Inhibition and activation of wheat embryo calcium-dependent protein kinase and inhibition of avian myosin light chain kinase by long chain aliphatic amphiphiles

Plant Science, 78 (1991) 165-175

165

Elsevier Scientific Publishers Ireland Ltd.

Inhibition and activation of wheat embryo calcium-dependent protein kinase and inhibition of avian myosin light chain kinase by long chain aliphatic amphiphiles W a n i d a Jinsart a'b, Bela Ternai a and Gideon M. Polya b aDepartment of Chemistry and hDepartment of Biochemistry, La Trobe University, Bundoora, Victoria 3083 (Australia) (Received April 9th.1991; accepted May 24th, 1991)

Wheat embryo~CaZ+-dependent protein kinase (CDPK) is inhibited by a variety of long chain amphiphilic compounds that also inhibit avian myosin light chain kinase (MLCK), namely by straight chain alkylamines, alkyltrimethylammonium halides and N-alkylN,N-dimethyl-3-ammonio-l-propane-sulfonates. These compounds also interact with calmodulin as assessed from enhancement of Ca2+-dependent dansyl-calmodulin fluorescence. A further common feature of these inhibitors is a greatly decreased inhibitory effectiveness at alkyl carbon chain length _< 12. Long chain acylcholines with acyl carbon chain length greater than 12 inhibit wheat germ CDPK but long chain acyl carnitines are ineffective. Acylcarnitines activate CDPK in the presence and absence of Ca 2+. DSphingosine and dihydrosphingosine inhibit both avian MLCK and plant CDPK. Sodium alkysulphates at about 10-4 M inhibit MLCK but not plant CDPK. Dihydrosphingosine and sodium alkylsulphates activate wheat germ CDPK in the presence and absence of Ca 2+.

Key words." calcium; protein kinase; amphiphiles

Introduction

Calmodulin and Ca2+-dependent protein kinase (CDPK) are involved in Ca2+-mediated transduction of external signals in plant cells [1] as in other eukaryotic systems [2]. While Ca2+-calmodufin activates a variety of plant enzymes a Ca2+-calmodulin-dependent protein kinase has not yet been resolved from plants [3]. Plant CDPKs are dependent on micromolar Ca 2+ concentration for activity [1,4-7]. While some partially purified plant CDPK preparations are activated by phospholipids (e.g. see [8]), extensively purified CDPKs from wheat germ [9] and soybean [7] are not lipid activated. However various plant CDPKs

Correspondence to: G.M. Polya, Department of Biochemistry, La Trobe University, Bundoora, Victoria 3083, Australia. Ca2+-dependent protein kinase; DMSO, dimethylsulphoxide; MLC, myosin light chain; MLCK, myosin light chain kinase; Zwittergent 3-16, Nhexadecyl-N,N-dimethyl-3-ammonio- l-propane-sulphonate.

Abbreviations." CDPK,

can be activated by fatty acids in the absence of Ca 2÷ [9-11], a property also exhibited by animal Ca 2÷- and phospholipid-dependent protein kinase C [12,131. Plant CDPKs are inhibited by a variety of calmodulin antagonists notably by phenothiazine derivatives, calmidazolium [14,15] and the naphthalene sulphonamide W7 [7]. Animal protein kinase C is also inhibited by phenothiazine calmodulin antagonists [16-18], calmidazolium [17,191 and by W7 [20]. Wheat germ CDPK is inhibited by a number of basic polypeptides that also bind to calmodulin [3]. The calmodulin-binding polypeptide melittin inhibits plant CDPKs [3] and also inhibits animal protein kinase C [20,211. Thus plant CDPKs and protein kinase C appear to possess domains that are calmodulin-like in the sense that these enzymes bind non-peptide and polypeptide calmodulin antagonists [3]. In addition plant CDPKs are inhibited by a variety of fatty acids [9-11,15] and other amphiphilic compounds [3,22].

