Vol.
187,
No.
September
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
16, 1992
Pages
DUAL MODULATION
OF PROTEIN Guillermo
KINASE
C ACTIVITY
635-640
BY SPHINGOSINE
Senisterra and Richard M. Epand’
McMaster University, Department of Biochemistry, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 325 Received
July
7,
1992
Sphingosine is one of a number of cationic amphiphiles that inhibit the activity of protein kinase C (PKC) in commonly used assay conditions. This inhibition occurs only at high concentrations of this amphiphile. In the presence of excess negative charge from oleic acid, the addition of sphingosine surprisingly leads to activation of PKC. The results are explicable in terms of the dual role of charge and lipid phase propensity. When the positive charge on sphingosine is compensated by the negative charge on oleic acid, sphingosine, a hexagonal phase promoting amphiphile, becomes an activator of PKC. This does not occur with a bilayer stabilizing cationic amphiphile, N,N,N-Trimethyl-N’-cholesteryl amidoethyl ammonium which is an inhibitor of PKC at all mol fractions, as well as in the presence of oleic acid. The results indicate that effects of sphingosine on more complex biological systems should be interpreted 0 1992 Academic press, Inc. with caution because of this dual role of the amphiphile.
Protein Kinase C (PKC) is involved in intracellular signal transduction [I]. This enzyme regulates many cell functions [2,3]. Both phosphatidylserine and 1,2-diacyl-sn-glycerol have been demonstrated to be required for the full activation of the enzyme [4].
PKC has also been
demonstrated to be the receptor for the tumour promoting phorbol esters which activate the enzyme by an interaction
at the l,Zdiacyl-sn-glycerol
site [5-81. As a result of many
investigations a number of compounds with diverse chemical structures have been shown to activate or inhibit substrate phosphorylation
by PKC [9,10]. In particular, charge has been
shown to play an important role in modulating enzyme function [lo]. A number of positively charged compounds inhibit polycationic substrate phosphorylation by PKC [9, lo]. Inhibitors of PKC are an important tool for understanding the specific functions of this enzyme in cells and animals. Sphingosine, a constituent of the sphingolipids,
is a PKC inhibitor
[l 1,121. Much
“To whom correspondence should be addressed. Ds: DOG, 1,2-dioleoyl-sn-glycerol; OA, oleic acid; PC, phosphatidylcholine; PS, phosphatidylserine; PKC, protein kinase C; POPC, 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine; POPS, 1-palmitoyl-2-oleoyl-sn-phosphatidylserine; SUV, small unilamellar vesicles.
635
All
Copyright 0 1992 rights of reproduction
0006-291X/92 $4.00 by Academic Press. Irtc. in any form reserved.
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
interest has been given to studying this effect [13] because of its possible physiological [14] and pathological relevance [ 151. In this work we show that sphingosine has two different mechanisms of action. Inhibition of PKC activity is caused by charge neutralization concentrations of sphingosine.
of phosphatidylserine
at relatively high
A second stimulatory effect of sphingosine occurs at relatively
lower concentrations when its charge is neutralized by the presence of oleic acid.
