Differential inhibition of plant calcium-dependent protein kinases by long chain fatty acids and other amphiphiles

Plant Science, 71 (1990)45--54

45

Elsevier ScientificPublishers Ireland Ltd.

Differential inhibition of plant calcium-dependent protein kinases by long chain fatty acids and other amphiphiles Gideon

M . P o l y a a, J o s e p h M i n i c h i e l l o a, R o s l y n N o t t L E l m a r K l u c i s " a n d P h i l i p J. K e a n e b

aDepartment of Biochemistry and bDepartment of Botany, La Trobe University, Bundoora, Victoria3083 (Australia)

(Received March 13th, 1990;revision receivedMay 16th, 1990;acceptedMay 17th, 1990) Ca2*-dependent protein kinase (CDPK) was partially purified from wheat (Triticum aestivum) embryoand from wheat and silver beet (Beta vulgaris) leaves by extensive protocols involvingCa2*-dependenthydrophobicchromatographyon phenyl-Sepharose CL4B, ion exchangechromatographyon DEAE-Sephaceland gel filtration as commonprocedures. Wheat leaf CDPK is similar to wheat embryo CDPK (purified by application of the same protocol) in Ca2÷-dependence, substrate specificity, molecular size (mol. wt. 81 000) and in inhibition by the calmodulin antagonist trifluoperazine. Both wheat leaf and wheat embryo CDPKs phosphorylate histones (III-S and II-AS preparations), bovine serum albumin, casein and myosin light chains but not the synthetic cyclicAMPdependent protein kinase substrate, Kemptide. Wheat and silver beet CDPKs are inhibited by amphipathic ligands including even carbon number long chain fatty acids (Ct+--C22),odd carbon number long fatty acids (C~--C2~), long chain aliphatic alcohols (C8 Ctt), amino acridines and sphingosine. Behenic acid and arachidic acid are potent inhibitors of wheat embryo CDPK (IC~0values 50 and 60/~M, respectively)but are poor inhibitors of the wheat leaf and silver beet leaf CDPKs. Key words: calcium; protein kinase; fatty acids; sphingosine

Introduction Calcium has a second messenger function in plant [5] as in animal cells [1,17], the effects o f transient elevation of cytosolic free Ca 2÷ concentration being transmitted through Ca 2÷ binding to calmodulin and to Ca2+-dependent protein kinases (CDPKs) [5]. Soluble CDPKs [4,13,20] and membranederived, solubilized CDPKs [11] have been partially purified from a number of plant sources. These enzymes have molecular weights in the range 50 000--90 000 and are inhibited by calmodulin antagonists [4,11,19--23]. While some partially-purified plant C D P K preparations are activated by phospholipids or dioleoylglycerol (for references see [13]), some extensively purified Abbreviations: CDPK, Ca2"-dependentprotein kinase; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EGTA, ethyleneglycol bis~-aminoethyl ether)-N,N,N',N'-tetraaceticacid).

plant CDPK preparations are not [11,13,20]. Dioleoylglycerol decreases the Ca 2÷ concentration requirement for animal Ca 2+- and phospholipidactivated protein kinase C [17]. However extensively purified C D P K preparations from wheat germ [13], oat leaves [15] and from silver beet leaves [11,20] are activated by fatty acids in the absence of Ca 2÷, a property also exhibited by animal Ca 2+- and phospholipid-activated protein kinase C [16,30]. Multiple forms o f protein kinase C are present in animal cells [1,17] and different types of plant CDPKs have been resolved from the same plant source [11,20]. Silver beet leaves contain two soluble CDPKs (I and II) [20] and two very similar and corresponding plasma membrane-associated CDPKs (PI and PII) [11] that differ in substrate specificity and activation by fatty acid s [10,11]. Silver beet leaf enzymes I and PI resemble wheat germ C D P K in being only modestly activated by unsaturated fatty acids in the absence of Ca 2÷ and

0168-9452/90/$03.50 © 1990Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

46 in utilizing bovine serum albumin as a substrate [11,13,20]. Bovine serum albumin is a poor substrate for silver beet leaf enzymes II and PII, which are much more substantially activated by unsaturated fatty acids than enzymes I and PI [I0,11,20]. As one approach to defining the multiplicity and function of plant CDPKs we have sought to define differentially acting inhibitors of such enzymes. The present paper describes the partial purification of a wheat leaf CDPK and the differential inhibition of this enzyme and CDPKs from wheat germ and silver beet leaves by fatty acids and other amphiphilic ligands. Materials and Methods