0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland ltd. Printed and Published in Ireland

166 In the present paper, we have sought to define the structural requirements for such inhibition of plant CDPKs by amphiphilic compounds. In order to delineate possible mechanisms of inhibition we have also examined the effects of such compounds on avian calmodulin-dependent myosin light chain kinase and on the Ca2+-dependent fluorescence of dansyl-calmodulin. In addition we also describe the Ca 2+ independent activation of CDPK by certain amphiphilic compounds. Materials and Methods

Purification of CDPK, calmodulin and MLCK Wheat germ CDPK was purified as described previously [9]. In this protocol, calmodulin is separated from CDPK by chromatography on Cibacron F3GA-Sepharose CL-6B, CDPK (but not calmodulin) binding to this matrix at pH 8.0. The calmodulin-containing filtrate was bound to 200 ml bed volume DEAE-Sephacel in 50 mM Tris (CI-, pH 8.0)/10 mM 2-mercaptoethanol (buffer A) and eluted in 0.5 M NaC1/buffer A. The eluate was concentrated by pressure filtration (Amicon YMI0 membrane) and chromatographed on an Ultrogel AcA44 column (7 cm 2 x 45 cm) in 0.2 M NaC1/buffer A. The calmodulin-containing fractions were pooled, heated to 85°/5 min and the solution clarified by centrifugation at 45 000 g/15 min. The calmodulin preparations were homogeneous on the basis of SDS-PAGE conducted in the presence of 0.1 mM CaCI2 or of 1.0 mM EGTA, yielding apparent molecular sizes of 14 or 18.1 kDa, respectively, as previously reported for wheat germ caimodulin [23]. Myosin light chain kinase was purified from freshly obtained chicken gizzards following the method of Walsh et al. [24] and stored at -70°C. Protein kinase determination Wheat germ CDPK was routinely assayed at 30°C in a reaction medium (125 gl), containing 50 mM Tris (CI-, pH 8.0), 8 mM MgC12, 8 mM dithiothreitol, 0.2 mM EGTA, 0.8 mM CaCI2, 40 mM NaCI, 2 mM 2-mercaptoethanol, 0.8 mg/ml lysine-rich histone III-S preparation, protein kinase and 24 ~tM ATP (specific activity of [3,-32p]ATP about 30 Ci/mol). Where specified the

protein substrate was MLC-derived synthetic peptide substrate (25 gM final). Chicken gizzard myosin light chain kinase was assayed at 30°C in a reaction mixture (125 #1) containing 6.4 mM Hepes (Na +, pH 7.0), 0.8 mM Mg-acetate, 0.1 mM CaCI2, 0.16 mg/ml bovine serum albumin, 0.02% Tween-80, 0.16 I~M calmodulin, 20 t~M MLC-derived synthetic peptide substrate (KKRAARATSNVFA-NH2), 0.4 mM potassium phosphate, 10 mM NaC1, 0.04 mM dithiothreitoi, MLCK and 20 /~M ATP (specific activity of [7-32p] ATP about 30 Ci/mol). Aliquots of 80/A of reaction mixture were applied to 4 cm 2 squares of phosphocellulose paper (Whatman P81) which was subsequently washed with 500 ml 75 mM H3PO4 (four times) and twice in absolute ethanol before drying and Cerenkov counting. In studies of protein kinase inhibition, inhibitors were added dissolved in DMSO (to give 16% v/v DMSO final concentration), and protein kinase rates were expressed as a percentage of the control rate (no inhibitor included). Protein kinase rates were routinely determined in duplicate with control rates being routinely determined in quadruplicate. The standard deviations associated with control protein kinase assays were about 5-10% of means.

Dansyl-calmodulin synthesis and flourescence measuremen ts Calmodulin was dansylated [25], dialyzed for 3 days against 2 1 of 20 mM Tris (CI-, pH 8.0)/0.25 M NaCI/5 mM MgCI2 and then dialyzed against 2 1 distilled water for 2 days. Dansyl-calmodulin fluorescence emission spectra were obtained at 30°C employing an Hitachi 650-10S fluorescence spectrophotometer (excitation slit width 5 nm and emission slit width 10 nm; excitation X 340 nm). Dansyl-calmodulin concentration was 0,4-10 gM in 10 mM Tris (CI-, pH 8.0)/1 mM EGTA or 10 mM Tris (CI-, pH 8,0)/1 mM EGTA/1 mM CaCI2. Materials Raw wheat (Triticum aestivum) germ was obtained locally. [7-32p] ATP was obtained from Bresa, Adelaide, Australia. Calf thymus histone preparation Ill-S, saturated n-alkylamines, alkylcholines, alkylcarnitines and Other alkyl-N-

167

trimethylammonium compounds were obtained from the Sigma Chemical Co., St. Louis, MO. Smooth muscle myosin light chain kinase synthetic peptide substrate (KKRAARATSNVFANH2) was obtained from Auspep, Melbourne, Australia. Oleylamine and alkylsulphates were obtained from Aldrich and zwittergents from Calbiochem.

