©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Interaction between Protein Kinase C and Lipid Cofactors Studied by Simultaneous Observation of Lipid and Protein Fluorescence (*)

(Received for publication, August 23, 1994; and in revised form, October 24, 1994)

Eward H. W. Pap Petra A. W. van den Berg Jan Willem Borst Antonie J. W. G Visser (§)

From the Department of Biochemistry, Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Protein kinase C (PKC) (^1)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 alphaPKC) 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 PKCbulletCabulletPSbulletDG complexation. In this model calcium stabilizes the binding of PKC to PS containing membranes (see also (2, 3, 4, 6) ). The interaction of the PKCbulletCabulletPS 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.


EXPERIMENTAL PROCEDURES

Materials

Bovine brain L-alpha-phosphatidylserine (PS), dioleoyl-PC, dioleoyl-PS, DG, Thesit (polyoxyethylene 9-lauryl ether), EGTA, phospholipase D from Streptomyces sp., phospholipase C from Bacillus cereus were supplied by Sigma.

Purification of PKC

Protein kinase C was purified from the cytosolic extract of homogenized Wistar rat brains similar to the procedure described by Huang and Huang (23) and consecutively by DEAE, phenyl-Sepharose, Sephacryl S200, and polylysine-agarose chromatography. The final PKC preparation (with isozyme composition as described elsewhere (9, 23) was essentially pure as demonstrated by silver staining of a polyacrylamide gel, and was stored at -70 °C in buffer (20 mM Tris, pH 7.9, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM beta-mercaptoethanol) with 25% glycerol (Merck, Darmstadt, Germany, fluorescence microscopy grade).

Synthesis of Dipyrenyl-labeled Phospholipids

Di(pyrenedecanoyl)PC (dipyr-PC) was synthesized by methods described previously(41) . Di(pyrenedecanoyl)-DG was synthesized by a phospholipase C-catalyzed hydrolysis of the diglyceride-phosphate linkage in dipyr-PC essentially as described by Myher and Kuksis(42) . Transphosphatidylation of dipyr-PC by phospholipase D yielded di(pyrenedecanoyl)-PS(43) . The synthesized lipids were purified with high performance liquid chromatography on a silicic acid column (240 times 10 mm, LiChroprep Si 60, Merck, Germany). Elution was performed with an increasing methanol gradient in chloroform. Di(pyrenedecanoyl)-PI and di(pyrenedecanoyl)-PIP were a kind gift of Drs. W. F. Nieuwenhuizen (University of Utrecht).

Preparation of Micelles

Mixed lipid micelles were prepared by drying the required amounts of lipids under a stream of nitrogen in a glass tube followed by solubilization in buffer (1 mM Thesit, 20 mM Tris/HCl (pH 7.5), 120 mM NaCl, and 50 µM EGTA) by vortexing and brief bath sonication. In the binding studies the Thesit concentration was 100 µM and the PS concentration was 10 µM (10 mol %). The fluorescent lipid concentration was 0.5 µM for the dipyrenyl-labeled lipids and 4 µM for the monopyrenyl lipids. The pyrene concentration was estimated by measuring the absorbance at 342 nm in ethanol/Me(2)SO (75:25 v/v) ( = 39,700 M cm). In the fluorescence experiments the PKC concentration was 400 nM.

Preparation of Vesicles

Small unilamellar vesicles were prepared by injecting 5 µl of lipid dissolved in ethanol/Me(2)SO through a Hamilton syringe into 0.5 ml of a magnetically stirred buffer solution (20 mM Tris/HCl (pH 7.5), 120 mM NaCl, and 50 µM EGTA) at room temperature(44) . For further details, see the previous section describing the preparation of micelles.

Methods

Resonance Energy Transfer

The tryptophan fluorescence of PKC is quenched by pyrene lipids according to the resonance energy transfer mechanism. This process involves transfer of excitation energy from the tryptophan residues to the pyrene moieties by a weak coupling of the transition moments involved(45, 46) . The rate of this nonradiative process is highly dependent on the separation of the donor and acceptor molecules(45) . Since the critical Förster distance for a pyrene-tryptophan couple is approximately 2.7 nm, we assume that the majority of the tryptophan quenching originates from pyrene lipids that directly interact with PKC(45) .

