activation of rat brain protein kinase c by lipid oxidation products

Vol. 155, No. 3, 1988

September 30, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages ]374-]380

ACTIVATION OF RAT BRAIN PROTEIN KINASE C BY LIPID OXIDATION PRODUCTS

Catherine A. O'Brian *1, Nancy E. Ward*, I. Bernard Weinstein ̂ , Arthur W. Bull§ 2, and Lawrence J. Marnettt

*Department of Cell Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030

^Department of Medicine and Public Health, Columbia University, College of Physicians and Surgeons,

New York, NY 10032

§Department of Internal Medicine, Wayne State University Medical School, Detroit, MI 48201

tDepartment of Chemistry, Wayne State University Detroit, MI 48202

Received August 15, 1988

The unsaturated fatty acid components of membrane lipids are susceptible to oxidation in vi tro and in vivo. The initial oxidation products are hydroperoxy fatty acids that are converted spontaneously or enzymatically to a variety of products. Hydroperoxy derivatives of oleic, linoleic, or arachidonic acids stimulate the activity of protein kinase C (PKC) purified from rat brain. The hydroperoxy acids satisfy the requirement of PKC for phospholipid (e.g., phosphatidylserine). Activation is observed in the presence or absence of 1 mM Ca 2+. Reduction of the hydroperoxides to alcohols or dehydration of the hydroperoxides to ketones increases the Ka for activation three- to fourfold but does not significantly reduce the maximal extent of PKC activation. The Ka's for activation by hydroperoxy acids are approximately half the values exhibited by the unoxidized fatty acids. Since oxidation of unsaturated fatty acids to hydroperoxides is the first event in lipid peroxidation, activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress. © 1988 Academic Press, Inc.

The Ca 2+- and phospholipid-dependent protein kinase (PKC) appears to

play a key role in the control of many cellular processes. The biological

actions of a number of structurally diverse growth factors, hormones, and

tumor promoters are mediated by activation of PKC (1,2). The requirement

of PKC for phospholipid renders it sensitive to modulation by a variety of

1. To whom correspondence should be addressed.

2. Present address, Department of Chemistry, Oakland University, Rochester, MI 48063

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. 1374

Vol. 155, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lipophilic compounds including polyunsaturated fatty acids.

Polyunsaturated fatty acids are readily oxidized under physiological

conditions to multiple oxygenated compounds that contain hydroperoxide,

epoxide, aldehyde, ketone, or alcohol functional groups (3,4). The primary

autoxidation products of unsaturated fatty acids are hydroperoxides that

can be reduced to alcohols or dehydrated to ketones. Hydroperoxy-,

hydroxy-, or keto-fatty acids or their counterparts are among the earliest

products of lipid peroxidation in biological membranes and may contribute

to the cellular effects induced by oxidative stress.

Previous studies of the effects of oxygenated fatty acids on PKC activity

have produced conflicting results. A crude mixture of autoxidation

products of arachidonic acid had no effect on the PKC activity of a detergent

extract of human neutrophils (5). In contrast, several hydroxy fatty acids

produced by enzymatic oxygenation of arachidonic acid stimulated a PKC

preparation purified from human placental cytosol (6). The effects of

hydroperoxy precursors of the hydroxy acids were not reported. In the

present investigation, we examined the effects of a series of hydroperoxy,

hydroxy, and keto fatty acids on PKC purified from rat brain. The results

indicate that hydroperoxy fatty acids, the initial products of lipid

peroxidation in biological membranes, are more potent activators of PKC

than hydroxy-, keto-, or unoxidized fatty acids.

EXPERIMENTAL Materials. (T32P) ATP was purchased from Amersham Corp (Arlington Hts, IL). Tris HC1, histone IIIS, ATP, (phosphatidylserine) PS, phenylmethylsulfonyl fluoride, G-25 Sephadex, and soybean trypsin inhibitor and soybean lipoxygenase were purchased from Sigma Chem. Co. (St. Louis, MO). Whatman phosphocellulose paper, grade p81, was from Fisher Scientific (Houston, TX). Leupeptin was a gift from the U.S.-Japan Cooperative Cancer Research Program. The BioRad protein assay solution was used for protein concentration determinations, employing bovine serum albumin as a standard. Synthesis and Purification of Hvdroxv- and Hydroperoxy-Fatty Acids. Soybean lipoxygenase was used to produce 13-hydroperoxyoctadeca-dienoic acid and 15-hydroperoxyeicosatetraenoic acid. The oxidized fatty acids were purified by silicic acid chromatography and characterized as previously described (7). Hydroxyoctadecadienoates were obtained by borohydride reduction of the respective hydroperoxides. Mixtures of 9-, and 10- octadecenoate were produced by photosensitized oxygenation of oleic acid. The ketone derived from these hydroperoxides was produced by dichromate oxidation of the corresponding alcohol. Purification and characterization were as previously described (7). In experiments utilizing unoxidized fatty acids, silicic acid chromatography was used to remove any autoxidation products prior to use in an assay. Enz3rme Assays. Protein kinase C was partially purified from frozen rat brains (Charles River Breeding Co., Wilmington, MA), to a specific activity of 230 nmol 32p/min/mg as previously described (8). In indicated experiments, PKC was purified to near homogeneity from frozen rat brains