100

80

§ 60

~ 4o 20

Results and Discussion

v

Inhibition of MLCK by long chain alkylamines and related compounds Long chain alkylamines inhibit avian MLCK (Fig. 1, Table I). The most effective inhibitors (IC50 values 6-19 /~M) are C13-C18 long chain alkylamines (compounds 8-13). There is a marked discontinuity in inhibitory effectiveness between these compounds and alkylamines of lower chain length. Thus, the IC50 values for tridecylamine (8), dodecylamine (5) and decylamine (4) are 19, 83 and 200 /zM respectively and n-octylamine (2) is not inhibitory at 160 /~M (Table I). The marked discontinuity between dodecylamine (Cl2) and

50 100 [n-Alkylamine]

v

150 (~M)

-

200

Fig. I. Inhibition of avian MLCK by n-alkylamines. Avian MLCK was assayed in the standard assay containing 16% (v/v) DMSO and increasing concentrations of n-alkylamines. MLCK is expressed as % of control (no added inhibitor). II, ndecylamine; El, n-dodecylamine: A, n-tridecylamine; A, nhexadecylamine; O, n-tetradecylamine; O, n-stearylamine (octadecylamine).

tridecylamine (C13) is noteworthy suggesting that complete occupancy of a Ci3-accommodation groove or cleft on either calmodulin or MLCK is required for maximal binding. In support of this

Table I.

Inhibition of wheat germ CDPK and avian MLCK by straight chain alkylamines. Avian MLCK and wheat germ CDPK were assayed in the standard assay systems with 25 ~,M MLC peptide and 0.8 mg/ml histone III-S as substrates, respectively, in the presence or absence of increasing concentrations of straight chain alkylamines. Alkylamines were added dissolved in DMSO ( 16% (v/v) DMSO final concentration). Concentrations for 50% inhibition (IC5o values) were determined by interpolation from plots of protein kinase activity versus inhibitor concentration. Protein kinase activity in the presence of 160 #M inhibitor is shown in parentheses as a percentage of the control rate activity (no inhibitor added) in those cases in which little inhibitory activity was observed. Compound

1 2 3 4 5 6 7 8 9 10 I1 12 13

Hexylamine Octylamine Dioctylamine Decylamine Dodecylamine Cyclododecylamine 1,12-Diaminododecane Tridecylamine Tetradecylamine Hexadecylamine Octadecylamine N-Methyloctadecylamine Oleylamine

Structure

C6HI3NH 2 CsHI7NH 2 (CsHIT)2NH CIoH21NH 2 CI2H25NH2 CI2H24NH NH2C~zH24NH 2 CI3H27NH 2 CI4H29NH 2 CI6H33NH 2 CIsH37NH 2 C18H37NHCH 3 CIsH35NH 2

IC50 (I~M) (or % control) MLCK

CDPK

(99) (90) 55 200 83 (93) 63 19 12 16 11 10 6

(103) ( 111 ) 40 125 140 (92) (80) 40 40 58 50 40 5

Table !I. Inhibition and activation of wheat germ CDPK and inhibition of avian MLCK by long chain amphiphiles and related compounds. Wheat germ CDPK and avian MLCK were assayed in the standard assay systems with 0.8 mg/ml histone Ill-S and 25 #M MLC peptide as substrates, respectively, in the presence or absence of increasing concentrations of inhibitors, lnhibitors were added dissolved in DMSO (16% (v/v) DMSO final concentration). Concentrations for 50% inhibition (IC50 values) were determined by the same method as for the compounds in Table I. Compound

IC50 (/~M) (or % control) MLCK

CDPK

(A) 14

D-Sphingosine

6

10

15

erythro- and threo-Dihydrosphingosine

8

10

Sodium dodecylsulphate Sodium tetradecylsulphate Sodium octadecylsulphate

49 38 43

(115) (115) (231)

Decamethonium bromide

(82)

(B) 16 17 18

(c) 19

(60% at 80 ~M)

(N,N'-decamethylenebis(trimethylammonium) dibromide) 20 21 22 23 24 25

Dodecyltrimethylammonium bromide Tetradecyltrimethylammonium bromide Hexadecyltrimethylammonium bromide I-Hexadecylpyridinium bromide Dimethyldioctadecylammonium bromide Trigonelline

