- Email: [email protected]
Effect of NaF and okadaic acid on the subcellular distribution of CTP: phosphocoline cytidylyltransferase activity in rat liver
314
Biochimrcu et Bloph_vsrcu Acru. 1042 (1990) 314-379 Elsevier
BBALIP
53324
Effect of NaF and okadaic acid on the subcellular distribution of CTP:phosphocholine cytidylyltransferase activity in rat liver Grant M. Hatch ‘, Tsz-Shan ’ Lipid and Lipoprotein Rewurch
2 and Dennis
Group und Department ofBiochemistry, Unic~ersri_vof Alberta and ’ FuJisawrr Pharmuceutiwl Company. Tokyo (Jupon)
(Revised
Key words:
Lam ‘, Y. Tsukitani
Okadaic
(Received 17 July 1989) manuscript received 23 October
acid; NaF; Cytidylyltransferase;
E. Vance ’
Edmonton,
Albertu (Ccrnudu)
1989)
Enzyme translocation:
(Rat liver)
The effect of preincubation of rat liver post-mitochondrial supernatant with NaF and okadaic acid on the subcellular distribution of CTP: phosphocholine cytidylyltransferase activity was investigated. NaF (20 mM) inhibited the time-dependent activation of cytidylyltransferase activity in post-mitochondrial supernatant. Subcellular fractionation of the post-mitochondrial supernatant revealed that cytidylyltransferase activity in the microsomal fraction was decreased and activity in the cytosolic fraction increased with time of preincubation with NaF compared to controls. Okadaic acid is a specific and potent inhibitor of type 1 and 2A phosphoprotein phosphatases. Preincubation of cytosol with 5 uM okadaic acid inhibited the time-dependent activation of cytosolic cytidylyltransferase activity. Preincubation of post-mitochondrial supernatants with 5 PM okadaic acid inhibited the time-dependent activation of cytidylyltransferase activity by 13% at 45 min and 16% at 60 min of preincubation compared to controls. Microsomal cytidylyltransferase activity was decreased 27% at 45 min and 31% at 60 min with a corresponding retention of cytosolic cytidylyltransferase activity of 21% at 45 min and 37% at 60 min of preincubation with okadaic acid compared to controls. We postulate that the activity of the type 1 and/or type 2A phosphoprotein phosphatases affect the subcellular distribution of CTP: phosphocholine cytidylyltransferase activity in rat liver.
Introduction
Correspondence: D.E. Vance, Lipid and Lipoprotein Research Group, 328 Heritage Medical Research Building. University of Alberta. Edmonton, Alberta, Canada, T6G 2S2.
somal fractions of rat liver homogenates [lo]. The rate of phosphatidylcholine biosynthesis is dependent on the subcellular distribution of the cytidylyltransferase between its inactive cytosolic form and active microsomal form [ll]. The inactive cytosolic form may be fully activated by anionic phospholipids and phosphatidylcholine:fatty acid vesicles [12-141. Phosphorylationdephosphorylation of the cytidylyltransferase is one proposed mechanism for the regulation of the enzyme’s activity [15]. The cytidylyltransferase was recently demonstrated to be phosphorylated on serine residue(s) by CAMP-dependent protein kinase [16]. It has been postulated that dephosphorylation of the cytosolic form of the cytidylyltransferase will promote the binding of the to the endoplasmic reticulum where it is enzyme activated by membrane phospholipids [ 171. Little information has been obtained on dephosphorylation of cytidylyltransferase and its subsequent effect on the subcellular distribution of the enzyme. Previously, the non-specific phosphoprotein phosphatase inhibitor NaF was demonstrated to retard the time-dependent activation of cytosolic cytidylyltransferase [17]. More recently, a decrease in cytosolic enzyme activity
OOOS-2760/90/$03.50
Division)
Phosphatidylcholine is the principle phospholipid in the mammalian liver [l]. The majority of phosphatidylcholine is synthesized by two distinct pathways in this organ. In the methylation pathway, phosphatidylethanolamine is progressively methylated to phosphatidylcholine with S-adenosylmethionine as the methyl group donor [2]. Phosphatidylethanolamine methyltransferase, recently purified to homogeneity [3], catalyzes all three methyl group transfers [3-51. Although the methylation pathway contributes significantly [20-30%] to phosphatidylcholine biosynthesis [6], the majority of phosphatidylcholine appears to be synthesized via the CDP-choline pathway in rat liver [7,8]. CTP:phosphocholine cytidylyltransferase, which catalyzes the rate-limiting step of the CDP-choline pathway [9], is located in both the cytosolic and micro-
‘0 1990 Elsevier Science Publishers
B.V. (Biomedical
375 with a corresponding increase in microsomal activity was observed in subcellular fractions prepared from post-mit~hondrial supernatants that had been incubated with alkaline phosphatase [16]. Phosphoprotein phosphatases-1 and -2A are the dominant serine/ threonine phosphatases responsible for the dephosphorylation of a wide variety of phosphoproteins in vivo [18]. It is not known if the type 1 and 2A serine/ threonine phosphoprotein phosphatases dephosphorylate cytidylyltransferase. Furthermore, the effect of the activities of these phosphatases on the subcellular distribution of the cytidylyltransferase is not known. Okadaic acid has been identified as a specific and potent inhibitor of protein phosphatases-1 and -2A [19,20]. Thus, the effect of okadaic acid on the subcellular distribution of CTP:phosphocholine cytidylyltransferase in rat liver post-mitochondrial supernatant was investigated and compared with that of NaF.
Materials and Methods
Choline chloride, phosphocholine chloride, cytidine 5’-diphosphocholine were purchased from Sigma. [Methyl- 3HICholine and aqueous counting scin tillant were obtained from Amersham International. Phospho [methyl- 3HIcholine was synthesized enzymatically from [methyl-3H]choline [21]. Okadaic acid was a generous gift from Dr. Y. Tsukitani, Fujisawa Pharmaceutical (Tokyo, Japan). All other biochemica~s were of analytical grade and were purchased from either Sigma or Fisher Scientific. Male Sprague-Dawley rats (125-175 g) were used throughout the study. They were maintained on Purina rat Chow and tap water, ad libitum, in a temperatureand light-controlled room. Rats were anaesthetized with diethyl ether and injected with pentobarbital (2.27 mg/kg body wt.). The liver was perfused with 0.9% NaCl through the portal vein for 335 min to allow for complete removal of the blood. Subsequent to perfusion, the liver was weighed and a 20% homogenate was prepared in buffer A (50 mM Tris-HCl (pH 7.4)/150 mM NaCl/2 mM dithiothreitol/l mM EDTA/O.O25% sodium azide). The homogenate was centrifuged at 12000 x g For 10 min. The resulting supernatant, designated the post-mitochondrial supernatant, was used in all preincubations. In some experiments the post-mitochondrial supernatant was centrifuged at 130000 X g for 60 min and the resulting supernatant, designated the cytosol, used for preincubation. The resulting pellet was resuspended in buffer A and designated the microsomal fraction. Okadaic acid was dissolved in 10% DMSO and a final DMSO concentration of 1.0% was present in both control and okadaic acid preincubations.
