Article |
Address correspondence to Michael Schaefer, Institut für Pharmakologie, Freie Universität Berlin, Thielallee 67-73, 14195 Berlin, Germany. Tel.: 49-30-8445-1863. Fax: 49-30-8445-1818. E-mail: schae{at}zedat.fu-berlin.de
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Abstract |
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Key Words: protein kinase C; diglycerides; calcium signaling; signal transduction; protein transport
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Introduction |
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In virtually each cell type and tissue, at least one member of the classical and one of the novel PKC isoforms are coexpressed (Wetsel et al., 1992; Dekker and Parker, 1994; Liu and Heckman, 1998). Stimulation of PLC-coupled receptors results in DAG formation and, via inositol-1,4,5-trisphosphate (InsP3) receptor activation, increases the [Ca2+]i. One might expect that classical and novel PKC isotypes are recruited to the plasma membrane in parallel. Nonetheless, upon receptor stimulation, an isotype-selective translocation of PKCs has been observed in several cell systems (Dekker and Parker, 1994). This isotype-specific membrane translocation of PKC isoenzymes must be based on mechanisms that take effect downstream of receptor activation. Although a number of PKC-interacting proteins sequester activated PKCs to various compartments (Jaken and Parker, 2000), little is known about the mechanisms that control the selectivity of the initial membrane recruitment of distinct PKC isoforms, which is most likely based on a proteinlipid interaction.
We have recently shown that a Ca2+-induced plasma membrane translocation of classical PKCs is a diffusion-driven and diffusion-limited binding process that is characterized by a high collisional coupling efficiency (Schaefer et al., 2001). Because both classical and novel PKCs share a DAG-binding C1 domain, the highly efficient Ca2+-driven plasma membrane association of classical PKCs suggests that, by finally competing for the same acceptor, classical PKCs may prevent translocation of coexpressed novel PKCs. To observe the interplay of PKC translocations in living cells, we generated cyan and yellow fluorescent PKC fusion proteins and applied a multivariate regression algorithm that reliably dissects signals arising from multiple, spectrally overlapping fluorochromes. We provide evidence that an agonist-induced selective membrane recruitment of classical and/or novel PKC isoenzymes can result from a Ca2+-controlled competitive binding to limiting plasma membrane concentrations of DAGs.
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Results |
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A possible Ca2+-controlled competition for the membrane anchorage of classical and novel PKCs was tested by coexpressing CFP- and YFP-tagged PKC fusion proteins and altering the intracellular Ca2+ concentrations. In HEK cells coexpressing PKCCFP and PKC
YFP, maximal stimulation of a coexpressed histamine receptor induced a similar translocation of PKC
(Fig. 4 A) as in cells that expressed PKC
alone (Fig. 3 B). The histamine-induced translocation of PKC
, however, was markedly delayed, and the maximal membrane attachment was reduced in cells that coexpressed PKC
as compared with cells that were not cotransfected with PKC
(Fig. 3 C; Fig. 4 A). In PKC
/PKC
-coexpressing cells, a pretreatment with BAPTA-AM reversed the selectivity of the receptor-induced PKC translocation. The histamine-induced translocation of PKC
was further suppressed, whereas the membrane association of coexpressed PKC
now remained as efficient as in cells that only expressed the PKC
fusion protein (Fig. 4 B as compared with Fig. 3, B and C). In another experiment, we coexpressed the H1 receptor together with PKC
CFP and only trace amounts of PKC
YFP (molar ratio between PKC
CFP and PKC
YFP
5:1). Under these conditions, the histamine-induced translocation of PKC
CFP displayed identical efficiencies and kinetics as shown for PKC
YFP without cotransfected PKC
(Fig. 3 C).
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This concept is corroborated by the finding that low concentrations (20100 µM) of the membrane-permeable di-octanoyl-s,n-glycerol (DOG) selectively recruited PKC to the plasma membrane, whereas PKC
required
10-fold higher DOG concentrations for a comparable plasma membrane association (Fig. 5). The concentration-dependent plasma membrane association was assessed by confocal measurement of transcellular concentration profiles of PKC isoforms 60 s after the addition of DOG (Fig. 5 A). The EC50 for the DOG-induced membrane association of PKC
YFP was
90 µM, and saturation was observed at concentrations >300 µM. The fluorescent PKC
fusion protein accumulated in the plasma membrane only at DOG concentrations >200 µM (Fig. 5 B). Thus, in living cells, the affinity of PKC
to bind either a membrane-permeable DAG analogue or the endogenous DAG species formed by PLC-ß or -
is indeed higher than that of the classical PKC
.
