Biochimica et Biophysica Acta, 1093 (1991) 72-79 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100183S
72
BBAMCR 12945
C a 2+
regulation of phosphatidylinositol turnover in the plasma membrane of tobacco suspension culture cells Yoshiaki Kamada and Shoshi Muto Institute of Applied Microbiology, Universityof Tokyo, Tokyo (Japan)
(Received 10 September 1990)
Key words: Phosphatidylinositol kinase: Phosphatidylinositol 4-phosphate kinase; Phospholipase C; Diacylglycerol kinase: Calcium ion regulation: Tobacco plasma membrane
The biochemical properties of the enzymes involved in phosphatidylinositol (PI) turnover in higher plants were investigated using the plasma membrane isolated from tobacco suspension culture cells by aqueous two-phase partitioning. Submicromolar concentrations of Caz+ inhibited PI kinase and phosphatidylinositoi 4-phosphate (PIP) kinase and stimulated phospholipase C. Diacyiglycerol (DG) kinase was inhibited by Caz+, but required a higher concentration than the physiological level. From the above results we postulate the following scheme: signal coupled activation of phospholipase C produces IP3 which induces Ca2+ release from the intracellular Caz+ compartment, the increased cytoplasmic Caz+ in turn activates phospholipase C and causes a further increase of the cytoplasmic Ca2+ level. This inhibits PI kinase and PIP kinase and brings about a limited supply of PIP2, the substr ¢c of phospholipase C. Consequently, IPa production decreases and Caz+ mobilization ceases. Then cytosolic Caz+ returns to the stationary level by the Caz+ pump at the plasma membrane and at the endoplasmic reticulum and CaZ+/H + antiporter at the plasma membrane and at the tonoplast.
Introduction
The importance of Pl turnover in transmembrane signaling in response to external stimuli has been established in animal cells. IP3 and DG, which are the products of PIP2 hydrolyzed by signal-coupled phospholipase C in the plasma membrane, are second messengers in the cell: IP3 mobilizes Ca 2+ from the internal Ca 2+ pool inducing Ca 2+-dependent reactions, and DG activates protein kinase C. It is assumed that certain signal transduction pathways in plant cells play important roles in growth and development or environmental adaptation of higher plants. Recent studies showed that inositol phospholipids and inositol phosphates exist in plant cells [1-5].
Abbreviations: BTP, i,3-bis(tris(hydroxymethyl)methylamino)propane: DG, diacylglycerol; GTP~,S, guanosine 5'-O-(thiotrisphosphate); IP, inositol l-phosphate; IP2, inositol 1,4--bisphosphate; IP3, inositol 1,4,5-trisphosphate; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate. Correspondence: y. Kamada, Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.
Involvement of PI turnover in signal transduction in higher plants has been suggested in several reports: inositol phospholipid breakdown is induced by light [6] and auxin [7,8]; the amounts of inositol phospholipids and inositol phosphates change depending on the stage of the growth cycle of suspension culture cells [9,10]; and IP3 stimulates Ca 2+ release from microsomal vesicles [11,12], tonoplast vesicles [13], and intact vacuoles [14,15]. However problems remain concerning the identification and quantification of inositol phosphates. Recently, Rinc6n et al. [16] reported that they failed to detect IP3 in in vivo study even with precise analyses and pointed out that Dowex column chromatography which, has been used by most investigators to identify inositol phosphates, was not suitable, and that some more accurate methods (e.g., electrophoresis, HPLC) were needed to identify inositol phosphates. The biochemical properties of enzymes involved in PI turnover such as PI kinase, PIP kinase, phospholipase C and DG kinase have been studied by several investigators using several plant materials [17-24]. One of the most interesting properties is the regulation of these enzymes by Ca 2+. Phospholipase C requires Ca 2+ for its activity [19-23]. PI kinase and PIP kinase of wheat were inhibited by 100 pM Ca 2+ [17,18]. DG
73 kinase purified from suspension culture Catharanthus roseus cells was inhibited by Ca ,'÷ [23]. However, the Ca ,'+ concentration examined in most ,,, these reports was higher than the physiological level. In the present study we characterized PI kinase, PIP kinase, DG kinase and phospholipase C in the plasma membrane purified from tobacco suspension culture cells, focusing on the effect of the physiological concentration of Ca -,+ on these enzyme activities. The results suggest that PI turnover in plant plasma membrane, which may induce Ca 2÷ mobilization from the intracellular store via IP3 production, is controlled by the physiological concentration of C a 2 + Material and Methods
Reagents. PI and diolein were purchased from Serdary Research Laboratories, PIP and PIP2 were from Sigma, [),-32p]ATP was from ICN Biochemical, [inositol-23H]PI, [inositol-2-aH]PIP, [inositol-2-3H]Pl~, [2-3H]in ositol, [inositol-2-aH]lP, [inositol-2- 3H]IP2, and [inositoi1-3H]IP3 were from New England Nuclear. All other reagents used were of the highest grade commercially available. Plant material. Tobacco suspension culture cells (Nicotiana tabacum L.cv.Bright Yellow-2 cell line BY-2) [25] were propagated according to Nemoto et al. [26]. To isolate the plasma membrane a 3-day-old culture was used. Isolation of plasma membrane. The plasma membrane was isolated and purified as described [27,28] with some modifications. All steps for membrane preparation were carried out at 0-4 ° C. Culture cells were harvested with filtration by suction, and suspended in an isolation medium containing 0.3 M sucrose, 50 mM Mes-Tris (pH 7.6), 5 mM EGTA, 5 mM EDTA, 20 mM NaF, 2.5 mM Na2S205, 1 mM dithiothreitol, 2 mM phenylmethanesulfonylfluoride, 4 mM salycylhydroxamic acid and 0.5% (w/v) bovine serum albumin at a medium-totissue ratio of 2. This suspension was homogenized with a French pressure cell at 140 kg/cm 2 and the homogenate was centrifuged for 10 min at 10000 x g. The resulting pellet was discarded and the supernatant was centrifuged for 30 min at 100000 x g to obtain the microsomal fraction, which was suspended in a suspension medium containing 0.25 M sucrose and 10 mM potassium phosphate (pH 7.8). The plasma membrane was then purified from the microsomal fraction by the aqueous two-phase partitioning with 5.4% Dextran T500, 5.4% poly(ethylene glycol) 3350 and 0.35% NaCI. An upper phase after the partition was diluted with 5 mM Hepes-BTP (pH 7.0), containing 0.25 M sucrose and 0.1 mM dithiothreitol, then centrifuged for 30 min at 160 000 × g. The resulting pellet was suspended in the same medium and used as the plasma membrane.
Protein was determined by the method of Bradford [29] using bovine serum albumin as the standard. Assay of marker enzymes. Vanadate-sensitive Mg 2+ ATPase, IDPase and NADPH-cytochrome-c reductase were chosen as the marker enzyme for plasma membrane, Golgi and endoplasmic reticulum, respectively and their activities were assayed as described [27]. PI kinase and PIP kinase activities. Plasma membrane (25 #g protein) was incubated for 3 min at 30°C in 200 /~1 of 50 mM Tris-HCl (pH 7.5) containing 10 mM MgCI 2, 5 mM EGTA, 0.05% (w/v) Triton X-100, 0.5 mM dithiothreitol, 20 #g PI or PIP and 1 mM ['y-32p]ATP (60 mCi/mmol). The reaction was started by the addition of ATP, and terminated by the addition of 1 ml of cold chloroform/methanol (1 : 2, v/v). After standing for 20 min at 0 ° C, 0.75 ml of chloroform and 0.75 ml of 1.2 M HC! were added to the reaction mixture. After mixing and phase separation, the lower phase was collected and washed with 1 ml c;f methanol/l.2 M HC! (10:9, v/v). The resulting lipid extracts were dried under a N, gas stream, then samples were chromatographed on silica gel 60 (Merck) impregnated with 1% potassium oxalate and dried at 110°C for 45 min prior to use. The solvent system was chloroform/ acetone/methanol/acetic acid/water (40 : 15 : 13:12:8, v/v). The plate was run for 100 min. Lipids were visualized by the method of Dittmer and Lester [30] and radioactive compounds were visualized by autoradiography. After development of autoradiograms, radioactive spots of PIP or PIP_, were scratched off and their radioactivities were determined by liquid scintillation counting. For precise separation, two-dimensional TLC was carried out using chloroform/methanol/water (30 : 70 : 30) and chloroform/acetone/methanol/acetic acid/water (40:15 : 13 • 12 : 8, v/v) for the first and the second dimensional solvents, respectively. The TLC plate was treated as described above. The plate was run for 100 min at each dimension. Both radioactive PIP and PIP2 spots were separated free from contamination of any other radioactive compounds (Fig. 1). DG kinase activity. Plasma membrane (10 #g protein) was incubated for 3 min at 300C in 100/~,1 of 50 mM Bistris-HCl (pH 6.5) containing 5 mM MgCI:, 1 mM dithiothreitol, 20 mM NaF, 0.02% (w/v) Triton X-100, 20 #g diolein and 0.5 mM [3,-a2p]ATP (30 mCi/mmol). The reaction was started by the addition of ATP, and terminated by the addition of 500 ~1 of chloroform/ methamol (1:2, v/v). Lipids were extracted as described for assays of PI and PIP kinases. Radioactivity of the lipid extract was measured by liquid scintillation counting. Separation of the phospholipids on TLC as described above showed that the majority of 32p-phosphorylated product was PA (> 95%). Phospholipase C activiO'. Plasma membrane (10 /.tg protein) was incubated for 3 min at 30°C in 80 #1 of 50 mM Bistris-HCi (pH 7.0) containing 1 mM dithio-
74 T A B L E II 0,4
Effects of various salts on Mg" +-A TPase in plasma membrane from tobacco suspension culture cells
-1-r O G# ~E
Addition
Mg 2+-ATPase activity (~)
None (control) KNO3 (50 mM) NaN 3 (1 mM) Na3VO4 (100/tM)
100 93 96 16
g
i
Results
tJ
t c ¢q
B
I st-*CHCI3
IMeOHI H20(30:70:30)
Fig. 1. Autoradiogram of phospholipids extracted from plasma membrane of tobacco suspension culture cells labeled for 3 rain with [-t-3'P]ATP and separated on two dimensional TLC as described under Material and Methods. The origin is denoted by a bar; PA, phosphatidic acid; LPA, lysophosphatidic acid; Pl, phosphatidylino,~itoi; PIP, phosphatidylinositol 4-monophosphate; PIP2, phosphatidylinositoi 4,5-bisphosphate. Radioactive Pl was not detected.