EXPERIMJZNTAL
PROCEDURES
Materials: All phospholipids and 1,2-Dioleoyl-sn-Glycerol (DOG) were purchased from Avanti Polar Lipids, Pelham, Al., and were pure as determined by TLC. Oleic acid was purchased from Calbiochem, San Diego, CA. Histone (type III-S) and bovine brain D-sphingosine were purchased from Sigma Chem. Co., St. Louis, MO. [Y-~*P] adenosine 5’-triphosphate was from NEN, Montreal, Quebec. Ultra pure grade Mg(NOJ, was obtained from Alpha Chem Co. Ward Hill, MA. Ultra pure grade Tris was obtained from BRL, Gaithersburg, MD. PKC was purified from rat brain as previously described [16]. Double distilled water was further purified by passage through a Gelman Science Water I filter. N,N,N-Trimethyl N’-cholesteryl amidoethyl ammonium (cholesterol derivative) was prepared as described [17]. Preparation of small unilamellar vesicles: A total amount of 1 pmol of phospholipid, from a dried lipid film was suspended in 1 ml of 50 mM Tris-HCl buffer (pH=7.4). The vesicle suspension was sonicated for 30 min using a water bath sonicator (Cole Palmer Ultrasonic Model 8849-00) for the preparation of small unilamellar vesicles (SUV). The vesicle composition was POPS/POPC (1:4) and when additives were added (OA, Sphingosine or Cholesterol derivative), the amount of POPC was reduced to maintain constant the mol fraction of POPS in the vesicles. Assay for PKC activity: A 25 ~1 aliquot of SUV suspension was used in a final assay volume of 250 ~1 containing 10 mM Mg(NO,),, 200 pg/ml histone III-S, 50 PM CaCl, (when Ca++ was not present, 25 ~1 1 mM EGTA was added) and 20 PM [7-32P] adenosine 5’-triphosphate. The total lipid concentration was 100 PM. To initiate the reaction, a 25 ~1 aliquot of PKC (60 ng) was added. After briefly vortexing, the tubes were incubated 10 min at 30°C. The reaction was terminated by adding 100 ~1 of cold 5 mg/ml BSA and 2 ml of cold 25% trichloroacetic acid. The samples were placed on ice for 15 min and filtered through Whatman G/FC glass tibre filters. The filters were washed 5 times with 2 ml each of ice cold 25% trichloroacetic acid. After drying, the filters were counted for their radioactivity.
RESULTS We compared the effects of two cationic amphiphiles, sphingosine and N,N,N-trimethylN’- cholesteryl amidoethyl ammonium (compound IV from ref 17) on PKC activity. Using SUV of POPS:POPC, 1:4 (Fig. 1) we find that the inhibitory effect of the cholesterol derivative is proportional to its concentration, while sphingosine has virtually no effect below 20 mol % but shows a potent inhibitory effect at 30 mol % (with respect to the total lipid concentration). At 20 mol % of cationic amphiphile
there is an approximate equivalence between the positive
charges from these amphiphiles and the negative charges from PS. Thus one requires an excess 636
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
27
7
I -
1
10 mol
20 % addltlve
CTRL
30
PUP
c
1XOOG 1XOOG 20XOA
P! i/PC 2, IXOA
-
PS/PI 20% 0 IOXSP
‘S/P !OXOA !OXSPH
Figure 1. PKC specific activity in the presence of 100 PM 1:4 POPSIPOPC SUV vesicles containing different concentrations of sphingosine ( q ) or Cholesterol derivate ( n ). Figure 2. PKC stimulation by POPYPOPC WV’s: Control in the absence of lipid and Ca++; PS/PC; PS/PC plus 1% 1,2-dioleoyl-sn-glycerol (DOG), PSlPC plus 1% DGG and 20% OA, PSIPC plus 20% OA , PSIPC plus 20% OA and 10% Sphingosine; PSIPC plus 20% OA and 20% Sphingosine.
of sphingosine over PS to induce inhibition,
which is not the case for the cholesterol
derivative. We investigated the effect of sphingosine on PKC activity when its charge is neutralized by oleic acid. This fatty acid has been described before as a potent PKC stimulatory agent in the presence [20] and in the absence [21, 221 of phospholipids. Unsaturated fatty acids are known to greatly potentiate the stimulation of PKC by DOG [20-271. We confirm this observation with the vesicle system we are using (Fig. 2). Sphingosine [lo] like DAG [28] is a hexagonal phase promoter. Hexagonal phase promoters which are not cationic are activators of PKC [lo].
