- Email: [email protected]
Purification of Diacylglycerol Kinase from Microsporum gypseum and Its Phosphorylation by the Catalytic Subunit of Protein Kinase A
Archives of Biochemistry and Biophysics Vol. 392, No. 2, August 15, pp. 219 –225, 2001 doi:10.1006/abbi.2001.2447, available online at http://www.idealibrary.com on
Purification of Diacylglycerol Kinase from Microsporum gypseum and Its Phosphorylation by the Catalytic Subunit of Protein Kinase A Ehtishamul Haq, Sadhna Sharma, and G. K. Khuller 1 Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh–160 012, India
Received November 17, 2000, and in revised form May 5, 2001; published online July 20, 2001
Diacylglycerol (DG) kinase (EC 2.7.1.107) was purified to homogeneity from the soluble extract of Microsporum gypseum, a dermatophyte. Purified enzyme showed a final specific activity of 2172 pmol/min/mg protein and its apparent molecular weight on SDS– PAGE was found to be 93 kDa. The activity of purified enzyme was inhibited in a dose-dependent manner in the presence of DG-kinase inhibitor (D5919, Sigma). DG-kinase activity was found to be stimulated in the presence of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin while the activity was alleviated in the presence of phosphatidic acid and arachidonic acid. Kinase activity was partially inhibited when assayed after prior treatment with alkaline phosphatase. Treatment of DG-kinase with the catalytic subunit of protein kinase A (PKA)-stimulated DGkinase activity in a dose-dependent manner. Incubation of DG-kinase with the catalytic subunit of PKA led to the phosphorylation of DG-kinase as revealed by autoradiography. The phosphorylated band disappeared completely in the presence of specific PKA inhibitor. Increased activity of DG-kinase on incubation with the catalytic subunit of PKA was possibly due to the phosphorylation of the former by the latter. Whether this in vitro phosphorylation and activation of DG-kinase occurs under physiological conditions remains to be elucidated. © 2001 Academic Press Key Words: DG-kinase; protein kinase A; phosphorylation; Microsporum gypseum.
Phospholipids are the key biological lipids having diverse and critical roles in cellular metabolism and function (1, 2). They are known to play important role 1 To whom correspondence should be addressed. Fax: ⫹91-0172-744 401 or 745 078. E-mail: [email protected] or [email protected].
0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
in growth, sporulation, replication, and germination in microorganisms including fungi (3, 4). Altered growth and environmental conditions have been reported to alter the rate of phospholipid biosynthesis in Microsporum gypseum, a dermatophyte, this in turn may help the fungus to adapt to the changed conditions (5). Evidence for the involvement of cAMP in cellular metabolism has been reported previously from our laboratory (6). It was reported that M. gypseum cells grown in the presence of aminophylline (4 mM), an inhibitor of phosphodiesterase, resulted in elevated levels of cAMP and concomitantly increased levels of phospholipids (7). While trying to unravel the mechanism behind the increased phospholipid levels seen in M. gypseum cells grown in the presence of aminophylline, we found a significant increase in the activity of diacylglycerol kinase (DG-kinase) 2 compared to control cells. DG-kinase (EC 2.7.1.107) catalyzes the phosphorylation of DG to generate phosphatidic acid (8). Recent evidence indicates that phosphatidic acid and its metabolite lysophosphatidic acid may act as second messengers (9 – 12). Thus DG-kinase has two important functions, first, to limit cellular levels of DG, which acts as an activator of PKC and second, to generate additional second messengers. Since cAMP mediates its effects mainly through cAMP-dependent protein kinase (PKA), which in turn phosphorylates many physiological substrates (13, 14), purification of both PKA and DG-kinase was carried out to find any correlation between enhanced DG-kinase activity and elevated cAMP levels. In this paper, we report the purification of DG-kinase and also evidence is being provided that the catalytic subunit of 2 Abbreviations used: DG-kinase, diacylglycerol kinase; PKA, protein kinase A; PMSF, phenylmethylsulfonyl fluoride; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; PKI, PKA inhibitor; BSA, bovine serum albumin.
219
220
HAQ, SHARMA, AND KHULLER TABLE I
Summary of the Purification of Diacylglycerol Kinase from M. gypseum
Purification step Cytosolic fraction DEAE–cellulose Cellulose–phosphate ATP–agarose
Activity (pmol/min)
Protein (mg)
Sp act (pmol/mg/min)
Purification fold
% Yield
6193 4132.8 3420 651.6
1533 28 6 0.3
4.04 147.6 570.5 2172
1 36.5 141.2 537.6
100 66.7 55.2 10.5
PKA does phosphorylate DG-kinase. The phosphorylation of DG-kinase may in turn be responsible for the enhanced DG-kinase activity seen in M. gypseum cells grown in the presence of aminophylline. MATERIALS AND METHODS [␥- 32P]ATP was purchased from Bhabha Atomic Research Centre (Mumbai, India). DEAE– cellulose, cellulose phosphate, DG-kinase inhibitor (D5919, Sigma), PMSF (phenylmethylsulfonyl fluoride), EDTA (ethylenediaminetetraacetic acid), ethylene glycol bis (-aminoethylether)-N,N,N⬘,N⬙-tetraacetic acid, ATP (adenosine 5⬘-triphosphate, disodium salt), DTT (dithiothreitol), and PKA inhibitor (5-24)peptide (PKI) were obtained from Sigma (St. Louis, MO). Centricon-5 concentrators were from Amicon (Beverly, MA). All other reagents were of the highest quality available. Organism and culture conditions. M. gypseum obtained from the Mycological Reference Laboratory, School of Hygiene and Tropical Medicine (London, UK), was used in the study. The organism was routinely maintained by periodic transfers on Sabouraud’s agar slants. It was grown as shake culture in Sabouraud’s broth (4% glucose, 1% peptone, pH 5.4) at 27°C. Purification of the catalytic subunit of PKA. The catalytic subunit of PKA was purified from M. gypseum following our earlier reported procedure (15). Assay of DG-kinase. During the course of enzyme purification the activity was measured under the standard incubation conditions using deoxycholate as reported by Kanoh et al. (16). The reaction mixture contained in a final volume of 125 l, 100 mM Tris–HCl (pH 7.4), 20 mM sodium fluoride (NaF), 1.0 mM deoxycholate, 0.5 mM DTT, 10 mM MgCl 2, 1.0 mM DG, 1.0 mM ATP containing ⬃100 cpm/pmol [␥- 32P]ATP and enzyme. Before incubations, diacylglycerol was sonicated in Tris–HCl buffer containing deoxycholate, NaF, and DTT. The reaction mixture was incubated for 10 min at 30°C and the reaction was stopped by addition of 50 l of concentrated HCl to the tubes, followed by 1.5 ml of water. The reaction products were then extracted with 1.0 ml of 1-butanol (17, 18). The radioactivity of the butanol extracts was, after being washed once with 1.0 ml of watersaturated butanol, measured in Bray’s scintillation fluid (19). The reaction products were analyzed by thin-layer chromatography with chloroform:methanol:4 M ammonia (50:50:14, v/v/v) as the developing solvent. It was found that the radioactivity of the butanol extracts was exclusively associated with carrier phosphatidic acid. Purification of DG-kinase. DG-kinase was purified from the cytosolic fraction of M. gypseum and all purification steps were performed at 4°C. Step 1: Preparation of M. gypseum cytosol. M. gypseum cells were harvested and washed with normal saline. They were sonicated using 25 mM Tris–HCl (pH 7.4) containing 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 50 M ATP (buffer A). The homogenates were centrifuged at 10,000g for 10 min, followed by 30,000g for 30 min.