In the NaF experiments, post-mitochondrial supernatant (1.5 mg protein) was preincubated at 37 “C for O-60 min with 20 mM Tris-HCl (pH 7.4)/2 mM dithiothreitol/4 mM MgCl,/O.$ mM ATP in the absence and presence of 20 mM NaF in a final volume of 600 ~1 as previously indicated [16]. Subsequently, a 70 ~1 aliquot was removed and the cytidylyltransferase activity assayed. The postmitochondrial supernatant was centrifuged at 350000 x g for 10 min in a Beckman TL-100 bench top ultracentrifuge with a TL-100.2 rotor. The supernatant from this centrifugation was designated the cytosolic fraction. The pellet was resuspended in 0.5 ml buffer A and designated the microsomai fraction. A 70 ~1 aliquot of the microsomal fraction was removed and cytidylyltransferase activity assayed. A 60 ~1 aliquot of the cytosohc fraction was removed and cytidylyltransferase activity assayed in the presence of 0.2 mM oleate : phosphatidylcholine (1 : 1 molar ratio) vesicles. The oleate : phosphatidylcholine vesicles were added to activate the inactive cytosolic enzyme. In the okadaic acid experiments post-mitochondrial supernatant (1.5 mg protein) was preincubated at 37°C for O-60 min with 20 mM Tris-HCl (pH 7.4)/2 mM dithiothreito1,‘O.l mM ATP in the absence and presence of 5 PM okadaic acid in a final volume of 600 ,ul_ Magnesium was omitted from the preincubation since it inhibits the time-dependent increase in cytidylyltransferase activity in rat liver cytosol incubated under similar conditions and is required for activation of protein phosphatase 2C [22]. The subcellular fractionations and enzyme activities were performed as above. Alkaline phosphatase activity was assayed as described [23], with the following modifications: 0.2 M Tris-HCl (pH 10) 10 mM MgCl,, and 110 pg microsomal protein in a final volume of 0.5 ml assayed at 37 o C for 10 min. CTP:phosphocholine cytidylyltransferase was purified as described previously 116,241. The cytidylyltransferase activity in the post-mitochondrial supernatant and both the cytosolic and microsomal fractions was assayed at 37°C with phospho[~eih_v~‘HIcholine as previously described [25]. A unit of enzyme activity was defined as that amount of enzyme which catalyzes the formation of 1 nmol of CDP-choline per min in the standard assay. Protein was determined by the method of Bradford [26]. Student’s t-test was used for the determination of significance. The level of significance was defined as P < 0.05. Results
The effect of NaF on cytidylyltransferase activity in rat liver post-mitochondrial supernatant and subcellular fractions Rat liver post-mitochondrial supernatant was preincubated at 37” C for O-60 min in the absence and
o-1 0
.
1 20
.
.
1 40
.
’
’ 60
Preincubation lime (min) Fig. 1. Effect of NaF on the time-dependent activation of cytidylyltransferase activity in post-mitochondrial supernatant at 37 o C. Rat liver post-mitochondrial supernatant was incubated for up to 60 min in the absence and presence of 20 mM NaF. Subsequently. cytidylyltransferase activity was assayed in the absence and presence of 0.2 mg/ml oleate:phosphatidylcholine vesicles. Enzyme activity: q, 1. control; 0, +, 20 mM NaF. Closed symbols, absence of oleate:phosphatidylcholine vesicles; open symbols, presence of oleate:phosphatidylcholine vesicles. This experiment was repeated twice with similar
0
20
40
60
Preincubation Time (min) Fig. 2. Effect of NaF on the time-dependent subcellular distribution of cytidylyltransferase activity at 37 o C. Rat liver post-mitochondrial supernatant was treated as in Fig. 1. Subsequently, the post-mitochondrial supernatant was separated into cytosolic and microsomal fractions and the fractions assayed for cytidylyltransferase activity. Enzyme activity; open symbols, cytosol+ oleate:phosphatidylcholine vesicles; closed symbols. microsomes. o, . . control; 0. l . 20 mM NaF. This experiment was repeated twice with similar results.
results.
presence of 20 mM NaF. This concentration of NaF was chosen since it has been shown to inhibit the time-dependent activation of cytidylyltransferase in rat liver cytosol [17]. Subsequent to preincubation, the cytidylyltransferase activity was determined. Cytidylyltransferase activity was increased with increasing time of preincubation at 37 o C (Fig. 1). The addition of NaF to the preincubation suspension caused a decrease in the time-dependent activation of cytidylyltransferase in post-mitochondrial supernatants at 30, 45 and 60 min of preincubation compared to controls (Fig. 1). There was no enhanced proteolysis of cytidylyltransferase in the presence of NaF, since maximal activation of enzyme activity by oleate:phosphatidylcholine vesicles was unchanged compared to controls (Fig. 1). The post-mitochondrial supernatant was separated into cytosolic and microsomal fractions and the cytidylyltransferase activity determined. Microsomal cytidylyltransferase activity was increased and a corresponding decrease of cytosolic cytidylyltransferase activity was observed with time of preincubation at 37 o C (Fig. 2). The addition of NaF caused a decrease in the time-dependent activation of microsomal cytidylyltransferase and a retention of cytosolic cytidylyltransferase activity compared to controls (Fig. 2). Thus, NaF inhibits the time-dependent activation of cytidylyltransferase in post-mitochondrial supernatants and the translocation of the enzyme from the cytosolic to the microsomal fraction.
The effect of okaduic acid on cytidylyltrunsferuse uctiuity in rut her cytosol To determine if phosphoprotein phosphatases-1 and -2A are involved in the activation of cytidylyltransferase, the effect of okadaic acid on the time-dependent activation of cytosolic cytidylyltransferase activity was determined. Rat liver cytosoi was preincubated at 37°C for O-60 min in the absence and presence of 5 PM okadaic acid. Preincubation of cytosol produced a time-dependent increase in cytidylyltransferase activity (Fig. 3). When okadaic acid was
Preincubation Time (min) Fig. 3. Effect of okadaic acid on the time-dependent activation of cytosolic cytidylyltransferase activity at 37 o C. Rat liver cytosol was preincubated at 37 o C for up to 60 min in the absence or presence of 5 PM okadaic acid. Subsequently, cytidylyltransferase activity was assayed. Enzyme activity: o. control; l , 5 PM okadaic acid. This experiment was repeated twice with similar results.