The apparent pK of the fatty acid in membranes is
approximately 7.5 [29], while that of sphingosine is approximately 8 [18]. At the pH used for the PKC assay, the oleic acid should be approximately half ionized while the sphingosine will be almost entirely protonated. Their exact states of ionization will vary somewhat occurring to their mol fractions because of changes in the surface charge of the membrane. With a two fold molar excess of fatty acid over sphingosine, the positive and negative charges contributed to the membrane by these amphiphiles should be largely equalized. Under these conditions sphingosine has a definite, albeit weak, stimulatory effect on PKC. At higher sphingosine concentration, however, when there is excess positive charge, this stimulatory effect is lost (Pig. 2). As sphingosine induces some stimulation of PKC when oleic acid is in the membrane, we determined the effect of sphingosine concentration on this activation and compare it with effects of the cholesterol derivative (Fig. 3). Between 3 and 10 mol % sphingosine there is a significant
stimulation
of PKC activity with vesicles composed of POPC:POPS,
containing 20 mol % oleic acid. No stimulation 637
4:l and
is observed at any concentration of the
Vol.
187,
No.
BIOCHEMICAL
2, 1992
OL-L 0
AND
10 mol
BIOPHYSICAL
20 % addltlve
AL 30
RESEARCH
COMMUNICATIONS
40
Figure 3. PKC specific activity in the presenceof 100 PM POPVPOPC vesicles plus 20% oleic acid with the percent of sphingosine (0) or cholesterol derivate (H) in the membrane.
cholesterol derivative where, as was found in the absence of fatty acid for this amphiphile (Fig. l), the inhibition is proportional to the concentration of cationic cholesterol derivative. DISCUSSION
Sphingosine is generally considered to be an inhibitor of PKC activity and it has been used in cell systems to test the involvement of this enzyme in signal transduction. The present study demonstrates that under certain circumstances sphingosine can activate PKC. The findings correlate, at least qualitatively,
with the generalizations that all cationic
amphiphiles are inhibitors of PKC and that neutral or zwitterionic hexagonal phase promoters are activators of this enzyme. Cationic amphiphiles may inhibit PKC by preventing binding of the cationic substrate, histone, to the membrane. This was proposed by Bazzi and Nelsestuen as the mechanism for the inhibition of PKC by sphingosine [19]. Even if the overall charge on the protein substrate was not cationic, the phosphorylation site on the protein substrate must have a high density of positively charged amino acid residues to be recognized by PKC. This phosphorylation site on the protein substrate would therefore be repelled if the membrane surface were cationic. We have previously shown that the inhibitory
effect of sphingosine could be
eliminated by raising the pH and thereby deprotonating this amphiphile 1181. The current study demonstrates that the inhibitory effect of sphingosine, resulting from its positive charge, can be overcome by an equal or greater concentration of a negatively charged amphiphile. relationship between amphiphile
The
concentration and membrane charge will also depend on the
effect of the membrane environment on the pK of the amphiphile and on charge interactions within the membrane. This demonstrates that positive and negative charges do not have to reside on the same zwitterionic molecule to affect changes in PKC activation. Of course, there are other factors in addition to charge neutralization
which will determine the extent of PKC
activation shown by sphingosine. Factors such as the lateral distribution of amphiphiles in the 638
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
membrane, their access to the vicinity of PKC and their effects on membrane physical properties will all contribute to the final observed effect. Thus, although oleic acid is not unique, other anionic amphiphiles or even other unsaturated fatty acid give comparable or lower activation of PKC when combined with sphingosine (unpublished results). Nevertheless our results clearly show that under certain circumstances sphingosine can act as an activator of PKC. If there is an excess of negative charge on the membrane, then the effect of cationic membrane additives on PKC activity maybe well correlated with the effects of these amphiphiles on the bilayer to hexagonal phase transition temperature of model membranes. Thus sphingosine, which is a hexagonal phase promoter does not inhibit PKC until high mol fractions, in contrast to the bilayer stabilizing cholesterol amphiphile which inhibits to some extent at all mol fractions (Figs. 1 and 3). Furthermore sphingosine, but not the cholesterol analogue, can activate PKC in the presence of oleic acid (Fig. 3). Thus the dual modulation of PKC activity by sphingosine is explicable in terms of its positive charge causing inhibition,
but under conditions of charge neutralization its hexagonal
phase promoting effect correlates with its observed activation of the enzyme. The current results suggest that effects of sphingosine observed in biological
systems, especially at low
concentrations, should be interpreted with caution as this amphiphile can have weak stimulatory as well as inhibitory effects on PKC.