Further centrifugation at 100,000g for 1 h was performed to obtain the cytosol. Step 2: DEAE– cellulose chromatography. The cytosol (1533 mg protein) was directly applied on a DEAE– cellulose column (2 ⫻ 12 cm) preequilibrated with buffer A. The column was washed with 200 ml of buffer A (fractions 1–15). The enzyme was then eluted with a discontinuous gradient of NaCl in buffer A, consisting of 50 mM NaCl (fractions 16 –32), 100 mM NaCl (fractions 32– 47), and 500 mM NaCl (fractions 48 – 69). Individual fractions were monitored for DG-kinase activity. It was found that the kinase activity eluted in 100 mM NaCl gradient. The active fractions were pooled and subjected to 80% ammonium sulfate precipitation. The precipitate obtained was dissolved in 100 mM sodium phosphate buffer (pH 6.5) containing 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 50 M ATP (buffer B) and dialyzed against the same buffer. Step 3: Cellulose phosphate column chromatography. The dialyzed protein (28 mg) was then loaded on cellulose phosphate column (1 ⫻ 14 cm), preequilibrated with buffer B. After washing the column with 50 ml of buffer B, the protein was eluted using 50 ml each of 100 mM NaCl, 200 mM NaCl, and 500 mM NaCl in buffer B. On monitoring DG-kinase activity, it was found that it eluted at 200 mM NaCl concentration. The active fractions were pooled and subjected to 80% ammonium sulfate precipitation. The precipitate obtained was dialyzed against 25 mM Tris–HCl (pH 7.4) containing 0.5 mM DTT, 10 M ATP, 1 mM EDTA, 1 mM MgCl 2, and 50 mM NaCl (buffer C). Step 4: ATP–agarose affinity chromatography. The dialyzed protein (6 mg) was then loaded on an ATP–agarose column (4 ml), which was preequilibrated with buffer C. The column was washed with 40 ml of buffer C (fractions 1–13). The column was then developed with 20 ml each of 50 – 450 mM NaCl in buffer C and approximately 0.5-ml fractions were collected. The fractions were assayed for DG-kinase activity, and the active fractions were pooled and concentrated using a Centricon-5 concentrator (Amicon). Protein determination. Protein was determined by the method of Bradford (20) or Lowry et al. (21) using BSA as standard. Alkaline phosphatase treatment of DG-kinase. DG-kinase (1 g) was incubated for 30 min with different concentrations of alkaline phosphatase (2– 40 units) in 50 mM Tris–HCl (pH 8.0) containing 10 mM MgCl 2 in a total volume of 30 l. The reaction mixture was then assayed for DG-kinase activity in 50 mM Tris–HCl (pH 7.4) containing 1 mM sodium orthovanadate, 20 mM NaF, 1 mM deoxycholate, 0.5 mM DTT, 1 mM dipalmitin, and 100 M ATP containing [␥- 32P]ATP (⬃100 cpm/pmol). Immediately before incubation, dipalmitin was sonicated in Tris buffer containing deoxycholate, NaF, and DTT. The reaction was continued for DG-kinase assay as described above. One unit of alkaline phosphatase hydrolyzes 1 mol of p-nitrophenyl phosphate per minute at 37°C. Effect of incubation of the catalytic subunit on the activity of DGkinase. DG-kinase (1 g) was incubated for 30 min with different concentrations of the catalytic subunit of PKA from M. gypseum (30, 50, 70, 90, and 120 ng) in Tris–HCl (pH 7.4) containing 10 mM MgCl 2
PHOSPHORYLATION OF DG-KINASE BY PROTEIN KINASE A FROM M. gypseum
221
FIG. 1. Elution and activity profile of DG-kinase of M. gypseum on a DEAE– cellulose column. Protein was eluted with 200 ml each of 50, 100, and 500 mM NaCl in buffer A, respectively, as discussed under Materials and Methods. The absorbance was monitored at 280 nm (F) and aliquots of fractions were assayed for DG-kinase activity (⫻). and 10 M ATP containing [␥- 32P]ATP (100 cpm/pmol) in a total volume of 25 l. The mixture was then incubated with 50 mM Tris–HCl (pH 7.4) containing 20 mM NaF, 1 mM deoxycholate, 0.5 mM DTT, 1 mM dipalmitin, and 2 M PKI (5–24), a specific inhibitor of PKA, and assayed for DG-kinase activity as described above. Gel electrophoresis and autoradiography. SDS–polyacrylamide gel electrophoresis was performed according to the method of Laemmli (22). For autoradiography, phosphorylated proteins were boiled for 5 min with 5⫻ sample buffer and subjected to SDS–PAGE. The gels were dried and autoradiography of dried gels was performed using Nieue Plus X-ray films (Japan) and an amplifying screen (Kodak).
RESULTS
Purification of DG-Kinase We report here the identification and purification of DG-kinase from M. gypseum. The enzyme was purified
up to 537-fold to a final specific activity of 2172 pmol/ min/mg protein (Table I). Initial resolution of DG-kinase was performed on a DEAE– cellulose column using a discontinuous NaCl gradient. Fractions containing DG-kinase activity (eluted at 100 mM NaCl) were pooled, concentrated, and dialyzed against buffer B (Fig. 1). The protein was then loaded on cellulose– phosphate column and DG-kinase eluted at 200 mM NaCl in buffer B (Fig. 2). The active fractions, after dialysis, were then resolved on an ATP–agarose affinity column. The column was developed with a continuous gradient of 50 – 450 mM NaCl in buffer C. DGkinase eluted as a single peak from this column (Fig. 3). Purification of DG-kinase is summarized in Table I. Analysis of the affinity-purified enzyme on SDS–PAGE
FIG. 2. Elution and activity profile of DG-kinase of M. gypseum on cellulose–phosphate column. The active fractions from a DEAE– cellulose column were pooled, concentrated, and chromatographed on a cellulose–phosphate column. DG-kinase was eluted with NaCl gradient as discussed under Materials and Methods. The absorbance was monitored at 280 nm (F) and aliquots of fractions were assayed for DG-kinase activity (⫻).
