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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Anesthesiology. Author manuscript; available in PMC 2010 July 1.
Published in final edited form as:
PMCID: PMC2747769
NIHMSID: NIHMS125728

Propofol Activates and Allosterically Modulates Recombinant Protein Kinase Cε

Peter J. Wickley, Ph.D.,* Ryo Yuge, M.D.,* Brad A. Martin, B.S., Jacob S. Meyer, B.S.,# and Derek S. Damron, Ph.D.

Abstract

Background

Myocardial protection by anesthetics is known to involve activation of protein kinase C epsilon (PKCε). A key step in the activation process is auto-phosphorylation of the enzyme at serine 729. Our objectives were to identify the extent to which propofol interacts with PKCε and to identify the molecular mechanism(s) of interaction.

Methods

Immuno-blot analysis of recombinant PKCε was used to assess auto-phosphorylation of PKCε at serine 729 before and after exposure to propofol. An enzyme-linked immuno-absorbant assay kit was used for measuring PKC activity. Spectral shifts in fluorescence emission maxima of the C1B subdomain of PKCε in combination with the fluorescent phorbol ester, sapintoxin D, was used to identify molecular interactions between propofol and the phorbol ester/diacylglycerol binding site on the enzyme.

Results

Propofol (1 μM) caused a 6-fold increase in immuno-detectable, serine 729 phosphorylated PKCε and increased catalytic activity of the enzyme in a dose-dependent manner. DOG- or phorbol myristic acetate-induced activation of recombinant PKCε activity was enhanced by preincubation with propofol. Both propofol and phorbol myristic acetate quenched the intrinsic fluorescence spectra of the PKCε C1B subdomain in a dose-dependent manner, and propofol caused a further leftward-shift in the fluorescence emission maxima of sapintoxin D following addition of the C1B subdomain.

Conclusions

These results demonstrate that propofol interacts with recombinant PKCε causing auto-phosphorylation and activation of the enzyme. Moreover, propofol enhances phorbol ester-induced catalytic activity suggesting propofol binds to a region near the phorbol ester binding site allowing for allosteric modulation of PKCε catalytic activity.

INTRODUCTION

Protein kinase C (PKC) has been identified as an important signal transduction molecule that regulates a variety of proteins involved in the regulation and maintenance of myocardial function. PKC exists as a family of isoforms, including the conventional PKC’s (α,β1, β2, γ), novel PKC’s (δ, ε, η, θ) and atypical PKC’s (λ,ζ)1. The role(s) of the individual isoforms in mediating cellular mechanisms of regulation in the heart are a subject of great debate. However, many studies have consistently identified that activation of PKCε plays a central role in the signaling pathways and cellular events that provide myocardial protection from ischemia and/or reperfusion injury.25

Activation of PKCε, as well as membrane targeting and substrate specificity, is regulated by several factors, including phosphorylation, diacylglycerol (DAG), and other lipids and anchoring proteins. PKCε is auto-phosphorylated at serine 729 in the hydrophobic motif of the enzyme, rendering it catalytically competent. Subsequent binding of diacylglycerol causes activation of the enzyme by releasing the pseudosubstrate domain from the catalytic site. The binding of diacylglycerol occurs at a tandem repeat of cysteine-rich zinc finger motifs located in the C1 subdomain (C1A and C1B) of the regulatory domain.6

Most volatile and many intravenous anesthetic agents routinely used in the clinical setting for anesthesia and/or analgesia are also capable of providing organ protection from ischemia-reperfusion injury via mechanisms involving activation of PKCε.711 In this study, we assessed whether propofol interacts with recombinant PKCε to activate or allosterically modulate catalytic activity. Specifically, we directly assessed the extent to which propofol binds to the C1B subdomain of recombinant PKCε causing auto-phosphorylation and/or activation of the enzyme. Our major findings are that propofol interacts with the C1B subdomain of PKCε causing auto-phosphorylation at serine 729 resulting in activation of the enzyme. Moreover, propofol enhances phorbol-ester-induced activation of recombinant PKCε indicating that propofol can allosterically modulate enzymatic activity.