Theory of Pyrene Excimer Formation

Next to their resonance energy acceptor ability, the pyrene-labeled lipids are excellent probes to study the organization and dynamics of lipid components in membranes through their ability to form excimers (a complex between a pyrene molecule in the excited state with a pyrene molecule in the ground state(47) . The excimer fluorescence is at higher wavelengths than the monomer. The ratio of excimer to monomer emission of pyrenyl probes is a measure for the collision frequency of the pyrene moieties and can be related to parameters describing membrane dynamics and organization (48, 49, 50) . In this study two kinds of pyrene lipids are employed to investigate PKC-lipid interaction: lipids with one pyrene decanoyl moiety (monopyrene) at the sn-2 position of the lipid and lipids with two pyrene decanoyl moieties (dipyrene) at the terminal of both acyl chains. The collision of monopyrene lipids is an intermolecular event and reports on the lateral lipid organization and dynamics, while intramolecular excimer formation of dipyrenyl lipids is directly related to the local motional freedom and dynamics of lipid acyl chains in the membrane.

Fluorescence Methods

Pyrene and tryptophan fluorescence intensities were monitored successively on a DMX-1000 steady state spectrofluorometer (SLM Aminco, Urbana, IL) with computer-driven excitation and emission monochromators. One measurement cycle consisted of registration of tryptophan fluorescence and of pyrene monomer and excimer fluorescence intensities. The tryptophan residues were excited at 285 nm and their fluorescence was detected at 340 nm. Subsequently, the excitation wavelength was advanced to 347 nm to monitor the pyrene monomer and excimer emission by setting the emission monochromator at 377 and 487 nm, respectively. The experiments were performed at 40 cycles/min. The measurements were corrected for background emission and spectral instrument characteristics. Excitation and emission band widths were set at 4 nm. The measurements were performed at room temperature (20 °C).


RESULTS

The Role of Calcium in the Binding of PKC to PS Containing Membranes

In order to evaluate the role of calcium in the interaction of PKC to PS, the excimer formation of pyr-PS or pyr-PC and their ability to quench tryptophanyl fluorescence of PKC was examined in Thesit/PS micelles and DOPC/PS vesicles. Both lipid systems yielded comparable calcium-independent and calcium-dependent effects on the tryptophan fluorescence and pyrene excimer formation. Fig. 1shows the dependence of the tryptophan fluorescence (Fig. 1A) and of the EM ratio (Fig. 1B) of 4 mol % of pyr-PS (solid line) or pyr-PC (dashed line) in Thesit micelles. In an earlier study it was found that one Thesit mixed micelle consists of approximately 330 surfactant molecules(51) . Thus each mixed micelle contains on the average 13 pyrene lipids. In the absence of PKC the EM values of pyr-PS and pyr-PC scatter around a value of 0.6 as a consequence of collisions between different pyrene lipids. Addition of PKC (at t = 50 s) to a final concentration of 400 nM yields a tryptophan fluorescence signal which is for convenience normalized to unity (the background fluorescence at the tryptophan emission wavelength in the absence of PKC is less than 2%). At this PKC concentration approximately equal molar ratios of PKC and micellar aggregates are present.


Figure 1: A, probing of PKC binding to Thesit micelles containing 10 mol % brain PS, 4 mol % pyr-PS (-) or pyr-PC (bulletbulletbullet) 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 (bulletbulletbullet) 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.

Effect of NaCl on Calcium Independent Binding of PKC to PS

In general several interaction forces are involved in protein-lipid association depending on the membrane or protein system. The range of these forces varies largely. Electrostatic forces dominate the interactions at relatively long distances and, generally, play a major role in peripheral binding of proteins to membranes. In this case both the protein and lipid surfaces carry oppositely charged groups. Hydrophobic peptide domains interacting with the phospholipid acyl chains may contribute to the interaction as well. Since low ionic strength enhances the electrostatic interactions between charged molecules and reduces the hydrophobic ones, evaluation of the tryptophan fluorescence of PKC and excimer formation of pyr-PS in calcium-free buffer at various concentrations of NaCl will yield information about the forces involved in the interaction between PKC and PS. As is shown in Fig. 2, the absence of NaCl leads to a stronger decrease of the EM ratio of pyr-PS and tryptophan fluorescence than in the presence of 120 mM NaCl (see also Fig. 1). The overall effect of a low ionic strength leads apparently to an enhancement of the total interaction between PKC and the micellar surface. This observation indicates that in the absence of NaCl, electrostatic interaction forces play a dominant role in the calcium-independent association.