threitol, 0.05% (w/v) sodium deoxycholate and 300/tM [3H]PIP2 (8000 dpm/nmol). The reaction was started by the addition of PIP2, and terminated by 20 ~tl of cold 25% (w/v) trichloroacetic acid. After centrifugation, the supernatant was washed four times with 800 t~l of diethyl ether. Released inositol phosphates were analyzed by HPLC. The samples were injected into a Partisil-10 SAX HPLC column (Whatman) and eluted with a three-step gradient of ammonium phosphate (pH 3.8) containing 1 mM EDTA. The gradient used was 10-208 mM over 10 rain for inositol and IP; 505-703 mM over 10 rain for IP2; and 1 M over 15 rain for IP3. The flow rate was 0.7 ml/min. Radioactivity in the eluate was measured by liquid scintillation counting.
Purity of plasma membrane Table I shows the distribution of marker enzymes in the microsomal and the plasma membrane fraction from the culture cells. Specific activities of NADPH-cytochrome-c reductase and IDPase of the plasma membrane were significantly lower than those of the microsome and the recovery of these enzymes were very low in the plasma membrane. On the other hand, the specific activity of VO3--sensitive ATPase increased significantly in the plasma membrane. These results indicate that contaminations of endoplasmic reticulum and Golgi in the plasma membrane are very low. To test the contaminations of tonoplasts and mitochondria in the plasma membrane the effects of various ions on Mg 2+ATPase were examined (Table If). While only slight inhibitions by NO; and N ; were found, the activity was markedly inhibited by VO43-. These results show that contaminations of tonoplasts and mitochondria are very low in the plasma membrane from tobacco culture cells. Characterization of PI kinase, PIP kinase and DG kinase Time courses of the enzyme reactions. Under the standard assay conditions, the reactions of these kinases proceeded linearly for 6 rain and the activities increased proportional to the amount of the plasma membrane up to 50/tg protein (data not shown). Thus, the following experiments were carried out for 3 min using 25 ~tg
TABLE i
Distribution of marker enzymes in microsome and plasma membrane fractions from tobacco suspension culture cells Marker enzymes
NADPH-cytochrolne-c reductase IDP~se V043--sensitive Mg 2+-ATPase
Specific activity (ttmol/mg protein per rain)
Total activity Qtmol/min)
MF a
PM
MF
PM
0.049 0.251
0.03 0.09
15.7 80.3
0.07 0.02
0.178
2.26
57.0
5.74
a MF, microsomal fraction; PM, plasma mcmbrane.
Percent recovered in plasma membrane
(~)
0.002 0.28 10.1
75 :°°° |
,
,
,
,
,
8
r
er / >-~ =:-,
ivc
A
/
|
_
o..
01
I
I
I
I
O•
I
0 0.02 0.05 0.1 1Hton X-IO0 (%)
0.2
protein for Pl and PIP kinases and 10 #g protein for DG kinase. Effect of Mg 2+. All the kinases required Mg 2+ for their activities. PI and PIP kinases were stimulated by as high as 10 mM Mg 2+, while DG kinase was stimulated by up to 5 mM and inhibited by higher concentrations of Mg 2+ (data not shown). Effect of Triton X-IO0. The kinases were markedly stimulated in the presence of Triton X-100 (Fig. 2). The optimal concentration of Triton X-100 was 0.05~ for PI kinase and PIP kinase, and 0.02% for DG kinase. pH optimum. The pH optimum was 7.5 for PI kinase and PIP kinase and 6.5 for D G kinase (Fig. 3). i
i
!
,
!
B
°~
g
B
.S 75O
kinase
, , , , , ,,i
e
D. O)
I
/
2|E
PIPkinase
v
I I
i
6
I
7
,
I
8
,
I
,
J
I
I
I
I
I
I
8
7
6
5
4
pCa
Fig. 2. Effect of Triton X-100 on Pl, PIP e and DG kinase activities. Experimental conditions are described under Material and Methods. Triton X-100 concentration of standard reaction mixture was changed as described.
1000 -'
'
5O
e-1oo
$
75-
i
9P H
Fig. 3. Effect of pH on PI kinase, PIP kinase and DG ldnase. (A) PI (closed symbol) and PIP kinase (open); and (B) DG kinase. The
buffer species of the standard reaction mixture were changed as follows; 50 mM Bistris-HCi (pH 6.5-7.0, O-o), Mops-KOH (pH 6.5-7.6, A-A) and Tris-HCl(pH 7.5-9.0,i-El).
Fig. 4. Effect of CaZ + on PI kinase, PIP kinase and DG kinase. Free Ca z+ concentrations were adjusted using EGTA-Ca 2+ buffer. Kca-EGTA used was l0 II [31]. All assays were normalized to 100% in the absence of CaC! 2.
Km for A TP and Vmax. The kinases showed Michaelis-Menten type saturation curves for ATP. Apparent K m values for ATP of PI kinase, PIP kinase and DG kinase were 0.68, 0.12 and 0.20 raM, and Vm.~x values were 1400, 190 and 12700 pmol A T P / m g protein per rain, respectively. DG kinase activity was much higher than the others. Effect of Ca: +. PI kinase and PIP kinase were inhibited by the physiological concentration of Ca 2+, i.e., their activities were decreased to 50~ between pCa 8 and 6. On the other hand, a Ca 2+ co,,centration approx. two orders of magnitude higher than the physiological concentration was required to inhibit DG kinase (Fig. 4). One should bear in mind that, if phospholipase C is activated by Ca 2+, the reaction products of PI kinase and PIP kinase would be hydrolyzed and the consequent decrease of PIP and PIP2 concentrations would lower the apparent activities of these kinases, in fact, phospholipase C was markedly activated by the physiological concentration of Ca :+ (see below). Disturbance of the kinase assays by phospholipase C can be prevented by inhibiting phospholipase C. Cruz-Rivera et al. [32] have reported that glycero-3-phospho-D-myo-inositol 4-phosphate is a specific inhibitor of phospholipase C from guinea-pig uteri, however, the inhibit)r had no effect on the tobacco enzyme. Thus, the inhibition of PI kinase and PIP kinase by Ca 2+ was re-examined in the presence of an excess amount of unlabeled reaction product in order to reduce hydrolysis of the labeled products by phospholipase C. In addition, the assays were done with 1 mM Mg 2+ instead of 10 mM to mimic the physiological condition. PI kinase and PIP kinase were also inhibited by a :ow concentration of Ca 2+ even in the presence of un-
76 1
e
|
I
i
|
,0t
400
pl hydrolysis
IP
IO0
v75
'
/I
,,
Fig, 5, Effect of Ca ~'+ . . . . . the presence oflmMMgCI2andl0~gofunlabeledPlP(Pikinase)orPlP2(PlP kinase), Free Ca" + concentrations were adjusted as described in Fig.