TABLE
I
Effecl of NaF and okadaic acid on the activity of purrfied
cytidyl-
ylrransferase CTP:phosphocholine cytidylyltransferase was purified as previously described [16,24] and 33 units of enzyme was assayed in the absence or presence of 20 mM NaF or 5 pM okadaic acid in the standard assay (251. Results are depicted as mean * SD. (No. of experiments). Activity No addition + 20 mM NaF + 5 PM okadaic
acid
(nmol/min)
2.95 F 0.20(3) 2.86+0.12(3) 3.03?0.12(3)
1.00 0
added to the preincubation mixture a decrease in the time-dependent activation of cytidylyltransferase was observed (Fig. 3). The difference was most pronounced after a 60 min preincubation. Neither 5 PM okadaic acid or 20 mM NaF added alone affected pure cytidylyltransferase activity in the assay system (Table I). Thus, the activity of the type 1 and/or 2A phosphoprotein phosphatases appear to be involved in the activation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol. The effect of okadaic acid on cytidylyltransferase activity in rat liver post-mitochondriai supernatant and subcellular fractions To determine if the activities of phosphoprotein phosphatases-1 and -2A are involved in the translocation of cytidylyltransferase, the effect of okadaic acid on the time-dependent translocation of cytidylyltransferase from the cytosolic to the microsomal fraction was investigated. Post-mitochondrial supernatant was preincubated at 37 o C for O-60 min in the absence or presence of 5 PM okadaic acid. A concentration of 5 PM okadaic acid was chosen since this concentration maximally inhibited cytidylyltransferase activity in post-mitochondrial supernatant after 60 min of preincubation. Subsequent to preincubation, the cytidylyltransferase activity was increased from O-30 min in both the control and okadaic-acid-treated post-mitochondrial supernatants (Fig. 4). However, there was a 13% and 16% decrease in cytidylyltransferase activity in post-mitochondrial supernatants preincubated with okadaic acid at 45 and 60 min respectively, compared to controls. No enhanced proteolysis of cytidylyltransferase in the presence of okadaic acid was detected, since maximal activation of the enzyme was unchanged compared to control (Fig. 4). The post-mitochondrial supernatant was separated into cytosolic and microsomal fractions and the cytidylyltransferase activity was determined in these fractions. Microsomal cytidylyltransferase activity was increased and cytosolic cytidylyltransferase activity decreased with time of preincubation in both control and okadaic acid
20 Preincubation
40
60
Time (min)
Fig. 4. Effect of okadaic acid on time-dependent activation of cytidylyltransferase activity in post-mitochondrial supernatant at 37 o C. Rat liver post-mitochondrial supernatant was incubated for up to 60 min in the absence and presence of 5 nM okadaic acid. Subsequently, cytidylyltransferase activity was assayed in the absence or presence of 0.2 mg/ml oleate : phosphatidylcholine vesicles. Enzyme activity: q, control; l , 5 PM okadaic acid; 0, control + oleate: phosphatidylcholine vesicles; ., 5 nM okadaic acid + oleate : phosphatidylchohne vesicles. The results represent the mean f SD. of four separate experi ments.
treated post-mitochondrial supernatants (Fig. 5). However, microsomal cytidylyltransferase activity was decreased 27% at 45 min and 31% at 60 min of preincubation with okadaic acid compared to controls (Fig. 5).
1
6.0 -
0
20 Preincubation
40
60
Time (min)
Fig. 5. Effect of okadaic acid on time-dependent subcellular distribution of cytidylyhransferase activity at 37 o C. Rat liver post-mitochondrial supernatant was treated as in Fig. 4. Subsequently, the postmitochondrial supematant was separated into cytosolic and microsomal fractions and cytidylyltransferase activity assayed in these fractions. Enzyme activity: Q control microsomes; 4, 5 pM okadaic acid microsomes; H, control cytosol + oleate:phosphatidylcholine vesicles; 0, 5 PM okadaic acid cytosol+ oleate:phosphatidylchohne vesicles. The results represent the mean f S.D. of four separate experiments.
378 TABLE
II
Microsomal alkaline phosphatase activity was assayed in the absence or presence of 20 mM NaF or 5 PM okadaic acid as described in Materials and Methods. Results are depicted as mean+S.D. (No. of experiments). Alkaline phosphatase activity (pmol/min per mg protein) Control + 20 mM NaF Control + .5 p M okadaic
6.97$0.19(3) 4.74 rl:0.43(3) *
acid
7.89 rt0.23(3) 7.74*0.18(3)
* D < 0.05
This corresponded with a retention of the cytosolic cytidylyltransferase activity of 21% at 45 min and 37% at 60 min of preincubation with okadaic acid compared to controls (Fig. 5). These data indicate that the activities of the type 1 and/or 2A phosphoprotein phosphatases may affect both the time-dependent activity and subcellular distribution of cytidylyltransferase in postmitochondrial supernatant. The ejject of NaF and okadaic acid on ulkaiine phosphutuse activity in rut liver microsomes Since alkaline phosphatase was demonstrated to dephosphorylate cytidylyltransferase in vitro [16], the effect of NaF and okadaic acid on alkaline phosphatase activity in rat liver microsomes was investigated. Microsomal alkaline phosphatase was assayed in the absence or presence of 20 mM NaF or 5 PM okadaic acid. Alkaline phosphatase activity was inhibited 32% by NaF but was unaffected by okadaic acid (Table II). The alkaline phosphatase activities in the okadaic acid experiments were slightly higher than those of the NaF experiments. This was probably due to the presence of DMSO in the assay. Thus, the okadaic acid induced time-dependent decrease in cytidy~yltransferase activity and change in subcellular distribution of the enzyme in post-mitochondrial supernatant was not due to inhibition of alkaline phosphatase activity. Discussion The objective of this study was to determine the effect of inhibition of phosphoprotein phosphatases on the subcellular distribution of CTP:phosphocholine cytidylyltransferase activity in rat liver. The inhibition of the time-dependent increase in cytidylyltransferase activity in post-mitochondrial supernatants and change in subcellular distribution of the enzyme by treatment with NaF and okadaic acid are consistent with phosphoprotein phosphatases affecting the dephosphoryla-