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) .

Calcium and PS Dependence of DG Binding to PKC

In the experiments described in previous sections the association of PKC with PS containing membranes was evaluated using a relatively high concentration of pyrene-labeled PS. Large effects are only observed when PKC interacts directly with the pyrene-labeled lipid. Since it has been reported that the cofactor DG interacts stoichiometrically with PKC (1:1)(4, 15, 21, 22) , evaluation of its binding to PKC and the role of calcium in this interaction, has to be approached slightly differently from the investigation of PKC-PS interaction. At a molar fraction of DG in the membranes comparable to that used with PS, only a minority of the DG molecules will interact with PKC and, consequently, the interaction will barely influence the EM ratio. At lower molar fractions, the intermolecular excimer formation of pyrene-labeled DG will be very low even in the absence of PKC and thus will not allow the detection of binding. Therefore, dipyrenyl-labeled DG (dipyr-DG) was used instead of a monopyrene analogue to characterize the interaction of PKC with this lipid messenger. The intramolecular excimer formation of these doubly labeled lipids enables detection of interaction with PKC at low mole fractions of labeled DG (1 mol %). Dipyr-DG dispersed in mixed micelles containing 10 mol % PS yielded a value of the EM ratio of approximately 0.54. Further dilution of dipyr-DG in the micelles by addition of an excess of Thesit detergent hardly reduced the EM values indicating that excimer formation mainly originates from intramolecular collisions between the two pyrenes on the same lipid. Analogous to the pyr-PS experiments, the measurements were performed sequentially in the absence of calcium, in the presence of calcium, in the presence of PKC, and then with saturating amounts of EGTA. The values of the EM ratio measured under various conditions are presented in the left panel of Fig. 3. The tryptophan fluorescence monitored in parallel to these pyrene EM measurements is displayed (normalized) in the right panel of Fig. 3. Initially, in the absence of calcium (50 µM EGTA) the values of the EM ratio scatter around a value of 0.54. Addition of calcium has no significant effect on these values. Subsequent addition of PKC results in a 40% reduction of the EM ratio of dipyr-DG when 10 mol % of unlabeled PS is present in the micelles, and leads to tryptophan fluorescence quenching (Fig. 3A). Addition of extra PKC did not give a further reduction of the EM ratio (data not shown), indicating that PKC is already present at saturating concentration. The effect of PKC on the value of the EM ratio of dipyr-DG can be largely, but not completely, reversed by removal of calcium with EGTA as is shown in the last column of Fig. 3A. At the same time, addition of EGTA leads to a significant increase in tryptophan fluorescence, indicating a removal of tryptophan quenching by resonance energy transfer from tryptophan in PKC to dipyr-DG.


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.

Effect of PKC-Membrane Interaction on the Excimer Formation of Pyrene-labeled Phosphoinositides and Phosphatidylserine