enzymes was greater under this assay condition: PI kinase in particular was inhibited 95% at a submicromolar concentration of Ca 2+. The addition of excess PIP or PIP2 to the reaction mixture did not inhibit the enzyme activities. These results indicate that both PI kinase and PIP kinase are really inhibited by the physiological concentration of Ca 2+. Effects of various compounds. We studied the effects of various compounds on the activities of kinases (Table Ill). R 59022 (50 #M), a specific inhibitor for DG kinase from human red blood cells [33], had no effect on DG kinase nor on Pl kinase and PIP kinase. A caimodulin inhibitor, compound 48/80 (0.02-0.1 mg/ml), was a strong inhibitor of Pl kinase and DG kinase, while it scarcely affected PIP kinase. Another calmodulin inhibitor, trifluoperazine (50 /~M), weakly inhibited PI kinase but not PIP kinase. Neither adenoTABLE III
Effectsof compounds on PI, PIP and D G kinases Com!~ounds
Control 50 ~tM R 59022 0,02 mg/ml compound 48/80 0.1 mg/ml compound 48/80 50 ItM trifluoperazine 5% ~tM adenosine 5O0 FM ADP 10 mM NaF 100 ~M GTPyS
Activities (~) P! kinase
PiP kinase DG kinase
100 99 70 29 82 93 97 68 95
100 72 97 87 107 94 90 53 107
100 85 54 21
100
6001
O 0
I
hydro
5
t ,~
" [1
10 ~ 15
20
25
1
30
Retention time (rain)
6.
Fig. Identification of the products of inositol phospholipid hydrolysis by HPLC as described m;der Material and Methods. The labeled products of hydlolysis were identified based on elution times of commercial standards; (A) Pl hydrolysis: (B) PIP hydroly~i~: {C) PI~ hydrolysis (each 10 rain incubation).
sine (500 #M) nor ADP (500 #M), which do inhibit some PI kinases [34], inhibited the tobacco PI kinase and PIP kinase. NaF (10 mM) which is reported to inhibit rat brain PI kinase and PIP kinase [35] inhibited the respective tobacco enzymes. GTPyS (100 #M) had no effect on PI kinase and PIP kinase.
Characterization of phospholipase C Analysis of reaction products. Inositol phosphates released from phosphoinositides were analyzed by HPLC to examine the presence in the plasma membrane of phospholipase C and D and phosphatases which hydrolyze inositol phosphates and inositol phospholipids (Fig. 6). The plasma membrane hydrolyzed exogenous PI, PIP and PIP2 and produced IP, IPa and IP3, respectively, indicating the presence of phospholipase C. When PIPa was incubated for more than 5 rain, an unknown peak appeared which eluted close to (but not co-eluting with) the IPa standard (Fig. 6C). This peak also appeared when [3H]IP3 was incubated with the plasma membrane (data not shown). These results suggest that the unknown peak is a dephosphorylated product of IP3 (inositol 1,5-bisphosphate or inositol 4,5-bisphosphate)
77 by IP3 phosphatase (IP3-4-phosphatase or IP3-1-phosphatase, respectively), or an unknown metabolized product of IP3. IP3-5-phosphatase of human platelets required Mg E+ [36], but we could not observe any activity in tobacco plasma membrane in the presence of Mg E+ (data not shown). The results above alternatively suggest that phospholipase D does not exist in the plasma membrane or it exists but does not hydrolyze phosphoinositides. Galliard [37] reported that phosphatidylinositol is apparently not a substrate for plant phospholipase D. IP2 was not produced when PIPE was substrate, indicating that phosphatase activity, which dephosphorylates PIP,, to PIP, did not exist or it was inactivated under the assay condition, because PIP was hydrolyzed to IP, by phospholipase C. To examine the contaminations of isomers of inositol phosphate in Each peak, all hydrolyzed products were reanalyzed by the more precise method of Dean and Moyer [38] using HPLC with a gentle gradient system which was established to separate inositoi phosphate isomers and all of them were free of contamination with other inositol phosphate isomers (data not shown). Hydrolysis of PI, PIP and PIP2 proceeded linearly for up to 5 min with each substrate (data not shown). The following experiments were carried out with [3HIPIP2 for 3 min. pH optimum. The pH optimum of phospholipase C was observed around 7.5 (Fig. 7). Substrate specificity. Apparent g m values of phospholipase C for Pl, PIP and PIP2 were 460, 107 and 97 #M and the Vmax values were 184, 108 a , d 102 nmol/mg protein per min, respectively.
I
I
I
I
I
I
60
80
!oo 2/" E
[..4o ! ,mr
20
t |
I
t
3
pCa
Fig. 8. Effect of Ca :+ on phospholipase C activity. [~H]PIP., was used as substrate. Various free Ca 2 + concentration were prepared as described in Fig. 4.
Effect of Ca -'+. The phospholipase C activity in the plasma membrane was markedly stimulated by Ca -'+ especially between pCa 8 and 6 (Fig. 8). Effect of GTP,/S. GTP binding protein is involved in the activation of phospholipase C by agonists in animal cells [39]. Recently, GTP binding protein was identified in the plasma membrane of higher plants [40]. The involvement of GTP binding protein in the activation of phospholipase C in tobacco plasma membrane was examined by adding GTP'yS to the reaction mixture. GTP~,S (100 #M) had no effect on the enzyme in the presence of Mg 2+ (1 or 10 mM). Discussk~n
.=_ E
~40 e
I
6
I
I
7
I
pH
I
8
I
I
9
Fig. 7. Effect of p H on phospholipase C activity using [3H]PIP2 as substrate. The buffers used are described in Fig. 3.
The properties of PI kinase and PIP kinase of the plasma membrane from tobacco suspension culture cells with respect to the time-course of the reaction and effects of Triton X-100 and pH on the activities are similar to those reported by Sommarin and Sandelius [17,18] with wheat plasma membrane. However, the affinity to ATP of the tobacco PI kinase is about 3-times lower than that of the wheat enzymes. Km (ATP) of the tobacco PI kinase is similar to that of type 3 kinase of bovine brain which was reported to be 0.74 mM [34]. Low sensitivity of the tobacco PI kinase to adenosine is also similar to the type 3 kinase. Inhibition of tobacco plasma membrane PI kinase and PIP kinase by a submicromolar range of Ca 2+ suggests that the two kinases are controlled by the change of cytoplasmic Ca 2+ concentration. Inhibitions of bovine PI kinase by a nanomolar range of Ca 2+ [41] have been reported and
Signal(lighlhormone,elicitor,etc.)
78
7", , ' ! I~eceyto.r L..-~G protein)
Plasmamembrane (
Pl
kinase ) - - ~ PiPklnese ~ ft~ ,PIP f ~ ~ , P I P 2 ~ D G
DGklnase ) f ~ .PA
Pl
ATP
AOP
ATP
(~%,. . . . . . . . . . . . . . .
AOP
~, ~ . . . . . . . . .
~
~_ i?, ATP
-2+
i[~At "--
.,P~
//'*
// I~'
r-_ . . . . .
AOP
"-I
I Protein I LklnaseC]
2+
Fig, 9. Regulation of PI t u r n o v e r by Ca 2 +. F o r details, see text.