78 11 1I 49 8 (100)

113 13 12 16 20 (100)

(D) 26

27

28

Merocyanine dye (Clo (4-(2-( I -hexadecyl- 1,4-dihydropyridin-4-ylidene) ethylidene) cyclohexa-2,5-dien- l-one) Merocyanine dye (CH3) (4-(2-(l-methyl-l,4-dihydropyridin-4-ylidene) ethylidene) cyclohexa-2,5-dien-1-one) Merocyanine dye (CH3) iodide

40

(103)

(125)

Zwittergent Zwittergent Zwittergent Zwittergent Zwittergent

(123) (97) (98) 78 110

(130) (113) (150) 175 175

(86% at 200/zM) 120 20 14 13

(114% at 200 #M) (105) 65 34 14

(85)

12

(132"/,, at 80 /~M)

(E) 29 30 31 32 33

3-8 (C8) 3-10 (C10) 3-12 (Ci2) 3-14 (C14) 3-16 (C~6)

(F) 34 35 36 37 38

Succinylcholine chloride Lauroylcholine iodide Myristoylcholine iodide Palmitoylcholine iodide Stearoylcholine iodide

(G) 39 40 41 42 43

DL-Hexanoylcarnitine chloride DL-Octanoylcarnitine chloride Myristoy[carnitine chloride Palmitoylcarnitine chloride Stearoylcarnitine chloride

(97) (89) (59) (61) (85)

(125) (113) (120) (187% at 200#M) (188)

169

model, dioctylamine (3) is much more effective than octylamine (2) as a MLCK inhibitor. NMethylation of octadecylamine (11) to yield Nmethyloctadecylamine (12) does not impair inhibitory activity and 1,12-diaminododecane (7) is as effective as dodecylamine (5). We conclude that the minimum requirement for potent inhibition of MLCK by compounds of this kind is a positively charged group and an alkyl chain of C13 or greater length. This generality has been further explored employing other long chain aliphatic amphiphiles (Table II). D-Sphingosine (14) and DL-dihydrosphingosine (15) are potent inhibitors of MLCK (IC50 values 6/~M and 8/zM respectively) (Table II). Sodium dodecylsulphate (16), sodium tetradecylsulphate (17) and sodium octadecylsulphate (18) also inhibit MLCK (IC50 values 49, 38 and 43 ttM, respectively) (Table II). A series of quaternary alkyl-ammonium compounds are potent inhibitors of MLCK (Table II). Dodecyltrimethylammonium bromide (20) (IC50 78 #M) is much less effective than tetradecyltrimethylammonium bromide (21) (IC50 11 #M) or hexadecyltrimethylammonium bromide (22) (IC50 11 /~M) (Table II) in agreement with the greatly reduced inhibitory effectiveness of straight chain alkylamines with chain lengths of Cl2 or less (cf. Table I). 1-Hexadecylpyridinium bromide (cetylpyridinium bromide) (23) is a poorer inhibitor (IC50 49 /~M) than hexadecyltrimethylammonium bromide (22) (IC50 I1/~M), suggesting that enhanced delocalization of the positive charge by the aromatic system (as opposed to aliphatic substituents) impairs inhibitory effectiveness. Indeed a range of zwitterionic aliphatic amphiphiles (Zwittergents 3-8 to 3-16) (29-33) are either not inhibitory or are relatively poor inhibitors (Table II), presumably through diminution of the positive charge on the tetraalkyl ammonium group. Trigonellin (N-methyl-3-carboxylate) (25) is not inhibitory, presumably due to the absence of an alkyl chain of suitable length (Table II). A Ci6 alkylated merocyanine dye (26) inhibits MLCK (IC50 40 #M) but analogues lacking a long alkyl chain (27,28) are not inhibitory (Table II). Zwittergent 3-16 (N-hexadecyl-N,N-dimethyl-

3-ammonio-l-propane-sulphonate) (33) and the tetradecyl analogue Zwittergent 3-14 (32) inhibit MLCK (IC50 values 110 and 78/~M, respectively) but lower chain length ( _ C12) analogues (29-31) are ineffective (Table II) as found in the straight chain alkylamine series (Table I) and the tetraalkyl ammonium bromide series (Table IIC). Succinylcholine (34) and lauroylcholine (35) are very poor inhibitors of MLCK compared to myristoylcholine (36), palmitoylcholine (37) and stearoylcholine (38) (Fig. 2, Table II) in agreement with the C14 to C~2 discontinuity in inhibitory effectiveness previously found with the Zwittergents, alkylammonium bromides (Table II) and straight chain alkylamines (Table I). The acylcarnitines, zwitterionic analogues of the acylcholines, are poor inhibitors of MLCK (Table IIG), in agreement with the low inhibitory effectiveness of the zwitterionic Zwittergents (Table liE) as compared to the tetralkylammonium bromides (Table IIC).