222
HAQ, SHARMA, AND KHULLER
FIG. 3. Affinity chromatography of DG-kinase on an ATP–agarose column. The active fractions from a cellulose–phosphate column were pooled, concentrated, and chromatographed as described under Materials and Methods using a sodium chloride gradient (—). The absorbance was monitored at 280 nm (‚) and aliquots of fractions were assayed for DG-kinase activity (⫻).
followed by silver staining revealed a single band of approximately 93 kDa (Fig. 4). When log molecular weight of standard markers was plotted against the Rf value, the molecular weight of purified DG-kinase was found to be 93.3 kDa.
decrease in the activity of the purified enzyme. Incubation of DG-kinase with 1, 3, 4, 6, 8, and 10 M concentrations of DG-kinase inhibitor resulted in 25, 40, 60, 70, 90, and 95% inhibition in the kinase activity, respectively.
Effect of DG-Kinase Inhibitor on Purified Enzyme
Effect of Lipid Molecules on DG-Kinase Activity
Incubation of the purified enzyme with DG-kinase inhibitor (D5919, Sigma) revealed a dose-dependent
Activity of DG-kinase was monitored in the presence of 250 M concentrations of different lipid molecules (Table II). It was seen that there was a decrease in the activity of DG-kinase in the presence of phosphatidic acid and arachidonic acid, while other lipid molecules, viz. Phosphatidylcholine, phosphatidylethanolamine, and cardiolipin, were found to have stimulatory effect on the DG-kinase activity. Effect of Alkaline Phosphatase Treatment on DG-Kinase Activity
FIG. 4. SDS–PAGE analysis of purified DG-kinase on a 12% gel. Lane 1, standard molecular weight markers; lane 2, aliquot from cytosolic fraction; lane 3, aliquot from DEAE– cellulose pooled fractions; lane 4, aliquot from cellulose–phosphate pooled fractions; lanes 5 and 6, aliquots from pooled DG-kinase-active fractions from an ATP–agarose affinity column.
Purified DG-kinase was incubated for 30 min with different concentrations of alkaline phosphatase; it was seen that there was a decrease in kinase activity with increase in alkaline phosphatase (Fig. 5). However, the decrease was not linear and almost 70% of the kinase activity persisted even when the alkaline phosphatase concentration was raised from 4 to 40 units. This result implies that almost 70% of kinase activity is phosphorylation independent.
PHOSPHORYLATION OF DG-KINASE BY PROTEIN KINASE A FROM M. gypseum TABLE II
Effect of Different Lipid Molecules on the Activity of Purified DG-Kinase from M. gypseum
Addition
Sp act (pmol/min/ mg protein)
Relative activity (%)
None Phosphatidic acid Phosphatidylcholine Arachidonic acid Phosphatidylethanolamine Cardiolipin
2140.5 1900.5 2994.5 1301.0 2975.5 3378.0
100 88.7 139.8 60.0 139.0 157.8
Note. Values are means of two independent observations.
Effect of Catalytic Subunit of PKA Treatment on DG-Kinase Activity Purified DG-kinase was incubated for 30 min with different concentrations of catalytic subunit of PKA and then the reaction mixture was assayed for DGkinase activity. It was found that with increase in the amount of catalytic subunit there was a concomitant increase in the DG-kinase activity (Fig. 6). Phosphorylation and Autoradiography of DG-Kinase DG-kinase was incubated for 30 min with catalytic subunit of PKA as mentioned under Materials and Methods and the mixture was then analyzed on a 10% SDS–PAGE and processed for autoradiography (Fig. 7). It was seen that when DG-kinase was incubated in the absence of the catalytic subunit of PKA no radio-
223
active band was detected on autoradiograph (Fig. 7, lane 1). However, when incubated in the presence of the catalytic subunit of PKA, DG-kinase became phosphorylated as observed on autoradiography (Fig. 7, lane 2). Further, the phosphorylated band disappeared completely when the reaction mixture contained PKI(5–24), a specific PKA inhibitor (Fig. 7, lane 3). DISCUSSION
The present study demonstrates the purification of DG-kinase from M. gypseum and its subsequent phosphorylation by the catalytic subunit of PKA. DG-kinase was purified up to 537-fold (Table I) and SDS–PAGE followed by silver staining revealed a single band of 93 kDa (approximately). DG-kinases have been purified or partially purified from various sources (23–26). These DG-kinases appear to differ with respect to their molecular weight, cofactor regulation, tissue distribution, and substrate specificity. Studies concerning DGkinase have revealed a markedly variable molecular weight of the enzyme, namely, 13 kDa in Escherichia coli (23), 79 kDa in pig brain cytosol (24), 60 kDa in pp60 (25), and apparently 120 kDa in rat liver cytosol (17). DG-kinase isoforms with different specificities toward molecular species of diacylglycerol and with distinct subcellular distributions have been identified, which differ in the Ca 2⫹ dependency, substrate specificities, and structural domains (27, 28). The activity of purified enzyme was almost completely inhibited in the presence a 10 M concentration of DG-kinase inhibitor (D5919, Sigma). This confirms that the purified enzyme is DG-kinase. M. gypseum
FIG. 5. Effect of alkaline phosphatase treatment on the activity of DG-kinase from M. gypseum. DG-kinase was incubated for 30 min with different concentrations of alkaline phosphatase and then the reaction mixture was assayed for DG-kinase activity as described under Materials and Methods.
224
HAQ, SHARMA, AND KHULLER
FIG. 6. Effect of incubation of catalytic subunit of PKA on the activity of DG-kinase from M. gypseum. DG-kinase was incubated for 30 min with different concentrations of the catalytic subunit of PKA and the reaction mixture was then assayed for DG-kinase activity as described under Materials and Methods.
DG-kinase behaved like DG-kinases from other sources in that its activity was inhibited in the presence of arachidonic acid (16), but stimulated in the
FIG. 7. Autoradiograph showing the phosphorylation of diacylglycerol kinase by PKA. Lane 1, DG-kinase (1 g) alone; lane 2, DG kinase (1 g) and C-subunit of PKA (1 g); lane 3, DG-kinase and C-subunit of PKA in the presence of PKI (5–24); lane 4, DG-kinase (1 g) and C-subunit (100 ng); lane 5, DG-kinase (1 g) and C-subunit (50 ng); lane 6, C-subunit alone.