Plant materials and chemicals Wheat seed (Triticum aestivum L. cultivar Avocet) was obtained from the Plant Breeding Institute (Castle Hill, N.S.W., 2154, Australia). Raw wheat germ and mature silver beet leaves were obtained locally. [),-a2p]ATP (3 Ci/mmol) was obtained from Amersham International (Amersham, U.K.). Fatty acids, sphingosine, acridines, proteins for gel filtration column calibration and protein kinase substrate proteins were obtained from the Sigma Chemical Co. (St. Louis, MO). The chicken phosvitin, salmon sperm protamine, bovine serum albumin and Kemptide preparations were homogeneous. The calf thymus histone preparations (types III-S and II-AS) and the dephosphorylated bovine casein were heterogeneous. The chicken muscle myosin light chain preparations contained only two major polypeptides ( M values 16 and 19 kDa). Alcohols were obtained from Aldrich. DEAE-cellulose (DE-52) and phosphocellulose (P-81) paper were obtained from Whatman, DEAE-Sephacel, Sephacryl S200 and phenyl-Sepharose CL-4B from Pharmacia and Ultrogel AcA44 from LKB Produkter. Partial purification of plant CDPKs Wheat leaf CDPK was partially purified by essentially the same procedure as employed to purify soluble silver beet leaf CDPKs I and II [20] except that the initial homogenization was by means of a Waring blender and chromatography

on Cibacron F3GA Sepharose CL-6B was omitted. Wheat germ CDPK was isolated by a similar procedure involving initial binding to DEAE-cellulose (DE-52) and CaE÷-dependent hydrophobic chromatography on phenyl Sepharose CL-6B followed by chromatography on DEAE-Sephacel, Cibacron F3GA-Sepharose 6B and Sephacryl S200 [20]. Protein was determined by the method of Sedmak and Grossberg [29].

Protein kinase assay Protein kinase was assayed radiochemically at 30°C in duplicate as previously described [19] with 1 mg/ml lysine-rich histone (Sigma catalogue specification: III-S) as substrate. 32p-labeIled protein was recovered on phosphocellulose (P-81) paper [19]. The standard reaction medium (125/al final volume) contained 50 mM Tris (Cl-, pH 8.0), 8 mM MgCI2, 8 mM DTT, 0.2 mM EGTA, 0.8-1.0 mM CaCI2, 2.0 mM 2-mercaptoethanol, 25/aM ATP (specific activity of [y-32p]ATP about 100 mCi/mmol), 1 mg/ml III-S and protein kinase. Assay cpm were corrected by subtraction of cpm from blanks conducted in the absence of added CDPK. Inhibitors of the protein kinase were routinely dissolved in DMSO (except where indicated otherwise) and were added to give 20070 (v/v) DMSO final concentration. Control rates of protein phosphorylation (assays conducted with no inhibitor added) were determined with 3--6-fold replication. Standard deviations of control rates were 6--10070 of mean values. Concentrations for 5007o inhibition of CDPKs (ICs0 values) were determined by assaying CDPKs in duplicate in the standard conditions with increasing concentrations of inhibitor. Results and Discussion

Properties of wheat leaf CDPK Soluble CDPK was partially purified from wheat leaves (2--3 weeks past sowing) as described in Materials and Methods by an extensive procedure involving homogenization, centrifugation, batchwise binding to DEAE-cellulose, CaE*-dependent binding to phenyl Sepharose CL6B, gradient elution from DEAE-cellulose and gel filtration on an Ultrogel AcA44 column (6 cm 2 x 45 cm) in buffer A (50 mM Tris (CI-, pH 8.0)--10

47

mM 2-mercaptoethanol) containing 0.2 M NaCl. The wheat leaf CDPK eluted as a single peak from DEAE-Sephacel (peak elution by 0.22 M NaCl in buffer A) and from Ultrogel AcA44 (data not shown). The CDPK elutes from Ultrogel AcA44 as a single peak of Ca2÷-dependent protein kinase that exactly copurifies with a peak o f Ca2÷-independent protein kinase activity (CaE÷-independent protein kinase activity with III-S as substrate was 2% of protein kinase activity measured with Ca 2÷ present and is likely to be associated with the CDPK itself). No other CDPK or Ca2÷-indepen dent protein kinase peaks were detected. The specific activity o f final preparations was 4.6 nmol min -1 mg protein -1 as compared to a specific activity of 0.009 nmol min -~ mg protein -l in the starting supernatant representing a purification o f 500fold. The yield of purified wheat leaf CDPK was 46 nmol min -1 kg fresh wt. -~ as compared to the starting amount of 198 nmol min -~ kg fresh wt. -~, representing a recovery of 23% from the starting homogenate. The specific activities of the wheat leaf CDPK preparations are of the same order as found for soluble and membrane-derived silver beet leaf CDPK preparations partially purified in a similar fashion (approx. 10 nmol min -~ mg -~) [11,20] but much lower than the specific activities of preparations of a single soluble CDPK purified from wheat germ as described by Lucantoni and Polya [13] (200 nmol min -~ mg -t) or o f a soybean CDPK purified to near-homogeneity but still containing additional proteins [4] (900 nmol min -~ mg-l). The apparent molecular weight of the wheat leaf CDPK (as determined from gel filtration in 0.2 M NaCl-buffer A on an Ultrogel AcA 44 column calibrated with proteins of known mol. wt.) is 81 000 _+ 4000 (mean _ S.D. from 4 determinations). This is similar to the molecular weight of soluble wheat embryo CDPK isolated in a similar fashion (86 000) [21] but higher than the molecular weights of soybean CDPK (65 000) [26], silver beet leaf soluble CDPK I (56 000) and CDPK II (57 000) [20] and silver beet leaf membranederived CDPKs PI (53 500) and PII (51 000) Ill]. It is not known whether these CDPKs, especially the lower molecular weight enzymes, are intact or have been subject to proteolysis during isolation.