In order to investigate the specificity of PKC for other dipyrene lipids, the EM values of dipyrene-labeled phosphoinositides PI (dipyr-PI) and PIP (dipyr-PIP) and of PS (dipyr-PS) were measured in micelles analogous to the dipyr-DG experiments described in the previous section. The EM values of these experiments are given in Fig. 5. In case of dipyr-PIP, relatively large effects of PKC were observed on the EM ratio (Fig. 5A). The reduction of this EM ratio is comparable with that found for dipyr-DG. Apparently, dipyr-PIP interacts with PKC to a similar extent as dipyr-DG. In the absence of unlabeled PS, no interaction is observed indicating that PS is a prerequisite for the interaction of PKC with dipyr-PIP (Fig. 5B) (see also (55) ). Stepwise addition of PKC in the absence of calcium leads to a concentration-dependent reduction of the EM ratio of dipyr-PIP (Fig. 5C) which is not observed when PKC is replaced by buffer. If then calcium is added, the EM ratio reduces to a similar one as obtained in the experiment described in Fig. 5A. The effect of PKC on the EM ratio in the absence of calcium indicates that PKC is able to change the intramolecular collision frequency of dipyr-PIP even without this cation. If the same experiment is performed with a dipyrene analogue of the natural precursor of PIP, dipyr-PI, only small effects are observed (Fig. 5D). When PKC, PS, and calcium are present, only a 7% reduction of the EM ratio is observed as compared with the 35% reduction observed when dipyr-PIP is used. The simplest explanation is that the affinity of PKC for labeled PI is lower than that for PIP. This is remarkable if one bears in mind that the only difference between PI and PIP is a (charged) phosphate group on the 4-position of the inositol head group which is apparently responsible for the binding of dipyr-PIP. The results obtained with dipyr-PS as labeled lipid showed similar effects as were obtained with dipyr-PI (Fig. 5D). Although it has been established in the experiments described in previous sections that PS is required for the interaction of PKC with dipyr-DG and dipyr-PIP, the effects are relatively small. This is probably because dipyr-PS, like dipyr-PI, has to compete with the 10-fold excess of unlabeled PS in the micelles for interaction sites on the protein surface.


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.



Competition Experiments with Unlabeled DG and Phorbol Ester

In order to investigate the ability of unlabeled DG to reverse the EM ratio of dipyr-DG in micelles by replacing this labeled analogue at the protein surface of PKC, a competition experiment was performed. The mole fraction of unlabeled DG in the mixed micelles was varied while keeping that of dipyr-DG constant to 1 mol %. The EM values obtained in the presence of PKC and calcium are presented in Fig. 6. Unlabeled DG is capable to compete with dipyr-DG, but especially at higher concentrations of DG, the EM values are highly scattered, and irreproducible, indicating that secondary effects like instability of lipid aggregates at high concentrations of DG may play a role in these experiments (note that DG is a bilayer-to-hexagonal phase promoter). The actual reason for this has to be investigated further systematically.


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.




DISCUSSION

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 (approx0.02 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.


FOOTNOTES

*
This research was supported by the Netherlands Foundation for Biophysics under the auspices of the Netherlands Organisation for Scientific Research (NWO) and by the Dutch Cancer Society (KWF). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Agricultural University, Dreijenlaan 3, NL-6703 HA Wageningen, The Netherlands. Tel.: 31-8370-82862; Fax: 31-8370-84801.

(^1)
The abbreviations used are: PKC, protein kinase C; DG, diacylglycerol; dipyr-DG, sn-1,2-(pyrenyldecanoyl)DG; dipyr-PC, sn-1,2-(pyrenyldecanoyl)PC; dipyr-PI, sn-1,2-(pyrenyldecanoyl)PI; dipyr-PIP, sn-1,2-(pyrenyldecanoyl)PIP; dipyr-PS, sn-1,2-(pyrenyldecanoyl)PS; DOPC, dioleoylphosphatidylcholine; EM, ratio of excimer-to-monomer fluorescence intensities; PC, phosphatidylcholine; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PMA, phorbol myristate acetate; PS, phosphatidylserine; pyr-PC, sn-2-(pyrenyldecanoyl)PC; pyr-PS, sn-2-(pyrenyldecanoyl)PS; Thesit, polyoxyethylene 9-lauryl ether.

(^2)
Pap, E. H. W., Ketelaars, M., Borst, J. W., van Hoek, A., and Visser, A. J. W. G., submitted for publication.

(^3)
Bastiaens, P. I. H., Pap, E. H. W., Widengren, J., Rigler, R., and Visser, A. J. W. G.(1994), J. Fluorescence, in press.