tion and translocation of cytidylyltransferase activity from the cytosolic to the microsomal fraction in rat liver. The effect of NaF on the inhibition of the time-dependent cytidylyltransferase activity in post-mitochondrial supernatants and change in subcellular distribution of the enzyme was more pronounced than that of okadaic acid. This was probably due to a non-specific inhibition of many phosphoprotein phosphatases by NaF [27]. We may have seen a larger effect of okadaic acid (Fig. 4), but there are other phosphatases present in the post-mitochol~drial supernatant (e.g., alkaline phosphatase) which would still be active and oppose the effect of the inhibited phosphoprotein phosphatases-1 and -2A. Alkaline phosphatase has been demonstrated to dephosphorylate cytidylyltransferase in vitro 1161 and its activity was inhibited by NaF but not okadaic acid. Substantial alkaline phosphatase activity has been demonstrated in liver microsomes [28]. However, the subcellular distribution of the majority of alkaline phosphatase activity. on the external side of the plasma membrane 1291, would preclude the importance of this enzyme in the dephosphorylation of cytidylyltransferase in vivo. It should be noted that the cytidylyltransferase activities in the post-mitochondrial fraction and subcellular fractions of the okadaic acid experiments were slightly higher than the activities obtained with the NaF experiments. This might be attributed to the omission of magnesium in the okadaic acid preincubations. Magnesium was omitted in the okadaic acid preincubations since it has been demonstrated to inhibit the time-dependent increase in cytidylyltransferase activity in rat liver cytosol incubated under similar conditions [17] and is required for activation of phosphoprotein phosphatase 2C [22]. Phosphoprotein phosphatases-1, -2A and -2C account for virtually all the phosphatase activity in skeletal muscle and liver towards at least 20 phosphoproteins that regulate several metabolic pathways and muscle contraction [30,31]. Recently. Haystead et al. [18] have postulated that protein phosphatases-1 and -2A rather than -2C are the dominant phosphatase catalytic subunits acting on a wide range of phosphoproteins in vivo. Okadaic acid is a potent and specific inhibitor of phosphoprotein phosphatases-1 and -2A 119,201. Thus, the effect of preincubation of post-lnitochondrial supernatant with okadaic acid on the subcellular distribution of cytidylyltransferase implies that phosphoprotein phosphatase-1 and/or -2A may be involved in the regulation of phosphatidylcho~ine biosynthesis via the CDP-choline pathway in rat liver. Whether only phosphoprotein phosphatase-1 or -2A was involved in the of cytidylyltransferase was not dephosphorylation investigated in this study. However, the involvement of both phosphatases and also type 2C phosphoprotein phosphatase cannot be eliminated as a considerable
319 number of phosphoprotein substrates have been demonstrated to be dephosphorylated by all three phosphatases in vitro [30]. Fatty acids have been demonstrated to promote the translocation of cytidylyltransferase from the cytosolic to the microsomal fraction [32,33]. Okadaic acid, a polyether derivative of a 3%carbon fatty acid [34], clearly did not promote the binding of cytidylyltransferase to the microsomal fraction. In fact, the effect was quite opposite to that observed with fatty acids. In addition, okadaic acid did not affect the activity of purified cytidylyltransferase when added to the assay system. Thus, the inhibitory effect of okadaic acid was most likely on phosphatase(s) that dephosphorylate cytidylyltransferase. If phosphoprotein phosphatases are indeed involved in the dephosphorylation of the cytidylyltransferase, it is not surprising that their inhibition does not have a more dramatic effect on the subcellular distribution of the enzyme. Incubation of rat liver hepatocytes with chlorophenylthio-CAMP, a stimulator of CAMP-dependent protein kinase, for 2 h produced a 34% decrease in microsomal cytidylyltransferase activity with a corresponding 30% decrease in the new synthesis of phosphatidylcholine [15]. Since the content and composition of phosphatidylcholine rarely change in a specific membrane, a gross alteration in the phosphorylation state and subcellular distribution of cytidylyltransferase would severely affect the level of phosphatidylcholine. Okadaic acid inhibited only type 1 and 2A phosphoprotein phosphatases present in the post-mitochondrial supernatant and the small change in the subcellular distribution of cytidylyltransferase may have been temporally related to a small change in the phosphorylation state of the enzyme. Thus, the phosphorylation-dephosphorylation of cytidylyltransferase and its subsequent subcellular distribution must be highly coordinated and under strict metabolic control. Acknowledgements This work was supported by a grant from the Medical Research Council of Canada. We are indebted to Sandi Ungarian and Drs. Haris Jamil and Lilian Tijburg for technical assistance and helpful discussions. T-S.L. is the recipient of a Faculty of Medicine Summer Studentship, University of Alberta. G.M.H. is the recipient of a Canadian Heart Foundation Fellowship. D.E.V. is a Medical Scientist of the Alberta Heritage Foundation for Medical Research. References 1
White, D.A. (1973) in Form (Ansell, 44-482,
and Function G.B., Hawthorne, J.N. and Dawson, Elsevier, Amsterdam.
of Phospholipids. R.M.C., eds.), pp.
D.M. (1961) Biochim. Biophys. Acta 2 Bremer, J. and Greenberg, 46, 205-216. N.D. and Vance, D.E. (1987) J. Biol. Chem. 262, 3 Ridgway. 17231-17239. 4 Schneider, W.J. and Vance, D.E. (1979) J. Biol. Chem. 254, 3886-3891. 5 Pajares, M.A., Alemany, S., Varela, I.. Marin Cao. D. and Mato. J.M. (1984) B&hem. J. 233, 61-66. Sundler, R. and Akesson, B. (1975) J. Biol. Chem. 250, 3359-3367. Kennedy, E.P. and Weiss, S.B. (1956) J. Biol. Chem. 22, 193-251. Kennedy, E.P. (1962) Harvey Lect. 57, 143-171. Pelech. S.L. and Vance. D.E. (1984) Biochim. Biophys. Acta 779, 217-251. 10 Schneider, W.C. (1963) J. Biol. Chem. 238, 3572-3578. 11 Vance. D.E. and Pelech. S.L. (1984) Trends Biochem. Sci. 9. 17-20. 12 Feldman, D.A.. Kovac, C.R., Dranginis. P.L. and Weinhold, P.A. (1978) J. Biol. Chem. 253. 4980-4986. 13 Feldman, D.A., Dietrich, J.W. and Weinhold. P.A. (1980) Biochim. Biophys. Acta 620, 603-611. 14 Feldman, D.A., Rounsifer, W.E. and Weinhold. P.A. (1985) Biochim. Biophys. Acta 429, 4299437. 15 Pelech, S.L., Pritchard, P.H. and Vance, D.E. (1981) J. Biol. Chem. 256, 8283-8286. 16 Sanghera. J.S. and Vance, D.E. (1989) J. Biol. Chem. 264, 1215-1223. 17 Pelech, S.L. and Vance. D.E. (1982) J. Biol. Chem. 257, 14198-14202. 18 Haystead, T.A.J., Sim, A.T.R.. Coding. R.C., Honnor, R.C., Tsukitani, Y., Cohen, P. and Hardie, D.G. (1989) Nature 337, 78-81. 19 Bialojan, C. and Takai. A. (1988) Biochem. J. 256, 283-290. 20 Hescheler, J., Mieskes. G., Ruegy. J.C., Takai, A. and Trantwein. T. (1988) Pfliigers Arch. Ges. Physiol. 412, 248-252. 21 Paddon, H.B. and Vance. D.E. (1977) Biochim. Biophys. Acta 488, 181-189. 22 Cohen, P. (1980) Eur. J. B&hem. 111. 563-574. 23 Hatch, G.M. and Choy, P.C. (1987) Lipids 22. 672-676. 24 Weinhold, P.A., Rounsifer. M.E. and Feldman, D.A. (1986) J. Biol. Chem. 261. 5104-5110. 25 Pelech, S.L. and Vance. D.E. (1982) J. Biol. Chem. 257, 14198-14202. 26 Bradford, M. (1976) Anal. Biochem. 72. 248-254. 27 Beg, Z.H.. Stonik. J.A. and Bremer. H.B., Jr. (1978) Proc. Natl. Acad. Sci. USA 75. 367883682. 28 Morton, R.K. (1954) Biochem. J. 57, 595-599. 29 Tokumitou, S.I. (1984) in Human Alkaline Phosphatases (Stigbrand, T. and Fishman. W.H.. eds.). pp. 189-197. Alan R. Liss, New York. 30 Ingebritsen, T.S. and Cohen, P. (1983) Science 221. 331-338. 31 Cohen. P. (1988) Proc. R. Sot. 234, 115-144. 32 Pelech. S.L.. Pritchard, P.H.. Brindley. D.N. and Vance, D.E. (1983) J. Biol. Chem. 258. 6782-6788. 33 Weinhold, P.A.. Rousifer. W.E., Williams, SE.. Brubaker. P. and Feldman. D.A. (1984) J. Biol. Chem. 259. 10315-10324. 34 Tachibaua. K. and Scheuer. P.J. (1981) J. Am. Chem. Sot. 103. 2469-2471.