ACKNOWLEDGMENTS We are grateful to Mr. Alan Stafford for his help in the purification of the PKC and to Dr. Leon van Gorkom for discussions.
REFERENCES 1. 2. 3.
Nishizuka, Y. (1984) Nature 308, 693-698. Kikkawa, U. and Nishizuka, Y. (1986) Ann. Rev. Cell. Biol. 2, 149-178. Lester, D. S. and Epand, R. M. (1992) “Protein Kinase C: Current Knowledge and Future Perspectives”, Ellis Horwood, in press. 4. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, V. and Nishizuka, Y. (1980) J. Biol. Chem. 255, 2273-2276. 5. Castagna, M., Takai, Y., Kaibuchi, K., Sana, K., Kikkawa, U. and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851. 6. Niedel, J. E., Kuhn, L. and Vandenbark, G. R. (1983) Proc. Natl. Acad. Sci. USA 80, 36-40. 7. Sharkey, N. A. and Blumberg, P. M. (1985) B&hem. Biophys. Res. Commun. 133, 1051-1056. 8. Bell, R. M. and Niedel, J. (1985) Proc. Natl. Acad. Sci. USA, 82, 815-819. 9. Weinstein, I. B. (1988) Mutation Research 202, 413-420. 10. Epand, R. M. (1987) Chem. Biol. Inter. 63, 239-257. 639
.,
I
VOI.
11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25 26. 27. 28. 29.
187,
No.2,1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Hannun, Y. A., Loomis, C. R., Merrill, A. H. and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609. Khan, W. A., Moscarello, S. W., Lewin, A. H., Wyrich, C. D., Carroll, F. I. and Hannun, Y. A. (1991) B&hem. J. 278, 387-392. Merrill, Jr. A. H. and Wang, E. (1986) B&him. Biophys. Acta. 1010, 131-139. Hannun, Y. A., Merrill, A. H. and Bell R. M. (1991) Methods in Enzymology 201, 316-328. Hannun, Y. A. and Bell, R. M. (1987) Science 235, 670-674. Huang, K. P., Chan, K. F. J., Singh, T. K., Nakahayashi, H. and Huang, F. L. (1986) J. Biol. Chem. 261, 12134-12140. Bottega, R. and Epand, R. M., submitted for publication. Bottega, R., Epand, R. M. and Ball, E. H. (1989) B&hem. Biophys. Res. Comm. 164, 102-107. Bazzi, M. D. and Nelsentuen, G. L. (1987) Biochim. Biophys. Res. Comm. 146,203-207. Chen, S. G. and Murakami, K. (1992) Biochem. J. 282, 33-39. Khan, W., Blobe, G. C and Hannun Y. (1992) J. Biol. Chem. 267, 36053612. El Touny, S., Khan, W. and Hannun, Y. (1990) J. Biol. Chem. 265, 16437-16444. Lester, D. S., Collins, C., Etchebrenigaray, R. and Alkon, D. (1991) B&hem. Biophys. Res Comm. 179, 1522-1528. Lester, D. (1990) Biochim Biophys Acta 1054, 297-303. Kitagawa, Y., Matsuo, Y., Minowada, J. and Nishizuka, Y. (1991) FEBS Letters 288, 37-40. Khan, W., El Touny, S. and Hannun, Y. (1991) FEBS Letters 292, 98-102. Murakami, K., Chan, S. and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429. Epand R. M. (1985) Biochemistry 24, 7092-7095. Small, D. M., Cab&, D. J., Cistola, D. P., Parks, J. J. and Hamilton, J. A. (1984) Hepatology 4, 775-795.
