tryptophan fluorescence quenching the pyrene EM
ratio of pyr-PS (-) or pyr-PC (
) was monitored.
As a control experiment, the EM values of pyr-PS in micelles were
measured in buffer only(- - -). The experiments were performed in 20
mM Tris buffer (pH 7.5, 120 mM NaCl, 5 µg/ml
leupeptine, and (initially) 50 µM EGTA) at 293
K.
In the absence of calcium (50 µM EGTA) the tryptophan fluorescence decays slowly to a level of 80% of the original intensity when pyr-PS is present in the micelles (Fig. 1A). This effect is not observed if unlabeled micelles are present (dashed line in Fig. 1A). Addition of extra EGTA to a final concentration of 200 µM (or even higher) does not affect the tryptophan emission. Upon PKC addition the EM ratio of pyr-PS decays from 0.61 to an almost steady level of 0.51 (Fig. 1B). This effect is not observed if buffer is added (dashed line in Fig. 1B) instead of PKC. It can thus be concluded that the decrease in intermolecular collision frequency of the pyr-PS molecules is a consequence of the interaction of PKC with the micelles. When pyr-PC is used instead of pyr-PS in the Thesit/PS micelles, a similar but less strong reduction of the EM ratio of pyr-PC is observed (dotted line in Fig. 1B). The calcium independent interaction with PKC apparently reduces the motional freedom of pyr-PS by direct interaction with these lipids. The fact that binding of PKC to unlabeled PS in micelles containing pyr-PC does not leave the intermolecular collisions of this pyrene lipid unaffected, indicates that noninteracting PC molecules in the micelle also trace the binding of PKC. Furthermore, the binding of PKC to pyr-PS containing micelles results in quenching of tryptophan fluorescence. Since the average distance between tryptophan donors of PKC in solution and pyr-PS in micelles is too large for effective energy transfer, the quenching of tryptophan fluorescence independently confirms a calcium-independent interaction of PKC with pyr-PS, as was concluded from the EM experiments. Pyr-PC is also able to quench the tryptophan fluorescence of PKC, but weaker than pyr-PS, indicating that pyr-PC is not interacting with PKC but is rather randomly distributed in the micelle. A rapid, further reduction of both tryptophan fluorescence and of the EM ratio is observed when calcium (0.4 mM) is added (see Fig. 1B). This observation suggests a calcium-dependent reduction of the lipid dynamics and a more efficient quenching of tryptophan residues in PKC. Upon addition of an excess of EGTA (5 mM), the calcium-induced reduction of both the EM ratio and tryptophan fluorescence is largely recovered to their values observed before addition of calcium. The calcium-dependent reduction and EGTA-induced recovery of both tryptophan fluorescence and EM ratios can be repeated several times per experiment, indicating that the action of EGTA is based primarily on the removal of calcium and that detergent-like effects of EGTA (52) play a minor role. The calcium dependence of the quenching and excimer characteristics can be explained by a calcium-dependent shift of the equilibrium of free and bound PKC to the bound form. Alternatively, it can be envisaged that addition of calcium does not affect the relative amount of PKC bound to micelles, but induces a (partial) insertion of PKC into the hydrophobic core of the micelle. This insertion leads to a closer average distance between the tryptophan donors in PKC and the pyrene acceptors in the micelle and thus to more efficient quenching and to simultaneous changes in the lipid dynamics.
Measurement of the calcium and phospholipid-dependent PKC activity essentially as described by Snoek et al.(53) , showed that in the absence of calcium, the enzyme is slightly stimulated by DOPC membranes that contain 10 mol % of dioleoylphosphatidylserine and 0.5 mol % of DG (data not shown). Apparently, the calcium-independent interaction of PKC with these membranes leads to partial activation of the enzyme. Subsequent addition of calcium induced a strong additional activation of the enzyme.
Figure 2: Dependence of the EM values of pyr-PS and of the tryptophan fluorescence of PKC on the NaCl concentration in the buffer solution. In the left panel the EM values are presented as determined in the presence of PKC at various concentrations of NaCl (&cjs2108;). During the experiments the PKC concentration was kept constant at 0.4 µM. The lowest bar in the left panel represents the EM ratio of pyr-PS in the absence of PKC (&cjs2113;). In the right panel the parallel monitored tryptophan fluorescence intensities of PKC are presented (&cjs2108;), normalized to the fluorescence intensity obtained with unlabeled micelles (&cjs2112;). See legend to Fig. 1for more experimental details.