presence of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin (29, 30). Cardiolipin has been shown to stimulate DG-kinase activity at concentrations as low as 1 mol% (31). The enhanced activity of DG-kinase in M. gypseum cells grown in the presence of aminophylline, (2.50 ⫹ 0.17 pmol/min/mg protein) compared to control cells (1.25 ⫹ 0.05 pmol/min/mg protein), prompted us to find whether this increase in DG-kinase activity has some correlation with the increased cAMP levels seen in these cells (7). We thus attempted the phosphorylation of DG-kinase by the catalytic subunit of PKA. We found that incubation of DG-kinase in the presence of alkaline phosphatase decreased the DG-kinase activity but complete inhibition was not attained. Earlier, Sanghera and Vance (32) have shown that treatment with alkaline phosphatase decreased the cytidyltransferase activity in the postmitochondrial supernatant, while the activity in the microsomal fraction increased by 2-fold. Again alkaline phosphatase catalyzed the dephosphorylation of 45kDa phosphatidic acid phosphatase and this resulted in 1.3-fold inhibition of the phosphatidic acid phosphatase activity (33). We noticed that the incubation of DG-kinase with increasing concentrations of the catalytic subunit of PKA enhanced the DG-kinase activity (Fig. 7). This observation is in agreement with that of Soeling et al. (34), who found that membrane bound DG-kinase in parotid glands was activated via phos-
PHOSPHORYLATION OF DG-KINASE BY PROTEIN KINASE A FROM M. gypseum
phorylation by cAMP- and Ca 2⫹/CaM-dependent protein kinase but not by PKC. In contrast Kanoh et al. (35) have reported in vitro phosphorylation of diacylglycerol kinase by PKC purified from pig thymus cytosol. We found that incubation of DG-kinase alone did not give any radioactive band; this is in agreement with the previous observations with pig brain enzyme (36). Incubation of the catalytic subunit of PKA with DGkinase resulted in phosphorylation of DG-kinase. Thus, the increase in the DG-kinase activity after its prior incubation with the catalytic subunit of PKA could be due to the phosphorylation of DG-kinase by PKA. The results thus suggest that the phosphorylation of DG-kinase increased its catalytic power and since we have found increased production of phosphatidic acid in aminophylline grown cells (data not shown), it implies that this leads to the increased phospholipid synthesis in aminophylline grown cells. In short, we have purified DG-kinase from M. gypseum and shown that its phosphorylation by the catalytic subunit of PKA results in the activation of DGkinase. Whether this phosphorylation of DG-kinase is responsible for the increased phospholipid levels seen in cAMP-stimulated cells remains to be elucidated. ACKNOWLEDGMENTS This work was supported by a grant from the Council of Scientific and Industrial Research, New Delhi, India, to Professor G. K. Khuller. E.H. is a recipient of fellowship from Postgraduate Institute of Medical Education and Research, Chandigarh, India.
REFERENCES 1. Chapman, D., and Lesline, R. B. (1970) in Membranes of Mitochondria and Chloroplast (Racker, E., Ed.), pp. 91–126, Van Nostrand–Reinhold, Princeton, NJ. 2. Fourcans, B., and Jain, M. K. (1974) Adv. Lipid Res. 12, 147–226. 3. Prasad, R. (1985) Adv. Lipid Res. 21, 187–242. 4. Loewy, B., Marczynski, G. T., Dingwall, A., and Shapiro, L. (1990) J. Bacteriol. 172, 5523–5530. 5. Khuller, G. K., and Manchanda, N. (1996) in Lipids of Pathogenic Fungi (Prasad, R., and Ghannoum, M. A., Eds.), pp. 169 – 198, CRC Press, Boca Raton, FL. 6. Bindra, A., and Khuller, G. K. (1992) Biochim. Biophys. Acta 1124, 185–189. 7. Bindra, A., and Khuller, G. K. (1991) Biochim. Biophys. Acta 1081, 61– 64. 8. Kanoh, H., Yamada, K., and Sakane, F. (1990) Trends Biochem. Sci. 15, 47–50.
225
9. van Corven, E. J., van Rijswijk, A., Jalink, K., van der Bend, R. L., Blitterswijk, W. J., and Moolenaar, W. H. (1990) Biochem. J. 281, 47–50. 10. Durieux, M. E., and Lynch, K. R. (1993) Trends Pharmacol. Sci. 14, 249 –254. 11. Knauss, T. C., Jaffer, F. E., and Abboud, H. E. (1990) J. Biol. Chem. 265, 14457–14463. 12. Moolenaar, W. H., Kruijer, W., Tilly, B. C., Verlaan, L., Bierman, A. J., and de Laat, S. W. (1986) Nature 323, 171–173. 13. Krebs, E. G., and Beavo, J. A. (1979) Annu. Rev. Biochem. 43, 923–959. 14. Taylor, S. S., Buechler, J. A., and Yonemoto, W. C. (1990) Annu. Rev. Biochem. 59, 971–1005. 15. Haq, E., Sharma, S., and Khuller, G. K. (2000) Biochim. Biophys. Acta 1474, 100 –106. 16. Kanoh, H., Kondoh, H., and Ono, T. (1983) J. Biol. Chem. 258, 1767–1774. 17. Kanoh, H., and Ohno, K. (1981) Arch. Biochem. Biophys. 209, 266 –275. 18. Bremer, J. (1963) J. Biol. Chem. 238, 2774 –2779. 19. Bray, G. A. (1960) Anal. Biochem. 1, 277–285. 20. Bradford, M. M. (1976) Anal. Biochem. 72, 248 –254. 21. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265–275. 22. Laemmli, U. K. (1976) Nature 227, 680 – 685. 23. Loomis, C. R., Walsh, J. P., and Bell, R. M. (1985) J. Biol. Chem. 260, 4091– 4097. 24. Kanoh, H., and Ono, T. (1984) J. Biol. Chem. 259, 11197–11202. 25. Sugimoto, Y., Whitman, M., Cantley, L. C., and Erickson, R. L. (1984) Proc. Natl. Acad. Sci. USA 81, 2117–2121. 26. Thomas, W. E., and Glomset, J. A. (1999) Biochemistry 38, 3320 – 3326. 27. Goto, K., and Kondo, H. (1996) Proc. Natl. Acad. Sci. USA 93, 11196 –11201. 28. Ding, L., Bunting, M., Topham, M. K., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (1997) Proc. Natl. Acad. Sci. 94, 5519 –5524. 29. Lin, C. H., Bishop, H., and Strickland, K. P. (1986) Lipids 21, 206 –211. 30. Kato, M., and Takenawa, T. (1990) J. Biol. Chem. 265, 794 – 800. 31. Walsh, J. P., and Bell, R. M. (1985) J. Biol. Chem. 261, 6239 – 6247. 32. Sanghera, J. S., and Vance, D. E. (1989) J. Biol. Chem. 264, 1215–1223. 33. Quinlan, J. J., Nickels, J. T., Jr., Wu, W., Lin, Y. P., Broach, J. R., and Carman, G. M. (1992) J. Biol. Chem. 267, 18013–18020. 34. Soeling, H. D., Fest, W., Schmidt, T., Esselman, H., and Bachmann, V. (1989) J. Biol. Chem. 264, 10643–10648. 35. Kanoh, H., Yamada, K., Sakane, F., and Imaizumi, T. (1989) Biochem. J. 258, 455– 462. 36. Kanoh, H., and Ono, T. (1986) FEBS Lett. 201, 97–100.