MATERIALS AND METHODS

Immuno-Blot Analysis of Recombinant PKCε

The recombinant PKCε protein was expressed using a baculovirus vector and purified from Sf9 insect cells that normally do not express any detectable endogenous PKC activity.12;13 Therefore, the recombinant PKCε protein used in these studies was free of contamination by any other PKC isoforms. Immuno-blot analysis was carried out on recombinant PKCε as previously described for PKC isoforms.14 Protein concentration was assessed using the Bradford method.15 Equal amounts of protein (50 μg) were electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes. Nonspecific binding was blocked with Tris-buffered saline solution (0.1% (v/v) Tween-20 in 20 mM tris base, 137 mM NaCl adjusted to pH 7.6 with HCl, containing 3% (w/v) bovine serum albumin for 1 hour at room temperature. Polyclonal antibodies against PKCε (holoenzyme), and the auto-phosphorylation site at serine 729 (PKCεpS729) were diluted 1:1000 in Tris-buffered saline solution containing 1% bovine serum albumin for immuno-blotting (2 hrs). After washing in Tris-buffered saline solution 3 times (10 min each), filters were incubated for 1 hr at room temperature with horseradish-peroxidase-linked secondary antibody (1:5,000 dilution in Tris-buffered saline solution containing 1% bovine serum albumin). Filters were again washed and bound antibody detected by the enhanced chemiluminescence method. The density of the individual bands was analyzed using National Institutes of Health Image software (National Institutes of Health, Washington D.C.).

Affinity Purification of PKCε Following Phosphatase Treatment

PKCε was immuno-purified from phosphatase-treated recombinant PKCε (pt-PKCε) using an Amino-Link Plus Immobilization Kit (Pierce, Biotechnology Inc., Rockford, IL) per manufacturer’s instructions. Briefly, 0.5–1.5 ml of cell lysate, diluted 1:1 in sample buffer (0.1 M phosphate, 0.15 NaCl, pH 7.2), was applied to an Amino-Link Plus-PKCε affinity column, washed, and equilibrated at room temperature. The column was then incubated at room temperature for 60 minutes or overnight at 4°C, washed and eluted with 8–10 ml of ImmunoPure IgG elution buffer. 0.5 ml elution fractions were collected and monitored by absorbance at 280 nm.

Recombinant PKCε Activity Assay

The activity of recombinant PKCε before and after treatment with propofol was measured using a colorimetric PKC activity assay kit (Stressgen Bioreagents, Victoria, British Columbia, Canada), per manufacturer’s instructions. Briefly, a readily PKC phosphorylated substrate (cyclic adenosine monophosphate response element binding protein) was precoated onto the wells of a PKC substrate microtiter plate provided in the kit. The recombinant protein was added to the wells and activation of the enzyme was initiated by addition of ATP in the presence of phosphatidylserine (200 μg/ml). After 90 min of incubation at 30°C, the reaction was terminated by emptying the contents of each well. A phospho-specific substrate antibody was then added to each well followed by a peroxidase conjugated anti-rabbit IgG secondary antibody. Following incubation (30 min, 23°C) and 4 washes, tetramethylbenzidine substrate was added to develop the reaction. The developing reaction was terminated after 45 min with acid stop solution (2 N HCl). The intensity of the color was measured on a microplate reader at 450 nm and the relative kinase activity (compared to untreated, baseline controls) of the samples was calculated from the absorbance measurements.