Stepwise titration of NaCl up to a concentration of 120 mM gradually changes both the EM ratio and tryptophan fluorescence to their values obtained initially in the presence of 120 mM NaCl. The lowest bar in the left panel represents the EM ratio of pyr-PS in absence of PKC. This ratio was essentially the same at the various NaCl concentrations, indicating that the increase of the EM ratio and of tryptophan fluorescence originates from reduction of the overall interaction forces between PKC and the micelles by NaCl. Further increase in the NaCl concentration (>120 mM) does not lead, however, to a further increase of both signals. Apparently, hydrophobic interaction forces are able to keep at least a part of the PKC population associated to the micellar surface at an ionic strength that approaches the physiological one, confirming the fluorescence studies of other research workers(2, 34, 54) .
Figure 3: A, C, and D, EM values (left panels) of dipyr-DG and dipyr-PC and of the parallel-monitored tryptophan fluorescence intensities of PKC (right panels) measured in the absence of PKC and calcium (&cjs2113;) and then after sequential addition of calcium (0.4 mM, &cjs2098;), PKC (0.4 µM, &cjs2108;), and EGTA (5 mM, &cjs2098;&cjs2108;). In B, PKC was added (&cjs2109;) before the addition of calcium (dark &cjs2108;). The dipyrene lipids were dispersed in Thesit micelles containing 10 mol % PS or PC. See legend to Fig. 1for more experimental details.
The tryptophan fluorescence quenching by dipyr-DG is relatively small as compared to that obtained with pyr-PS since the concentration of dipyr-DG in the micelles is four times lower than in pyr-PS experiments. When this experiment is repeated with addition of buffer instead of PKC, no effect on the excimer formation is observed (data not shown). Like the pyr-PS experiment in the previous section, the interaction of PKC with dipyr-DG changes the motional properties of the lipid chains and results in quenching of the tryptophan fluorescence. The reduction of excimer formation of dipyr-DG is independent of the sequence of addition of the various compounds as is shown in the experiment described in Fig. 3B. When PKC is added in the absence of calcium, the excimer formation is only slightly reduced, indicating that dipyr-DG hardly traces the PKC which interacts in a calcium independent fashion with PS lipids of the micelle. Addition of calcium to this sample with PKC then leads to a further reduction of the excimer formation to similar values as obtained in the experiment described in Fig. 3A. Furthermore, if unlabeled PS (10 mol %) in these micelles is replaced by PC (10 mol %), no effect is observed even when both PKC and calcium are present in both excimer formation and tryptophan fluorescence experiments (Fig. 3C). Similarly, experiments with dipyr-PC instead of dipyr-DG also did not give a change, even when 1 mol % of unlabeled DG is present in the micelles (Fig. 3D). It is thus clear that the excimer formation of dipyr-DG, but not that of dipyr-PC, is only affected when the three components calcium, PS, and PKC are simultaneously present, independent of the sequence of addition. This effect can only be explained by a physical interaction between PKC and dipyr-DG which is not established if calcium or PS are lacking. This conclusion is confirmed by the quenching of tryptophan fluorescence which accompanies the effect on excimer formation. All experiments presented in Fig. 3were also performed in vesicles instead of in micelles. The EM values obtained in these membrane bilayers were fully comparable with those obtained in micelles, but the standard errors of the EM values are larger, which is probably due to a lower reproducibility of the vesicle preparation. Two sets of sequential experiments with dipyr-DG and dipyr-PC in DOPC/PS vesicles (8:2) are presented in Fig. 4.
Figure 4: EM values of dipyr-DG and dipyr-PC dispersed in DOPC/PS (8:2) vesicles in the absence of PKC and calcium (&cjs2113;) and then after sequential addition of PKC (0.4 µM, &cjs2109;), calcium (0.4 mM, &cjs2108;), and EGTA (5 mM, dark &cjs2098;&cjs2108;). The total lipid concentration was 20 µM and the probe:lipid ratio was 1:50. See legend to Fig. 1for more experimental details.
Both explanations proposed for the calcium dependence of PKC-PS binding (Fig. 1) may fit to the calcium modulation of DG binding observed in Fig. 3and Fig. 4. When calcium simply enhances the binding of PKC to PS lipids, the probability of finding a PKC molecule at the micellar surface is larger when calcium is present than when it is not. Consequently, the probability of interaction of PKC with dipyr-DG is enhanced. Alternatively, calcium may facilitate the binding of dipyr-DG to PKC more directly by changing the protein conformation in such a way that its binding to dipyr-DG is altered.