The wheat leaf CDPK has an almost absolute dependence on added Ca 2÷ (Table I) as found for other soluble CDPKs [4,11,19--21]. However the wheat leaf CDPK requires higher free Ca 2÷ concentrations for activation than other plant CDPKs. The free Ca 2÷ concentrations for halfmaximal activation of silver beet leaf CDPKs I and II and wheat embryo CDPK are 0.4 laM, 0.4 /aM [18] and 0.8/aM [13], respectively. In contrast, the wheat leaf CDPK, which is fully activated at 250/aM free Ca 2÷, is only slightly activated at 3/aM free Ca 2÷ (3% of maximum activity). (Free Ca 2÷ concentrations in the presence of E G T A were determined as described previously [19]). The wheat leaf CDPK preparation is virtually completely dependent on added protein substrate for activity, as are the wheat embryo and silver beet leaf CDPK preparations [13,20]; activity with no added substrate protein (including possible autophosphorylation [4]) is less than 1% of activity with 1 mg/ml histone III-S as substrate. Histone preparation III-S (a histone HI-rich preparation from calf thymus) is the best protein sub-

Table I. Substrate specificity o f wheat leaf C D P K . W h e a t leaf C D P K was assayed in the standard conditions with 1 m g / ml protein substrate (except for Kemptide and myosin light chains, present at 1.25 and 0.8 m g / m l , respectively) in the presence of Ca ~" (0.25 m M E G T A a n d 1.25 m M CaCI 2 present) or in the absence o f Ca ~÷ (0.25 m M E G T A present). Protein kinase activity is presented as ~/0 of control rate with histone IlLS in the presence of Ca ~÷ (100o70). Millimolar concentrations o f pure protein substrates are given in parenthesis.

Protein substrate

Calf t h y m u s III-S histones Bovine serum albumin (0.015 raM) Calf t h y m u s type II-AS histones Chicken myosin light chains Salmon protamine (0.2 mM) Chicken phosvitin (0.04 mM) Bovine dephosphorylated casein Kemptide (1.0 m M )

Protein kinase (07o control) _ Ca2*

+ Ca ~÷

2.3

100

0.2

27

0.4 -0.4 0.6 0 0.1

6 5 2 2 2 0

48 strate yet found for soybean [4], wheat embryo [13,23] and silver beet leaf [18] CDPKs. Calf thymus III-S historic preparation is also the best substrate for the wheat leaf CDPK but Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly - - a good substrate for cyclic AMP-dependent protein kinase) [7] is not significantly phosphorylated in the presence or absence of Ca 2÷ (Table I). Bovine serum albumin is a good substrate for wheat leaf CDPK (Table I), as found for silver beet leaf CDPKs I and PI [11,20] but not for silver beet leaf CDPKs II and PII [11,20]. In comparison, bovine serum albumin (0.5 mg/ml) is phosphorylated at 40070 of the rate with 0.5 mg/ml III-S by wheat embryo CDPK in the standard protein kinase assay conditions (cf. Table I). Chicken myosin light chains (1.25 mg/ml) are phosphorylated by the wheat leaf CDPK (phosphorylation rate 5 0 of that with 1 mg/ml III-S) (Table I) and by wheat embryo CDPK (the rate with 0.5 mg/ml chicken myosin light chains is 0.3°70 of the rate with 0.5 mg/ml IIIS). It should be noted that myosin light chain phosphorylation by Ca2+-regulated protein kinases is involved in initiation of contraction in animal muscle systems [1,31] and in plant systems the contractile system involved in cytoplasmic streaming is regulated by elevated free Ca 2÷ concentrations of approx. 10-5 M [32], i.e., by Ca 2÷ concentrations commensurate with those required to fully activate plant CDPKs [ 11,13,19--21,26]. The wheat leaf CDPK is inhibited by trifluoperazine (IC50 0.3 mM), a phenothiazine-based calmodulin antagonist which also inhibits protein kinase C (IC50 0.05 mM) [27,28]. Such phenothiazinc-derived calmodulin antagonists also inhibit other plant CDPKs, albeit also at relatively high concentrations [11,19--22]. The interactions of these and other amphipathic compounds with calmodulin- and Ca 2÷- and phospholipid-activated protein kinases have been explained in terms of interaction with functional hydrophobic domains on these proteins [6,27] and a similar model may apply to such interactions with the hydrophobic plant CDPKs. The interactions of wheat leaf CDPK and other plant CDPKs with other amphipathic ligands are described below.