Mechanism of inhibition of MLCK by long chain aklylamines Long chain ( C 1 2 - C I 6 ) alkyltrimethylammonium bromides inhibit calmodulin-activated cyclic nucleotide phosphodiesterase [26]. Cetyltrimethylammonium bromide while not inhibiting basal (calmodulin unactivated) activity, can (in conjunction with an alkyl sulphate) mimic calmodulin in

100

8C 6C 0

o...1 I~ 20 0"

20

40

60 80 [Acylcholine]

100 (pM)

120

140

160

Fig. 2. Inhibition of avian MLCK by acylcholine iodides. Avian MLCK was assayed in the standard assay containing 16% (v/v) DMSO and increasing concentrations of acylcholine iodides. MLCK is expressed as % control (no added inhibitor). &, lauroylcholine iodide; •, myristoylcholine iodide; O, palmitoylcholine iodide; O, stearoylcholine iodide.

170

activating phosphodiesterase and interacts directly with calmodulin as determined from analysis of fluorescence emission spectral changes of dansylcalmodulin [26]. In contrast, sodium tetradecylsulphate does not alter the dansyi-calmodulin emission spectrum, implying that it does not bind specifically to calmodulin [26]. In the present study, changes in dansylcalmodulin fluorescence have indicated direct interaction of long chain alkylamines with calmodulin. Thus 0.6 t~M N-methyl-n-octadecylamine enhances the Ca2+-dependent fluorescence emission of dansyl-calmodulin (Fig. 3A). Dodecyl trimethylammonium bromide (25 tzM), stearoylcholine (0.6 ~M) and stearoylcarnitine (2.5 /~M) also enhance dansyl-calmodulin fluorescence (Fig. 3B-D). Zwittergents have been previously shown to enhance dansyl-calmodulin fluorescence [26]. It is therefore likely that the straight chain alkylamines, alkyl trimethylammonium bromides and zwittergents are inhibiting MLCK through interaction with calmodulin. Inhibition of wheat germ CDPK by long chain amphiphiles Long straight chain alkylamines inhibit wheat germ CDPK-catalysed phosphorylation of III-S (Table I). As found with avian MLCK, oleylamine (13) is the most effective inhibitor (IC50 5 /zM) (Table I), there is a discontinuity with respect to inhibitory effectiveness between tridecylamine (8) (IC50 40/~M) and dodecylamine (5) (IC50 140 ~M) and while octylamine (2) is not inhibitory at 160 /~M, dioctylamine (3) is inhibitory (IC50 55 and 40 /~M for MLCK and CDPK, respectively) (Table I). Other long chain amphiphiles inhibit plant CDPK. Thus, long chain fatty acids inhibit wheat and silver beet CDPKs [22] and oat leaf CDPK [15] and decreasing chain length decreases inhibitory effectiveness as found with long chain alkylamines (Table I). With long chain fatty acids, a discontinuity with respect to inhibition of wheat germ CDPK occurs between myristic acid (Cl4; IC50 1 mM) and tridecanoic acid (not inhibitory at 1 mM) [3]. A similar discontinuity occurs with respect to inhibition of wheat leaf CDPK and silver beet leaf CDPKs I and II [3]. With oat leaf CDPK (which is inhibited by long chain saturated