Purification of Diacylglycerol Kinase from Microsporum gypseum and Its Phosphorylation by the Catalytic Subunit of Protein Kinase A Ehtishamul Haq, Sadhna Sharma, and G. K. Khuller 1 Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh–160 012, India
Received November 17, 2000, and in revised form May 5, 2001; published online July 20, 2001
Diacylglycerol (DG) kinase (EC 2.7.1.107) was purified to homogeneity from the soluble extract of Microsporum gypseum, a dermatophyte. Purified enzyme showed a final specific activity of 2172 pmol/min/mg protein and its apparent molecular weight on SDS– PAGE was found to be 93 kDa. The activity of purified enzyme was inhibited in a dose-dependent manner in the presence of DG-kinase inhibitor (D5919, Sigma). DG-kinase activity was found to be stimulated in the presence of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin while the activity was alleviated in the presence of phosphatidic acid and arachidonic acid. Kinase activity was partially inhibited when assayed after prior treatment with alkaline phosphatase. Treatment of DG-kinase with the catalytic subunit of protein kinase A (PKA)-stimulated DGkinase activity in a dose-dependent manner. Incubation of DG-kinase with the catalytic subunit of PKA led to the phosphorylation of DG-kinase as revealed by autoradiography. The phosphorylated band disappeared completely in the presence of specific PKA inhibitor. Increased activity of DG-kinase on incubation with the catalytic subunit of PKA was possibly due to the phosphorylation of the former by the latter. Whether this in vitro phosphorylation and activation of DG-kinase occurs under physiological conditions remains to be elucidated. © 2001 Academic Press Key Words: DG-kinase; protein kinase A; phosphorylation; Microsporum gypseum.
Phospholipids are the key biological lipids having diverse and critical roles in cellular metabolism and function (1, 2). They are known to play important role 1 To whom correspondence should be addressed. Fax: ⫹91-0172-744 401 or 745 078. E-mail: [email protected] or [email protected].
0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
in growth, sporulation, replication, and germination in microorganisms including fungi (3, 4). Altered growth and environmental conditions have been reported to alter the rate of phospholipid biosynthesis in Microsporum gypseum, a dermatophyte, this in turn may help the fungus to adapt to the changed conditions (5). Evidence for the involvement of cAMP in cellular metabolism has been reported previously from our laboratory (6). It was reported that M. gypseum cells grown in the presence of aminophylline (4 mM), an inhibitor of phosphodiesterase, resulted in elevated levels of cAMP and concomitantly increased levels of phospholipids (7). While trying to unravel the mechanism behind the increased phospholipid levels seen in M. gypseum cells grown in the presence of aminophylline, we found a significant increase in the activity of diacylglycerol kinase (DG-kinase) 2 compared to control cells. DG-kinase (EC 2.7.1.107) catalyzes the phosphorylation of DG to generate phosphatidic acid (8). Recent evidence indicates that phosphatidic acid and its metabolite lysophosphatidic acid may act as second messengers (9 – 12). Thus DG-kinase has two important functions, first, to limit cellular levels of DG, which acts as an activator of PKC and second, to generate additional second messengers. Since cAMP mediates its effects mainly through cAMP-dependent protein kinase (PKA), which in turn phosphorylates many physiological substrates (13, 14), purification of both PKA and DG-kinase was carried out to find any correlation between enhanced DG-kinase activity and elevated cAMP levels. In this paper, we report the purification of DG-kinase and also evidence is being provided that the catalytic subunit of 2 Abbreviations used: DG-kinase, diacylglycerol kinase; PKA, protein kinase A; PMSF, phenylmethylsulfonyl fluoride; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; PKI, PKA inhibitor; BSA, bovine serum albumin.
219
220
HAQ, SHARMA, AND KHULLER TABLE I
Summary of the Purification of Diacylglycerol Kinase from M. gypseum
Purification step Cytosolic fraction DEAE–cellulose Cellulose–phosphate ATP–agarose
Activity (pmol/min)
Protein (mg)
Sp act (pmol/mg/min)
Purification fold
% Yield
6193 4132.8 3420 651.6
1533 28 6 0.3
4.04 147.6 570.5 2172
1 36.5 141.2 537.6
100 66.7 55.2 10.5
PKA does phosphorylate DG-kinase. The phosphorylation of DG-kinase may in turn be responsible for the enhanced DG-kinase activity seen in M. gypseum cells grown in the presence of aminophylline. MATERIALS AND METHODS [␥- 32P]ATP was purchased from Bhabha Atomic Research Centre (Mumbai, India). DEAE– cellulose, cellulose phosphate, DG-kinase inhibitor (D5919, Sigma), PMSF (phenylmethylsulfonyl fluoride), EDTA (ethylenediaminetetraacetic acid), ethylene glycol bis (-aminoethylether)-N,N,N⬘,N⬙-tetraacetic acid, ATP (adenosine 5⬘-triphosphate, disodium salt), DTT (dithiothreitol), and PKA inhibitor (5-24)peptide (PKI) were obtained from Sigma (St. Louis, MO). Centricon-5 concentrators were from Amicon (Beverly, MA). All other reagents were of the highest quality available. Organism and culture conditions. M. gypseum obtained from the Mycological Reference Laboratory, School of Hygiene and Tropical Medicine (London, UK), was used in the study. The organism was routinely maintained by periodic transfers on Sabouraud’s agar slants. It was grown as shake culture in Sabouraud’s broth (4% glucose, 1% peptone, pH 5.4) at 27°C. Purification of the catalytic subunit of PKA. The catalytic subunit of PKA was purified from M. gypseum following our earlier reported procedure (15). Assay of DG-kinase. During the course of enzyme purification the activity was measured under the standard incubation conditions using deoxycholate as reported by Kanoh et al. (16). The reaction mixture contained in a final volume of 125 l, 100 mM Tris–HCl (pH 7.4), 20 mM sodium fluoride (NaF), 1.0 mM deoxycholate, 0.5 mM DTT, 10 mM MgCl 2, 1.0 mM DG, 1.0 mM ATP containing ⬃100 cpm/pmol [␥- 32P]ATP and enzyme. Before incubations, diacylglycerol was sonicated in Tris–HCl buffer containing deoxycholate, NaF, and DTT. The reaction mixture was incubated for 10 min at 30°C and the reaction was stopped by addition of 50 l of concentrated HCl to the tubes, followed by 1.5 ml of water. The reaction products were then extracted with 1.0 ml of 1-butanol (17, 18). The radioactivity of the butanol extracts was, after being washed once with 1.0 ml of watersaturated butanol, measured in Bray’s scintillation fluid (19). The reaction products were analyzed by thin-layer chromatography with chloroform:methanol:4 M ammonia (50:50:14, v/v/v) as the developing solvent. It was found that the radioactivity of the butanol extracts was exclusively associated with carrier phosphatidic acid. Purification of DG-kinase. DG-kinase was purified from the cytosolic fraction of M. gypseum and all purification steps were performed at 4°C. Step 1: Preparation of M. gypseum cytosol. M. gypseum cells were harvested and washed with normal saline. They were sonicated using 25 mM Tris–HCl (pH 7.4) containing 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 50 M ATP (buffer A). The homogenates were centrifuged at 10,000g for 10 min, followed by 30,000g for 30 min.