Fluorescence Emission Spectra

The PKCε C1B subdomain was synthesized and purified by high performance liquid chromatography by the Molecular Biotechnology Core facility located in the Lerner Research Institute at Cleveland Clinic. Fluorescence measurements were performed on a Photon Technology International (Birmingham, NJ) spectrofluorometer equipped with temperature and stirringcontrol for cuvette (1.5-ml quartz) based studies. Recombinant pt-PKCε, C1B subdomain and sapintoxin D (SAPD; at varying molar ratios) weremixed in a buffer (50 mM Tris, pH 7.2) at a final volume of 1 ml with gentle stirring (20 min; 23°C). The molecular interactions between SAPD (0.2 μM) and the C1B subdomain (2 μM) were assessed by evaluating shifts in the SAPD fluorescence emission spectra. Deconvolution of the spectra obtained under these conditions (10:1 molar ratio of protein to SAPD) was utilized to delineate differences in the peaks for the protein bound SAPD (405 nm) from the aqueous SAPD (437 nm).16 To assess allosteric modulation of SAPD binding to the C1B subdomain by propofol, the molar ratio of protein to SAPD was reduced from 10:1 to 2:1 (2 μM C1B subdomain; 1 μM SAPD). For both protocols, emission spectra were acquired from 375 to 575 nm at an excitation wavelength of 355 nm. The C1B subdomain contains 1 tryptophan (Trp 264) affording intrinsic fluorescence to the protein. To assess the effects of propofolor phorbol myristic acid (PMA) on the fluorescence emission spectra of the C1B subdomain, 2-μl aliquots of propofol or PMA (1 μM stock solutions) were titrated against the C1B subdomain (1 μM). Spectra were recordedafter incubation (30 min) with slow stirring. Emission spectra were acquired from 310 nm to 480 nm at an excitation wavelength of 290 nm.

Statistical Analysis

Changes in pt-PKCe auto-phosphorylation, activity or relative fluorescence were compared to baseline values (normalized to 100%) using one-way analysis of variance with repeated measures and the Bonferroni post hoc test. Differences were considered statistically significant at p < 0.05. All results are expressed as means ± SD. Statistical analysis was conducted using NCSS software (Kaysville, UT).

Experimental Protocols

Protocol 1. Effect of Propofol on Auto-Phosphorylation of Recombinant PKCε

Western blot analysis of recombinant PKCε before and after treatment (37°C, 30 min) with PMA (1 μM), propofol (0.1 – 10 μM) or intralipid (IL; 0.1–10 μM) was performed using an antibody (PKCεpS729) recognizing the auto-phosphorylation site (serine 729) on PKCε. Because of significant auto-phosphorylation and constituitive activity of the holoenzyme under baseline conditions (see fig. 1), all subsequent protocols were performed after the recombinant PKCε was subjected to λ phosphatase 200 units, 30 min, 30°C) to remove the phosphate from serine 729 which is( required for catalytic activity of the enzyme. Therefore, λ phosphatase treatment significantly reduces baseline activity of the recombinant protein in the absence of any putative activators allowing for a greater difference in our ability to assess an increase in catalytic activity by the interventions when compared to baseline. PKCε was then immuno-purified by affinity chromatography and referred to as pt-PKCε throughout the text. Baseline auto-phosphorylation in the absence of any intervention was considered the control value and was normalized to 100%. Summarized data are expressed as the ratio of total PKCε to pPKCε (percent of baseline control).

Figure 1
Effect of Propofol on Auto-Phosphorylation of Recombinant PKCε at Ser729 Before and After Treatment with λ Phosphatase

Protocol 2. Effect of Propofol on pt-PKCε Activity

Activity of the immuno-purified pt-PKCε (50 ng) was assessed by colorimetric determination of cyclic adenosine monophosphate response element binding protein (CREB). Propofol, IL, or the PKC activators DOG (50 μM) or PMA (0.1 μM) were added alone or in combination to pt-PKCε and CREB (90 min). pt-PKCε activity was assessed on a microplate reader by assessing absorbance of the colored derivative at 450 nm. Baseline pt-PKCε activity in the absence of any intervention was considered the control value and was normalized to 100%. Summarized data are expressed as a percent of baseline control.

Protocol 3. Effect of PKCε C1B Sub-domain on Fluorescence Emission Spectra of SAPD

The fluorescence emission spectra of SAPD (0.2 μM) was assessed in 50 mM Tris buffer (pH 7.4), before and after addition of a 10 fold excess of PKCε C1B subdomain (2 μM; 30 min). SAPD was excited at 355 nm and emission spectra were collected from 375 nm to 575 nm. Control experiments were also performed to assess the effect of denaturing by boiling (90°C, 20 min) the PKCε C1B subdomain on SAPD fluorescence emission spectra.