Figure 5: EM values of dipyrenyl phosphoinositides and dipyr-PS dispersed in Thesit micelles containing 10 mol % PS or PC. These values were obtained in the absence of PKC and calcium (&cjs2113;), then after sequential addition of PKC (0.4 µM, &cjs2109;), calcium (0.4 mM, &cjs2108;), and EGTA (5 mM, dark &cjs2098;&cjs2108;). See legend to Fig. 1for more experimental details.
Figure 6: EM values of dipyr-DG dispersed in Thesit micelles containing 10 mol % PS and various mole fractions of unlabeled DG. The values were obtained in the presence of PKC (0.4 µM) and calcium (0.4 mM). See legend to Fig. 1for more experimental details.
It has previously been reported that DG and PMA have a common binding site(14, 56) . When unlabeled phorbol ester (PMA) is added to a micellar solution containing PKC and dipyr-DG, the reduced EM ratio is instantaneously restored to the original value measured in the absence of PKC (Fig. 7A). Apparently, PMA binds to PKC and is able to completely reverse the effect of PKC on dipyr-DG. This can be explained by assuming that PMA and dipyr-DG are not able to bind simultaneously to the same PKC molecule. This inverse binding relationship might originate from a shared binding site of DG and PMA or allosteric effects like, for instance, a PMA stabilized PKC conformation which is not able to interact with dipyr-DG. Since in similar binding experiments dipyr-PIP and dipyr-DG were almost equally affected by PKC, it is of interest to see if PMA is able to replace dipyr-PIP. A similar replacement would provide some evidence for a shared binding site of dipyr-DG and dipyr-PIP. However, no significant recovery of the EM values upon addition of PMA is observed (Fig. 7B). This indicates that the binding of PMA to PKC does not induce a dissociation of dipyr-PIP from the PKC surface, and both molecules are able to bind simultaneously to PKC.
Figure 7: EM values of dipyr-DG and dipyr-PIP dispersed in Thesit micelles containing 10 mol % PS. The values were obtained in the absence of PKC and calcium (&cjs2113;), then after sequential addition of PKC (0.4 µM, &cjs2109;), calcium (0.4 mM, &cjs2098;), and PMA (0.5 µM, &cjs2098;&cjs2108;). See legend to Fig. 1for more experimental details.
In this paper the binding of PKC to PS and DG is re-evaluated
by simultaneous monitoring of quenching of PKC tryptophan fluorescence
by resonance energy transfer to pyrene lipids and of excimer-to-monomer
fluorescence intensity ratio of the pyrene lipids. The simultaneous
registration methods of PKC-lipid interaction reduces experimental
uncertainties and evaluates the binding event from both lipid and
protein viewpoints. In addition, fast and sensitive detection and the
use of close analogues of natural lipid cofactors allows rapid on-line
observation of specific PKC-lipid interactions. Other studies that
evaluated the translational and rotational diffusion rates of the
micelles as function of various buffer and lipid components showed that
only PKC influenced the micelle motional properties. From
these results we conclude that the various buffer and lipid components
only effect the observables of the current binding study when PKC is
present and do themselves not have an impact on the interpretation of
observed effects.
Several mechanisms may contribute to the reduction
of intermolecular excimer formation of pyr-PS and pyr-PC observed in
the presence of PKC (Fig. 1). It can be envisaged that the
interaction with PKC with the vesicular or micellar membranes will
affect the lateral organization of the pyrene lipids. Bazzi and
Nelsestuen (36) observed that PKC induced lateral segregation
of nitrobenzoxadiazolyl-labeled phosphatidic acid in membranes.