Further centrifugation at 100,000g for 1 h was performed to obtain the cytosol. Step 2: DEAE– cellulose chromatography. The cytosol (1533 mg protein) was directly applied on a DEAE– cellulose column (2 ⫻ 12 cm) preequilibrated with buffer A. The column was washed with 200 ml of buffer A (fractions 1–15). The enzyme was then eluted with a discontinuous gradient of NaCl in buffer A, consisting of 50 mM NaCl (fractions 16 –32), 100 mM NaCl (fractions 32– 47), and 500 mM NaCl (fractions 48 – 69). Individual fractions were monitored for DG-kinase activity. It was found that the kinase activity eluted in 100 mM NaCl gradient. The active fractions were pooled and subjected to 80% ammonium sulfate precipitation. The precipitate obtained was dissolved in 100 mM sodium phosphate buffer (pH 6.5) containing 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 50 M ATP (buffer B) and dialyzed against the same buffer. Step 3: Cellulose phosphate column chromatography. The dialyzed protein (28 mg) was then loaded on cellulose phosphate column (1 ⫻ 14 cm), preequilibrated with buffer B. After washing the column with 50 ml of buffer B, the protein was eluted using 50 ml each of 100 mM NaCl, 200 mM NaCl, and 500 mM NaCl in buffer B. On monitoring DG-kinase activity, it was found that it eluted at 200 mM NaCl concentration. The active fractions were pooled and subjected to 80% ammonium sulfate precipitation. The precipitate obtained was dialyzed against 25 mM Tris–HCl (pH 7.4) containing 0.5 mM DTT, 10 M ATP, 1 mM EDTA, 1 mM MgCl 2, and 50 mM NaCl (buffer C). Step 4: ATP–agarose affinity chromatography. The dialyzed protein (6 mg) was then loaded on an ATP–agarose column (4 ml), which was preequilibrated with buffer C. The column was washed with 40 ml of buffer C (fractions 1–13). The column was then developed with 20 ml each of 50 – 450 mM NaCl in buffer C and approximately 0.5-ml fractions were collected. The fractions were assayed for DG-kinase activity, and the active fractions were pooled and concentrated using a Centricon-5 concentrator (Amicon). Protein determination. Protein was determined by the method of Bradford (20) or Lowry et al. (21) using BSA as standard. Alkaline phosphatase treatment of DG-kinase. DG-kinase (1 g) was incubated for 30 min with different concentrations of alkaline phosphatase (2– 40 units) in 50 mM Tris–HCl (pH 8.0) containing 10 mM MgCl 2 in a total volume of 30 l. The reaction mixture was then assayed for DG-kinase activity in 50 mM Tris–HCl (pH 7.4) containing 1 mM sodium orthovanadate, 20 mM NaF, 1 mM deoxycholate, 0.5 mM DTT, 1 mM dipalmitin, and 100 M ATP containing [␥- 32P]ATP (⬃100 cpm/pmol). Immediately before incubation, dipalmitin was sonicated in Tris buffer containing deoxycholate, NaF, and DTT. The reaction was continued for DG-kinase assay as described above. One unit of alkaline phosphatase hydrolyzes 1 mol of p-nitrophenyl phosphate per minute at 37°C. Effect of incubation of the catalytic subunit on the activity of DGkinase. DG-kinase (1 g) was incubated for 30 min with different concentrations of the catalytic subunit of PKA from M. gypseum (30, 50, 70, 90, and 120 ng) in Tris–HCl (pH 7.4) containing 10 mM MgCl 2
PHOSPHORYLATION OF DG-KINASE BY PROTEIN KINASE A FROM M. gypseum
221
FIG. 1. Elution and activity profile of DG-kinase of M. gypseum on a DEAE– cellulose column. Protein was eluted with 200 ml each of 50, 100, and 500 mM NaCl in buffer A, respectively, as discussed under Materials and Methods. The absorbance was monitored at 280 nm (F) and aliquots of fractions were assayed for DG-kinase activity (⫻). and 10 M ATP containing [␥- 32P]ATP (100 cpm/pmol) in a total volume of 25 l. The mixture was then incubated with 50 mM Tris–HCl (pH 7.4) containing 20 mM NaF, 1 mM deoxycholate, 0.5 mM DTT, 1 mM dipalmitin, and 2 M PKI (5–24), a specific inhibitor of PKA, and assayed for DG-kinase activity as described above. Gel electrophoresis and autoradiography. SDS–polyacrylamide gel electrophoresis was performed according to the method of Laemmli (22). For autoradiography, phosphorylated proteins were boiled for 5 min with 5⫻ sample buffer and subjected to SDS–PAGE. The gels were dried and autoradiography of dried gels was performed using Nieue Plus X-ray films (Japan) and an amplifying screen (Kodak).
RESULTS
Purification of DG-Kinase We report here the identification and purification of DG-kinase from M. gypseum. The enzyme was purified
up to 537-fold to a final specific activity of 2172 pmol/ min/mg protein (Table I). Initial resolution of DG-kinase was performed on a DEAE– cellulose column using a discontinuous NaCl gradient. Fractions containing DG-kinase activity (eluted at 100 mM NaCl) were pooled, concentrated, and dialyzed against buffer B (Fig. 1). The protein was then loaded on cellulose– phosphate column and DG-kinase eluted at 200 mM NaCl in buffer B (Fig. 2). The active fractions, after dialysis, were then resolved on an ATP–agarose affinity column. The column was developed with a continuous gradient of 50 – 450 mM NaCl in buffer C. DGkinase eluted as a single peak from this column (Fig. 3). Purification of DG-kinase is summarized in Table I. Analysis of the affinity-purified enzyme on SDS–PAGE
FIG. 2. Elution and activity profile of DG-kinase of M. gypseum on cellulose–phosphate column. The active fractions from a DEAE– cellulose column were pooled, concentrated, and chromatographed on a cellulose–phosphate column. DG-kinase was eluted with NaCl gradient as discussed under Materials and Methods. The absorbance was monitored at 280 nm (F) and aliquots of fractions were assayed for DG-kinase activity (⫻).