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Up to this point, the competition for DAG binding was observed at high PKC expression levels. This competitive mechanism may also operate at more physiological conditions with lower PKC expression levels and weaker PLC stimulation. We therefore generated a stably transfected HEKPKCYFP cell line that typically expresses
10-fold lower amounts of PKC
YFP per cell than transiently transfected cells. Cells were stimulated with carbachol (100 µM) acting on an endogenously expressed M1 family muscarinic acetylcholine receptor. This treatment caused a markedly weaker plasma membrane association of PKC
YFP as compared with maximal stimulation via a cotransfected H1 receptor (Fig. 7 A). Another estimate to quantify the phosphatidyl-4,5-bisphosphate (PIP2) breakdown by stimulation of the endogenously expressed M1 receptor was based on the membrane dissociation of a PIP2- and InsP3-binding YFP-fused PLC
1 PH domain. The results confirm a remaining 20% PIP2 hydrolysis as compared with stimulation of the transiently coexpressed H1 receptor (unpublished data). As expected from the positive local feedback of InsP3 and Ca2+ at InsP3 receptors, the reduced input into the PLC signaling cascade was still sufficient to generate large [Ca2+]i signals with peak values of 300700 nM (Fig. 7 B), which clearly exceed threshold values for the Ca2+-driven translocation of PKC
. HEKPKC
YFP cells were again transfected with various amounts of a PKC
CFP-encoding cDNA plasmid. Furthermore, the molar ratios between stably expressed PKC
YFP and transiently expressed PKC
CFP were determined at the single-cell level. The transient coexpression of PKC
CFP concentration-dependently suppressed the carbachol-induced plasma membrane docking of PKC
YFP. This reduction was
70% at an
1:1 molar ratio, and saturation of the inhibitory effect was evident at a threefold molar excess of PKC
CFP over PKC
YFP (Fig. 7 C).
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Discussion |
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Imaging of fluorescent PKC fusion proteins has provided a more realistic impression of the dynamics of these signaling molecules (Sakai et al., 1997; Feng and Hannun, 1998; Feng et al., 1998; Almholt et al., 1999; Codazzi et al., 2001). Most strikingly, the repetitive translocation of classical PKCs during oscillatory [Ca2+]i responses was only detectable using these techniques (Oancea and Meyer, 1998; Dale et al., 2001; Schaefer et al., 2001). Currently available data on subtype-specific translocations of PKC isoforms predominantly rely on Western blot analyses of particulate and soluble cell fractions. This approach, however, does not necessarily reflect the distribution of PKCs at a given time point. In particular, during subcellular fractionation, classical PKCs may lose their membrane attachment (Kiley et al., 1990; Fig. 8). A secondary loss of membrane contact of classical PKCs during fractionation may result from (a) release of classical PKCs that are bound solely through a Ca2+-dependent interaction with phosphatidylserine and/or (b) a low affinity of classical PKCs for DAG binding, which is not sufficient to maintain the membrane-bound state if additional stabilization by Ca2+ is lost. The contribution of both mechanisms may be indirectly inferred from the capability of Ca2+-bound classical PKCs to prevent the DAG binding of the novel isoforms. Indeed, when [Ca2+]i was clamped to resting concentrations (5080 nM), translocation of PKC was impaired whereas PKC
appeared to access DAGs more easily.