pituitary PIP kinase by a micromolar range of Ca 2+ [42]. However, the latter was investigated using purified plasma membrane, without considering the activation of phospholipase C by Ca 2+. Compound 48/80 inhibited PI kinase, PIP kinase and DG kinase and trifluoperazine inhibited PI kinase, but it is not probable that they associate with calmodulin because these enzymes were inhibited by Ca 2+. Though PI kinase and PIP kinase had very similar biochemical properties, they showed different behavior to compound 48/80 suggesting that they are different_ enzymes. The existence of isozymes of respective kinases can be assumed. DO kinase has been solubilized and purified from membrane fractions of Catharanthus roseus suspension culture cells and characterized [23]. The characteristics of tobacco DG kinase, such as optimal pH, apparent Km (ATP), stimulation by Mg 2+ and inhibition by Ca 2+, are similar to those of C. roseus enzyme. Since the effective concentration of Ca 2+ was higher than 10 /~M, DG kinase may not be controlled by cytoplasmic Ca 2+. DG kinase activity was so high that most of Pi incorporated from ATP into plasma membrane lipids was PA. This occurred without exogenous substrate, indicating that DG may be accumulated during the isolation of the plasma membrane. Though it is unknown whether the DO level is also high in vivo, it is likely that DG kinase may function to keep the DG concentration at a low level. Phospholipase C of tobacco plasma membrane hydrolyzed three inositol phospholipids. Phosphoinositide specific phospholipase C has been reported in the plasma membranes of soybean [19], wheat [20] and Arena [22]. Phospholipase C from tobacco as well as from soybean
and wheat preferentially hydrolyzed PIP and PIP2 more than PI. The tobacco enzyme has a very similar specificity to PIP and PIP2, while soybean and wheat enzymes had different specificities to PIP and PIP2. The tobacco phospholipase C was stimulated by a low concentration of Ca 2+. Phospholipase C from various higher plants req,.,.ired Ca 2+, among them only a few [20,22,23] were stimulated by a micromolar range of Ca 2+. Stimulation by guanine nucleotides of inositol phosphate release from membrane isolated from Acer culture cells has been reported [43]. Zbell and Walter [8] observed that the stimulation of phosphoinositide breakdown by auxin plus GTPyS occurred in microsomal membrane of carrot suspension culture cells. However, many investigators failed to observe guanine nucleotide stimulation of inositol phospholipid breakdown using purified plasma membrane. GTPTS was also ineffective on the tobacco membrane. We observed that IP3 was metabolized by the plasma membrane. Joseph et al. [44] reported that a soluble fraction prepared from tobacco suspension culture cells hydrolyzed inositol phosphates and its activity was stimulated by 1.8/~M Ca 2+. It hydrolyzed IP3 to form inositol 1,4-bisphosphate and inositol 4,5-bisphosphate. Fig. 9 schematically represents regulation of PI turnover by Ca 2+. Cytoplasmic C a 2+ concentration is kept at the submicromolar range or lower [45]. The maintenance of this low level is due to sequestration of Ca 2+ into the intracellular pool and extrusion of Ca 2+ through the plasma membrane by the Ca2+-pump and Ca2+/H + antiporter [27]. Coupled to signals, phospholipase C hydrolyzes PIP2 and produces IP.~ which induces Ca 2+ mobilization from vacuole (and endo-
79 plasmic reticulum?) raising the Ca 2+ concentration in the cytoplasm. Ca 2+ in turn activates various Ca2+-de pendent enzymes, especially phospholipase C itself. Consequently, more IP3 is synthesized and causes an increase of cytosolic Ca 2+. On the other hand, PI kinase and PIP kinase are inhibited by Ca = ~ and less PIP2 is synthesized. Thus activated phospholipase C is limited in its substrate and produces less IP3. In this way Ca 2+ works as a feedback inhibitor for regulation of cytoplasmic Ca 2+ concentration via PI turnover. This scheme might give a plausible explanation for the oscillation in the increase of internal Ca 2+ found, for example, in the guard cell [46]. To adapt this scheme to in vivo conditions, we are currently investigating PI turnover in the intact cells.
Acknowledgements We thank Prof. T. Kuroiwa and Prof. T. Nagata of the Department of Botany, University of Tokyo for the generous gift of the strain of tobacco suspension culture cell. We also thank Dr. N. Sato of the Department of Botany, University of Tokyo for his valuable advice on the analytical method of phospholipids, respectively. This work was supported by a Grant-in-Aid for General Scientific Research 01480010 from the Japanease Ministry of Education, Science and Culture.
References 1 Boss, W.F. and Massel, M.O. (1985) Biochem. Biophys. Res, Commun. 132, 1018-1023. 2 Wheeler, J.J. and Boss, W.F. (1987) Plant Physiol. 85, 389-392. 3 Heim, S., Bauleke, A, Wylegalla, C. and Wagner, K.G. (1987) Plant Sci. 49, 159-165. 4 Irvine, R.F., Letcher, AJ., Lander, D.J, Drobak, B.K., Dawson, A.P. and Musgrave, A. (1989) Plant Physiol. 89, 888-892, 5 Cot6, G.G., DePass, A.L., Quarmby, L.M., Tare, B.F., Morse, M.J., Satter, R.L. and Crain, R.C. (1989) Plant Physiol. 90, 14221428. 6 Morse, M.J., Crain, R.C. and Satter, R.L. (1987) Proc. Natl. Acad. Sci. USA 84, 7075-7078. 7 Ettlinger, C. and Lehle, L. (1988) Nature 331, 176-178. 8 Zbell, B. and Walter, C. (1987) in Plant Hormone Recepters (Kliimt, D., ed.), pp. 141-153, Springer-Verlag, Berlin. 9 Heim, S. and Wagner, K.G. (1986) Biochem. Biophys. Res. Commun, 134, 1175-1181. 10 Heim, S. and Wagner, K.G. (1989) Plant Sci. 63, 159-165. 11 DrobaL B.K. and Ferguson, I.B. (1985) Biochem. Biophys. Res. Commun. 130, 1241-1246. 12 geddy, A.S.N. and Poovaiah, B.W. (1987) J. Biochem. (Tokyo) 101, 569-573. 13 Schumaker, K.S. and Sze, H. (1987) J. Biol. Chem. 262, 3944-3946.
14 Ranjeva, R., Carrasco, A. and Boudet, A.M. (1988) FEBS Lett. 230, 137-141. 15 Alexandre, J., Lassalles, J.P. and Kado, R.T. (1990) Nature 343, 567-570. 16 Rinc6n, M., Chen, Q. and Boss, W.F. (1989) Plant Physiol. 89, 126-132. 17 Sandelius, A.S. and Sommarin, M. (1986) FEBS Lett. 201,282-286. 18 Sommarin, M. and Sandelius, A.S. (1988) Biochim. Biophys. Acta 958, 268-278. 19 Pfaffmann, H., Hartmann, E., Brightman, A.O. and Morre, D.J. (1987) Plant Physiol. 85, 1151-1155. 20 Melin, P.-M., Sommarin, M., Sandelius, A.S. and Jergil, B. (1987) FEBS Lett. 223, 87-91. 21 McMurray, W.C. and Irvine, R.F. (1.988) Biochem. J. 249, 877881. 22 Tate, B.F., Schaller, G.E., Sussman, M.R. and Crain, R.C. (1989) Plant Physiol. 91, 1275-1279. 23 Biffen, M. and Hanke, D.E. (1990) Plant Sci. 69, 147-155. 24 Wissing, J., Heim, S. and Wagner, K.G. (1989) Plant Physiol. 90, 1546-1551. 25 Kato, K., ,qhiozawa, Y., Yamada, A., Nishida, K, and Noguchi. M. (1972) Agr. Biol. Chem. 36, 899-902. 26 Nemoto, Y., Kawano, S., Nakamura, S., Mita, T., Nagata, T. and Kuroiwa, T. (1988) Plant Cell Physiol. 29, 167-177. 27 Kasai, M. and Muto, S. (1990) J. Membr, Biol. 114, 133-142. 28 Yoshida, S., Kawata, T., Uemura, M. and Niki, T. (1986) Plant Physiol. 80, 152-160. 29 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 30 Dittmer, C. and [,ester, L. (1964) J. Lipid Res. 5, 126-127. 31 Raaflaub, J. (1960) Methods Biochem. Anal. 3, 301-325. 32 Cruz-Rivera, M., Bennett, C.F. and Crooke, S.T. (1990) Biochim. Biophys. Acta 1042, 113-118. 33 De Chaffoy de Courcelles, D., Roevens, P. and Van Belle, H. (1985) J. Biol. Chem. 260, 15762-15770. 34 Endemann, G., Dunn, S.N. and Cantley, L.C. (1987) Biochemistry 26, 6845-6852. 35 Claro, E., Wallace, M.A. and Fain, J.N. (1990) Biochem. J. 268, 733-737. 36 Connolly, T.M., Bross, T.E. and Majerus, P.W. (1985) J. Biol. Chem. 260, 7868-7874. 37 Galliard, T. (1980) in The Biochemistry of Plants (Stumpf, P,K., ed.), Vol. 4, pp. 85-116, Academic Press, New York. 38 Dean, N.M. and Moyer, J.D. (1987) Biochem. J. 242, 361-366. 39 Gilman, A.G. (1987) Annu. Rev. Biochem. 56, 615-649. 40 Blum, W., Hinsch, K.-D., Schultz, G. and Weiler, E.W. (1988) Biochem. Biophys. Res. Commun. 156, 954-959, 41 Husebye, E.S., Letcher, A.J., Lander, D.J. and Flatmark, T. (1990) Biochim. Biophys. Acta 1042, 330-337. 42 lmai, A., Rebecchi, M.J. and Gershengorn, M.C. (1986) Biochem. J. 240, 341-348. 43 Dillenschneider, M, Hetherington, A., Graziana, A., Aliberi, G., Berta, P, Haiech, J. and Ranjeva, R. (1986) FEBS Lett. 208. 413-417. 44 Joseph, S.K., Esch, T. and Bonner, Jr., W.D. (1989) Biochem. J. 264, 851-856. 45 Gilroy, S., l[ughes, W.A. and Trewavas, A.J. (1986) FEBS Lett. 199, 217-221. 46 McAinsh, M.R., Brownlee, C. and Hetherington, A.M. (1990) Nature 343, 186-188.