222
HAQ, SHARMA, AND KHULLER
FIG. 3. Affinity chromatography of DG-kinase on an ATP–agarose column. The active fractions from a cellulose–phosphate column were pooled, concentrated, and chromatographed as described under Materials and Methods using a sodium chloride gradient (—). The absorbance was monitored at 280 nm (‚) and aliquots of fractions were assayed for DG-kinase activity (⫻).
followed by silver staining revealed a single band of approximately 93 kDa (Fig. 4). When log molecular weight of standard markers was plotted against the Rf value, the molecular weight of purified DG-kinase was found to be 93.3 kDa.
decrease in the activity of the purified enzyme. Incubation of DG-kinase with 1, 3, 4, 6, 8, and 10 M concentrations of DG-kinase inhibitor resulted in 25, 40, 60, 70, 90, and 95% inhibition in the kinase activity, respectively.
Effect of DG-Kinase Inhibitor on Purified Enzyme
Effect of Lipid Molecules on DG-Kinase Activity
Incubation of the purified enzyme with DG-kinase inhibitor (D5919, Sigma) revealed a dose-dependent
Activity of DG-kinase was monitored in the presence of 250 M concentrations of different lipid molecules (Table II). It was seen that there was a decrease in the activity of DG-kinase in the presence of phosphatidic acid and arachidonic acid, while other lipid molecules, viz. Phosphatidylcholine, phosphatidylethanolamine, and cardiolipin, were found to have stimulatory effect on the DG-kinase activity. Effect of Alkaline Phosphatase Treatment on DG-Kinase Activity
FIG. 4. SDS–PAGE analysis of purified DG-kinase on a 12% gel. Lane 1, standard molecular weight markers; lane 2, aliquot from cytosolic fraction; lane 3, aliquot from DEAE– cellulose pooled fractions; lane 4, aliquot from cellulose–phosphate pooled fractions; lanes 5 and 6, aliquots from pooled DG-kinase-active fractions from an ATP–agarose affinity column.
Purified DG-kinase was incubated for 30 min with different concentrations of alkaline phosphatase; it was seen that there was a decrease in kinase activity with increase in alkaline phosphatase (Fig. 5). However, the decrease was not linear and almost 70% of the kinase activity persisted even when the alkaline phosphatase concentration was raised from 4 to 40 units. This result implies that almost 70% of kinase activity is phosphorylation independent.
PHOSPHORYLATION OF DG-KINASE BY PROTEIN KINASE A FROM M. gypseum TABLE II
Effect of Different Lipid Molecules on the Activity of Purified DG-Kinase from M. gypseum
Addition
Sp act (pmol/min/ mg protein)
Relative activity (%)
None Phosphatidic acid Phosphatidylcholine Arachidonic acid Phosphatidylethanolamine Cardiolipin
2140.5 1900.5 2994.5 1301.0 2975.5 3378.0
100 88.7 139.8 60.0 139.0 157.8
Note. Values are means of two independent observations.
Effect of Catalytic Subunit of PKA Treatment on DG-Kinase Activity Purified DG-kinase was incubated for 30 min with different concentrations of catalytic subunit of PKA and then the reaction mixture was assayed for DGkinase activity. It was found that with increase in the amount of catalytic subunit there was a concomitant increase in the DG-kinase activity (Fig. 6). Phosphorylation and Autoradiography of DG-Kinase DG-kinase was incubated for 30 min with catalytic subunit of PKA as mentioned under Materials and Methods and the mixture was then analyzed on a 10% SDS–PAGE and processed for autoradiography (Fig. 7). It was seen that when DG-kinase was incubated in the absence of the catalytic subunit of PKA no radio-
223
active band was detected on autoradiograph (Fig. 7, lane 1). However, when incubated in the presence of the catalytic subunit of PKA, DG-kinase became phosphorylated as observed on autoradiography (Fig. 7, lane 2). Further, the phosphorylated band disappeared completely when the reaction mixture contained PKI(5–24), a specific PKA inhibitor (Fig. 7, lane 3). DISCUSSION
The present study demonstrates the purification of DG-kinase from M. gypseum and its subsequent phosphorylation by the catalytic subunit of PKA. DG-kinase was purified up to 537-fold (Table I) and SDS–PAGE followed by silver staining revealed a single band of 93 kDa (approximately). DG-kinases have been purified or partially purified from various sources (23–26). These DG-kinases appear to differ with respect to their molecular weight, cofactor regulation, tissue distribution, and substrate specificity. Studies concerning DGkinase have revealed a markedly variable molecular weight of the enzyme, namely, 13 kDa in Escherichia coli (23), 79 kDa in pig brain cytosol (24), 60 kDa in pp60 (25), and apparently 120 kDa in rat liver cytosol (17). DG-kinase isoforms with different specificities toward molecular species of diacylglycerol and with distinct subcellular distributions have been identified, which differ in the Ca 2⫹ dependency, substrate specificities, and structural domains (27, 28). The activity of purified enzyme was almost completely inhibited in the presence a 10 M concentration of DG-kinase inhibitor (D5919, Sigma). This confirms that the purified enzyme is DG-kinase. M. gypseum
FIG. 5. Effect of alkaline phosphatase treatment on the activity of DG-kinase from M. gypseum. DG-kinase was incubated for 30 min with different concentrations of alkaline phosphatase and then the reaction mixture was assayed for DG-kinase activity as described under Materials and Methods.
224
HAQ, SHARMA, AND KHULLER
FIG. 6. Effect of incubation of catalytic subunit of PKA on the activity of DG-kinase from M. gypseum. DG-kinase was incubated for 30 min with different concentrations of the catalytic subunit of PKA and the reaction mixture was then assayed for DG-kinase activity as described under Materials and Methods.
DG-kinase behaved like DG-kinases from other sources in that its activity was inhibited in the presence of arachidonic acid (16), but stimulated in the
FIG. 7. Autoradiograph showing the phosphorylation of diacylglycerol kinase by PKA. Lane 1, DG-kinase (1 g) alone; lane 2, DG kinase (1 g) and C-subunit of PKA (1 g); lane 3, DG-kinase and C-subunit of PKA in the presence of PKI (5–24); lane 4, DG-kinase (1 g) and C-subunit (100 ng); lane 5, DG-kinase (1 g) and C-subunit (50 ng); lane 6, C-subunit alone.