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by the method of Huang et al. through the polylysine chromatography step (9). The resultant PKC preparation had a specific activity of 1300 nmol 32p/min/mg. Both enzyme preparations had phosphotransferase activities which were stimulated from 10- to 30-fold by 1 mM Ca 2+ and 30 ~g/ml PS. PKC activity was assayed by our standard procedure (10). Reaction mixtures (120 p]) contained 20 mM Tris-HC1 at pH 7.5, 5 mM 2-

mercaptoethanol, 10 mM MgC12, 1 mM CaC12 (or 1 mM EGTA), 30 pg/ml

PS (or none), 70 ~ (~32p) ATP (150-400 cpm/pmol), 0.67 mg/ml histone III- S, and 1-4 ~g isolated rat brain PKC. In indicated experiments, fatty acids were used in place of PS to stimulate PKC activity. The fatty acids were stored in methanol at -70°C and used within one month. Stock solutions of the fatty acids were prepared daily by drying the fatty acids under N2 and then suspending them in 10 mM sodium phosphate at pH 7.5 containing 10% DMSO. All phosphotransferase reactions were incubated for periods of five to ten minutes at 30°C, which is in the linear phase of the time course. Reactions were terminated on phosphocellulose paper, and the radioactivity incorporated into histone was measured as previously described (10). Removal of 2-mercaptoethanol from Partially Purified PKC. 400 ~1 PKC was desalted on a 2 ml G-25 Sephadex column equilibrated in 20 mM Tris HC1, 5 mM EDTA, 5 mM EGTA at pH 7.5. PKC activity eluted in the first 600 pl of the eluant, and greater than 95% of the 2-mercaptoethanol was removed according to the absorbance of the thiolate anion generated from 2- 2' dithiopyridine at 343 nm.

RF~ULTS

Figure 1 illustrates the activation of partially purified PKC by I mM

Ca 2+ and 13-hydroperoxyoctadecadienoic acid, in the absence of PS. This

hydroperoxy fatty acid acted as a surrogate for PS in the activation of PKC. Half-maximal activation of the enzyme (Ka) occurred at 16 ~g/ml

hydroperoxy fatty acid. We next compared the capacities of hydroperoxy

fatty acids, hydroxy fatty acids, and unoxygenated fatty acids to serve as

lipid cofactors for PKC. The hydroperoxy fatty acids activated PKC with similar Ka's that were approximately twice as low as those measured for

three related unoxygenated fatty acids and about four times lower than the Ka's observed for two related hydroxy fatty acids (Table I).

In order to ascertain whether contaminants in the PKC preparation

were affecting the observed lipid cofactor potencies, each fatty acid was tested at its Ka for activation of a nearly homogeneous PKC preparation. No

differences in the extent of activation were observed between partially

purified and nearly homogeneous PKC preparations. Both the partially

purified and highly purified PKC preparations contained 2-

mercaptoethanol, which is known to reduce hydroperoxides to alcohols.

Therefore, we removed over 95% of the 2-mercaptoethanol from the PKC

preparation by gel filtration through a G-25 column (see Methods). The

1376

50

1 0

4 O _c

n 30

"6 E

20

10 20 30 40 50

jug 13-hydroperoxyoctadecadienoic acid/ml

Figure 1:


The Activation of PKC by I mM Ca 2+ and 13-hydroperoxyoctadecadienoic acid. The capacity of 13-hydroperoxyoctadecadienoic acid to replace

the requirement for phospholipid in the activation of PKC was

determined in the presence of 1 mM Ca 2+. Pmol 32p/rain

represents the rate of the Ca 2+- and lipid-dependent transfer of 32p from [~2p] ATP to histone III-S. See Methods for

experimental details.