72
BBAMCR 12945
C a 2+
regulation of phosphatidylinositol turnover in the plasma membrane of tobacco suspension culture cells Yoshiaki Kamada and Shoshi Muto Institute of Applied Microbiology, Universityof Tokyo, Tokyo (Japan)
(Received 10 September 1990)
Key words: Phosphatidylinositol kinase: Phosphatidylinositol 4-phosphate kinase; Phospholipase C; Diacylglycerol kinase: Calcium ion regulation: Tobacco plasma membrane
The biochemical properties of the enzymes involved in phosphatidylinositol (PI) turnover in higher plants were investigated using the plasma membrane isolated from tobacco suspension culture cells by aqueous two-phase partitioning. Submicromolar concentrations of Caz+ inhibited PI kinase and phosphatidylinositoi 4-phosphate (PIP) kinase and stimulated phospholipase C. Diacyiglycerol (DG) kinase was inhibited by Caz+, but required a higher concentration than the physiological level. From the above results we postulate the following scheme: signal coupled activation of phospholipase C produces IP3 which induces Ca2+ release from the intracellular Caz+ compartment, the increased cytoplasmic Caz+ in turn activates phospholipase C and causes a further increase of the cytoplasmic Ca2+ level. This inhibits PI kinase and PIP kinase and brings about a limited supply of PIP2, the substr ¢c of phospholipase C. Consequently, IPa production decreases and Caz+ mobilization ceases. Then cytosolic Caz+ returns to the stationary level by the Caz+ pump at the plasma membrane and at the endoplasmic reticulum and CaZ+/H + antiporter at the plasma membrane and at the tonoplast.
Introduction
The importance of Pl turnover in transmembrane signaling in response to external stimuli has been established in animal cells. IP3 and DG, which are the products of PIP2 hydrolyzed by signal-coupled phospholipase C in the plasma membrane, are second messengers in the cell: IP3 mobilizes Ca 2+ from the internal Ca 2+ pool inducing Ca 2+-dependent reactions, and DG activates protein kinase C. It is assumed that certain signal transduction pathways in plant cells play important roles in growth and development or environmental adaptation of higher plants. Recent studies showed that inositol phospholipids and inositol phosphates exist in plant cells [1-5].
Abbreviations: BTP, i,3-bis(tris(hydroxymethyl)methylamino)propane: DG, diacylglycerol; GTP~,S, guanosine 5'-O-(thiotrisphosphate); IP, inositol l-phosphate; IP2, inositol 1,4--bisphosphate; IP3, inositol 1,4,5-trisphosphate; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate. Correspondence: y. Kamada, Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.
Involvement of PI turnover in signal transduction in higher plants has been suggested in several reports: inositol phospholipid breakdown is induced by light [6] and auxin [7,8]; the amounts of inositol phospholipids and inositol phosphates change depending on the stage of the growth cycle of suspension culture cells [9,10]; and IP3 stimulates Ca 2+ release from microsomal vesicles [11,12], tonoplast vesicles [13], and intact vacuoles [14,15]. However problems remain concerning the identification and quantification of inositol phosphates. Recently, Rinc6n et al. [16] reported that they failed to detect IP3 in in vivo study even with precise analyses and pointed out that Dowex column chromatography which, has been used by most investigators to identify inositol phosphates, was not suitable, and that some more accurate methods (e.g., electrophoresis, HPLC) were needed to identify inositol phosphates. The biochemical properties of enzymes involved in PI turnover such as PI kinase, PIP kinase, phospholipase C and DG kinase have been studied by several investigators using several plant materials [17-24]. One of the most interesting properties is the regulation of these enzymes by Ca 2+. Phospholipase C requires Ca 2+ for its activity [19-23]. PI kinase and PIP kinase of wheat were inhibited by 100 pM Ca 2+ [17,18]. DG
73 kinase purified from suspension culture Catharanthus roseus cells was inhibited by Ca ,'÷ [23]. However, the Ca ,'+ concentration examined in most ,,, these reports was higher than the physiological level. In the present study we characterized PI kinase, PIP kinase, DG kinase and phospholipase C in the plasma membrane purified from tobacco suspension culture cells, focusing on the effect of the physiological concentration of Ca -,+ on these enzyme activities. The results suggest that PI turnover in plant plasma membrane, which may induce Ca 2÷ mobilization from the intracellular store via IP3 production, is controlled by the physiological concentration of C a 2 + Material and Methods
Reagents. PI and diolein were purchased from Serdary Research Laboratories, PIP and PIP2 were from Sigma, [),-32p]ATP was from ICN Biochemical, [inositol-23H]PI, [inositol-2-aH]PIP, [inositol-2-3H]Pl~, [2-3H]in ositol, [inositol-2-aH]lP, [inositol-2- 3H]IP2, and [inositoi1-3H]IP3 were from New England Nuclear. All other reagents used were of the highest grade commercially available. Plant material. Tobacco suspension culture cells (Nicotiana tabacum L.cv.Bright Yellow-2 cell line BY-2) [25] were propagated according to Nemoto et al. [26]. To isolate the plasma membrane a 3-day-old culture was used. Isolation of plasma membrane. The plasma membrane was isolated and purified as described [27,28] with some modifications. All steps for membrane preparation were carried out at 0-4 ° C. Culture cells were harvested with filtration by suction, and suspended in an isolation medium containing 0.3 M sucrose, 50 mM Mes-Tris (pH 7.6), 5 mM EGTA, 5 mM EDTA, 20 mM NaF, 2.5 mM Na2S205, 1 mM dithiothreitol, 2 mM phenylmethanesulfonylfluoride, 4 mM salycylhydroxamic acid and 0.5% (w/v) bovine serum albumin at a medium-totissue ratio of 2. This suspension was homogenized with a French pressure cell at 140 kg/cm 2 and the homogenate was centrifuged for 10 min at 10000 x g. The resulting pellet was discarded and the supernatant was centrifuged for 30 min at 100000 x g to obtain the microsomal fraction, which was suspended in a suspension medium containing 0.25 M sucrose and 10 mM potassium phosphate (pH 7.8). The plasma membrane was then purified from the microsomal fraction by the aqueous two-phase partitioning with 5.4% Dextran T500, 5.4% poly(ethylene glycol) 3350 and 0.35% NaCI. An upper phase after the partition was diluted with 5 mM Hepes-BTP (pH 7.0), containing 0.25 M sucrose and 0.1 mM dithiothreitol, then centrifuged for 30 min at 160 000 × g. The resulting pellet was suspended in the same medium and used as the plasma membrane.
Protein was determined by the method of Bradford [29] using bovine serum albumin as the standard. Assay of marker enzymes. Vanadate-sensitive Mg 2+ ATPase, IDPase and NADPH-cytochrome-c reductase were chosen as the marker enzyme for plasma membrane, Golgi and endoplasmic reticulum, respectively and their activities were assayed as described [27]. PI kinase and PIP kinase activities. Plasma membrane (25 #g protein) was incubated for 3 min at 30°C in 200 /~1 of 50 mM Tris-HCl (pH 7.5) containing 10 mM MgCI 2, 5 mM EGTA, 0.05% (w/v) Triton X-100, 0.5 mM dithiothreitol, 20 #g PI or PIP and 1 mM ['y-32p]ATP (60 mCi/mmol). The reaction was started by the addition of ATP, and terminated by the addition of 1 ml of cold chloroform/methanol (1 : 2, v/v). After standing for 20 min at 0 ° C, 0.75 ml of chloroform and 0.75 ml of 1.2 M HC! were added to the reaction mixture. After mixing and phase separation, the lower phase was collected and washed with 1 ml c;f methanol/l.2 M HC! (10:9, v/v). The resulting lipid extracts were dried under a N, gas stream, then samples were chromatographed on silica gel 60 (Merck) impregnated with 1% potassium oxalate and dried at 110°C for 45 min prior to use. The solvent system was chloroform/ acetone/methanol/acetic acid/water (40 : 15 : 13:12:8, v/v). The plate was run for 100 min. Lipids were visualized by the method of Dittmer and Lester [30] and radioactive compounds were visualized by autoradiography. After development of autoradiograms, radioactive spots of PIP or PIP_, were scratched off and their radioactivities were determined by liquid scintillation counting. For precise separation, two-dimensional TLC was carried out using chloroform/methanol/water (30 : 70 : 30) and chloroform/acetone/methanol/acetic acid/water (40:15 : 13 • 12 : 8, v/v) for the first and the second dimensional solvents, respectively. The TLC plate was treated as described above. The plate was run for 100 min at each dimension. Both radioactive PIP and PIP2 spots were separated free from contamination of any other radioactive compounds (Fig. 1). DG kinase activity. Plasma membrane (10 #g protein) was incubated for 3 min at 300C in 100/~,1 of 50 mM Bistris-HCl (pH 6.5) containing 5 mM MgCI:, 1 mM dithiothreitol, 20 mM NaF, 0.02% (w/v) Triton X-100, 20 #g diolein and 0.5 mM [3,-a2p]ATP (30 mCi/mmol). The reaction was started by the addition of ATP, and terminated by the addition of 500 ~1 of chloroform/ methamol (1:2, v/v). Lipids were extracted as described for assays of PI and PIP kinases. Radioactivity of the lipid extract was measured by liquid scintillation counting. Separation of the phospholipids on TLC as described above showed that the majority of 32p-phosphorylated product was PA (> 95%). Phospholipase C activiO'. Plasma membrane (10 /.tg protein) was incubated for 3 min at 30°C in 80 #1 of 50 mM Bistris-HCi (pH 7.0) containing 1 mM dithio-
74 T A B L E II 0,4
Effects of various salts on Mg" +-A TPase in plasma membrane from tobacco suspension culture cells
-1-r O G# ~E
Addition
Mg 2+-ATPase activity (~)
None (control) KNO3 (50 mM) NaN 3 (1 mM) Na3VO4 (100/tM)
100 93 96 16
g
i
Results
tJ
t c ¢q
B
I st-*CHCI3
IMeOHI H20(30:70:30)
Fig. 1. Autoradiogram of phospholipids extracted from plasma membrane of tobacco suspension culture cells labeled for 3 rain with [-t-3'P]ATP and separated on two dimensional TLC as described under Material and Methods. The origin is denoted by a bar; PA, phosphatidic acid; LPA, lysophosphatidic acid; Pl, phosphatidylino,~itoi; PIP, phosphatidylinositol 4-monophosphate; PIP2, phosphatidylinositoi 4,5-bisphosphate. Radioactive Pl was not detected.