Protocol 4: Effect of Propofol on Intrinsic Fluorescence of PKCε C1B-Subdomain

We monitored the intrinsic fluorescence of the PKCε C1B subdomain during titration with propofol, intralipid or PMA as an indicator of direct binding to the protein. Fluorescence emission spectra of the PKCε C1B subdomain (1 μM) were assessed in 50 mM Tris buffer (pH 7.4) before and after successive additions of PMA (0.1 μM– 1 μM), propofol (0.1–1.0 μM) or intralipid (IL; 1 μM). Excitation was 290 nm and emission spectra were collected from 310 to 480 nm.

Protocol 5: Effect of Propofol on SAPD Binding to PKCε C1B Subdomain

Experiments were performed as described under Protocol 3 except the molar ratio of SAPD to PKCε C1B subdomain was reduced from 10:1 to 2:1. Fluorescence emission spectra for SAPD alone (1 μM) with and without propofol (1 μM) and/or the PKCε C1B subdomain (2 μM) were recorded as described in Protocol 3. Control experiments were also performed to assess the effect of intralipid on the fluorescence emission spectra for SAPD alone and for SAPD in the presence of PKCε C1B subdomain.

Materials

Recombinant PKCε expressed and purified from insect Sf9 cells was obtained from Panvera (Carlsbad, CA). Monoclonal antibodies for PKCε and PKCεpS729 were purchased from Cell Signaling (Beverly, MA). PMA and DOG were purchased from Sigma Chemical Co. (St. Louis, MO). SAPD was obtained from Alexis (San Diego, CA). The PKCε C1B subdomain was synthesized and purified by high performance liquid chromatography by the Molecular Biotechnology Core facility (Lerner Research Institute at Cleveland Clinic, Cleveland Ohio). Propofol and the IL vehicle were obtained from Cleveland Clinic Pharmacy.

RESULTS

Effect of Propofol on Auto-Phosphorylation of Recombinant PKCe

Western blot analysis of recombinant PKCε before and after treatment (37°C, 30 min) with IL, PMA or propofol was performed using anti-PKCεpS729. In the absence of any intervention, recombinant PKCε exhibited some degree of auto-phosphorylation (fig. 1, panel A). PMA and propofol, but not IL, all increased the amount of detectable recombinant PKCεpS729, compared to baseline. Because of the high degree of baseline auto-phosphorylation of recombinant PKCε, we incubated recombinant PKCε with λ phosphatase 200 units, 30 min, 30°C) and the pt-PKC ( ε was then immuno-purified using affinity chromatography. The pt-PKCε exhibited minimal auto-phosphorylation (fig. 1, panel B). IL, PMA and propofol markedly increased auto-phosphorylation of pt-PKCε. The summarized data for the effects of IL, PMA, and propofol on auto-phosphorylation of PKCε or pt-PKCε are presented in panels C and D, respectively.

Effect of Propofol on PKCe Activity

We assessed the extent to which propofol, IL and classical PKC activators increase activity of pt-PKCε. Activation of pt-PKCε was assessed measuring the extent to which the enzyme phosphorylates CREB. Propofol, IL, or the PKC activators DOG or PMA were added alone, and in combination, with pt-PKCε into the wells containing the substrate, CREB. Propofol caused a dose-dependent increase in phosphorylation of CREB (fig. 2, panel A). IL alone also caused modest phosphorylation of CREB at high concentrations. PMA and DOG increased phosphorylation of CREB to a similar extent (fig. 2, panel B). Pretreatment with DOG or PMA potentiated propofol-stimulated (1 μM) pt-PKCε-dependent phosphorylation of CREB.

Figure 2
Effect of Propofol on Phosphatase-Treated PKCε (pt-PKCε) Activity

Effect of PKCε C1B Subdomain on Fluorescence Emission Spectra of SAPD

In 50 mM Tris buffer (pH 7.4), SAPD (0.2 μM) has an emission maximum (λmax) of 437 nm (fig. 3, trace A). Addition of the PKCε C1B subdomain (2 μM) to SAPD (fig. 3, trace B) caused a shift in the emission spectra that could be deconvoluted into two components with a λmax of 405 nm and 437 nm representing bound and free SAPD, respectively (fig. 3, traces C and D). Boiling (90°C; 30 min) the C1B:SAPD solution resulted in an emission spectra with a single λmax centered at 437 nm (data not shown). Similarly, a single λmax of 437 nm was observed when the C1B subdomain was boiled (90°C, 20 min) prior to mixing with SAPD (see inset, fig. 3).