and unsaturated fatty acids), decreasing chain length also decreases inhibitory effectiveness with a marked discontinuity occurring between myristic (Cl4) and lauric (Cl3) acids (concentrations for half maximal inhibition 40 and > 2000 t~M respectively) [15]. Long chain alcohols show a different pattern of inhibition of wheat germ CDPK and of other plant CDPKs. C8-Cll chain length aliphatic alcohols are inhibitory but long chain alcohols with carbon chain length number greater than 11 are ineffective [22]. 1-Nonanol, l-decanol and 9-decen-l-ol (IC50 values 4 mM, 3 mM and 3 mM respectively) are the most effective long chain alcohol inhibitors of wheat germ CDPK, other shorter and longer chain length alcohols being markedly ineffective in comparison [22]. It is noteworthy that decylamine (CI0; IC50 125 #M) is an inhibitor of wheat germ CDPK whereas n-octylamine (C8) is not inhibitory at 160 t~M (Table I). Decylamine and the C9-CI0 aliphatic alcohols evidently satisfy the inhibitory motif in which C9-CI0 amphiphiles (excluding long chain fatty acids) are inhibitors of CDPK. Small chain length amino alcohols 5-amino-l-pentanol and 6-amino-l-hexanol (both at 160 I~M) are not inhibitory (< 20% inhibition) and neither are 2-amino-heptane (200/~M) and/3phenylethylamine (160 #M). As found with MLCK, aIkyltrimethylammonium compounds are potent inhibitors of CDPK (Table IIC). As found with the straight chain alkylamines there is a discontinuity in inhibitory effectiveness between the CI4 (ICs0 13 tzM) and Cl2 (IC50 113 /~M) analogues (Table IIC). Zwittergents 3-16 and 3-14 are also inhibitors of wheat germ CDPK (Table liE) and a discontinuity in inhibitory effectiveness is observed between the C14analogue (IC50 175/~M) and the Cl2 analogue (not inhibitory at 160/zM) (Table IIE). A variety of long chain acylcholine halides also inhibit wheat germ CDPK (Fig. 4, Table IIF), again with a discontinuity in inhibitory effectiveness occurring between myristoylcholine iodide (Cl4; IC50 65 #M) and lauroylcholine iodide (C12; no inhibition at 160 t~M) (Fig. 4, Table IIF). Long chain acylcholine halides may well be acting in the same fashion as the analogous alkyltrimethylammonium halides (Table IIC).

171 5O

70

[A]

[B] (1)

60

(I}

40 50

E

" 30

40

E

=o

8{o 30 o i";-

o

(4)

20

o=

20.

IT

10.

0 390

40

s4o 5~o

630

0 --

~,(nm)

5O

fi0

40" [C]

450

390

570

630

570

630

[D]

,0]

(1)

510 X(nm)

(I)

" 30. E

8

8 20"

=o

,7

E

10,

\\\\ 390

450

510 X(nm)

570

630

0 390

450

510 ~(nm)

Fig. 3. Effect of long chain amphiphiles on fluorescence emission of dansyl calmodulin. Fluorescence emission spetra of dansylcalmodulin in 20 m M Tris (CI-, pH 8.0)/1 m M E G T A were obtained at 30°C as described in Materials and Methods in the absence of Ca 2÷ (4 m M E G T A present) or the presence of Ca 2÷ (1 m M E G T A and 1 m M CaCI 2 present). (A) 0.5 #M dansylcalmodulin present. (1) and (2), 0.6 t~M N-Me-n-octadecylamine in the presence (1) and absence (2) of Ca2+; (3) and (4), no added inhibitor in the presence (3) and absence (4) of Ca2+; (5) no dansylcalmoduli:n. (B) 0.25 #M dansylcalmodulin present. (1) and (2), 25 #M dodecyltrimethyl-ammonium bromide in the presence (1) or absence (2) of Ca2+; (3) and (4), no added inhibitor in the presence (3) or absence (4) of Ca 2+. (C) 0.4/zM dansylcalmodulin present. (1) and (2), 0.6 #M stearoylcholine in the presence (1) or absence (2) of Ca2+; (3) and (4), no inhibitor added in the presence (3) or absence (4) of Ca2+; (5), no dansyl-calmodulin but 1 m M CaCI 2 or 1 m M CACI2/0.6 #M stearoylcholine present. (D) 0.4 I~M dansylcalmodulin present. (1) and (2), 2.5 I~M stearoylcarnitine in the presence (1) or absence (2) of Ca2+; (3) and (4), no inhibitor added in the presence (3) or absence (4) of Ca2+; (5), no dansyl calmodulin added and 1 m M CaCI 2 present.

172 120 100 ~" 80 E o

o~ 6o h~ n

40" 20' 0

50

100 [Acylcholine] (~tM)

150

200

Fig 4. Inhibition of wheat germ CDPK by acylcholines. CDPK was assayed with 0.8 mg/ml III-S histone as protein substrate in the standard assay containing 16'¼,(v/v) DMSO and increasing concentrations of acylcholine iodides• CDPK activity is expressed as % of control (no added inhibitor). O, stearoylcholine iodide (C18); O, palmitoylcholine iodide (C16); Zk, myristoylcholine iodide (Ci4); A lauroylcholine iodide (C12).