threitol, 0.05% (w/v) sodium deoxycholate and 300/tM [3H]PIP2 (8000 dpm/nmol). The reaction was started by the addition of PIP2, and terminated by 20 ~tl of cold 25% (w/v) trichloroacetic acid. After centrifugation, the supernatant was washed four times with 800 t~l of diethyl ether. Released inositol phosphates were analyzed by HPLC. The samples were injected into a Partisil-10 SAX HPLC column (Whatman) and eluted with a three-step gradient of ammonium phosphate (pH 3.8) containing 1 mM EDTA. The gradient used was 10-208 mM over 10 rain for inositol and IP; 505-703 mM over 10 rain for IP2; and 1 M over 15 rain for IP3. The flow rate was 0.7 ml/min. Radioactivity in the eluate was measured by liquid scintillation counting.
Purity of plasma membrane Table I shows the distribution of marker enzymes in the microsomal and the plasma membrane fraction from the culture cells. Specific activities of NADPH-cytochrome-c reductase and IDPase of the plasma membrane were significantly lower than those of the microsome and the recovery of these enzymes were very low in the plasma membrane. On the other hand, the specific activity of VO3--sensitive ATPase increased significantly in the plasma membrane. These results indicate that contaminations of endoplasmic reticulum and Golgi in the plasma membrane are very low. To test the contaminations of tonoplasts and mitochondria in the plasma membrane the effects of various ions on Mg 2+ATPase were examined (Table If). While only slight inhibitions by NO; and N ; were found, the activity was markedly inhibited by VO43-. These results show that contaminations of tonoplasts and mitochondria are very low in the plasma membrane from tobacco culture cells. Characterization of PI kinase, PIP kinase and DG kinase Time courses of the enzyme reactions. Under the standard assay conditions, the reactions of these kinases proceeded linearly for 6 rain and the activities increased proportional to the amount of the plasma membrane up to 50/tg protein (data not shown). Thus, the following experiments were carried out for 3 min using 25 ~tg
TABLE i
Distribution of marker enzymes in microsome and plasma membrane fractions from tobacco suspension culture cells Marker enzymes
NADPH-cytochrolne-c reductase IDP~se V043--sensitive Mg 2+-ATPase
Specific activity (ttmol/mg protein per rain)
Total activity Qtmol/min)
MF a
PM
MF
PM
0.049 0.251
0.03 0.09
15.7 80.3
0.07 0.02
0.178
2.26
57.0
5.74
a MF, microsomal fraction; PM, plasma mcmbrane.
Percent recovered in plasma membrane
(~)
0.002 0.28 10.1
75 :°°° |
,
,
,
,
,
8
r
er / >-~ =:-,
ivc
A
/
|
_
o..
01
I
I
I
I
O•
I
0 0.02 0.05 0.1 1Hton X-IO0 (%)
0.2
protein for Pl and PIP kinases and 10 #g protein for DG kinase. Effect of Mg 2+. All the kinases required Mg 2+ for their activities. PI and PIP kinases were stimulated by as high as 10 mM Mg 2+, while DG kinase was stimulated by up to 5 mM and inhibited by higher concentrations of Mg 2+ (data not shown). Effect of Triton X-IO0. The kinases were markedly stimulated in the presence of Triton X-100 (Fig. 2). The optimal concentration of Triton X-100 was 0.05~ for PI kinase and PIP kinase, and 0.02% for DG kinase. pH optimum. The pH optimum was 7.5 for PI kinase and PIP kinase and 6.5 for D G kinase (Fig. 3). i
i
!
,
!
B
°~
g
B
.S 75O
kinase
, , , , , ,,i
e
D. O)
I
/
2|E
PIPkinase
v
I I
i
6
I
7
,
I
8
,
I
,
J
I
I
I
I
I
I
8
7
6
5
4
pCa
Fig. 2. Effect of Triton X-100 on Pl, PIP e and DG kinase activities. Experimental conditions are described under Material and Methods. Triton X-100 concentration of standard reaction mixture was changed as described.
1000 -'
'
5O
e-1oo
$
75-
i
9P H
Fig. 3. Effect of pH on PI kinase, PIP kinase and DG ldnase. (A) PI (closed symbol) and PIP kinase (open); and (B) DG kinase. The
buffer species of the standard reaction mixture were changed as follows; 50 mM Bistris-HCi (pH 6.5-7.0, O-o), Mops-KOH (pH 6.5-7.6, A-A) and Tris-HCl(pH 7.5-9.0,i-El).
Fig. 4. Effect of CaZ + on PI kinase, PIP kinase and DG kinase. Free Ca z+ concentrations were adjusted using EGTA-Ca 2+ buffer. Kca-EGTA used was l0 II [31]. All assays were normalized to 100% in the absence of CaC! 2.
Km for A TP and Vmax. The kinases showed Michaelis-Menten type saturation curves for ATP. Apparent K m values for ATP of PI kinase, PIP kinase and DG kinase were 0.68, 0.12 and 0.20 raM, and Vm.~x values were 1400, 190 and 12700 pmol A T P / m g protein per rain, respectively. DG kinase activity was much higher than the others. Effect of Ca: +. PI kinase and PIP kinase were inhibited by the physiological concentration of Ca 2+, i.e., their activities were decreased to 50~ between pCa 8 and 6. On the other hand, a Ca 2+ co,,centration approx. two orders of magnitude higher than the physiological concentration was required to inhibit DG kinase (Fig. 4). One should bear in mind that, if phospholipase C is activated by Ca 2+, the reaction products of PI kinase and PIP kinase would be hydrolyzed and the consequent decrease of PIP and PIP2 concentrations would lower the apparent activities of these kinases, in fact, phospholipase C was markedly activated by the physiological concentration of Ca :+ (see below). Disturbance of the kinase assays by phospholipase C can be prevented by inhibiting phospholipase C. Cruz-Rivera et al. [32] have reported that glycero-3-phospho-D-myo-inositol 4-phosphate is a specific inhibitor of phospholipase C from guinea-pig uteri, however, the inhibit)r had no effect on the tobacco enzyme. Thus, the inhibition of PI kinase and PIP kinase by Ca 2+ was re-examined in the presence of an excess amount of unlabeled reaction product in order to reduce hydrolysis of the labeled products by phospholipase C. In addition, the assays were done with 1 mM Mg 2+ instead of 10 mM to mimic the physiological condition. PI kinase and PIP kinase were also inhibited by a :ow concentration of Ca 2+ even in the presence of un-
76 1
e
|
I
i
|
,0t
400
pl hydrolysis
IP
IO0
v75
'
/I
,,
Fig, 5, Effect of Ca ~'+ . . . . . the presence oflmMMgCI2andl0~gofunlabeledPlP(Pikinase)orPlP2(PlP kinase), Free Ca" + concentrations were adjusted as described in Fig.