Inhibition of plant CDPKs by fatty acids Histone preparation III-S (highly enriched in

histone HI) is the best substrate yet found for the wheat embryo [13,23,24], silver beet leaf [20], oat leaf [15] and wheat leaf (Table I) CDPKs and historic HI is an endogenous substrate for plant CDPK [ 13]. Accordingly histone preparation III-S was used as a protein substrate in the studies on differential inhibition of plant CDPKs. No other comparable endogenous CDPK substrates have been isolated from plants as yet. Wheat leaf CDPK is inhibited by a number of long chain saturated fatty acids, notably by myristic (C14), pentadecanoic (C15), palmitic (C16), heptadecanoic (C17), stearic (C18), nonadecanoic acid (C19), arachidic (C20) and behenic (C22) acids (Table II). Longer and shorter chain unsaturated fatty acids at 1 mM concentration are not inhibitory and a similar pattern of inhibition is observed with silver beet soluble CDPKs I and II and with wheat embryo CDPK (Table II). However a notable difference is that of these CDPKs only the wheat embryo CDPK is markedly inhibited by low concentrations of behenic acid (C22) (Table III). Repeated titrations with a range of concentrations of behenic acid have confirmed that the wheat leaf CDPK is relatively insensitive to behenic acid (IC50 0.9 mM) while behenic acid is one of the most potent inhibitors yet found for wheat embryo CDPK (IC50 50/aM) (Fig. 1; Table III). A similar marked relative insensitivity of the wheat leaf (as compared to wheat embryo) CDPK to inhibition by fatty acids is observed with arachidic, heptadecanoic, palmitic and pentadecanoic acids (Table III). Given the extensive purification of the wheat leaf and wheat embryo CDPKs, this provides evidence that these enzymes, while having many properties in common, are nevertheless distinct. Whether the wheat leaf and wheat embryo CDPKs derive from distinct genes or whether these differences derive from post-translational modification is not known. The inhibitory effectiveness of long chain saturated fatty acids with respect to the wheat embryo CDPK increases with increasing chain length (Figs. 1A,B). Thus with respect to IC50 values for the wheat embryo CDPK, behenic < arachidic < stearic < palmitic < myristic acid (Table III). The marked differential inhibition by saturated fatty acids of the wheat embryo CDPK as compared to the wheat leaf and silver beet leaf enzymes repre-

49 Table 11. Inhibition of plant CDPKs by saturated fatty acids. Plant CDPKs were assayed in the standard assay conditions in the presence of 200/o (v/v) DMSO and in the presence or absence of I mM fatty acid (data asterisked derive from addition of 0.8 mM fatty acid). Protein kinas¢ activity is expressed as % of control (no added compound). Saturated fatty acid carbon number is indicated in parenthesis. Addition

None Caproic acid (C6) Heptanoic acid (C7) Caprylic acid (C8) Nonanoic acid (C9) Capric acid (C 10) Undecanoic acid (CI 1) Laurie acid (C12) Tridecanoic acid (C 13) Myristic acid (C 14) Pentadecanoic acid (C 15) Palmitic acid (C 16) Heptadecanoic acid (C 17) Stearic acid (C18) Nonadecanoic acid (C 19) Arachidic acid (C20) Heneicosanoic acid (C21) Behenic acid (C22) Lignoceric acid (C24)

Protein kinase (% control) Wheat embryo

Wheat leaf

Silver beet 1

Silver beet II

100 106 103 103 102 103 107 130 107 67 24 13 45 4 12 2 22 6 75

100 93 96 95 95 106 81 120 75 67 63 64 55 29 38 10 58 39 87

100 94 95 91 87 73 68 75 93 77 84 37 33 23 6 20* 78 95" 96

100 97 91 99 90 96 90 100 110 89 92 19 45 19 6 19* 65 83" 104

Table IlL Differential inhibition of plant CDPKs. Plant CDPKs were assayed in the presence of increasing concentrations of inhibitors in the standard assay conditions as described in Materials and Methods and concentrations for 50°70 inhibition of activity (ICs0 values) determined. Compound

IC5o(mM) Wheat embryo

Wheat leaf

Silver beet I

Silver beet ll

Behenic acid (C22) Heneicosanoic acid (C21) Arachidic acid (C20) Nonadecanoic acid (C19) Stearic acid (C18) Heptadecanoic acid (C17) Palmitic acid (C16) Pentadecanoic acid (C 15) Myristic acid (C14)

0.05 0.4 0.06 0.3 0.14 0.3 0.27 0.4 1.0

0.9 > 1 0.5 0.6

>1 >1 0.6 0.3 0.7 0.7

> 1 > 1 0.4 0.3 0.6 1

1-Octanol l-Nonanol

4

2-Nonanol l-Decanol 9-Decen- l-ol Acridine orange Acridine yellow G Sphingosine

3 3 0.8 0.7 0.2

> 1 > 1 5 2 3 6 2 0.7 0.8 0.1

> 1 > 1 6 4 4 4 2

> > > >

1 1 1 1 6 14 4 0.2 0.7 0.1

0.1

50 I

I

I

I

/,!,

)

1

A _

I

0

05

1.0

t."