REFERENCES

  1. Nishizuka, Y. (1989) J. Am. Med. Assoc. 262, 1826-1833
  2. Bazzi, M. D. & Nelsestuen, G. L. (1988) Biochemistry 27, 7589-7593 [Medline] [Order article via Infotrieve]
  3. Bazzi, M. D. & Nelsestuen, G. L. (1989) Biochemistry 28, 3577-3585 [Medline] [Order article via Infotrieve]
  4. Brockerhoff, H. (1986) FEBS Lett. 201, 1-4 [CrossRef][Medline] [Order article via Infotrieve]
  5. Newton, A. C. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 1-25 [CrossRef][Medline] [Order article via Infotrieve]
  6. Nishizuka, Y. (1984) Nature 308, 693-697 [Medline] [Order article via Infotrieve]
  7. Zidovetzki, R. & Lester, D. S. (1992) Biochim. Biophys. Acta 1134, 261-272 [Medline] [Order article via Infotrieve]
  8. Huang, K. P., Huang, F. L., Nakabayashi, H. & Yoshida, Y. (1988) J. Biol. Chem. 263, 14839-14845 [Abstract/Free Full Text]
  9. Sekiguchi, K., Tsukuda, M., Ase, K., Kikkawa, U. & Nishizuka, Y. (1988) J. Biochem. (Tokyo) 103, 759-765 [Abstract]
  10. Burns, D. J., Bloomenthal, J., Lee, M. H. & Bell, R. M. (1990) J. Biol. Chem. 265, 12044-12051 [Abstract/Free Full Text]
  11. Lee, M. H. & Bell, R. M. (1989) J. Biol. Chem. 264, 14797-14805 [Abstract/Free Full Text]
  12. Orr, J. W. & Newton, A. C. (1992) Biochemistry 31, 4661-4667 [Medline] [Order article via Infotrieve]
  13. Orr, J. W. & Newton, A. C. (1992) Biochemistry 31, 4667-4673 [Medline] [Order article via Infotrieve]
  14. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. & Nishizuka, Y. (1982) Biol. Chem. 257, 7847-7851 [Abstract/Free Full Text]
  15. Hannun, Y. A., Loomis, C. R. & Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043 [Abstract/Free Full Text]
  16. Kaibuchi, K., Takai, Y. & Nishizuka, Y. (1981) J. Biol. Chem. 256, 7146-7149 [Abstract/Free Full Text]
  17. Takai, Y., Kishimoto, A., Kikkawa, U., Mori, T. & Nishizuka, Y. (1979) Biochem. Biophys. Res. Commun. 91, 1218-1224 [CrossRef][Medline] [Order article via Infotrieve]
  18. Boni, L. T. & Rando, R. (1985) J. Biol. Chem. 260, 10819-10825 [Abstract/Free Full Text]
  19. Rando, R. R. & Young, N. (1984) Biochem. Biophys. Res. Commun. 122, 818-823 [Medline] [Order article via Infotrieve]
  20. Burns, D. & Bell, R. (1991) J. Biol. Chem. 266, 18330-18338 [Abstract/Free Full Text]
  21. Ganong, B., Loomis, C., Hannun, Y. & Bell, R. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1184-1188 [Abstract]
  22. Gschwendt, M., Kittstein, W. & Marks, F. (1991) Trends Biochem. Sci. 16, 167-169 [CrossRef][Medline] [Order article via Infotrieve]
  23. Huang, K. P. & Huang, F. L. (1986) J. Biol. Chem. 261, 14781-14787 [Abstract/Free Full Text]
  24. Bazzi, M. D. & Nelsestuen, G. L. (1987) Biochemistry 26, 115-122 [Medline] [Order article via Infotrieve]
  25. Bell, R. M. (1986) Cell 45, 631-632 [Medline] [Order article via Infotrieve]
  26. House, C. & Kemp, B. E. (1987) Science 238, 1726-1728 [Medline] [Order article via Infotrieve]
  27. Makowske, M. & Rosen, O. M. (1989) J. Biol. Chem. 264, 16155-16159 [Abstract/Free Full Text]
  28. Mosior, M. & Mclaughlin, S. (1991) Biophys. J. 60, 149-159 [Abstract]
  29. Orr, J. W., Keranen, L. M. & Newton, A. C. (1992) J. Biol. Chem. 267, 15263-15266 [Abstract/Free Full Text]