The zwitterionic long chain acylcarnitine chlorides are not inhibitors of wheat germ C D P K . Indeed palmitoylcarnitine chloride and stearoylcarnitine chloride both stimulate wheat germ C D P K in the presence of Ca 2+ (Fig. 5, Table 200 180 160

~ c o

Inhibition and activation of wheat germ CDPK by sphingosine and other long chain amphiphiles

140 120.

0

100: 20:

0

IIG). Palmitoylcarnitine (about 100 I~M) elicits significant C D P K activity in the absence of Ca 2+ (Fig. 5). Palmitoylcarnitine inhibits both protein kinase C (ICs0 8 - 4 0 I~M) [17,27-29] and CaZ+-calmodulin activated protein kinase (ICs0 2 3 - 4 5 # M ) [27]. As discussed above in relation to inhibition of M L C K , long chain alkyl amphiphiles, namely long chain alkylamines, a l k y l t r i m e t h y l a m m o n i u m halides and zwittergents can bind to calmodulin. Stearoylcholine (0.6 I~M) can bind to calmodulin as determined from enhancement of Ca2+-dep endent dansyl-calmodulin fluorescence (Fig. 3C). Given the evidence for a domain on plant C D P K that is calmodulin-like in the sense of binding nonpeptide [14] and polypeptide [3] calmodulin antagonists, it seems likely that these long alkyl chain amphiphiles inhibit plant C D P K by binding to such a calmodulin-like site. The inhibition of plant C D P K by long chain acylcholines raises an interesting possibility in relation to the occurrence in plants of choline acetyl transferase and acetyl cholinesterase [30]. While evidence for an acetylcholine-based signalling system in plants is equivocal [30], the choline acyltransferases and acylcholinesterases present in plants could conceivably be involved in the synthesis and degradation, respectively, of long chain fatty acylcholine inhibitors of signal-mediating plant C D P K s .

50

100

150

[Acylcarnitine] (~M)

Fig. 5. Activation of wheat germ CDPK by acylcarnitines in the presence or absence of Ca 2÷. Wheat germ CDPK was assayed with 0.8 mg/ml IlI-S histone as protein substrate in the standard assay conditions with either 0.8 mM EGTA present (closed symbols) or 0.2 mM EGTA and 0.8 mM CaCI2 present (open symbols) and with increasing concentrations of acylcarnitines. 0,0, stearoylcarnitine; /x,&, palmitoylcarnitine; 1711, myristoyl carnitine. CDPK activity is expressed as % of control (no added inhibitor; CaCI 2 added)•

D-Sphingosine inhibits wheat e m b r y o C D P K (ICs0 10 ~M) (Fig. 6). We have previously shown that sphingosine inhibits wheat germ, wheat leaf and silver beet leaf C D P K s [22]. Sphingosine also inhibits animal protein kinase C [31,32]. H o w e v e r a mixture of the threo and erythro isomers o f dihydrosphingosine is inhibitory at low concentrations (ICs0 10 ~M) but stimulates at high concentrations ( > 2 0 0 t~M) (Fig. 6). Further, this dihydrosphingosine mixture activates wheat germ C D P K in the absence of Ca 2+, 100 I~M eliciting 30% of the activity observed in the presence of Ca 2+ (Fig. 6). In contrast, in the absence of Ca 2+, 200 t~M D-sphingosine fails to activate C D P K . Both erythro and threo dihydrosphingosine at 40

173

350

250

/

200' 150

250 "~ 200 ,¢ O

121

[A]

300

150 100 5O

s'o 0 ]Fig, 6.

50

100

150 [Inhibitor]

200 (i.tM)

250

Inhibition and activation of wheat germ CDPK by

1~o

2~o

250

260

25o

(~tM)

50 ~

40-

sphingosine and dihydrosphingosinein the presence and absence of Ca 2+. Wheat germ CDPK was assayed in the standard assay conditionswith 0.8 mg/ml histone III-S as substrate in the absence of Ca 2+ (0.8 mM EGTA present; open symbols)or the

"~ R'E 30 ~ 20 '¢ 0..

presence of Ca 2+ (0.2 mM EGTA and 0.8 mM CaCI 2 present; closed symbols) and with increasing concentrations of sphingosine (0,0) or erythro- and threo-dihydrosphingosine (A,'t). CDPK activity is expressed as % of control (no added inhibitor; CaCI 2 added).