Figure 3
Effect of PKCε C1B Sub-domain on Fluorescence Emission Spectra of Sapintoxin D

Effect of Propofol on Intrinsic Fluorescence of PKCε C1B-Subdomain

The PKCε C1B subdomain contains a single tryptophan (Trp-264) which showed a λmax of 348 nm (fig. 4, panel A, left). The intrinsic fluorescence of PKCε C1B subdomain (1 μM) was quenched by successive addition of 0.1 μM propofol (panel A, left, lower traces) in a concentration dependent manner. Summarized data for the concentration dependent effects of propofol (0.1–1.0 μM) and the effect of intralipid (1 μM) are depicted in panel A, right.

Figure 4
Effect of Propofol and PMA on Intrinsic Fluorescence of PKCε C1B Subdomain

Similarly, the intrinsic fluorescence of PKCε C1B subdomain (1 μM) was quenched by successive addition of 0.1 μM PMA (fig. 4, panel B, left) in a concentration dependent manner. Summarized data for the concentration dependent effects of PMA (0.1–1.0 μM) are depicted in panel B, right.

Effect of Propofol on SAPD binding to PKCε C1B Subdomain

As shown in figure 3, the emission spectra of SAPD in the presence of a 10 fold excess of protein consists of overlapping free and bound contributions with a λmax of 437 and 405, respectively. However, when we performed experiments in which the C1B (2 μM) to SAPD (1 μM) molar ratio was reduced from 10:1 to 2:1, the composite peaks were more symmetrical and exhibited a λmax of about 437 nm for the free and 425 nm for the bound (fig. 5, trace A and C, respectively). Propofol did not have any effect on the SAPD emission spectra in the absence of the PKCε C1B subdomain. (fig. 5, trace B). However, in the presence of the PKCε C1B subdomain, addition of propofol (1 μM) caused more SAPD to bind resulting in an even further leftward shift that had a λmax of about 415 nm (fig. 5, trace D). In contrast, intralipid (1 μM) had no effect on the SAPD λmax alone or in the presence of the PKCε C1B subdomain (see inset, fig. 5). Summarized data for the effects of propofol (1 μM), intralipid (1μM) and PKCε C1B subdomain on SAPD emission spectra are depicted in panel B.

Figure 5
Effect of Propofol on SAPD binding to PKCε C1B Sub-domain

DISCUSSION

This is the first study to directly assess the effects of propofol on molecular mechanisms regulating activation of purified, recombinant PKCε. Previous studies, using purified rat brain PKC under various assay conditions have demonstrated that propofol and halothane can stimulate PKC activity,17 however the PKC isoforms affected and molecular mechanisms of activation have not been identified. These studies also did not address the potential for anesthetic-induced, allosteric modulation of PKC activity by phorbol esters and/or other lipid activators such as DOG or free fatty acids.17 A recent study from our laboratory demonstrated that propofol activates PKCα, PKCδ, PKCε and PKCζ, resulting in their translocation to distinct intracellular sites in cardiomyocytes.14 In addition, previous studies from our laboratory utilizing pharmacological inhibitors of PKC have demonstrated propofol-induced, PKC-dependent alterations in cardiomyocyte intracellular free Ca2+ concentration and shortening,18 α adrenoreceptor19 and β adrenoreceptor20 signal transduction, phosphorylation of contractile proteins21, Na+-H+-activity and intracellular pH and myofilament Ca2+ sensitivity.22 The key finding of the current study is that propofol activates and allosterically modulates activation of purified, recombinant PKCε via a molecular interaction with PKCε at or near the phorbol ester/diacylglycerol binding domain (C1A and/or C1B subdomain). A schematic diagram depicting domains of the PKCε holoenzyme and proposed molecular interaction between propofol and the PKCε C1B subdomain is shown in figure 6.