In living cells, several lines of evidence point to a higher DAG binding affinity of PKC as compared with PKC
. Low concentrations of membrane-permeable DOG preferentially recruited PKC
to the plasma membrane. Second, receptor stimulation in BAPTA-loaded cells effectively translocated PKC
, but not coexpressed PKC
, and, in vascular smooth muscle cells, PMA treatment resulted in a complete translocation of endogenous PKC
, whereas the redistribution of PKC
was only partial. The DAG binding of recombinantly expressed and purified PKC isoenzymes has been determined using phorbol esters as DAG surrogates. Both absolute and relative binding affinities of different PKC isoforms, however, strongly depend on the lipid composition and the dispersion technique to generate artificial membranes (Kazanietz et al., 1993; Dimitrijevic et al., 1995). Therefore, reconstitution experiments do not necessarily reflect the DAG binding of PKC isoforms in living cells. Despite its inability to estimate absolute affinities, simultaneous imaging of PKC isoforms in living cells can be applied to compare the DAG binding of different PKC isoenzymes within their physiological environment. Another advantage of imaging versus extraction experiments is the coverage of the entire time course of a receptor-induced PKC redistribution. Co-imaging of classical and novel PKCs now revealed that the translocation of the classical isoforms precedes the maximum of the membrane anchorage of novel isoforms. For a given cell type and agonist, the observation of selective translocations of PKC isoforms is, therefore, not only influenced by extraction techniques but also by the choice of the incubation time.
The Ca2+-controlled competitive translocation of classical and novel PKCs represents a mechanism by which signal diversification may emerge downstream of receptor activation. Although stimulation of PLC-coupled receptors commonly induces both [Ca2+]i and DAG signals, their relative contribution to the PKC translocation underlies additional regulation. The catalytic activity of phosphoinositide-specific PLCs cleaves PIP2 to form equimolar amounts of DAGs and InsP3. In contrast to the direct interaction of DAGs with PKCs, InsP3 action additionally depends on the filling state and responsiveness of internal Ca2+ stores. Refractory Ca2+ stores have been demonstrated to result from InsP3 receptor downregulation (Wojcikiewicz and Nahorski, 1991; Bokkala and Joseph, 1997; Willars et al., 2001) or from a cGMP-dependent phosphorylation of IRAG, an InsP3 receptor-associated cGMP kinase substrate (Schlossmann et al., 2000). Furthermore, owing to the biphasic effect of Ca2+ and ATP on InsP3 receptor gating, InsP3 can only operate at certain cytosolic conditions (Mak et al., 1998, 1999).
A competition for DAG binding of classical and novel PKCs requires that the number of accessible DAG molecules is limiting. Our data demonstrate that the Ca2+-induced displacement of DOG-bound PKC by PKC
is restricted to low concentrations of the membrane-permeable DAG analogue. The number of DAG molecules that are formed upon receptor stimulation can be roughly estimated. The InsP3 receptor is fully activated in the presence of 10-8 to 10-7 M InsP3 (Mak et al., 1998), which corresponds to a number of 12,000120,000 molecules in a given cell of 2 picoliter volume. In various cell types, phorbol ester binding experiments have revealed a number of 40,000800,000 binding sites per cell (Trilivas and Brown, 1989; Obeid et al., 1990; Combadière et al., 1993). Although these binding sites presumably include other DAG receptors, such as PKD, chimaerins, Munc13-1, or Ras-GRP, the total number of DAG acceptors, including classical and novel PKCs, appears not to be in vast excess over the number of DAGs formed during receptor stimulation.
From measurements of the total cellular DAG, one can calculate that a single cell contains 5 x 105 to 108 DAG molecules (Kiley et al., 1991; Baldassare et al., 1992). The high basal values and the modest increases upon receptor stimulation indicate that a major fraction of the total DAG content cannot be accessible for PKCs. A more detailed analysis has revealed that in resting cells, DAGs with saturated or monounsaturated fatty acids are predominant, whereas PLC activity mainly forms polyunsaturated DAG species, and the hypothesis that distinct DAG species, rather than changes in the total DAG mass, regulate PKC has been raised (Eskildsen-Helmond et al., 1998; Ivanova et al., 2001). A delayed formation of DAGs in the absence of [Ca2+]i signals has been attributed to the catalytic activities of phosphatidylcholine-specific PLCs or PLDs. Whether this second wave of DAG formation leads to PKC activation is still under debate (Pettitt et al., 1997). Furthermore, PKC, via a noncatalytic pathway, is regarded as an activator of PLD activity rather than a target of PLD-induced DAG formation (Singer et al., 1996; Zhang et al., 1999). Consistent with the idea that second messengers formed by PLC activity may be scavenged by heterologously expressed proteins, a modified InsP3 receptor fragment, the "InsP3 sponge," has only recently been introduced as a transfectable tool that intracellularly captures InsP3 and prevents the agonist-induced Ca2+ mobilization from internal stores (Uchiyama et al., 2002). Kinase-dead PKC isoforms have been considered as dominant negative modulators of endogenous pathways (Ohba et al., 1998; Matsumoto et al., 2001; Braz et al., 2002). Although competition of kinase-dead and wild-type PKC for receptor for activated C-kinases binding has been demonstrated (Pass et al., 2001), the dominant negative mechanism is far from being clarified. Our data suggest that dominant negative effects of overexpressed PKCs may also result from scavenging the accessible DAG molecules.