presence of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin (29, 30). Cardiolipin has been shown to stimulate DG-kinase activity at concentrations as low as 1 mol% (31). The enhanced activity of DG-kinase in M. gypseum cells grown in the presence of aminophylline, (2.50 ⫹ 0.17 pmol/min/mg protein) compared to control cells (1.25 ⫹ 0.05 pmol/min/mg protein), prompted us to find whether this increase in DG-kinase activity has some correlation with the increased cAMP levels seen in these cells (7). We thus attempted the phosphorylation of DG-kinase by the catalytic subunit of PKA. We found that incubation of DG-kinase in the presence of alkaline phosphatase decreased the DG-kinase activity but complete inhibition was not attained. Earlier, Sanghera and Vance (32) have shown that treatment with alkaline phosphatase decreased the cytidyltransferase activity in the postmitochondrial supernatant, while the activity in the microsomal fraction increased by 2-fold. Again alkaline phosphatase catalyzed the dephosphorylation of 45kDa phosphatidic acid phosphatase and this resulted in 1.3-fold inhibition of the phosphatidic acid phosphatase activity (33). We noticed that the incubation of DG-kinase with increasing concentrations of the catalytic subunit of PKA enhanced the DG-kinase activity (Fig. 7). This observation is in agreement with that of Soeling et al. (34), who found that membrane bound DG-kinase in parotid glands was activated via phos-
PHOSPHORYLATION OF DG-KINASE BY PROTEIN KINASE A FROM M. gypseum
phorylation by cAMP- and Ca 2⫹/CaM-dependent protein kinase but not by PKC. In contrast Kanoh et al. (35) have reported in vitro phosphorylation of diacylglycerol kinase by PKC purified from pig thymus cytosol. We found that incubation of DG-kinase alone did not give any radioactive band; this is in agreement with the previous observations with pig brain enzyme (36). Incubation of the catalytic subunit of PKA with DGkinase resulted in phosphorylation of DG-kinase. Thus, the increase in the DG-kinase activity after its prior incubation with the catalytic subunit of PKA could be due to the phosphorylation of DG-kinase by PKA. The results thus suggest that the phosphorylation of DG-kinase increased its catalytic power and since we have found increased production of phosphatidic acid in aminophylline grown cells (data not shown), it implies that this leads to the increased phospholipid synthesis in aminophylline grown cells. In short, we have purified DG-kinase from M. gypseum and shown that its phosphorylation by the catalytic subunit of PKA results in the activation of DGkinase. Whether this phosphorylation of DG-kinase is responsible for the increased phospholipid levels seen in cAMP-stimulated cells remains to be elucidated. ACKNOWLEDGMENTS This work was supported by a grant from the Council of Scientific and Industrial Research, New Delhi, India, to Professor G. K. Khuller. E.H. is a recipient of fellowship from Postgraduate Institute of Medical Education and Research, Chandigarh, India.
REFERENCES 1. Chapman, D., and Lesline, R. B. (1970) in Membranes of Mitochondria and Chloroplast (Racker, E., Ed.), pp. 91–126, Van Nostrand–Reinhold, Princeton, NJ. 2. Fourcans, B., and Jain, M. K. (1974) Adv. Lipid Res. 12, 147–226. 3. Prasad, R. (1985) Adv. Lipid Res. 21, 187–242. 4. Loewy, B., Marczynski, G. T., Dingwall, A., and Shapiro, L. (1990) J. Bacteriol. 172, 5523–5530. 5. Khuller, G. K., and Manchanda, N. (1996) in Lipids of Pathogenic Fungi (Prasad, R., and Ghannoum, M. A., Eds.), pp. 169 – 198, CRC Press, Boca Raton, FL. 6. Bindra, A., and Khuller, G. K. (1992) Biochim. Biophys. Acta 1124, 185–189. 7. Bindra, A., and Khuller, G. K. (1991) Biochim. Biophys. Acta 1081, 61– 64. 8. Kanoh, H., Yamada, K., and Sakane, F. (1990) Trends Biochem. Sci. 15, 47–50.
225
9. van Corven, E. J., van Rijswijk, A., Jalink, K., van der Bend, R. L., Blitterswijk, W. J., and Moolenaar, W. H. (1990) Biochem. J. 281, 47–50. 10. Durieux, M. E., and Lynch, K. R. (1993) Trends Pharmacol. Sci. 14, 249 –254. 11. Knauss, T. C., Jaffer, F. E., and Abboud, H. E. (1990) J. Biol. Chem. 265, 14457–14463. 12. Moolenaar, W. H., Kruijer, W., Tilly, B. C., Verlaan, L., Bierman, A. J., and de Laat, S. W. (1986) Nature 323, 171–173. 13. Krebs, E. G., and Beavo, J. A. (1979) Annu. Rev. Biochem. 43, 923–959. 14. Taylor, S. S., Buechler, J. A., and Yonemoto, W. C. (1990) Annu. Rev. Biochem. 59, 971–1005. 15. Haq, E., Sharma, S., and Khuller, G. K. (2000) Biochim. Biophys. Acta 1474, 100 –106. 16. Kanoh, H., Kondoh, H., and Ono, T. (1983) J. Biol. Chem. 258, 1767–1774. 17. Kanoh, H., and Ohno, K. (1981) Arch. Biochem. Biophys. 209, 266 –275. 18. Bremer, J. (1963) J. Biol. Chem. 238, 2774 –2779. 19. Bray, G. A. (1960) Anal. Biochem. 1, 277–285. 20. Bradford, M. M. (1976) Anal. Biochem. 72, 248 –254. 21. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265–275. 22. Laemmli, U. K. (1976) Nature 227, 680 – 685. 23. Loomis, C. R., Walsh, J. P., and Bell, R. M. (1985) J. Biol. Chem. 260, 4091– 4097. 24. Kanoh, H., and Ono, T. (1984) J. Biol. Chem. 259, 11197–11202. 25. Sugimoto, Y., Whitman, M., Cantley, L. C., and Erickson, R. L. (1984) Proc. Natl. Acad. Sci. USA 81, 2117–2121. 26. Thomas, W. E., and Glomset, J. A. (1999) Biochemistry 38, 3320 – 3326. 27. Goto, K., and Kondo, H. (1996) Proc. Natl. Acad. Sci. USA 93, 11196 –11201. 28. Ding, L., Bunting, M., Topham, M. K., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (1997) Proc. Natl. Acad. Sci. 94, 5519 –5524. 29. Lin, C. H., Bishop, H., and Strickland, K. P. (1986) Lipids 21, 206 –211. 30. Kato, M., and Takenawa, T. (1990) J. Biol. Chem. 265, 794 – 800. 31. Walsh, J. P., and Bell, R. M. (1985) J. Biol. Chem. 261, 6239 – 6247. 32. Sanghera, J. S., and Vance, D. E. (1989) J. Biol. Chem. 264, 1215–1223. 33. Quinlan, J. J., Nickels, J. T., Jr., Wu, W., Lin, Y. P., Broach, J. R., and Carman, G. M. (1992) J. Biol. Chem. 267, 18013–18020. 34. Soeling, H. D., Fest, W., Schmidt, T., Esselman, H., and Bachmann, V. (1989) J. Biol. Chem. 264, 10643–10648. 35. Kanoh, H., Yamada, K., Sakane, F., and Imaizumi, T. (1989) Biochem. J. 258, 455– 462. 36. Kanoh, H., and Ono, T. (1986) FEBS Lett. 201, 97–100.