1.5

.2<)

I

I

3

4

5

[Fatty acid] (raM) I

1

--

I

I

I

B

OI 0

I

I

I

t

20

40

60

80

I

I00

I'Fatty aci(~ (.uM)

Fig. 1. Inhibition of wheat embryo CDPK by long chain fatty acids. CDPK was assayed as described in Materials and Methods in the standard assay containing 20*/o (v/v) DMSO and with increasing concentrations of fatty acids. Protein kinase is presented as percentage of control activity (no added inhibitor). (A)/x--ZX, lignoceric acid; A - - A , myristic acid; O - - O , palmitic acid; • - - 0 , stearic acid; (B) O - - O , arachidic acid; • - - O, behenic acid.

51 sents a first step in the detection and devising of selective potent inhibitors of specific plant CDPKs. The wheat embryo CDPK and silver beet leaf CDPKs I, II, PI and PII are activated by unsaturated fatty acids in the absence of Ca 2÷ [10,11,13] with silver beet leaf enymes II and PII exhibiting very substantial activations [10,11]. Similarly, differences in activation of animal protein kinase C isozymes by unsaturated fatty acids have been reported [30]. The wheat leaf CDPK differs from the wheat embryo CDPK in not exhibiting activation by arachidonic, linoleic and stearic acids in the absence of Ca 2÷. Thus rates of phosphorylation of III-S by wheat leaf CDPK in the absence of Ca 2+ (0.2 mM E G T A present) but with 1 mM arachidonic acid, 1 mM stearic acid or l mM linoleic acid present are 87070, 27°70 and 2070, respectively of the rate with no fatty acid and no C a 2÷ added. In contrast, wheat embryo CDPK is markedly activated by these fatty acids in these conditions [13]. Further, while 0.7 mM linoleic acid does not inhibit wheat embryo C D P K in the presence of Ca 2÷ [13], linoleic acid is a strong inhibitor of the wheat leaf CDPK (ICs0 in the absence of DMSO, 0.12 mM). Further evidence that the wheat embryo and wheat leaf CDPKs are distinct enzymes comes from differential inhibition of these enzymes by basic polypeptides [25]. It is conceivable that such differences between plant CDPKs in sensitivity to fatty acids could derive from contaminating components in the two preparations. Thus, for example, it appears that the inhibition by CTP of very crude wheat embryo chromatin-derived CDPK preparations [19] and crude silver beet leaf CDPK preparations [23] (but not of much more extensively purified soluble wheat germ [21] or silver beet leaf CDPK [20] preparations) both derive from a CTP-binding inhibitory factor that can be removed by an incisive Ca2+-dependent chromatography step on phenyl-Sepharose CL-4B [9] yielding CDPK preparations that are not inhibited by C T P [9,20,21]. However the CDPK preparations used in the present study have all been very extensively purified by procedures including CaE+-dependent hydrophobic chromatography. This latter step excludes hydrophilic contaminants and only retains those

hydrophobic proteins binding to phenyl-Sepharose CL-4B in a Ca2+-dependent fashion. Further, the concentration of leaf and embryo CDPK preparation protein in the protein kinase assays (approx. 1 /ag CDPK preparation protein added, corresponding to a maximal 0.1 laM concentration of any 100-kDa protein in the assay) is much lower than the effective inhibitor concentrations employed (10 /aM to mM). Thus the possibility that the poor inhibition of wheat leaf CDPK by long chain fatty acids derives from different levels in the wheat leaf CDPK preparations used of a protein that stoichiometrically binds fatty acids can be ruled out since such a protein would be present at levels at least 100 times lower than the fatty acid inhibitor concentrations used. While wheat embryo CDPK is much more sensitive to behenic acid inhibition (ICs0 50/aM) than the wheat leaf CDPK (ICs0 0.9 mM), we have found that oat leaf CDPK isolated in essentially the same fashion is also very sensitive to inhibition by behenic acid (ICso 20 ~M) [15].