  30. Souvignet, C., Pelosin, J.-M., Daniel, S., Chambaz, E. M., Ransac, S. & Verger, R. (1991) J. Biol. Chem. 266, 40-44 [Abstract/Free Full Text]
  31. Lester, D., Doll, L., Brumfeld, V. & Miller, I. (1990) Biochim. Biophys. Acta 1039, 33-41 [Medline] [Order article via Infotrieve]
  32. Bazzi, M. D. & Nelsestuen, G. L. (1990) Biochemistry 29, 7624-7630 [Medline] [Order article via Infotrieve]
  33. Bazzi, M. D. & Nelsestuen, G. L. (1991) Biochemistry 30, 971-979 [Medline] [Order article via Infotrieve]
  34. Brumfeld, V. & Lester, D. S. (1990) Arch. Biochem. Biophys. 227, 318-323
  35. Trudell, J. R., Costa, A. K. & Csemansky, C. A. (1989) Biochem. Biophys. Res. Commun. 162, 45-50 [Medline] [Order article via Infotrieve]
  36. Bazzi, M. D. & Nelsestuen, G. L. (1991) Biochemistry 30, 7961-7969 [Medline] [Order article via Infotrieve]
  37. Seelig, J. (1990) Cell Biol. Int. Rep. 14, 353-360 [Medline] [Order article via Infotrieve]
  38. Shah, J. & Shipley, G. G. (1992) Biochim. Biophys. Acta 1119, 19-26 [Medline] [Order article via Infotrieve]
  39. Epand, R. M., Stafford, A. R. & Lester, D. S. (1992) Eur. J. Biochem. 208, 327-332 [Abstract]
  40. Nishizuka, Y. (1986) Science 233, 305-312 [Medline] [Order article via Infotrieve]
  41. Patel, K. M., Morrisett, J. D. & Sparrow, J. T. (1979) J. Lipid Res. 20, 674-677 [Abstract]
  42. Myher, J. J. & Kuksis (1984) J. Biochem. Cell Biol. 62, 352-356
  43. Comfurius, P., Bevers, E. M. & Zwaal, R. F. A. (1990) J. Lipid Res. 31, 1719-1721 [Abstract]
  44. Kremer, J. M. H., van de Esker, M. W. J., Pathmamanoharan, C. & Wiersema, P. H. (1977) Biochemistry 16, 3932-3935 [Medline] [Order article via Infotrieve]
  45. Förster, T. (1948) Ann. Physik. 2, 55-75
  46. Stryer, L. (1978) Annu. Rev. Biochem. 47, 819-846 [CrossRef][Medline] [Order article via Infotrieve]
  47. Förster, T. & Kasper, K. (1955) Z. für Elektrochem. 59, 976-980
  48. Galla, H. J. & Sackman, E. (1974) Biochim. Biophys. Acta 339, 103-115 [Medline] [Order article via Infotrieve]
  49. Sassaroli, M., Vaukonen, M., Perry, D. & Eisinger, J. (1990) Biophys. J. 57, 281-290 [Abstract]
  50. Vanderkooi, J. M. & Gallis, J. B. (1974) Biochemistry 13, 4000-4007 [Medline] [Order article via Infotrieve]
  51. Bastiaens, P. I. H., Pap, E. H. W., Borst, J. W., van Hoek, A., Kulinski, T., Rigler, R. & Visser, A. J. W. G. (1993) Biophys. Chem. 48, 183-191 [CrossRef][Medline] [Order article via Infotrieve]
  52. Wallach, D. F. H. & Zingler, R. J. (1974) in Evolving Strategies and Tactics in Membrane Research , pp. 74-78, Springer-Verlag, Berlin
  53. Snoek, G. T., Feijen, A., Halem, W. J., Rotterdam, W. V. & De Laat, S. W. (1988) Biochem. J. 255, 629-637 [Medline] [Order article via Infotrieve]
  54. Banno, Y., Nakashima, T., Kumada, T., Ebisawa, K., Nonomura, Y. & Nozawa, Y. (1992) J. Biol. Chem. 267, 6488-6494 [Abstract/Free Full Text]
  55. Pap, E. H. W., Bastiaens, P. I. H., Borst, J. W., van den Berg, P. A. W., van Hoek, A., Snoek, G. T., Wirtz, K. W. A. & Visser, A. J. W. G. (1993) Biochemistry 32, 13310-13317 [Medline] [Order article via Infotrieve]
  56. Chauhan, A., Chauhan, V. P. S., Deshmukh, D. S. & Brockerhoff, H. (1989) Biochemistry 28, 4952-4956 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.