Figure 6
Schematic Diagram Depicting the Domains of PKCε Holoenzyme and Proposed Molecular Interaction Between Propofol and the PKCε C1B Sub-domain

Effect of Propofol on Auto-Phosphorylation of Recombinant PKCε

A key step in the activation process of PKC isoforms is the binding of endogenous mediators (diacylglycerol, free fatty acids) at a tandem repeat of cysteine-rich zinc finger motifs (C1A and C1B) contained in the regulatory domain.6 Binding of diacylglycerol causes activation by the enzyme by releasing the pseudo-substrate domain from the catalytic site and allowing for auto-phosphorylation of the enzyme on serine 729 in the C terminal hydrophobic motif.23 We identified that the recombinant PKCε used in these studies exhibits a significant degree of auto-phosphorylation at serine 729 that is markedly reduced by treatment with λ phosphatase. Moreover, the extent to which PMA and propofol stimulate auto-phosphorylation of recombinant PKCε is significantly enhanced following dephosphorylation of serine 729 with λ phosphatase. The IL at concentrations 10X higher than propofol was also capable of stimulating some auto-phosphorylation. This is likely due to the presence of fatty acids (soy bean oil) and phospholipids (lecithin) which create the lipid emulsion facilitating solubility of propofol. Fatty acids are known to activate some isoforms of PKC and phospholipids serve as co-factors for activation.24 These data provide molecular evidence for an interaction between propofol and recombinant PKCε and support our previous pharmacological studies demonstrating propofol-induced, PKC-dependent alterations in cardiomyocyte signal transduction and function.14;2022;25

Effect of Propofol on PKCε Activity

Auto-phosphorylation of PKCε at serine 729 is an important step in the activation of PKCε.23 In order to confirm that the propofol-induced increase in auto-phosphorylation at serine 729 correlated with catalytic activity of the enzyme, we directly measured pt-PKCε activity by assessing the ability of propofol to stimulate PKCε-dependent phosphorylation CREB. Although time course studies were not performed, we have identified that propofol stimulates a dose-dependent increase in catalytic activity of recombinant pt-PKCε, providing a direct correlation between propofol-induced auto-phosphorylation at serine 729 and catalytic activity of the enzyme. The intralipid vehicle also stimulates some catalytic activity at high concentrations consistent with the modest auto-phosphorylation observed at serine 729. Moreover, propofol potentiated PMA and DOG-induced recombinant pt-PKCε activity suggesting that propofol can allosterically modulate catalytic activity induced by other known classical activators of most PKC isoforms, including PKCε. Taken together with the auto-phosphorylation data, these results indicate that propofol increases pt-PKCε activity via an intermolecular interaction with the recombinant protein. Moreover, propofol potentiates pt-PKCε activity following pretreatment with the classical activators, DOG or PMA.

Effect of PKCε C1B Subdomain on Fluorescence Emission Spectra of SAPD

In an effort to establish the efficacy of our experimental methodology with regards to accurately measuring changes in fluorescence emission spectra, we first assessed the effect of PKCε C1B Subdomain on fluorescence emission spectra of sapintoxin D (SAPD). Using SAPD, we determined that the λ max for the emission spectrum of this fluorescent phorbol ester was quenched and exhibited a left shift upon incubation with the PKCε C1B subdomain protein, and that could be deconvoluted into two peaks representing bound and free SAPD. The data in figure 3 demonstrate an intermolecular interaction between SAPD and the PKCε C1B subdomain, confirming the sensitivity of the experimental approach to detect changes in the fluorescence emission spectra of SAPD.

Effect of Propofol on Intrinsic Fluorescence of PKCε C1B-Subdomain

We also took advantage of the tryptophan (Trp 264) that affords fluorescence to the C1B subdomain protein and examined the extent to which propofol or PMA (nonfluorescent) modify the C1B subdomain fluorescence emission spectra. Our findings indicate that propofol and PMA exert similar quenching effects on the emission spectra of the C1B subdomain. These data support a intermolecular interaction of propofol with the C1B subdomain similar to that observed with PMA, and suggest this is the likely mechanism to explain propofol-induced activation of PKCε. These findings are also consistent with a study suggesting the interaction of general anesthetic alcohols with the C1B subdomain of PKCδ, 16 which has greater than 60% conserved homology with the PKCε C1B subdomain. We propose a molecular interaction between the hydrophobic isopropyl groups of propofol with hydrophobic residues on the C1B subdomain near Tyr 250 or Trp 264. The ribbon diagram for the C1B subdomain (fig. 6) illustrates a potential binding pocket for propofol in close proximity to the Tyr 250 and Trp 264 residues where interactions between the hydrophobic side chains could occur resulting in activation of PKCε or allosteric modulation of diacylglycerol binding.