Biochimrcu et Bloph_vsrcu Acru. 1042 (1990) 314-379 Elsevier
BBALIP
53324
Effect of NaF and okadaic acid on the subcellular distribution of CTP:phosphocholine cytidylyltransferase activity in rat liver Grant M. Hatch ‘, Tsz-Shan ’ Lipid and Lipoprotein Rewurch
2 and Dennis
Group und Department ofBiochemistry, Unic~ersri_vof Alberta and ’ FuJisawrr Pharmuceutiwl Company. Tokyo (Jupon)
(Revised
Key words:
Lam ‘, Y. Tsukitani
Okadaic
(Received 17 July 1989) manuscript received 23 October
acid; NaF; Cytidylyltransferase;
E. Vance ’
Edmonton,
Albertu (Ccrnudu)
1989)
Enzyme translocation:
(Rat liver)
The effect of preincubation of rat liver post-mitochondrial supernatant with NaF and okadaic acid on the subcellular distribution of CTP: phosphocholine cytidylyltransferase activity was investigated. NaF (20 mM) inhibited the time-dependent activation of cytidylyltransferase activity in post-mitochondrial supernatant. Subcellular fractionation of the post-mitochondrial supernatant revealed that cytidylyltransferase activity in the microsomal fraction was decreased and activity in the cytosolic fraction increased with time of preincubation with NaF compared to controls. Okadaic acid is a specific and potent inhibitor of type 1 and 2A phosphoprotein phosphatases. Preincubation of cytosol with 5 uM okadaic acid inhibited the time-dependent activation of cytosolic cytidylyltransferase activity. Preincubation of post-mitochondrial supernatants with 5 PM okadaic acid inhibited the time-dependent activation of cytidylyltransferase activity by 13% at 45 min and 16% at 60 min of preincubation compared to controls. Microsomal cytidylyltransferase activity was decreased 27% at 45 min and 31% at 60 min with a corresponding retention of cytosolic cytidylyltransferase activity of 21% at 45 min and 37% at 60 min of preincubation with okadaic acid compared to controls. We postulate that the activity of the type 1 and/or type 2A phosphoprotein phosphatases affect the subcellular distribution of CTP: phosphocholine cytidylyltransferase activity in rat liver.
Introduction
Correspondence: D.E. Vance, Lipid and Lipoprotein Research Group, 328 Heritage Medical Research Building. University of Alberta. Edmonton, Alberta, Canada, T6G 2S2.
somal fractions of rat liver homogenates [lo]. The rate of phosphatidylcholine biosynthesis is dependent on the subcellular distribution of the cytidylyltransferase between its inactive cytosolic form and active microsomal form [ll]. The inactive cytosolic form may be fully activated by anionic phospholipids and phosphatidylcholine:fatty acid vesicles [12-141. Phosphorylationdephosphorylation of the cytidylyltransferase is one proposed mechanism for the regulation of the enzyme’s activity [15]. The cytidylyltransferase was recently demonstrated to be phosphorylated on serine residue(s) by CAMP-dependent protein kinase [16]. It has been postulated that dephosphorylation of the cytosolic form of the cytidylyltransferase will promote the binding of the to the endoplasmic reticulum where it is enzyme activated by membrane phospholipids [ 171. Little information has been obtained on dephosphorylation of cytidylyltransferase and its subsequent effect on the subcellular distribution of the enzyme. Previously, the non-specific phosphoprotein phosphatase inhibitor NaF was demonstrated to retard the time-dependent activation of cytosolic cytidylyltransferase [17]. More recently, a decrease in cytosolic enzyme activity
OOOS-2760/90/$03.50
Division)
Phosphatidylcholine is the principle phospholipid in the mammalian liver [l]. The majority of phosphatidylcholine is synthesized by two distinct pathways in this organ. In the methylation pathway, phosphatidylethanolamine is progressively methylated to phosphatidylcholine with S-adenosylmethionine as the methyl group donor [2]. Phosphatidylethanolamine methyltransferase, recently purified to homogeneity [3], catalyzes all three methyl group transfers [3-51. Although the methylation pathway contributes significantly [20-30%] to phosphatidylcholine biosynthesis [6], the majority of phosphatidylcholine appears to be synthesized via the CDP-choline pathway in rat liver [7,8]. CTP:phosphocholine cytidylyltransferase, which catalyzes the rate-limiting step of the CDP-choline pathway [9], is located in both the cytosolic and micro-
‘0 1990 Elsevier Science Publishers
B.V. (Biomedical
375 with a corresponding increase in microsomal activity was observed in subcellular fractions prepared from post-mit~hondrial supernatants that had been incubated with alkaline phosphatase [16]. Phosphoprotein phosphatases-1 and -2A are the dominant serine/ threonine phosphatases responsible for the dephosphorylation of a wide variety of phosphoproteins in vivo [18]. It is not known if the type 1 and 2A serine/ threonine phosphoprotein phosphatases dephosphorylate cytidylyltransferase. Furthermore, the effect of the activities of these phosphatases on the subcellular distribution of the cytidylyltransferase is not known. Okadaic acid has been identified as a specific and potent inhibitor of protein phosphatases-1 and -2A [19,20]. Thus, the effect of okadaic acid on the subcellular distribution of CTP:phosphocholine cytidylyltransferase in rat liver post-mitochondrial supernatant was investigated and compared with that of NaF.
Materials and Methods
Choline chloride, phosphocholine chloride, cytidine 5’-diphosphocholine were purchased from Sigma. [Methyl- 3HICholine and aqueous counting scin tillant were obtained from Amersham International. Phospho [methyl- 3HIcholine was synthesized enzymatically from [methyl-3H]choline [21]. Okadaic acid was a generous gift from Dr. Y. Tsukitani, Fujisawa Pharmaceutical (Tokyo, Japan). All other biochemica~s were of analytical grade and were purchased from either Sigma or Fisher Scientific. Male Sprague-Dawley rats (125-175 g) were used throughout the study. They were maintained on Purina rat Chow and tap water, ad libitum, in a temperatureand light-controlled room. Rats were anaesthetized with diethyl ether and injected with pentobarbital (2.27 mg/kg body wt.). The liver was perfused with 0.9% NaCl through the portal vein for 335 min to allow for complete removal of the blood. Subsequent to perfusion, the liver was weighed and a 20% homogenate was prepared in buffer A (50 mM Tris-HCl (pH 7.4)/150 mM NaCl/2 mM dithiothreitol/l mM EDTA/O.O25% sodium azide). The homogenate was centrifuged at 12000 x g For 10 min. The resulting supernatant, designated the post-mitochondrial supernatant, was used in all preincubations. In some experiments the post-mitochondrial supernatant was centrifuged at 130000 X g for 60 min and the resulting supernatant, designated the cytosol, used for preincubation. The resulting pellet was resuspended in buffer A and designated the microsomal fraction. Okadaic acid was dissolved in 10% DMSO and a final DMSO concentration of 1.0% was present in both control and okadaic acid preincubations.