Table I

Activation of PKC by Fatty Acids in the Presence of i mM Ca 2+

% Maximal %Maximal Fatty Acid K a (~g/ml) Activation Activation/K a

13 hydroperoxyoctadecadienoate 16 ± 2

9+10 hydroperoxyoctadecadienoate 13 + 2

15 hydroperoxyeicosatetraenoate 17 + 2

13 hydroxyoctadecadienoate

9+10 keto octadecenoate

34 _+ 2 2.10

36 ± 4 2.77

23 +_ 2 1.35

60 + 3 25 + 3 0.42

51 ± 5 41 + 5 0.80

oleate 29 ± 3 61 _+ 4 2.10

linoleate 24 ± 2 48 + 3 2.00

arachidonate 28 _+ 2 36-+ 1 1.29

"% Maximal Activation" represents the maximal percentage of activation

achieved when the indicated fatty acid served as a lipid cofactor of the

enzyme in the presence of I mM Ca 2+. 100% activation is the activation . achieved by 30 ttg/ml PS in the presence of I mM Ca 2+. Each K a value

represents the concentration of the indicated lipid cofactor which

stimulated PKC activity to 50% of the maximal activation achieved with that lipid cofacter. K a values were determined graphically. % Maximal

Activation/K a represents the % maximal activation divided by the K a.

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Table II

Activation of PKC by Oxidized Fatty Acids in the Absence of Ca 2+

Fatty Acid (100 ~g/ml) % Activation

15 hydroperoxyeicosatetraenoate 9+10 hydroperoxyoctadecadienoate 13 hydroperoxyoctadecadienoate 9+10 keto octadecenoate 13 hydroxyoctadecadienoate

31+2 32_+6 16_+ 1 15_+2 4_+1

"% Activation" represents the percentage of activation of PKC achieved when the indicated fatty acid served as a lipid cofactor of the enzyme in the absence of 1 mM Ca 2+. 100% activation is the level of activation achieved by 30 ~tg/ml PS in the presence of 1 mM Ca 2+.

resultant PKC preparation was activated at similar concentrations and to

similar extents by hydroperoxy fatty acids (data not shown).

The value of the maximal activation of PKC observed with a given lipid

cofactor divided by the corresponding Ka provides an overall measure of the

efficacy of the lipid as a PKC activator. By this criterion, the hydroperoxy

and unoxygenated fat ty acids activated PKC with similar efficacies,

whereas the hydroxy fat ty acids were much weaker PKC cofactors (Table I).

Thus, introduction of a hydroperoxide group into unsatura ted fat ty acids

may increase PKC activation at low fatty acid concentrations, whereas

reduction of the hydroperoxide to an alcohol may significantly diminish the

extent of enzyme activation.

Arachidonic acid and other unsatura ted fatty acids can also stimulate

PKC activity in the absence of added Ca 2+ (11). We tested the hydroxy and

hydroperoxy fat ty acids as activators of PKC in the absence of added Ca 2+.

These fatty acids activated PKC in the absence of added Ca 2+, but were

generally less potent PKC cofactors under these conditions than in the

presence of 1 mM Ca 2+ (Table II).

DISCUSSION

The experiments in this report demonstrate tha t several oxidation

products of unsatura ted fat ty acids directly activate PKC. Furthermore, the

hydroperoxide derivatives were active at lower concentrations than the

unoxidized parent fat ty acids. In contrast, reduction of the hydroperoxide

moiety to an alcohol or dehydration to a ketone raised the fat ty acid

concentration necessary for activation of PKC.

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Unsaturated fatty acids have previously been shown to be activators of

PKC with effective concentrations between 100 and 300 ~M (5). Similar

values were obtained in the present study. On the other hand, the

hydroperoxy fatty acids were found to activate PKC at concentrations which

were about one-half those of the unoxidized parent compounds. The

mechanism responsible for the activation of PKC by hydroperoxy fatty acids

may involve oxidation of a component of the enzyme rather than simply

binding to the protein, since reduction of the hydroperoxide to an alcohol

raised the effective concentration needed for activation. A similar

observation has been made for the activation of guanylate cyclase by

hydroperoxy but not hydroxy fatty acids (12).