Effect of Propofol on SAPD binding to PKCε C1B Subdomain

We performed experiments to further assess the possibility that propofol allosterically modulates enzymatic activity by examining whether propofol alters SAPD binding to the PKCε C1B subdomain. Our data indicate that when the enzyme to SAPD molar ratio was reduced from 10:1 to 2:1, addition of propofol caused a further leftward shift in the λmax for SAPD. In addition, propofol had no effect on SAPD fluorescence emission spectra in the absence of the PKCε C1B subdomain. These data suggest that propofol allosterically modulates SAPD binding to the PKCε C1B subdomain. Moreover, these data also suggest that propofol likely interacts at a discrete allosteric binding site on the PKCε C1B subdomain and synergistically enhances the binding of SAPD. This may explain our observations that propofol potentiates PMA- or diacylglycerol-induced activity of recombinant PKCε Moreover, because the intralipid had no effect on the emission spectra suggests that the ability of the intralipid to activate recombinant PKCε or cause auto-phosphorylation at Ser 729 is likely due to a molecular interaction of the intralipid at yet another discrete binding site on the PKCε holoenzyme. It should be noted that the data in this study does not fully characterize whether the molecular interaction between propofol and PKCε is direct or allosterically mediated. Our results indicate a molecular interaction between propofol and PKCε, but additional studies incorporating time-resolved analysis of anisotropy would be needed to further delineate the precise nature of this interaction.

Summary and Conclusions

These results demonstrate that propofol interacts with the C1B subdomain of PKCε resulting in auto-phosphorylation and activation of the enzyme. Moreover, propofol enhances phorbol ester binding to the C1B sub-domain indicating propofol can also allosterically modulate enzymatic activity.

Acknowledgments

This study was supported by a National Heart, Lung and Blood Institute (Bethesda, Maryland) Grant HL-65701 (DSD).

Footnotes

Presented in part at the Annual Meeting of the American Society of Anesthesiology, October 14–18, 2006, Chicago, Illinois.

Summary Statement: Propofol binds to the C1B subdomain of recombinant protein kinase C epsilon causing auto-phosphorylation at serine 729 and catalytic activation of the enzyme.