In the NaF experiments, post-mitochondrial supernatant (1.5 mg protein) was preincubated at 37 “C for O-60 min with 20 mM Tris-HCl (pH 7.4)/2 mM dithiothreitol/4 mM MgCl,/O.$ mM ATP in the absence and presence of 20 mM NaF in a final volume of 600 ~1 as previously indicated [16]. Subsequently, a 70 ~1 aliquot was removed and the cytidylyltransferase activity assayed. The postmitochondrial supernatant was centrifuged at 350000 x g for 10 min in a Beckman TL-100 bench top ultracentrifuge with a TL-100.2 rotor. The supernatant from this centrifugation was designated the cytosolic fraction. The pellet was resuspended in 0.5 ml buffer A and designated the microsomai fraction. A 70 ~1 aliquot of the microsomal fraction was removed and cytidylyltransferase activity assayed. A 60 ~1 aliquot of the cytosohc fraction was removed and cytidylyltransferase activity assayed in the presence of 0.2 mM oleate : phosphatidylcholine (1 : 1 molar ratio) vesicles. The oleate : phosphatidylcholine vesicles were added to activate the inactive cytosolic enzyme. In the okadaic acid experiments post-mitochondrial supernatant (1.5 mg protein) was preincubated at 37°C for O-60 min with 20 mM Tris-HCl (pH 7.4)/2 mM dithiothreito1,‘O.l mM ATP in the absence and presence of 5 PM okadaic acid in a final volume of 600 ,ul_ Magnesium was omitted from the preincubation since it inhibits the time-dependent increase in cytidylyltransferase activity in rat liver cytosol incubated under similar conditions and is required for activation of protein phosphatase 2C [22]. The subcellular fractionations and enzyme activities were performed as above. Alkaline phosphatase activity was assayed as described [23], with the following modifications: 0.2 M Tris-HCl (pH 10) 10 mM MgCl,, and 110 pg microsomal protein in a final volume of 0.5 ml assayed at 37 o C for 10 min. CTP:phosphocholine cytidylyltransferase was purified as described previously 116,241. The cytidylyltransferase activity in the post-mitochondrial supernatant and both the cytosolic and microsomal fractions was assayed at 37°C with phospho[~eih_v~‘HIcholine as previously described [25]. A unit of enzyme activity was defined as that amount of enzyme which catalyzes the formation of 1 nmol of CDP-choline per min in the standard assay. Protein was determined by the method of Bradford [26]. Student’s t-test was used for the determination of significance. The level of significance was defined as P < 0.05. Results
The effect of NaF on cytidylyltransferase activity in rat liver post-mitochondrial supernatant and subcellular fractions Rat liver post-mitochondrial supernatant was preincubated at 37” C for O-60 min in the absence and
o-1 0
.
1 20
.
.
1 40
.
’
’ 60
Preincubation lime (min) Fig. 1. Effect of NaF on the time-dependent activation of cytidylyltransferase activity in post-mitochondrial supernatant at 37 o C. Rat liver post-mitochondrial supernatant was incubated for up to 60 min in the absence and presence of 20 mM NaF. Subsequently. cytidylyltransferase activity was assayed in the absence and presence of 0.2 mg/ml oleate:phosphatidylcholine vesicles. Enzyme activity: q, 1. control; 0, +, 20 mM NaF. Closed symbols, absence of oleate:phosphatidylcholine vesicles; open symbols, presence of oleate:phosphatidylcholine vesicles. This experiment was repeated twice with similar
0
20
40
60
Preincubation Time (min) Fig. 2. Effect of NaF on the time-dependent subcellular distribution of cytidylyltransferase activity at 37 o C. Rat liver post-mitochondrial supernatant was treated as in Fig. 1. Subsequently, the post-mitochondrial supernatant was separated into cytosolic and microsomal fractions and the fractions assayed for cytidylyltransferase activity. Enzyme activity; open symbols, cytosol+ oleate:phosphatidylcholine vesicles; closed symbols. microsomes. o, . . control; 0. l . 20 mM NaF. This experiment was repeated twice with similar results.
results.
presence of 20 mM NaF. This concentration of NaF was chosen since it has been shown to inhibit the time-dependent activation of cytidylyltransferase in rat liver cytosol [17]. Subsequent to preincubation, the cytidylyltransferase activity was determined. Cytidylyltransferase activity was increased with increasing time of preincubation at 37 o C (Fig. 1). The addition of NaF to the preincubation suspension caused a decrease in the time-dependent activation of cytidylyltransferase in post-mitochondrial supernatants at 30, 45 and 60 min of preincubation compared to controls (Fig. 1). There was no enhanced proteolysis of cytidylyltransferase in the presence of NaF, since maximal activation of enzyme activity by oleate:phosphatidylcholine vesicles was unchanged compared to controls (Fig. 1). The post-mitochondrial supernatant was separated into cytosolic and microsomal fractions and the cytidylyltransferase activity determined. Microsomal cytidylyltransferase activity was increased and a corresponding decrease of cytosolic cytidylyltransferase activity was observed with time of preincubation at 37 o C (Fig. 2). The addition of NaF caused a decrease in the time-dependent activation of microsomal cytidylyltransferase and a retention of cytosolic cytidylyltransferase activity compared to controls (Fig. 2). Thus, NaF inhibits the time-dependent activation of cytidylyltransferase in post-mitochondrial supernatants and the translocation of the enzyme from the cytosolic to the microsomal fraction.
The effect of okaduic acid on cytidylyltrunsferuse uctiuity in rut her cytosol To determine if phosphoprotein phosphatases-1 and -2A are involved in the activation of cytidylyltransferase, the effect of okadaic acid on the time-dependent activation of cytosolic cytidylyltransferase activity was determined. Rat liver cytosoi was preincubated at 37°C for O-60 min in the absence and presence of 5 PM okadaic acid. Preincubation of cytosol produced a time-dependent increase in cytidylyltransferase activity (Fig. 3). When okadaic acid was
Preincubation Time (min) Fig. 3. Effect of okadaic acid on the time-dependent activation of cytosolic cytidylyltransferase activity at 37 o C. Rat liver cytosol was preincubated at 37 o C for up to 60 min in the absence or presence of 5 PM okadaic acid. Subsequently, cytidylyltransferase activity was assayed. Enzyme activity: o. control; l , 5 PM okadaic acid. This experiment was repeated twice with similar results.