In contrast to experimental settings, physiological responses to hormones or paracrine stimuli have to decode slight changes in agonist concentrations that cause only submaximal receptor occupancy and weaker DAG formation. Therefore, competitive processes between DAG-binding signaling molecules are likely to occur in vivo. The highly efficient Ca2+-driven plasma membrane association of classical PKCs and the superior DAG binding affinity of the novel PKC represent a finely tuned system where [Ca2+]i serves as a switch for the subtype-selective activation of PKCs. As a consequence, the competitive and/or sequential isotype-specific PKC activation is one of the mechanisms through which specific physiological responses may emerge from the signaling network of PKCs.
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Materials and methods |
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Generation of fluorescent PKC fusion proteins and fluorescence imaging
PKC, ß1, and
were COOH-terminally fused to YFP in a custom-made pcDNA3-YFP fusion plasmid as described earlier (Schaefer et al., 2001). CFP fusions were generated analogous to the YFP-fused PKCs but using a custom-made pcDNA3-CFP vector that contains the open reading frame of enhanced CFP (Invitrogen) instead of enhanced YFP. Fluorescence imaging was performed with a monochromator and a cooled CCD camera (TILL-Photonics) connected to an inverted epifluorescence microscope (Axiovert 100; Carl Zeiss MicroImaging, Inc.). A 505-nm dichroic mirror (inflection point) with extended reflectivity (300500 nm) was combined with a 510-nm long pass filter allowing coimaging of fura-2, CFP, and YFP signals. All imaging experiments were performed in a Hepes-buffered solution (HBS) containing 128 mM NaCl, 6 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5.5 mM glucose, 10 mM Hepes (pH 7.4), and 0.2% (wt/vol) BSA. Excitation spectra (320490 nm) of single fluorochromes were taken either in untransfected HEK cells loaded with 4 µM fura-2AM (Molecular Probes) or in cells transiently expressing either CFP or YFP. Fura-2loaded cells were equilibrated for 3 h in HBS supplemented with 10 µM ionomycin and 10 mM Ca2+ or 10 mM EGTA instead of Ca2+ in order to record excitation spectra of Ca2+-bound fura-2 or free fura-2, respectively. Spectra were taken for each fluorochrome, dichroic mirror, and objective used in this study. Reference spectra were stored after subtracting background signals and normalizing the data to the maximal intensity of each dye. For coimaging of fluorescent proteins and [Ca2+]i, HEK cells were loaded with 24 µM fura-2AM for 30 min at 37°C. To obtain a measure for the membrane translocation of PKC isoenzymes, regions of interest were defined over the outer border (typical thickness of regions, 12 µm) and over the cytosol of a cell as shown in Fig. 3 A. Fluorescence intensities over the expected localization of the plasma membrane were averaged (Fmemb) and divided by the mean fluorescence intensity over the cytosol of the same cell (Fcyt). To detect agonist-induced changes in the plasma membrane association, the resulting ratios were normalized to the initial values.
Confocal imaging of CFP- and YFP-fused PKCs was performed with an LSM510 inverted laser scanning microscope and a 63x/1.4 Plan-Apochromat objective (Carl Zeiss MicroImaging, Inc.). CFP was excited with the 458-nm line of an argon laser, and emitted light was collected with a 470500-nm band pass filter. YFP was excited with the 488-nm laser line, and emission was recorded with a 530560-nm band pass filter. For photolysis of caged Ca2+, cells were loaded with 10 µM o-nitrophenyl-EGTA-AM (Molecular Probes; 40 min at 25°C), washed, and incubated for another 30 min in HBS before the experiment. Caged Ca2+ was photolyzed in defined areas by briefly switching the 364-nm line of an argon laser to maximal intensity. Pinholes were adjusted to yield optical sections of 0.71.4 µm.