References

1. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature Lond. 1988;334:661–5. [PubMed]
2. Ping P, Zhang J, Pierce WM, Bolli R. Functional proteomic analysis of protein kinase Cε signaling complexes in the normal heart and during cardioprotection. Circ Res. 2001;88:59–62. [PubMed]
3. Ping P, Song C, Zhang J, Guo Y, Cao X, Li RC, Wu W, Vondriska TM, Pass JM, Tang XL, Pierce WM, Bolli R. Formation of protein kinase C(epsilon)-Lck signaling modules confers cardioprotection. J Clin Invest. 2002;109:499–507. [PMC free article] [PubMed]
4. Vondriska TM, Zhang J, Song C, Tang XL, Cao X, Baines CP, Pass JM, Wang S, Bolli R, Ping P. Protein kinase C epsilon-Src modules direct signal transduction in nitric oxide-induced cardioprotection: Complex formation as a means for cardioprotective signaling. Circ Res. 2001;88:1306–13. [PubMed]
5. Zhang J, Baines CP, Zong C, Cardwell EM, Wang G, Vondriska TM, Ping P. Functional proteomic analysis of a three-tier PKCε-Akt-eNOS signaling module in cardiac protection. Am J Physiol Heart Circ Physiol. 2004;288:H954–H961. [PubMed]
6. Giorgione J, Hysell M, Harvey DF, Newton AC. Contribution of the C1A and C1B domains to the membrane interaction of protein kinase C. Biochemistry. 2003;42:11194–202. [PubMed]
7. Chiari PC, Pagel PS, Tanaka K, Krolikowski JG, Ludwig LM, Trillo RA, Puri N, Kersten JR, Warltier DC. Intravenous emulsified halogenated anesthetics produce acute and delayed preconditioning against myocardial infarction in rabbits. Anesthesiology. 2004;101:1160–6. [PubMed]
8. Kevin LG, Katz P, Camara AK, Novalija E, Riess ML, Stowe DF. Anesthetic preconditioning: Effects on latency to ischemic injury in isolated hearts. Anesthesiology. 2003;99:385–91. [PubMed]
9. Kato R, Foex P. Myocardial protection by anesthetic agents against ischemia-reperfusion injury: An update for anesthesiologists. Can J Anaesth. 2002;49:777–91. [PubMed]
10. Kokita N, Hara A, Abiko Y, Arakawa J, Hashizume H, Namiki A. Propofol improves functional and metabolic recovery in ischemic reperfused isolated rat hearts. Anesth Analg. 1998;86:252–8. [PubMed]
11. Ko SH, Yu CW, Lee SK, Choe H, Chung MJ, Kwak YG, Chae SW, Song HS. Propofol attenuates ischemia-reperfusion injury in the isolated rat heart. Anesth Analg. 1997;85:719–24. [PubMed]
12. Burns DJ, Bloomenthal J, Lee M-H, Bell RM. Expression of the α, βII, and γ protein kinase C isozymes in the baculovirus-insect cell expression system: Purification and characterization of the individual isoforms. J Biol Chem. 1990;265:12044–51. [PubMed]
13. Schaap D, Parker PJ. Expression, purification and characterization of protein kinase C-ε J Biol Chem. 1990;265:7301–7. [PubMed]
14. Wickley PJ, Murray PA, Damron DS. Propofol-induced activation of protein kinase C isoforms in adult rat ventricular myocytes. Anesthesiology. 2006;104:970–7. [PubMed]
15. Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. [PubMed]
16. Das J, Addona GH, Sandberg WS, Husain SS, Stehle T, Miller KW. Identification of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase Cδ J Biol Chem. 2001;279:37964–72. [PubMed]
17. Hemmings HC, Jr, Adamo AIB. Effects of halothane and propofol on purified brain protein kinase C activation. Anesthesiology. 1994;81:147–55. [PubMed]
18. Kanaya N, Gable B, Wickley PJ, Murray PA, Damron DS. Experimental conditions are important determinants of cardiac inotropic effects of propofol. Anesthesiology. 2005;103:1026–34. [PubMed]
19. Gable BD, Shiga T, Murray PA, Damron DS. Propofol increases contractility during α1a-adrenoreceptor activation in adult rat cardiomyocytes. Anesthesiology. 2005;103:335–43. [PubMed]
20. Kurokawa H, Murray PA, Damron DS. Propofol attenuates β-adrenoreceptor-mediated signal transduction via a protein kinase C-dependent pathway in cardiomyocytes. Anesthesiology. 2002;96:688–98. [PubMed]
21. Kanaya N, Gable B, Murray PA, Damron DS. Propofol increases phosphorylation of troponin I and myosin light chain 2 via protein kinase C activation in cardiomyocytes. Anesthesiology. 2003;98:1363–71. [PubMed]
22. Kanaya N, Murray PA, Damron DS. Propofol increases myofilament Ca2+ sensitivity and intracellular pH via activation of Na+-H+ exchange in rat ventricular myocytes. Anesthesiology. 2001;94:1096–104. [PubMed]
23. Cenni V, Doppler H, Sonnenburg ED, Marlad N, Newton AC, Toker A. Regulation of novel protein kinase Cε by phosphorylation. Biochem J. 2002;363:537–45. [PubMed]
24. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992;258:607–14. [PubMed]
25. Wickley PJ, Shiga T, Murray PA, Damron DS. Propofol decreases myofilament Ca2+ sensitivity via a PKC-, nitric oxide synthase-dependent pathway in diabetic cardiomyocytes. Anesthesiology. 2006;104:978–87. [PubMed]