TABLE
I
Effecl of NaF and okadaic acid on the activity of purrfied
cytidyl-
ylrransferase CTP:phosphocholine cytidylyltransferase was purified as previously described [16,24] and 33 units of enzyme was assayed in the absence or presence of 20 mM NaF or 5 pM okadaic acid in the standard assay (251. Results are depicted as mean * SD. (No. of experiments). Activity No addition + 20 mM NaF + 5 PM okadaic
acid
(nmol/min)
2.95 F 0.20(3) 2.86+0.12(3) 3.03?0.12(3)
1.00 0
added to the preincubation mixture a decrease in the time-dependent activation of cytidylyltransferase was observed (Fig. 3). The difference was most pronounced after a 60 min preincubation. Neither 5 PM okadaic acid or 20 mM NaF added alone affected pure cytidylyltransferase activity in the assay system (Table I). Thus, the activity of the type 1 and/or 2A phosphoprotein phosphatases appear to be involved in the activation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol. The effect of okadaic acid on cytidylyltransferase activity in rat liver post-mitochondriai supernatant and subcellular fractions To determine if the activities of phosphoprotein phosphatases-1 and -2A are involved in the translocation of cytidylyltransferase, the effect of okadaic acid on the time-dependent translocation of cytidylyltransferase from the cytosolic to the microsomal fraction was investigated. Post-mitochondrial supernatant was preincubated at 37 o C for O-60 min in the absence or presence of 5 PM okadaic acid. A concentration of 5 PM okadaic acid was chosen since this concentration maximally inhibited cytidylyltransferase activity in post-mitochondrial supernatant after 60 min of preincubation. Subsequent to preincubation, the cytidylyltransferase activity was increased from O-30 min in both the control and okadaic-acid-treated post-mitochondrial supernatants (Fig. 4). However, there was a 13% and 16% decrease in cytidylyltransferase activity in post-mitochondrial supernatants preincubated with okadaic acid at 45 and 60 min respectively, compared to controls. No enhanced proteolysis of cytidylyltransferase in the presence of okadaic acid was detected, since maximal activation of the enzyme was unchanged compared to control (Fig. 4). The post-mitochondrial supernatant was separated into cytosolic and microsomal fractions and the cytidylyltransferase activity was determined in these fractions. Microsomal cytidylyltransferase activity was increased and cytosolic cytidylyltransferase activity decreased with time of preincubation in both control and okadaic acid
20 Preincubation
40
60
Time (min)
Fig. 4. Effect of okadaic acid on time-dependent activation of cytidylyltransferase activity in post-mitochondrial supernatant at 37 o C. Rat liver post-mitochondrial supernatant was incubated for up to 60 min in the absence and presence of 5 nM okadaic acid. Subsequently, cytidylyltransferase activity was assayed in the absence or presence of 0.2 mg/ml oleate : phosphatidylcholine vesicles. Enzyme activity: q, control; l , 5 PM okadaic acid; 0, control + oleate: phosphatidylcholine vesicles; ., 5 nM okadaic acid + oleate : phosphatidylchohne vesicles. The results represent the mean f SD. of four separate experi ments.
treated post-mitochondrial supernatants (Fig. 5). However, microsomal cytidylyltransferase activity was decreased 27% at 45 min and 31% at 60 min of preincubation with okadaic acid compared to controls (Fig. 5).
1
6.0 -
0
20 Preincubation
40
60
Time (min)
Fig. 5. Effect of okadaic acid on time-dependent subcellular distribution of cytidylyhransferase activity at 37 o C. Rat liver post-mitochondrial supernatant was treated as in Fig. 4. Subsequently, the postmitochondrial supematant was separated into cytosolic and microsomal fractions and cytidylyltransferase activity assayed in these fractions. Enzyme activity: Q control microsomes; 4, 5 pM okadaic acid microsomes; H, control cytosol + oleate:phosphatidylcholine vesicles; 0, 5 PM okadaic acid cytosol+ oleate:phosphatidylchohne vesicles. The results represent the mean f S.D. of four separate experiments.
378 TABLE
II
Microsomal alkaline phosphatase activity was assayed in the absence or presence of 20 mM NaF or 5 PM okadaic acid as described in Materials and Methods. Results are depicted as mean+S.D. (No. of experiments). Alkaline phosphatase activity (pmol/min per mg protein) Control + 20 mM NaF Control + .5 p M okadaic
6.97$0.19(3) 4.74 rl:0.43(3) *
acid
7.89 rt0.23(3) 7.74*0.18(3)
* D < 0.05
This corresponded with a retention of the cytosolic cytidylyltransferase activity of 21% at 45 min and 37% at 60 min of preincubation with okadaic acid compared to controls (Fig. 5). These data indicate that the activities of the type 1 and/or 2A phosphoprotein phosphatases may affect both the time-dependent activity and subcellular distribution of cytidylyltransferase in postmitochondrial supernatant. The ejject of NaF and okadaic acid on ulkaiine phosphutuse activity in rut liver microsomes Since alkaline phosphatase was demonstrated to dephosphorylate cytidylyltransferase in vitro [16], the effect of NaF and okadaic acid on alkaline phosphatase activity in rat liver microsomes was investigated. Microsomal alkaline phosphatase was assayed in the absence or presence of 20 mM NaF or 5 PM okadaic acid. Alkaline phosphatase activity was inhibited 32% by NaF but was unaffected by okadaic acid (Table II). The alkaline phosphatase activities in the okadaic acid experiments were slightly higher than those of the NaF experiments. This was probably due to the presence of DMSO in the assay. Thus, the okadaic acid induced time-dependent decrease in cytidy~yltransferase activity and change in subcellular distribution of the enzyme in post-mitochondrial supernatant was not due to inhibition of alkaline phosphatase activity. Discussion The objective of this study was to determine the effect of inhibition of phosphoprotein phosphatases on the subcellular distribution of CTP:phosphocholine cytidylyltransferase activity in rat liver. The inhibition of the time-dependent increase in cytidylyltransferase activity in post-mitochondrial supernatants and change in subcellular distribution of the enzyme by treatment with NaF and okadaic acid are consistent with phosphoprotein phosphatases affecting the dephosphoryla-