640
187,
No.
September
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
16, 1992
Pages
DUAL MODULATION
OF PROTEIN Guillermo
KINASE
C ACTIVITY
635-640
BY SPHINGOSINE
Senisterra and Richard M. Epand’
McMaster University, Department of Biochemistry, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 325 Received
July
7,
1992
Sphingosine is one of a number of cationic amphiphiles that inhibit the activity of protein kinase C (PKC) in commonly used assay conditions. This inhibition occurs only at high concentrations of this amphiphile. In the presence of excess negative charge from oleic acid, the addition of sphingosine surprisingly leads to activation of PKC. The results are explicable in terms of the dual role of charge and lipid phase propensity. When the positive charge on sphingosine is compensated by the negative charge on oleic acid, sphingosine, a hexagonal phase promoting amphiphile, becomes an activator of PKC. This does not occur with a bilayer stabilizing cationic amphiphile, N,N,N-Trimethyl-N’-cholesteryl amidoethyl ammonium which is an inhibitor of PKC at all mol fractions, as well as in the presence of oleic acid. The results indicate that effects of sphingosine on more complex biological systems should be interpreted 0 1992 Academic press, Inc. with caution because of this dual role of the amphiphile.
Protein Kinase C (PKC) is involved in intracellular signal transduction [I]. This enzyme regulates many cell functions [2,3]. Both phosphatidylserine and 1,2-diacyl-sn-glycerol have been demonstrated to be required for the full activation of the enzyme [4].
PKC has also been
demonstrated to be the receptor for the tumour promoting phorbol esters which activate the enzyme by an interaction
at the l,Zdiacyl-sn-glycerol
site [5-81. As a result of many
investigations a number of compounds with diverse chemical structures have been shown to activate or inhibit substrate phosphorylation
by PKC [9,10]. In particular, charge has been
shown to play an important role in modulating enzyme function [lo]. A number of positively charged compounds inhibit polycationic substrate phosphorylation by PKC [9, lo]. Inhibitors of PKC are an important tool for understanding the specific functions of this enzyme in cells and animals. Sphingosine, a constituent of the sphingolipids,
is a PKC inhibitor
[l 1,121. Much
“To whom correspondence should be addressed. Ds: DOG, 1,2-dioleoyl-sn-glycerol; OA, oleic acid; PC, phosphatidylcholine; PS, phosphatidylserine; PKC, protein kinase C; POPC, 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine; POPS, 1-palmitoyl-2-oleoyl-sn-phosphatidylserine; SUV, small unilamellar vesicles.
635
All
Copyright 0 1992 rights of reproduction
0006-291X/92 $4.00 by Academic Press. Irtc. in any form reserved.
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
interest has been given to studying this effect [13] because of its possible physiological [14] and pathological relevance [ 151. In this work we show that sphingosine has two different mechanisms of action. Inhibition of PKC activity is caused by charge neutralization concentrations of sphingosine.
of phosphatidylserine
at relatively high
A second stimulatory effect of sphingosine occurs at relatively
lower concentrations when its charge is neutralized by the presence of oleic acid.