Regression-based spectral evaluation of multiple fluorochromes
At any combination of excitation and emission wavelengths i, the background-corrected signal of the probe Fi is additively composed of fluorescences emitted from m different dyes fi,m: Fi = (fi,1 ... fi,m). Because fi,m scales with both the unknown relative concentration of the dye cm and its normalized fluorescence intensity at the chosen wavelength settings xi,m (Fig. 1 A), the term fi,m = cmxi,m dissects known (xi,m) and unknown (cm) parts. The signal of the probe can be described as Fi =
(c1xi,1 ... cmxi,m) (Fig. 1, B and C). The determination of fluorescence intensities of the probe at n different combinations of excitation and/or emission wavelengths provides n independent solutions for Fi. The resulting linear systems of equations can be solved if the number of different spectral settings n equals or exceeds the number of unknown relative dye concentrations c1..cm (provided that linear dependence between the solutions can be excluded). For best reliability, we solved overdetermined (n > m) linear systems of equations by means of multivariate, constrained (c1..cm
0), linear regression analyses:
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As a result, the relative concentrations of free (cfura-2) and the Ca2+-bound fura-2 (cCafura-2) are known, and interfering signals of GFP variants are eliminated. Therefore, the equation [Ca2+]i = 224 nM x cCa-fura-2/cfura-2 can be applied to calibrate [Ca2+]i. The regression analysis was repeated for each image pixel at any time point resulting in a spatially and/or temporally resolved calibration of the [Ca2+]i and in a dissection of signals arising from coexpressed CFP- and YFP-fused proteins. Furthermore, the molar ratio between CFP- and YFP-tagged fusion proteins can be assessed at the single-cell level. Based on the equimolar expression of CFP and YFP in an intramolecularly fused CFPYFP tandem protein, a compensation for the lower fluorescence intensity of CFP could be calibrated. In our optical system used for coimaging of [Ca2+]i, CFP, and YFP, the fluorescence of YFP (cYFP) appeared 8.1-fold brighter than for the intramolecularly coupled CFP (cCFP; as detected after complete disruption of fluorescence resonance energy transfer by YFP photobleaching). Thus, the term cYFP:8.1 x cCFP corresponds to the molar ratio between coexpressed YFP- and CFP-fused proteins.
Cell fractionation and immunoblotting
For detection of PKCs in cytosolic and particulate fractions, cells were homogenized by two 5-s pulses of sonification (Branson sonifier) in an ice-cold lysis buffer containing 25 mM Tris/HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 200 µM phenylmethylsulfonyl fluoride and leupeptin/aprotinin (20 µg/ml each). Particulate material was removed (30,000 g, 20 min at 4°C), and supernatants representing the soluble PKC fractions were collected. The pellets were resuspended in lysis buffer supplemented with 1% (wt/vol) Triton X-100, sonified (5 s), and placed on a rocking platform (20 min at 4°C). Supernatants of a second centrifugation (30,000 g, 20 min at 4°C) were assigned as particulate PKC fraction. For immunoblotting, samples were diluted in Laemmli buffer containing 10% ß-mercaptoethanol and subjected to a 10% SDS-PAGE. Separated proteins were electroblotted on nitrocellulose membranes, blocked in PBS containing 5% nonfat dry milk, and probed with polyclonal rabbit anti-PKC or anti-PKC
antisera (1:1,000 in PBS; BD Biosciences) and a secondary, peroxidase-coupled antirabbit IgG antibody (Sigma-Aldrich). Chemiluminescence was detected with a Lumiglo reagent (New England Biolabs, Inc.).
The expression levels of endogenously expressed PKC isoenzymes and of heterologously expressed PKC fusion proteins were assessed in cytosolic fractions prepared from quiescent cells. 20 µg of protein or diluted samples of the transiently overexpressing cells were loaded on the gel. The transfection efficiency in these experiments was >50%, as detected by counting the total number and number of fluorescent cells.
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Footnotes |
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Acknowledgments |
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This study was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
Submitted: 11 March 2002
Revised: 19 September 2002
Accepted: 19 September 2002
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References |
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