These data have several implications for the regulation of PKC activity

and the response of cells to oxidative stress. The complex series of reactions

comprising lipid peroxidation have been implicated in numerous

pathological processes (13,14). The primary products of lipid peroxidation

are fatty acid hydroperoxides and it is likely that the decomposition

products of biologically derived hydroperoxides are responsible for some of

the activities associated with lipid peroxidation. However, there is a

distinct difference between extensive peroxidation of polyunsaturated fatty

acids, with the accumulation of degradation products, and low level

production of hydroperoxy fatty acids by enzymatic or spontaneous

reactions. The results of the present investigation suggest that alteration of

biological responses can occur even in the presence of low levels of lipid

oxidation products which would not be overtly toxic.

PKC has been strongly implicated in the process of tumor promotion, in

part because the phorbol ester tumor promoters are excellent activators of

the enzyme (15,16). Another component of tumor promotion may involve

the formation of oxidants associated with the reductive metabolism of

molecular oxygen (17-19). It is, therefore, conceivable that the results of the

present study provide a link between oxidative stress within a cell and

promotion of tumorigenesis. Specifically, the production of oxidized fatty

acids could enhance the activation of PKC and an initiation of the cascade of

events that comprise tumor promotion. In support of this suggestion, it has

been shown that hydroperoxy fatty acids and other organic peroxides

possess many of the properties of tumor promoters (7,20).

The results of the current study plus the reports of other investigators

(6) demonstrate additional factors of potential importance in the regulation

of PKC activity. At present it is not clear whether the oxidation products of

unsaturated fatty acids are involved in normal cellular processes or

mediate pathological responses to environmental stress. In either case, it

is clearly important to unravel the role of oxidized fatty acids in the complex

regulatory mechanism for this key enzyme.

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Vol. 155, No. 3, 1988 BIOCHEMICADAND BIOPHYSICAL RESEARCH COMMUN CAT ONS

ACKNOWLEDGE~NTS This work was supported by research grants from the National

Institutes of Health (CA 47479).

REFERENCES

1. Weinstein, I.B. (1987) J. Cell Biochem. 33, 213-224. 2. Nishizuka, Y. (1986) Science 233, 305-312. 3. Esterbauer, H., Cheeseman, K.H., Dianzani, M.U., Poli, G., and

Slater, T.F. (1982) Biochem. J. 208, 129-140. 4. Porter, N.A., Lehman, L.S., Weber, B.A., and Smith, K.J. (1981) J.

Amer. Chem. Soc. 103, 6447-6455. 5. McPhail, L.C. Clayton, C.C., and Snyderman, R. (1984) Science 224,

622-625. 6. Hansson, A., Serhan, C.N., Haeggstrom, J., IngeIman-Sundberg,

M., and Samuelsson, B. (1986) Biochem. Biophys. Res. Commun. 134, 1215-1222.

7. Bull, A.W., Nigro, N.D., and Marnett, L.J. (1988) Cancer Res. 48, 1771-1776.

8. O'Brian, C.A., Arthur, W.L., and Weinstein, I.B. (1987) FEBS Letts. 214, 339-342.

9. Huang, K.-P., Chan, K.-F., Singh, T.J., Nakabayashi, H., and Huang, F.L. (1986) J. Biol. Chem. 261, 12134-12140.

10. O'Brian, C.A., Lawrence, D.S., Kaiser, E.T., and Weinstein, I.B. (1984) Biochem. Biophys. Res. Commun. 124, 296-302.

11. Murakami, K. and Routtenberg, A. (1985) FEBS Letts. 192, 189-193. 12. Graff, G., Stephenson, J.H., Glass, D.B., Haddox, M.K., and

Goldberg, N.D. (1978) J. Biol. Chem. 253, 7662-7676.13. Ernster, L., Nordenbrand, K., and Orrenius, S. (1982) in Lipid Peroxides in Biology and Medicine (Yagi, K., ed.) Academic Press, pp. 55-79.

14. Plaa, G.L. and Witschi, H. (1976) Ann Rev. Pharm. Toxicol. 16, 125- 141.

15. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851.

16. Arcoleo, J.P., Weinstein, I.B. (1985) Carcinogenesis. 6, 2133-217. 17. Kensler, T.W. and Trush, M.A. (1984) Environ. Mut. 6, 593-616. 18. Cerrutti, P.A. (1985) Science 227, 375-381. 19. Fischer, S.M., Baldwin, J.K., Jasheway, D.W., Patrick, K.E., and

Cameron, G.S. (1988) Cancer Res. 48, 658-664. 20. Slaga, T.J., Klein-Szanto, A.J.P., Tl~plett, L.L., Yotti, L.P., and

Trosko, J.E. (1981) Science 213, 1023-1025.

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activation of rat brain protein kinase c by lipid oxidation products

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