enzymes was greater under this assay condition: PI kinase in particular was inhibited 95% at a submicromolar concentration of Ca 2+. The addition of excess PIP or PIP2 to the reaction mixture did not inhibit the enzyme activities. These results indicate that both PI kinase and PIP kinase are really inhibited by the physiological concentration of Ca 2+. Effects of various compounds. We studied the effects of various compounds on the activities of kinases (Table Ill). R 59022 (50 #M), a specific inhibitor for DG kinase from human red blood cells [33], had no effect on DG kinase nor on Pl kinase and PIP kinase. A caimodulin inhibitor, compound 48/80 (0.02-0.1 mg/ml), was a strong inhibitor of Pl kinase and DG kinase, while it scarcely affected PIP kinase. Another calmodulin inhibitor, trifluoperazine (50 /~M), weakly inhibited PI kinase but not PIP kinase. Neither adenoTABLE III
Effectsof compounds on PI, PIP and D G kinases Com!~ounds
Control 50 ~tM R 59022 0,02 mg/ml compound 48/80 0.1 mg/ml compound 48/80 50 ItM trifluoperazine 5% ~tM adenosine 5O0 FM ADP 10 mM NaF 100 ~M GTPyS
Activities (~) P! kinase
PiP kinase DG kinase
100 99 70 29 82 93 97 68 95
100 72 97 87 107 94 90 53 107
100 85 54 21
100
6001
O 0
I
hydro
5
t ,~
" [1
10 ~ 15
20
25
1
30
Retention time (rain)
6.
Fig. Identification of the products of inositol phospholipid hydrolysis by HPLC as described m;der Material and Methods. The labeled products of hydlolysis were identified based on elution times of commercial standards; (A) Pl hydrolysis: (B) PIP hydroly~i~: {C) PI~ hydrolysis (each 10 rain incubation).
sine (500 #M) nor ADP (500 #M), which do inhibit some PI kinases [34], inhibited the tobacco PI kinase and PIP kinase. NaF (10 mM) which is reported to inhibit rat brain PI kinase and PIP kinase [35] inhibited the respective tobacco enzymes. GTPyS (100 #M) had no effect on PI kinase and PIP kinase.
Characterization of phospholipase C Analysis of reaction products. Inositol phosphates released from phosphoinositides were analyzed by HPLC to examine the presence in the plasma membrane of phospholipase C and D and phosphatases which hydrolyze inositol phosphates and inositol phospholipids (Fig. 6). The plasma membrane hydrolyzed exogenous PI, PIP and PIP2 and produced IP, IPa and IP3, respectively, indicating the presence of phospholipase C. When PIPa was incubated for more than 5 rain, an unknown peak appeared which eluted close to (but not co-eluting with) the IPa standard (Fig. 6C). This peak also appeared when [3H]IP3 was incubated with the plasma membrane (data not shown). These results suggest that the unknown peak is a dephosphorylated product of IP3 (inositol 1,5-bisphosphate or inositol 4,5-bisphosphate)
77 by IP3 phosphatase (IP3-4-phosphatase or IP3-1-phosphatase, respectively), or an unknown metabolized product of IP3. IP3-5-phosphatase of human platelets required Mg E+ [36], but we could not observe any activity in tobacco plasma membrane in the presence of Mg E+ (data not shown). The results above alternatively suggest that phospholipase D does not exist in the plasma membrane or it exists but does not hydrolyze phosphoinositides. Galliard [37] reported that phosphatidylinositol is apparently not a substrate for plant phospholipase D. IP2 was not produced when PIPE was substrate, indicating that phosphatase activity, which dephosphorylates PIP,, to PIP, did not exist or it was inactivated under the assay condition, because PIP was hydrolyzed to IP, by phospholipase C. To examine the contaminations of isomers of inositol phosphate in Each peak, all hydrolyzed products were reanalyzed by the more precise method of Dean and Moyer [38] using HPLC with a gentle gradient system which was established to separate inositoi phosphate isomers and all of them were free of contamination with other inositol phosphate isomers (data not shown). Hydrolysis of PI, PIP and PIP2 proceeded linearly for up to 5 min with each substrate (data not shown). The following experiments were carried out with [3HIPIP2 for 3 min. pH optimum. The pH optimum of phospholipase C was observed around 7.5 (Fig. 7). Substrate specificity. Apparent g m values of phospholipase C for Pl, PIP and PIP2 were 460, 107 and 97 #M and the Vmax values were 184, 108 a , d 102 nmol/mg protein per min, respectively.
I
I
I
I
I
I
60
80
!oo 2/" E
[..4o ! ,mr
20
t |
I
t
3
pCa
Fig. 8. Effect of Ca :+ on phospholipase C activity. [~H]PIP., was used as substrate. Various free Ca 2 + concentration were prepared as described in Fig. 4.
Effect of Ca -'+. The phospholipase C activity in the plasma membrane was markedly stimulated by Ca -'+ especially between pCa 8 and 6 (Fig. 8). Effect of GTP,/S. GTP binding protein is involved in the activation of phospholipase C by agonists in animal cells [39]. Recently, GTP binding protein was identified in the plasma membrane of higher plants [40]. The involvement of GTP binding protein in the activation of phospholipase C in tobacco plasma membrane was examined by adding GTP'yS to the reaction mixture. GTP~,S (100 #M) had no effect on the enzyme in the presence of Mg 2+ (1 or 10 mM). Discussk~n
.=_ E
~40 e
I
6
I
I
7
I
pH
I
8
I
I
9
Fig. 7. Effect of p H on phospholipase C activity using [3H]PIP2 as substrate. The buffers used are described in Fig. 3.
The properties of PI kinase and PIP kinase of the plasma membrane from tobacco suspension culture cells with respect to the time-course of the reaction and effects of Triton X-100 and pH on the activities are similar to those reported by Sommarin and Sandelius [17,18] with wheat plasma membrane. However, the affinity to ATP of the tobacco PI kinase is about 3-times lower than that of the wheat enzymes. Km (ATP) of the tobacco PI kinase is similar to that of type 3 kinase of bovine brain which was reported to be 0.74 mM [34]. Low sensitivity of the tobacco PI kinase to adenosine is also similar to the type 3 kinase. Inhibition of tobacco plasma membrane PI kinase and PIP kinase by a submicromolar range of Ca 2+ suggests that the two kinases are controlled by the change of cytoplasmic Ca 2+ concentration. Inhibitions of bovine PI kinase by a nanomolar range of Ca 2+ [41] have been reported and
Signal(lighlhormone,elicitor,etc.)
78
7", , ' ! I~eceyto.r L..-~G protein)
Plasmamembrane (
Pl
kinase ) - - ~ PiPklnese ~ ft~ ,PIP f ~ ~ , P I P 2 ~ D G
DGklnase ) f ~ .PA
Pl
ATP
AOP
ATP
(~%,. . . . . . . . . . . . . . .
AOP
~, ~ . . . . . . . . .
~
~_ i?, ATP
-2+
i[~At "--
.,P~
//'*
// I~'
r-_ . . . . .
AOP
"-I
I Protein I LklnaseC]
2+
Fig, 9. Regulation of PI t u r n o v e r by Ca 2 +. F o r details, see text.