EXPERIMJZNTAL
PROCEDURES
Materials: All phospholipids and 1,2-Dioleoyl-sn-Glycerol (DOG) were purchased from Avanti Polar Lipids, Pelham, Al., and were pure as determined by TLC. Oleic acid was purchased from Calbiochem, San Diego, CA. Histone (type III-S) and bovine brain D-sphingosine were purchased from Sigma Chem. Co., St. Louis, MO. [Y-~*P] adenosine 5’-triphosphate was from NEN, Montreal, Quebec. Ultra pure grade Mg(NOJ, was obtained from Alpha Chem Co. Ward Hill, MA. Ultra pure grade Tris was obtained from BRL, Gaithersburg, MD. PKC was purified from rat brain as previously described [16]. Double distilled water was further purified by passage through a Gelman Science Water I filter. N,N,N-Trimethyl N’-cholesteryl amidoethyl ammonium (cholesterol derivative) was prepared as described [17]. Preparation of small unilamellar vesicles: A total amount of 1 pmol of phospholipid, from a dried lipid film was suspended in 1 ml of 50 mM Tris-HCl buffer (pH=7.4). The vesicle suspension was sonicated for 30 min using a water bath sonicator (Cole Palmer Ultrasonic Model 8849-00) for the preparation of small unilamellar vesicles (SUV). The vesicle composition was POPS/POPC (1:4) and when additives were added (OA, Sphingosine or Cholesterol derivative), the amount of POPC was reduced to maintain constant the mol fraction of POPS in the vesicles. Assay for PKC activity: A 25 ~1 aliquot of SUV suspension was used in a final assay volume of 250 ~1 containing 10 mM Mg(NO,),, 200 pg/ml histone III-S, 50 PM CaCl, (when Ca++ was not present, 25 ~1 1 mM EGTA was added) and 20 PM [7-32P] adenosine 5’-triphosphate. The total lipid concentration was 100 PM. To initiate the reaction, a 25 ~1 aliquot of PKC (60 ng) was added. After briefly vortexing, the tubes were incubated 10 min at 30°C. The reaction was terminated by adding 100 ~1 of cold 5 mg/ml BSA and 2 ml of cold 25% trichloroacetic acid. The samples were placed on ice for 15 min and filtered through Whatman G/FC glass tibre filters. The filters were washed 5 times with 2 ml each of ice cold 25% trichloroacetic acid. After drying, the filters were counted for their radioactivity.
RESULTS We compared the effects of two cationic amphiphiles, sphingosine and N,N,N-trimethylN’- cholesteryl amidoethyl ammonium (compound IV from ref 17) on PKC activity. Using SUV of POPS:POPC, 1:4 (Fig. 1) we find that the inhibitory effect of the cholesterol derivative is proportional to its concentration, while sphingosine has virtually no effect below 20 mol % but shows a potent inhibitory effect at 30 mol % (with respect to the total lipid concentration). At 20 mol % of cationic amphiphile
there is an approximate equivalence between the positive
charges from these amphiphiles and the negative charges from PS. Thus one requires an excess 636
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
27
7
I -
1
10 mol
20 % addltlve
CTRL
30
PUP
c
1XOOG 1XOOG 20XOA
P! i/PC 2, IXOA
-
PS/PI 20% 0 IOXSP
‘S/P !OXOA !OXSPH
Figure 1. PKC specific activity in the presence of 100 PM 1:4 POPSIPOPC SUV vesicles containing different concentrations of sphingosine ( q ) or Cholesterol derivate ( n ). Figure 2. PKC stimulation by POPYPOPC WV’s: Control in the absence of lipid and Ca++; PS/PC; PS/PC plus 1% 1,2-dioleoyl-sn-glycerol (DOG), PSlPC plus 1% DGG and 20% OA, PSIPC plus 20% OA , PSIPC plus 20% OA and 10% Sphingosine; PSIPC plus 20% OA and 20% Sphingosine.
of sphingosine over PS to induce inhibition,
which is not the case for the cholesterol
derivative. We investigated the effect of sphingosine on PKC activity when its charge is neutralized by oleic acid. This fatty acid has been described before as a potent PKC stimulatory agent in the presence [20] and in the absence [21, 221 of phospholipids. Unsaturated fatty acids are known to greatly potentiate the stimulation of PKC by DOG [20-271. We confirm this observation with the vesicle system we are using (Fig. 2). Sphingosine [lo] like DAG [28] is a hexagonal phase promoter. Hexagonal phase promoters which are not cationic are activators of PKC [lo].