tion and translocation of cytidylyltransferase activity from the cytosolic to the microsomal fraction in rat liver. The effect of NaF on the inhibition of the time-dependent cytidylyltransferase activity in post-mitochondrial supernatants and change in subcellular distribution of the enzyme was more pronounced than that of okadaic acid. This was probably due to a non-specific inhibition of many phosphoprotein phosphatases by NaF [27]. We may have seen a larger effect of okadaic acid (Fig. 4), but there are other phosphatases present in the post-mitochol~drial supernatant (e.g., alkaline phosphatase) which would still be active and oppose the effect of the inhibited phosphoprotein phosphatases-1 and -2A. Alkaline phosphatase has been demonstrated to dephosphorylate cytidylyltransferase in vitro 1161 and its activity was inhibited by NaF but not okadaic acid. Substantial alkaline phosphatase activity has been demonstrated in liver microsomes [28]. However, the subcellular distribution of the majority of alkaline phosphatase activity. on the external side of the plasma membrane 1291, would preclude the importance of this enzyme in the dephosphorylation of cytidylyltransferase in vivo. It should be noted that the cytidylyltransferase activities in the post-mitochondrial fraction and subcellular fractions of the okadaic acid experiments were slightly higher than the activities obtained with the NaF experiments. This might be attributed to the omission of magnesium in the okadaic acid preincubations. Magnesium was omitted in the okadaic acid preincubations since it has been demonstrated to inhibit the time-dependent increase in cytidylyltransferase activity in rat liver cytosol incubated under similar conditions [17] and is required for activation of phosphoprotein phosphatase 2C [22]. Phosphoprotein phosphatases-1, -2A and -2C account for virtually all the phosphatase activity in skeletal muscle and liver towards at least 20 phosphoproteins that regulate several metabolic pathways and muscle contraction [30,31]. Recently. Haystead et al. [18] have postulated that protein phosphatases-1 and -2A rather than -2C are the dominant phosphatase catalytic subunits acting on a wide range of phosphoproteins in vivo. Okadaic acid is a potent and specific inhibitor of phosphoprotein phosphatases-1 and -2A 119,201. Thus, the effect of preincubation of post-lnitochondrial supernatant with okadaic acid on the subcellular distribution of cytidylyltransferase implies that phosphoprotein phosphatase-1 and/or -2A may be involved in the regulation of phosphatidylcho~ine biosynthesis via the CDP-choline pathway in rat liver. Whether only phosphoprotein phosphatase-1 or -2A was involved in the of cytidylyltransferase was not dephosphorylation investigated in this study. However, the involvement of both phosphatases and also type 2C phosphoprotein phosphatase cannot be eliminated as a considerable
319 number of phosphoprotein substrates have been demonstrated to be dephosphorylated by all three phosphatases in vitro [30]. Fatty acids have been demonstrated to promote the translocation of cytidylyltransferase from the cytosolic to the microsomal fraction [32,33]. Okadaic acid, a polyether derivative of a 3%carbon fatty acid [34], clearly did not promote the binding of cytidylyltransferase to the microsomal fraction. In fact, the effect was quite opposite to that observed with fatty acids. In addition, okadaic acid did not affect the activity of purified cytidylyltransferase when added to the assay system. Thus, the inhibitory effect of okadaic acid was most likely on phosphatase(s) that dephosphorylate cytidylyltransferase. If phosphoprotein phosphatases are indeed involved in the dephosphorylation of the cytidylyltransferase, it is not surprising that their inhibition does not have a more dramatic effect on the subcellular distribution of the enzyme. Incubation of rat liver hepatocytes with chlorophenylthio-CAMP, a stimulator of CAMP-dependent protein kinase, for 2 h produced a 34% decrease in microsomal cytidylyltransferase activity with a corresponding 30% decrease in the new synthesis of phosphatidylcholine [15]. Since the content and composition of phosphatidylcholine rarely change in a specific membrane, a gross alteration in the phosphorylation state and subcellular distribution of cytidylyltransferase would severely affect the level of phosphatidylcholine. Okadaic acid inhibited only type 1 and 2A phosphoprotein phosphatases present in the post-mitochondrial supernatant and the small change in the subcellular distribution of cytidylyltransferase may have been temporally related to a small change in the phosphorylation state of the enzyme. Thus, the phosphorylation-dephosphorylation of cytidylyltransferase and its subsequent subcellular distribution must be highly coordinated and under strict metabolic control. Acknowledgements This work was supported by a grant from the Medical Research Council of Canada. We are indebted to Sandi Ungarian and Drs. Haris Jamil and Lilian Tijburg for technical assistance and helpful discussions. T-S.L. is the recipient of a Faculty of Medicine Summer Studentship, University of Alberta. G.M.H. is the recipient of a Canadian Heart Foundation Fellowship. D.E.V. is a Medical Scientist of the Alberta Heritage Foundation for Medical Research. References 1
White, D.A. (1973) in Form (Ansell, 44-482,
and Function G.B., Hawthorne, J.N. and Dawson, Elsevier, Amsterdam.
of Phospholipids. R.M.C., eds.), pp.
D.M. (1961) Biochim. Biophys. Acta 2 Bremer, J. and Greenberg, 46, 205-216. N.D. and Vance, D.E. (1987) J. Biol. Chem. 262, 3 Ridgway. 17231-17239. 4 Schneider, W.J. and Vance, D.E. (1979) J. Biol. Chem. 254, 3886-3891. 5 Pajares, M.A., Alemany, S., Varela, I.. Marin Cao. D. and Mato. J.M. (1984) B&hem. J. 233, 61-66. Sundler, R. and Akesson, B. (1975) J. Biol. Chem. 250, 3359-3367. Kennedy, E.P. and Weiss, S.B. (1956) J. Biol. Chem. 22, 193-251. Kennedy, E.P. (1962) Harvey Lect. 57, 143-171. Pelech. S.L. and Vance. D.E. (1984) Biochim. Biophys. Acta 779, 217-251. 10 Schneider, W.C. (1963) J. Biol. Chem. 238, 3572-3578. 11 Vance. D.E. and Pelech. S.L. (1984) Trends Biochem. Sci. 9. 17-20. 12 Feldman, D.A.. Kovac, C.R., Dranginis. P.L. and Weinhold, P.A. (1978) J. Biol. Chem. 253. 4980-4986. 13 Feldman, D.A., Dietrich, J.W. and Weinhold. P.A. (1980) Biochim. Biophys. Acta 620, 603-611. 14 Feldman, D.A., Rounsifer, W.E. and Weinhold. P.A. (1985) Biochim. Biophys. Acta 429, 4299437. 15 Pelech, S.L., Pritchard, P.H. and Vance, D.E. (1981) J. Biol. Chem. 256, 8283-8286. 16 Sanghera. J.S. and Vance, D.E. (1989) J. Biol. Chem. 264, 1215-1223. 17 Pelech, S.L. and Vance. D.E. (1982) J. Biol. Chem. 257, 14198-14202. 18 Haystead, T.A.J., Sim, A.T.R.. Coding. R.C., Honnor, R.C., Tsukitani, Y., Cohen, P. and Hardie, D.G. (1989) Nature 337, 78-81. 19 Bialojan, C. and Takai. A. (1988) Biochem. J. 256, 283-290. 20 Hescheler, J., Mieskes. G., Ruegy. J.C., Takai, A. and Trantwein. T. (1988) Pfliigers Arch. Ges. Physiol. 412, 248-252. 21 Paddon, H.B. and Vance. D.E. (1977) Biochim. Biophys. Acta 488, 181-189. 22 Cohen, P. (1980) Eur. J. B&hem. 111. 563-574. 23 Hatch, G.M. and Choy, P.C. (1987) Lipids 22. 672-676. 24 Weinhold, P.A., Rounsifer. M.E. and Feldman, D.A. (1986) J. Biol. Chem. 261. 5104-5110. 25 Pelech, S.L. and Vance. D.E. (1982) J. Biol. Chem. 257, 14198-14202. 26 Bradford, M. (1976) Anal. Biochem. 72. 248-254. 27 Beg, Z.H.. Stonik. J.A. and Bremer. H.B., Jr. (1978) Proc. Natl. Acad. Sci. USA 75. 367883682. 28 Morton, R.K. (1954) Biochem. J. 57, 595-599. 29 Tokumitou, S.I. (1984) in Human Alkaline Phosphatases (Stigbrand, T. and Fishman. W.H.. eds.). pp. 189-197. Alan R. Liss, New York. 30 Ingebritsen, T.S. and Cohen, P. (1983) Science 221. 331-338. 31 Cohen. P. (1988) Proc. R. Sot. 234, 115-144. 32 Pelech. S.L.. Pritchard, P.H.. Brindley. D.N. and Vance, D.E. (1983) J. Biol. Chem. 258. 6782-6788. 33 Weinhold, P.A.. Rousifer. W.E., Williams, SE.. Brubaker. P. and Feldman. D.A. (1984) J. Biol. Chem. 259. 10315-10324. 34 Tachibaua. K. and Scheuer. P.J. (1981) J. Am. Chem. Sot. 103. 2469-2471.