Inhibition of plant CDPKs by long chain alcohols, acridines and sphingosine Since the CDPKs are inhibited by long chain fatty acids the effects of alcohols including long chain alcohols - - on CDPK activity was examined. Low molecular weight alcohols inhibit wheat embryo CDPK, albeit at very high concentrations. Thus such compounds (dissolved in H20) affect wheat embryo CDPK activity (expressed as 070 of control with no addition) as follows: 4.9 M methanol, 35070; 0.5 M methanol, 114°70; 3.4 M ethanol, 8°70; 0.3 M ethanol, 110070; 2.7 M n-propanol, 0070; 0.3 M n-propanol, 63070; 2.6 M isopropanol, 0070; 0.3 M isopropanol, 100°70; 2.2 M n-butanol, 2°70; 0.2 M n-butanol, 3°7o; 2.2 M 2-butanol, 9070; 0.2 M 2-butanol, 3070; 2.2 M 2-methyl-l-propanol, 0°7o; 0.2 M 2-methyl-l-propanol, 3°70; 1.3 M octan-1ol, 14070; 0.13 M octan-l-ol, 42070. The ICs0 for nbutanol is 100 mM with respect to the wheat embryo CDPK. Similar inhibition by high concentrations of low molecular weight alcohols and other polarity decreasing compounds is observed with the silver beet leaf enzymes. Thus such compounds affect silver beet leaf C D P K I activity (ex-

-

52

greater than 12 are ineffective at 4 mM as inhibitors of all 4 CDPKs tested (~ 25070 inhibition), namely 1-tetradecanol (C14), 2-tetradecanol (C14), 1,2-tetradecanediol (C14), 1-pentadecanol (C15), 1,2-hexanediol (C16), 1-heptadecanol (C17), 1-octadecanol (C18), l-nonadecanol (Ci9), 1-eicosanol (C20) and 1-docosanol (C22). Thus long chain alcohols contrast with the corresponding long chain saturated fatty acid analogues (Tables II and III) in being inactive as inhibitors of the plant CDPKs and indeed the only effective alcohols (C8--C10) have relatively high, millimolar ICs0 values (Table III). Plant growth inhibition by long chain fatty acids and aliphatic alcohols have been reported (for review see [13]). Thus, for example, unsaturated fatty acids (C4--C12 but not C18) and related compounds (including 1octanol) at 1 mM concentration inhibit Avena coleoptile growth [18]. However interpretation of such in vivo effects may be complicated by the issue of differential entry of applied compounds [18]. While longer chain alcohols are ineffective as plant CDPK inhibitors (Table IV), sphingosine -an amphipathic long chain alcohol -- is an inhibitor of wheat embryo CDPK and of the leaf CDPKs (IC~0 values 0.1--0.2 raM) (Table III). Sphingosine is also a potent inhibitor of animal protein kinase C (IC50 0.1 mM) [3,14]. Aminoacri-

pressed as 070 of control with no additions) as follows: 4.9 M methanol, 25°70; 3.4 M ethanol, 2.3070; 2.7 M glycerol, 64070; 2.6 M dimethylformamide, 9070; 2.8 M dimethylsulfoxide, 78070; and affect CDPK II thus: 4.9 M methanol, 42070; 3.4 M ethanol, 7070; 2.7 M glycerol, 70070; 2.6 M dimethylformamide, 15070; 2.8 M dimethylsulfoxide, 75°70. The silver beet leaf CDPKs I and II are similar to the wheat embryo CDPK in being more sensitive to inhibition by ethanol than by methanol. The silver beet leaf CDPKs are not substantially inhibited by high concentrations of DMSO as compared to high concentrations of other solvent polarity decreasing agents such as dimethylformamide, ethanol and methanol. Similarly the wheat embryo and wheat leaf CDPK activities are 92070 and 30% of control, respectively, in the presence of 2.8 M DMSO. While low molecular weight alcohols are only inhibitory at very high concentrations, the plant CDPKs are inhibited by much lower concentrations of some long chain alcohols. Of the alcohols tested at 4 mM, 1-octanol, 1-nonanol, 2-nonanol, 1-decanol and 9-decen-l-ol are the most effective (Tables III--V). The amino alcohols 3-amino-1propanol, 4-amino-l-butanol and 5-amino-l-pentanol at 4 mM do not substantially inhibit either wheat embryo or wheat leaf CDPKs (~ 13070 inhibition). Long chain alcohols with carbon numbers

Table IV. Effect of alcohols on plant CDPKs. Plant CDPKs were assayed in the standard conditions in the presence of 20°70 (v/v) DMSO and in the presence or absence of 4 mM added compound. Protein kinase is expressed as 070of control (no added compound). Alcohol carbon chain number is indicated in parenthesis.