The apparent pK of the fatty acid in membranes is
approximately 7.5 [29], while that of sphingosine is approximately 8 [18]. At the pH used for the PKC assay, the oleic acid should be approximately half ionized while the sphingosine will be almost entirely protonated. Their exact states of ionization will vary somewhat occurring to their mol fractions because of changes in the surface charge of the membrane. With a two fold molar excess of fatty acid over sphingosine, the positive and negative charges contributed to the membrane by these amphiphiles should be largely equalized. Under these conditions sphingosine has a definite, albeit weak, stimulatory effect on PKC. At higher sphingosine concentration, however, when there is excess positive charge, this stimulatory effect is lost (Pig. 2). As sphingosine induces some stimulation of PKC when oleic acid is in the membrane, we determined the effect of sphingosine concentration on this activation and compare it with effects of the cholesterol derivative (Fig. 3). Between 3 and 10 mol % sphingosine there is a significant
stimulation
of PKC activity with vesicles composed of POPC:POPS,
containing 20 mol % oleic acid. No stimulation 637
4:l and
is observed at any concentration of the
Vol.
187,
No.
BIOCHEMICAL
2, 1992
OL-L 0
AND
10 mol
BIOPHYSICAL
20 % addltlve
AL 30
RESEARCH
COMMUNICATIONS
40
Figure 3. PKC specific activity in the presenceof 100 PM POPVPOPC vesicles plus 20% oleic acid with the percent of sphingosine (0) or cholesterol derivate (H) in the membrane.
cholesterol derivative where, as was found in the absence of fatty acid for this amphiphile (Fig. l), the inhibition is proportional to the concentration of cationic cholesterol derivative. DISCUSSION
Sphingosine is generally considered to be an inhibitor of PKC activity and it has been used in cell systems to test the involvement of this enzyme in signal transduction. The present study demonstrates that under certain circumstances sphingosine can activate PKC. The findings correlate, at least qualitatively,
with the generalizations that all cationic
amphiphiles are inhibitors of PKC and that neutral or zwitterionic hexagonal phase promoters are activators of this enzyme. Cationic amphiphiles may inhibit PKC by preventing binding of the cationic substrate, histone, to the membrane. This was proposed by Bazzi and Nelsestuen as the mechanism for the inhibition of PKC by sphingosine [19]. Even if the overall charge on the protein substrate was not cationic, the phosphorylation site on the protein substrate must have a high density of positively charged amino acid residues to be recognized by PKC. This phosphorylation site on the protein substrate would therefore be repelled if the membrane surface were cationic. We have previously shown that the inhibitory
effect of sphingosine could be
eliminated by raising the pH and thereby deprotonating this amphiphile 1181. The current study demonstrates that the inhibitory effect of sphingosine, resulting from its positive charge, can be overcome by an equal or greater concentration of a negatively charged amphiphile. relationship between amphiphile
The
concentration and membrane charge will also depend on the
effect of the membrane environment on the pK of the amphiphile and on charge interactions within the membrane. This demonstrates that positive and negative charges do not have to reside on the same zwitterionic molecule to affect changes in PKC activation. Of course, there are other factors in addition to charge neutralization
which will determine the extent of PKC
activation shown by sphingosine. Factors such as the lateral distribution of amphiphiles in the 638
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
membrane, their access to the vicinity of PKC and their effects on membrane physical properties will all contribute to the final observed effect. Thus, although oleic acid is not unique, other anionic amphiphiles or even other unsaturated fatty acid give comparable or lower activation of PKC when combined with sphingosine (unpublished results). Nevertheless our results clearly show that under certain circumstances sphingosine can act as an activator of PKC. If there is an excess of negative charge on the membrane, then the effect of cationic membrane additives on PKC activity maybe well correlated with the effects of these amphiphiles on the bilayer to hexagonal phase transition temperature of model membranes. Thus sphingosine, which is a hexagonal phase promoter does not inhibit PKC until high mol fractions, in contrast to the bilayer stabilizing cholesterol amphiphile which inhibits to some extent at all mol fractions (Figs. 1 and 3). Furthermore sphingosine, but not the cholesterol analogue, can activate PKC in the presence of oleic acid (Fig. 3). Thus the dual modulation of PKC activity by sphingosine is explicable in terms of its positive charge causing inhibition,
but under conditions of charge neutralization its hexagonal
phase promoting effect correlates with its observed activation of the enzyme. The current results suggest that effects of sphingosine observed in biological
systems, especially at low
concentrations, should be interpreted with caution as this amphiphile can have weak stimulatory as well as inhibitory effects on PKC.