pituitary PIP kinase by a micromolar range of Ca 2+ [42]. However, the latter was investigated using purified plasma membrane, without considering the activation of phospholipase C by Ca 2+. Compound 48/80 inhibited PI kinase, PIP kinase and DG kinase and trifluoperazine inhibited PI kinase, but it is not probable that they associate with calmodulin because these enzymes were inhibited by Ca 2+. Though PI kinase and PIP kinase had very similar biochemical properties, they showed different behavior to compound 48/80 suggesting that they are different_ enzymes. The existence of isozymes of respective kinases can be assumed. DO kinase has been solubilized and purified from membrane fractions of Catharanthus roseus suspension culture cells and characterized [23]. The characteristics of tobacco DG kinase, such as optimal pH, apparent Km (ATP), stimulation by Mg 2+ and inhibition by Ca 2+, are similar to those of C. roseus enzyme. Since the effective concentration of Ca 2+ was higher than 10 /~M, DG kinase may not be controlled by cytoplasmic Ca 2+. DG kinase activity was so high that most of Pi incorporated from ATP into plasma membrane lipids was PA. This occurred without exogenous substrate, indicating that DG may be accumulated during the isolation of the plasma membrane. Though it is unknown whether the DO level is also high in vivo, it is likely that DG kinase may function to keep the DG concentration at a low level. Phospholipase C of tobacco plasma membrane hydrolyzed three inositol phospholipids. Phosphoinositide specific phospholipase C has been reported in the plasma membranes of soybean [19], wheat [20] and Arena [22]. Phospholipase C from tobacco as well as from soybean
and wheat preferentially hydrolyzed PIP and PIP2 more than PI. The tobacco enzyme has a very similar specificity to PIP and PIP2, while soybean and wheat enzymes had different specificities to PIP and PIP2. The tobacco phospholipase C was stimulated by a low concentration of Ca 2+. Phospholipase C from various higher plants req,.,.ired Ca 2+, among them only a few [20,22,23] were stimulated by a micromolar range of Ca 2+. Stimulation by guanine nucleotides of inositol phosphate release from membrane isolated from Acer culture cells has been reported [43]. Zbell and Walter [8] observed that the stimulation of phosphoinositide breakdown by auxin plus GTPyS occurred in microsomal membrane of carrot suspension culture cells. However, many investigators failed to observe guanine nucleotide stimulation of inositol phospholipid breakdown using purified plasma membrane. GTPTS was also ineffective on the tobacco membrane. We observed that IP3 was metabolized by the plasma membrane. Joseph et al. [44] reported that a soluble fraction prepared from tobacco suspension culture cells hydrolyzed inositol phosphates and its activity was stimulated by 1.8/~M Ca 2+. It hydrolyzed IP3 to form inositol 1,4-bisphosphate and inositol 4,5-bisphosphate. Fig. 9 schematically represents regulation of PI turnover by Ca 2+. Cytoplasmic C a 2+ concentration is kept at the submicromolar range or lower [45]. The maintenance of this low level is due to sequestration of Ca 2+ into the intracellular pool and extrusion of Ca 2+ through the plasma membrane by the Ca2+-pump and Ca2+/H + antiporter [27]. Coupled to signals, phospholipase C hydrolyzes PIP2 and produces IP.~ which induces Ca 2+ mobilization from vacuole (and endo-
79 plasmic reticulum?) raising the Ca 2+ concentration in the cytoplasm. Ca 2+ in turn activates various Ca2+-de pendent enzymes, especially phospholipase C itself. Consequently, more IP3 is synthesized and causes an increase of cytosolic Ca 2+. On the other hand, PI kinase and PIP kinase are inhibited by Ca = ~ and less PIP2 is synthesized. Thus activated phospholipase C is limited in its substrate and produces less IP3. In this way Ca 2+ works as a feedback inhibitor for regulation of cytoplasmic Ca 2+ concentration via PI turnover. This scheme might give a plausible explanation for the oscillation in the increase of internal Ca 2+ found, for example, in the guard cell [46]. To adapt this scheme to in vivo conditions, we are currently investigating PI turnover in the intact cells.
Acknowledgements We thank Prof. T. Kuroiwa and Prof. T. Nagata of the Department of Botany, University of Tokyo for the generous gift of the strain of tobacco suspension culture cell. We also thank Dr. N. Sato of the Department of Botany, University of Tokyo for his valuable advice on the analytical method of phospholipids, respectively. This work was supported by a Grant-in-Aid for General Scientific Research 01480010 from the Japanease Ministry of Education, Science and Culture.
References 1 Boss, W.F. and Massel, M.O. (1985) Biochem. Biophys. Res, Commun. 132, 1018-1023. 2 Wheeler, J.J. and Boss, W.F. (1987) Plant Physiol. 85, 389-392. 3 Heim, S., Bauleke, A, Wylegalla, C. and Wagner, K.G. (1987) Plant Sci. 49, 159-165. 4 Irvine, R.F., Letcher, AJ., Lander, D.J, Drobak, B.K., Dawson, A.P. and Musgrave, A. (1989) Plant Physiol. 89, 888-892, 5 Cot6, G.G., DePass, A.L., Quarmby, L.M., Tare, B.F., Morse, M.J., Satter, R.L. and Crain, R.C. (1989) Plant Physiol. 90, 14221428. 6 Morse, M.J., Crain, R.C. and Satter, R.L. (1987) Proc. Natl. Acad. Sci. USA 84, 7075-7078. 7 Ettlinger, C. and Lehle, L. (1988) Nature 331, 176-178. 8 Zbell, B. and Walter, C. (1987) in Plant Hormone Recepters (Kliimt, D., ed.), pp. 141-153, Springer-Verlag, Berlin. 9 Heim, S. and Wagner, K.G. (1986) Biochem. Biophys. Res. Commun, 134, 1175-1181. 10 Heim, S. and Wagner, K.G. (1989) Plant Sci. 63, 159-165. 11 DrobaL B.K. and Ferguson, I.B. (1985) Biochem. Biophys. Res. Commun. 130, 1241-1246. 12 geddy, A.S.N. and Poovaiah, B.W. (1987) J. Biochem. (Tokyo) 101, 569-573. 13 Schumaker, K.S. and Sze, H. (1987) J. Biol. Chem. 262, 3944-3946.
14 Ranjeva, R., Carrasco, A. and Boudet, A.M. (1988) FEBS Lett. 230, 137-141. 15 Alexandre, J., Lassalles, J.P. and Kado, R.T. (1990) Nature 343, 567-570. 16 Rinc6n, M., Chen, Q. and Boss, W.F. (1989) Plant Physiol. 89, 126-132. 17 Sandelius, A.S. and Sommarin, M. (1986) FEBS Lett. 201,282-286. 18 Sommarin, M. and Sandelius, A.S. (1988) Biochim. Biophys. Acta 958, 268-278. 19 Pfaffmann, H., Hartmann, E., Brightman, A.O. and Morre, D.J. (1987) Plant Physiol. 85, 1151-1155. 20 Melin, P.-M., Sommarin, M., Sandelius, A.S. and Jergil, B. (1987) FEBS Lett. 223, 87-91. 21 McMurray, W.C. and Irvine, R.F. (1.988) Biochem. J. 249, 877881. 22 Tate, B.F., Schaller, G.E., Sussman, M.R. and Crain, R.C. (1989) Plant Physiol. 91, 1275-1279. 23 Biffen, M. and Hanke, D.E. (1990) Plant Sci. 69, 147-155. 24 Wissing, J., Heim, S. and Wagner, K.G. (1989) Plant Physiol. 90, 1546-1551. 25 Kato, K., ,qhiozawa, Y., Yamada, A., Nishida, K, and Noguchi. M. (1972) Agr. Biol. Chem. 36, 899-902. 26 Nemoto, Y., Kawano, S., Nakamura, S., Mita, T., Nagata, T. and Kuroiwa, T. (1988) Plant Cell Physiol. 29, 167-177. 27 Kasai, M. and Muto, S. (1990) J. Membr, Biol. 114, 133-142. 28 Yoshida, S., Kawata, T., Uemura, M. and Niki, T. (1986) Plant Physiol. 80, 152-160. 29 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 30 Dittmer, C. and [,ester, L. (1964) J. Lipid Res. 5, 126-127. 31 Raaflaub, J. (1960) Methods Biochem. Anal. 3, 301-325. 32 Cruz-Rivera, M., Bennett, C.F. and Crooke, S.T. (1990) Biochim. Biophys. Acta 1042, 113-118. 33 De Chaffoy de Courcelles, D., Roevens, P. and Van Belle, H. (1985) J. Biol. Chem. 260, 15762-15770. 34 Endemann, G., Dunn, S.N. and Cantley, L.C. (1987) Biochemistry 26, 6845-6852. 35 Claro, E., Wallace, M.A. and Fain, J.N. (1990) Biochem. J. 268, 733-737. 36 Connolly, T.M., Bross, T.E. and Majerus, P.W. (1985) J. Biol. Chem. 260, 7868-7874. 37 Galliard, T. (1980) in The Biochemistry of Plants (Stumpf, P,K., ed.), Vol. 4, pp. 85-116, Academic Press, New York. 38 Dean, N.M. and Moyer, J.D. (1987) Biochem. J. 242, 361-366. 39 Gilman, A.G. (1987) Annu. Rev. Biochem. 56, 615-649. 40 Blum, W., Hinsch, K.-D., Schultz, G. and Weiler, E.W. (1988) Biochem. Biophys. Res. Commun. 156, 954-959, 41 Husebye, E.S., Letcher, A.J., Lander, D.J. and Flatmark, T. (1990) Biochim. Biophys. Acta 1042, 330-337. 42 lmai, A., Rebecchi, M.J. and Gershengorn, M.C. (1986) Biochem. J. 240, 341-348. 43 Dillenschneider, M, Hetherington, A., Graziana, A., Aliberi, G., Berta, P, Haiech, J. and Ranjeva, R. (1986) FEBS Lett. 208. 413-417. 44 Joseph, S.K., Esch, T. and Bonner, Jr., W.D. (1989) Biochem. J. 264, 851-856. 45 Gilroy, S., l[ughes, W.A. and Trewavas, A.J. (1986) FEBS Lett. 199, 217-221. 46 McAinsh, M.R., Brownlee, C. and Hetherington, A.M. (1990) Nature 343, 186-188.