Addition

1-Octanol (C8) 2-Octanol (C8) 3-Octanol (C8) 1-Octen-3-ol (C8) l-Nonanol (C9) 2-Nonanol (C9) l-Decanol (CI0) 9-Decen- 1-ol (C 10) l-Undecanol (C 11) 1-Dodecanol (C12) 2-Dodecanol (C 12) l-Tetradecanol (C 14)

Protein kinase(07ocontrol) Wheat embryo

Wheat leaf

88 96 89 86 50 83 18 22 78 123 87 102

69 72 85 97 58 55 70 49 63 70 68 80

Silver beet I

Silver beet II

55

70

89 40 42 61 24 99 l 11 101 108

85 56 51 58 22 87 105 120 119

53 Table V.

Inhibition of plant CDPKs by acridines. Plant CDPKs were assayed in the standard conditions in the presence of 20070 (v/v) DMSO and in the presence or absence of l mM added compound. Protein kinase is expressed as 070 of control (no added compound). Addition

Acridine orange Acridine yellow G Acridine red 9-Aminoacridine

Protein kinase (°70control) Wheat embryo

Wheat leaf

Silver beet I

Silver beet II

34 6 82 97

6 8 88 105

22 31 63 70

3 5 7l 65

dines are amphipathic compounds that are also inhibitors of animal protein kinase C [2]. Acridine orange (1 mM) and acridine yellow G (but not aniline red or 9-aminoacridine) substantially inhibit the plant CDPKs (Table V), although relatively high concentrations (0.2--0.8 mM) are required for 500/0 inhibition (Table III). In comparison, the ICs0 values for these compounds with respect to protein kinase C are about 0.1 mM [2]. Adriamycin-like the amino acridines a DNA-binding compound and an inhibitor of protein kinase C (ICs0 about 0.1 mM) [6] -- does not inhibit the wheat embryo, wheat leaf or silver beet leaf CDPKs (< 25 070inhibition at 1 mM concentration). General Discussion

Plant CDPKs are similar to animal protein kinase C in exhibiting Ca2÷-independent activation by unsaturated fatty acids [10,11,13] and in being inhibited by amphipathic compounds such as polyamines and phenothiazine derivatives known to inhibit calmodulin function [4,8,22]. The present paper shows that, in addition, various plant CDPKs are inhibited by other amphipathic compounds known to inhibit protein kinase C, namely aminoacridines [2] and sphingosine [3,14]. The inhibition of protein kinase C by such compounds has been related to interference with the phospholipid-activation of the enzyme at a hydrophobic site [8]. The inhibition of plant CDPKs by such compounds and the activation of these enzymes by certain fatty acids similarly suggests the existence on these plant CDPKs of one or more hydrophobic domains involved in regulation of catalytic

activity. This is further supported by the specific and differential inhibition of CDPKs by long chain fatty acids (Fig. 1; Tables II and III). Behenic and arachidic acids are potent inhibitors of wheat germ CDPK (and indeed are the best inhibitors of this enzyme yet found) but not of wheat leaf CDPK or silver beet leaf CDPKs assayed in the same conditions (Table III). It is not known whether these fatty acids may act as physiological regulators of CDPK in vivo. However, potent amphipathic inhibitors acting at such sites can be used to distinguish between otherwise very similar plant CDPKs such as the wheat embryo and wheat leaf CDPK as shown in the present work. Specific CDPK inhibitors will be useful in defining the involvement of particular CDPKs in plant cellular responses to external signals. References 1

2

3

4

5 6

A.M. Edelman, D.K. Blumenthal and E.G. Krebs, Protein serine/threonine kinases. Annu. Rev. Biochem., 56 (1987) 567--613. Y.A. Hannun and R.M. Bell, Aminoacridines, potent inhibitors of protein kinase C. J. Biol. Chem., 263 (1988) 5124--5131. Y.A. Hannun, C.R. Loomis, A.H. Merrill and R.M. Bell, Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J. Biol. Chem., 261 (1986) 12604--12609. A.C. Harmon, C. Putnam-Evans and M.J. Cormier, A calcium-dependent but calmodulin-independent protein kinase from soybean. Plant Physiol., 83 0987) 830--837. P.K. Hepler and R.O. Wayne, Calcium and plant development. Annu. Rev. Plant Physiol., 36 0985) 397--439. N. Katoh, B.C. Wise, R.W. Wrenn and J.F. Kuo, Inhibition by adriamycin of calmodulin-sensitive and phospholipid-sensitive calcium dependent phosphorylation of