~ 10 ¢.)

#M and 80 #M, respectively, activate C D P K in the absence of Ca 2+ to 15-25% of the control C D P K activity observed in the absence of these compounds and the presence of Ca 2+. Sodium alkylsulphates do not inhibit C D P K at 160 #M and indeed, like palmitoylcarnitine and stearoylcarnitine, can stimulate activity in the presence of Ca 2+ (Table liB). Like dihydrosphingosine (Fig. 6), sodium alkylsulphates can activate wheat germ C D P K in both the presence and the absence of Ca 2÷ (Fig. 7), the C D P K activity in the absence of Ca 2+ increasing to a level of activity of the same order as that obtaining in the presence of Ca 2+ (Fig. 7B). We have previously shown that a variety of saturated and unsaturated fatty acids can variously inhibit plant CDPKs in the presence of Ca 2+ or activate plant CDPKs in the absence of Ca 2+ [7-11,151. Unsaturated fatty acids (oleic, linoleic and arachidonic acids) activate wheat germ C D P K [91 and silver beet leaf soluble [10] and membranederived [11] CDPKs. A variety of saturated ~Ind unsaturated fatty acids activate oat leaf C D P K in

1~o

[Sodium alkylsulfate]

0

[B] J

s'o

16o

1~o

[Sodium alkylsulfate] (gM) Fig. 7. Activation of wheat germ CDPK by sodium alkylsulphates in the presence or absence of Ca 2+. Wheat germ CDPK was assayed with 0.8 mg/ml III-S histone as protein substrate in the standard assay conditions with 0.2 mM EGTA and 0.8 mM CaCI 2 present (A) or with 0.8 mM EGTA present and no added CaC12 (B) and with increasing concentrations of sodium alkylsulphates. O/, sodium dodecylsulphate (Cl2): A sodium tetradecylsulphate (Ci4); O, sodium octadecylsulphate

(Cis).

the absence of Ca 2+ [15]. Fatty acid-activated C D P K activity can represent a substantial proportion of maximal Ca2+-activated activity [9,11]. Animal protein kinase C isozymes can also be activated by fatty acids in the absence of Ca 2+ [12,13]. The Ca2+-independent activation of plant C D P K by sodium alkylsulphates, dihydrosphingosine and fatty acids is observed at about 10 -4 M and is unlikely to be due to micelle formation which occurs at 10-3-10 -2 M of these types of compounds [33]. Arachidonic acid produced by the fungus Phytophthora infestans acts as an elicitor of antifungal phytoalexins by infected potato tuber cells [34]. The activation of plant CDPKs by

174

arachidonic acid in the absence of Ca2+has suggested that plant CDPKs may be the amplifying targets for such elicitors in vivo [15]. Arachidonic acid, while present in fungal [34] and animal cells [35] and very abundant in mosses [36] and ferns [37], is a rare fatty acid in higher plant systems [38]. This differential distribution of arachidonic acid in eukaryote systems makes it appropriate as a signalling molecule for the activation of plant cells by other plant cells or by non-plant cells as in the situation in which arachidonic acid serves as a fungal pathogen-derived elicitor of plant antifungal defences [ 3 4 ] . Similarly, while sphingosine-containing lipids are abundant in animal [32] and fungal [39,40] systems, lipids containing sphingosine are of relatively low abundance in plants [38], although inositol-containing phyto-glycophospholipids containing phytosphingosine (4-hydroxysphingosine) or dehydrophytosphingosine (4-hydroxy-8-sphingenine) have been isolated from plants [42]. Sphingosine and its derivatives could also serve as signalling molecules in plants. Both arachidonic acid and sphingosine (and their derivatives) have such signalling functions in animal systems [32,35]. The in vitro Ca2+-independent activation of plants CDPK by arachidonic acid [9-11] and by dihydrosphingosine (Fig. 6) suggest that plants CDPK may be activated by such signalling molecules in vivo.

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11

12

13

Acknowledgements This work was supported by a grant from the Australian Research Council. W. Jinsart is grateful for a La Trobe University Postgraduate Scholarship while on leave from Scientific & Technology Research Equipment Centre, Chulalongkorn University, Bangkok, Thailand.

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