ACKNOWLEDGMENTS We are grateful to Mr. Alan Stafford for his help in the purification of the PKC and to Dr. Leon van Gorkom for discussions.
REFERENCES 1. 2. 3.
Nishizuka, Y. (1984) Nature 308, 693-698. Kikkawa, U. and Nishizuka, Y. (1986) Ann. Rev. Cell. Biol. 2, 149-178. Lester, D. S. and Epand, R. M. (1992) “Protein Kinase C: Current Knowledge and Future Perspectives”, Ellis Horwood, in press. 4. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, V. and Nishizuka, Y. (1980) J. Biol. Chem. 255, 2273-2276. 5. Castagna, M., Takai, Y., Kaibuchi, K., Sana, K., Kikkawa, U. and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851. 6. Niedel, J. E., Kuhn, L. and Vandenbark, G. R. (1983) Proc. Natl. Acad. Sci. USA 80, 36-40. 7. Sharkey, N. A. and Blumberg, P. M. (1985) B&hem. Biophys. Res. Commun. 133, 1051-1056. 8. Bell, R. M. and Niedel, J. (1985) Proc. Natl. Acad. Sci. USA, 82, 815-819. 9. Weinstein, I. B. (1988) Mutation Research 202, 413-420. 10. Epand, R. M. (1987) Chem. Biol. Inter. 63, 239-257. 639
.,
I
VOI.
11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25 26. 27. 28. 29.
187,
No.2,1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Hannun, Y. A., Loomis, C. R., Merrill, A. H. and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609. Khan, W. A., Moscarello, S. W., Lewin, A. H., Wyrich, C. D., Carroll, F. I. and Hannun, Y. A. (1991) B&hem. J. 278, 387-392. Merrill, Jr. A. H. and Wang, E. (1986) B&him. Biophys. Acta. 1010, 131-139. Hannun, Y. A., Merrill, A. H. and Bell R. M. (1991) Methods in Enzymology 201, 316-328. Hannun, Y. A. and Bell, R. M. (1987) Science 235, 670-674. Huang, K. P., Chan, K. F. J., Singh, T. K., Nakahayashi, H. and Huang, F. L. (1986) J. Biol. Chem. 261, 12134-12140. Bottega, R. and Epand, R. M., submitted for publication. Bottega, R., Epand, R. M. and Ball, E. H. (1989) B&hem. Biophys. Res. Comm. 164, 102-107. Bazzi, M. D. and Nelsentuen, G. L. (1987) Biochim. Biophys. Res. Comm. 146,203-207. Chen, S. G. and Murakami, K. (1992) Biochem. J. 282, 33-39. Khan, W., Blobe, G. C and Hannun Y. (1992) J. Biol. Chem. 267, 36053612. El Touny, S., Khan, W. and Hannun, Y. (1990) J. Biol. Chem. 265, 16437-16444. Lester, D. S., Collins, C., Etchebrenigaray, R. and Alkon, D. (1991) B&hem. Biophys. Res Comm. 179, 1522-1528. Lester, D. (1990) Biochim Biophys Acta 1054, 297-303. Kitagawa, Y., Matsuo, Y., Minowada, J. and Nishizuka, Y. (1991) FEBS Letters 288, 37-40. Khan, W., El Touny, S. and Hannun, Y. (1991) FEBS Letters 292, 98-102. Murakami, K., Chan, S. and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429. Epand R. M. (1985) Biochemistry 24, 7092-7095. Small, D. M., Cab&, D. J., Cistola, D. P., Parks, J. J. and Hamilton, J. A. (1984) Hepatology 4, 775-795.
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