54

7

8

9

10

11

12

13

14

15

16

17 18

19

endogenous proteins from heart. Biochem. J., 198 (1981) 199--205. B.E. Kemp, Phosphorylation of synthetic peptide analogues of rabbit cardiac troponin inhibitory subunit by the cyclic AMP-dependent protein kinase. J. Biol. Chem., 254 (1979) 2638--2642. U. Kikkawa and Y. Nishizuka, Protein kinase C, in: P.D. Boyer and E.G. Krebs (Eds.), The Enzymes, Vol. 17, Academic Press, New York, 1986, pp. 167--189. E. Klucis, A. Lucantoni, M. Haritou and G.M. Polya, Multiple soluble and particulate calcium-dependent protein kinases in silver beet leaf tissue. Proc. Aust. Biochem. Soc., 19 (1987) 61. E. Klucis and G.M. Polya, Calcium-independent activation of two plant leaf calcium-regulated protein kinases by unsaturated fatty acids. Biochem. Biophys. Res. Commun., 147 (1987) 1041--1047. E. Klucis and G.M. Polya, Localization, solubilization and characterization of plant membrane-associated calcium-dependent protein kinases. Plant Physiol., 88 (1988) 164--171. D.S. Letham, Naturally-occurring plant growth regulators other than the principal hormones of higher plants, in: D.L. Letham, P.D. Goodwin and T.J.V. Higgins (Eds.), Phytohormones and Related Compounds - - A Comprehensive Treatise, Elsevier, New York, 1978, pp. 349--417. A. Lucantoni and G.M. Polya, Activation of wheat embryo calcium-regulated protein kinase by unsaturated fatty acids in the presence and absence of calcium. FEBS Lett., 221 (1987) 33--36. A.H. Merrill and V.L. Stevens, Modulation of protein kinase C and diverse cell functions by sphingosine - - a pharmacologically interesting compound linking sphingolipids and signal transduction. Biochim. Biophys. Acta, 1010 (1989) 131--139. J. Minichiello, G.M. Polya and P.J. Keane, Inhibition and activation of oat leaf calcium-dependent protein kinase by fatty acids. Plant Sci., 65 (1989) 143--152. K. Murakami and A. Routtenberg, Direct activation of purified protein kinase C by unsaturated fatty acids (oleate and arachidonate) in the absence of phospholipids and Ca 2÷. FEBS Lett., 192 (1985) 189--193. Y. Nishizuka, Studies and perspectives of protein kinase C. Science, 233 (1986) 305--312. M. Ohkawa and Y. Nishikawa, Plant growth inhibitory activity of fatty acids and the related compounds by the Avena coleoptile test. Plant Sci., 53 (1987) 35--38. G.M. Polya, J.R. Davies and V. Micucci, Properties of a calmodulin-activated Ca2*-dependent protein kinase from wheat germ. Biochim. Biophys. Acta, 761 (1983) 1--12.

20

21

22

23

24

25

26

27 28

29

30

31

32

G.M. Polya, E. Klucis and M. Haritou, Resolution and characterization of two soluble calcium-dependent protein kinases from silver beet leaves. Biochim. Biophys. Acta, 931 (1987) 68--77. G.M. Polya and V. Micucci, Partial purification and characterization of a second calmodulin-activated Ca 2÷dependent protein kinase from wheat germ. Biochim. Biophys. Acta, 785 (1984)68--74. G.M. Polya and V. Micucci, Interaction of wheat germ Ca2÷-dependent protein kinases with calmodulin antagonists and polyamines. Plant Physiol., 79 (1985) 968--972. G.M. Polya, V, Micucci, S. Basiliadis, T. Lithgow and A. Lucantoni, Plant leaf calcium-dependent protein kinases, in: A.J. Trewavas (Ed.), Molecular and Cellular Aspects of Calcium in Plant Development. Plenum Publ. Corp., New York, 1986, pp. 75--82. G.M. Polya, N. Morrice and R.E.H. Wettenhall, Substrate specificity of wheat embryo calcium-dependent protein kinase. FEBS Lett., 253 (1989) 137--140. G.M. Polya, R. Nott, E. Klucis, J. Minichiello and S. Chandra, Inhibition of plant calcium-dependent protein kinases by basic polypeptides. Biochim. Biophys. Acta, 1037 (1990) 259--262. C.L. Putnam-Evans, A.C. Harmon and M.J. Cormier, Calcium-dependent protein phosphorylation in suspension-cultured soybean cells, in: A.J. Trewavas (Ed.), Molecular and Cellular Aspects of Calcium in Plant Development. Plenum Publ. Corp., New York, 1986, pp. 99--106. R.R. Rando, Regulation of protein kinase C activity by lipids. FASEB J., 2 (1988) 2348--2355. R.C. Schatzmann, B.C. Wise and J.F. Kuo, Phospholipid-sensitive calcium-dependent protein kinase: inhibition by antipsychotic drugs• Biochem. Biophys. Res. Commun., 98 (1981) 669--676. J.J. Sedmak and S.E. Grossberg, A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G250. Anal. Biochem., 79 (1977) 544--552. K. Sekiguchi, M. Tsukuda, K. Ogita, U. Kikkawa and Y. Nishizuka, Three distinct forms of rat brain protein kinase C: differential response to fatty acids. Biochem. Biophys. Res. Commun., 145 (1987) 797--802. J.T. Stull, M.H. Nunnally and C.H. Michnof, Calmodulin-dependent protein kinases, in: P.D. Boyer and E.G. Krebs (Eds.), The Enzymes, Vol. 17, Academic Press, 1986, pp. 113--166. R.E. Williamson and C.C. Ashley, Free Ca 2~ and cytoplasmic streaming in the alga Chara. Nature, 296 (1982) 647--651.