Formation of such lipid domains enriched with anionic lipids would
lead, however, to an increase of intermolecular excimer formation of
pyr-PS. Since we observe rather a decrease of excimer formation in both
micelles and vesicles when PKC is added, other mechanisms will dominate
the effects of lipid domain formation, e.g. 1) pyrene lipids
adjacent to inserted protein elements are surrounded by a reduced
number of lipid neighbors and therefore have a reduced probability to
form an excimer. Since a pyrene moiety is separated 10 carbon atoms
from the lipid head group region, segments of PKC have to insert
considerably into the membrane hydrophobic region before a pyrene
moiety is partly shielded from its chain neighbors. In addition, 2)
lateral diffusion of the pyrene lipids within the micelle, and 3)
rotational diffusion of the pyrene moieties in the vicinity of the PKC
surface may be reduced considerably. If these diffusion rates become
smaller than the average fluorescence decay rate of an excited pyrene
(0.02 ns
), the probability of excimer formation
of a pyrene lipid at the protein surface even approaches zero. By
comparing the results of the intermolecular excimer formation of pyr-PS
and pyr-PC with those obtained with dipyr-DG one can draw conclusions
about the motional properties that are influenced by the binding of PKC
to the membranes. The only processes which govern the intramolecular
excimer formation of the dipyrene lipids are the rotational diffusion
and freedom of the pyrene acyl chains. Since calcium independent
association can be probed in intermolecular excimer forming experiments (Fig. 1) but not (or hardly) in the intramolecular excimer
forming experiments (Fig. 3), it can be concluded that the
calcium independent association of PKC mainly affects the lipid-lipid
lateral organization and lipid lateral diffusion within the micelles,
but not the acyl chain rotational order and dynamics. Peripheral
binding of PKC to the membrane surface could, for instance, decrease
the lipid lateral diffusion of the monopyrene lipids. The pyrene lipids
that directly interact with the protein (like pyr-PS) will be laterally
immobilized during the pyrene emission lifetime. Pyrene lipids that do
not interact directly with PKC (like pyr-PC) will still be hindered in
their lateral motions by the interaction of surrounding lipids with
PKC.
When calcium is added in the presence of PKC a strong reduction of the excimer formation of pyr-PS and dipyr-DG is observed in vesicles and micelles. Apparently, in the absence of calcium, loose accommodation of the pyrene lipids in the lattice of surrounding lipid and nonionic detergent molecules allows more extended and/or faster chain motions than in the presence of calcium. The calcium dependence of the quenching and excimer characteristics can be explained by two alternative mechanisms. The first explanation is that in the absence of calcium only part of the PKC population loosely interacts with the micellar surface. Addition of calcium shifts the equilibrium of free and bound PKC to the bound form. This enhanced binding will simply intensify the fluorescence quenching and reduction of the excimer formation of pyr-PS which were already induced by the weaker binding in the absence of calcium. In the absence of calcium, PKC is bound loosely to the micelles, dynamically exchanging between buffer and micellar surface. Under these binding conditions, the time that the protein is retained at the micellar surface is too short for interaction with DG. When calcium is added, the protein becomes more firmly bound to the micellar surface. As a consequence of increased probability of finding the protein at the membrane surface, the probability of the interaction of PKC with dipyr-DG is enlarged. Alternatively, it can be envisaged that in the absence of calcium the majority of PKC molecules interacts peripherally with the anionic micelles. This surface bound PKC is not able to interact with dipyr-DG. Addition of calcium does not affect the relative amount of PKC bound to micelles but affects the protein in such a way that its binding to dipyr-DG and PS are dramatically changed. In this context one might hypothesize that only the calcium bound form of PKC is able to bind dipyr-DG. Calcium may induce an insertion of PKC segments into the hydrophobic region of the micelles. This insertion leads to a closer tryptophan-pyrene distance and thus to more efficient quenching. Accompanied changes in the lipid organization and dynamics induced by protein insertion will lead to an additional reduction of excimer formation of pyr-PS and pyr-PC. Furthermore, direct interaction between PKC and dipyr-DG leads to a large reduction of the acyl chain dynamics of dipyr-DG. Unfortunately, based on the experiments described in this paper, no conclusive distinction can be made between the two different concepts of PKC-membrane interaction. In the presence of calcium, PKC reduces the excimer formation of dipyr-PIP and dipyr-DG considerably, while for the dipyrene analogues of PC, PS, and PI only small effects were observed. In addition, both dipyr-PIP and dipyr-DG do not interact with PKC in the absence of unlabeled PS (see also (55) ). The fact that in competition experiments PMA displaces dipyr-DG, but not dipyr-PIP, from PKC indicates, however, that the PIP analogue does not bind at the cofactor site of PKC. Its binding to PKC is more analogous to PS since both dipyr-PIP and pyr-PS interacts with PKC in the absence of calcium. The fact that the PKC induced reduction of dipyr-PIP excimer formation clearly exceeds that of dipyr-PS suggests that PKC has a higher affinity for PIP than for PS (55) . The failure of binding to dipyr-PIP in the absence of (unlabeled) PS may thus simply be due to insufficient anionic lipid density in the micelles.