Partial purification and characterization of a second Calmodulin-activated Ca2+-dependent protein kinase from wheat germ

68

Biochimica et Biophysica Acta, 785 (1984) 68-74 Elsevier

BBA31834

PARTIAL PURIFICATION AND CHARACTERIZATION OF A SECOND CALMODULIN-ACTIVATED Ca 2+-DEPENDENT PROTEIN KINASE F R O M WHEAT GERM GIDEON M. POLYA * and VITO MICUCCI Department of Biochemistry, La Trobe University, Bundoora, Victoria 3083 (Australia) (Received October 19th, 1983)

Key words: Calmodulin; Protein kinase," Ca 2 + dependence," (Wheat germ)

A soluble Ca2+-dependent protein kinase was partially purified from wheat germ by a procedure involving adsorption to DEAE-cellulose, Ca2+-dependent binding to phenyl-Sepharose, (NH 4)2504 precipitation and gel filtration. The protein kinase (M F86 000) catalyzes the phosphorylation of histones, casein and phosvitin. Calmodulin activates the phosphorylation of histones catalyzed by the protein kinase. The protein is largely dependent upon Ca2+ for activity. The rate of casein phosphorylation is half-maximal at 0.3 pM free Ca2+ and maximal at 3 pM free Ca2+; much higher free Ca2+ concentrations are required for half-maximal (60 pM) and maximal (500 pM) rates of histone phosphorylation. Miilimolar Mg 2+ is required in addition to Ca2+ for maximal activity of the enzyme and millimolar Mn 2+ can substitute for the (Ca2++ Mg 2+) requirement. The protein kinase is inhibited by the calmodulin antagonists, chlorpromazine and fluphenazine. The K m for ATP is 16 pM and the enzyme phosphorylates serine and threonine residues of casein. The enzyme is very similar to a Ca2+- and calmodulin-activated protein kinase isolated from wheat-germ chromatin, but differs from this enzyme in various properties, notably apparent molecular size and insensitivity to nucleotides that inhibit the chromatin-derived enzyme.

Introduction Increase in cytosolic free Ca 2+ concentration represents the key initial response of animal cells to certain neurotransmitter, hormonal and electrical signals [1-3]. A major consequence of such increases in cytosolic free Ca 2+ concentration is the activation of Ca2+-calmodulin-activated or Ca2+-phospholipid-activated protein kinases, with consequent changes to cellular processes through the phosphorylation of specific proteins. Animal enzymes other than protein kinases can also be * To whom reprint requests should be addressed. Abbreviations: EGTA, ethyleneglycol bis(fl-aminoethyl ether)N,N,N'N'-tetraacetic acid; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid; Mes, 2-(N-morpholino)propanesulfonic acid. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

modified by interaction with Ca2+-calmodulin [1-3]. In higher plants cytosolic free Ca 2+ concentration increases greatly in response to electrical stimulation [4] and changes in cytosolic Ca 2÷ concentration have been inferred to be involved in various plant hormone-, fungal elicitor- and red light-stimulated processes [5-8]. Higher plants contain calmodulin, various Ca2+-calmodulin activated enzymes [5,9,10] and CaZ+-dependent protein kinases [11-14]. A membrane-associated protein kinase from pea shoots is fully activated by 3/~M free Ca 2+ and is inhibited by the calmodulin antagonist trifluoperazine but is not activated significantly by added calmodulin [11]. A soluble Ca 2+- and Ca 2+calmodulin-activated protein kinase has been partially purified from a wheat-germ chromatin extract [12,14]. This enzyme is half-maximally

69 activated by 0.3 /~M free Ca2+, is inhibited by phenothiazine-derived calmodulin antagonists and is activated by micromolar calmodulin [14]. The nature of the endogenous substrates of these CaE+-dependent protein kinases has yet to be determined. However, it has been recently shown that quinate:NAD + oxidoreductase from carrot cells is activated by phosphorylation catalyzed by Ca 2+-dependent, fluphenazine-sensitive protein kinase [13]. In the process of isolating calmodulin from wheat germ by Ca2+-dependent hydrophobic chromatography on phenyl-Sepharose CL-4B it was found that protein kinase activity is retained together with calmodulin in the presence of Ca 2+ and is eluted with calmodulin in the absence of Ca 2+ [15]. This protein kinase activity can be resolved into two components by subsequent chromatography on casein-Sepharose 4B, namely a calmodulin-activated, Ca2+-independent protein kinase that binds to casein-Sepharose 4B at low ionic strength [15] and a Ca2+-dependent protein kinase that does not bind to casein-Sepharose 4B in these conditions. The present paper describes the properties of this soluble Ca 2+-dependent protein kinase. This enzyme is similar to but quite distinct from the previously described Ca2+-depen dent protein kinase resolved from a wheat-germ chromatin extract [14]. Thus, wheat germ contains two distinct (but possibly related) Ca 2+-dependent protein kinases.

liter 0.5 M NaC1/buffer A at room temperature. The eluate was brought to 5 mM CaCl2 concentration, phenyl-Sepharose CL-4B (200 ml bed volume; 40/xmol ligand per ml gel bed) was added and the suspension was stirred intermittently for 60 rain at room temperature. The phenyl-Sepharose was recovered by filtration on a Biichner funnel and was washed successively with 1 liter 0.5 M NaCl/5 mM CaClJbuffer A and 50 mM Tris-HC1 (pH 8.0)/0.1 mM CaC12. The phenyl-Sepharose was packed into a column and the protein kinase was eluted (together with the calcium-binding regulator protein, calmodulin) in 50 mM Tris-HC1 (pH 8.0)/1 mM EGTA. The fractions were pooled, applied to a column (7 cm2 x 7 cm) of caseinSepharose equilibrated with 50 mM Tris-HC1 (pH 8.0)/10 mM 2-mercaptoethanol (buffer B) and the CaE+-dependent protein kinase was eluted in the same buffer. It should be noted that a CaE+-independent protein kinase is retained by the caseinSepharose at this stage and can be recovered by elution in 0.5 M NaC1/buffer B [15]. The fractions containing Ca2+-dependent protein kinase were pooled and brought to 55% (NH4)2SO 4 saturation, and the resulting precipitate was collected by centrifugation and dissolved in 2 ml buffer C (50 mM Tris-HCl (pH 8.0)/10 mM 2-mercaptoethanol/1 mM EGTA). The Ca 2+-dependent protein kinase was finally chromatographed on a column (4.9 cm2 x 84 cm) of Sephacryl S-200 in buffer C. The partially purified CaE+-dependent protein kinase was routinely stored at 4°C.

Materials and Methods

Partial purification of the Ca2-dependent protein kinase 500 g wheat germ was suspended in 2 liters 50 mM Tris-HC1 (pH 8.0)/0.1 mM EGTA/1 mM 2-mercaptoethanol (buffer A) containing 0.1 mM phenylmethylsulfonyl fluoride and 0.2 % (v/v) ethanol. The suspension was homogenized for 2 min at 4°C using an Ultra-Turrax blender (Janke and Kunkel, Staufen, F.R.G.) and the homogenate filtered through muslin. 50 g of preswollen DEAE-cellulose (Whatman DE-52) was added to the filtrate and the suspension was stirred intermittently for 15 rain. The DEAE-cellulose was collected by filtration and washed with 1 liter buffer A and the protein kinase was eluted with 1

Enzyme and protein assays and analysis of protein phosphorylation Protein kinase was assayed at 30°C by precipitation of 3zP-labelled phosphorylated protein onto paper disks in 15% trichloroacetic acid [16]. The standard assay medium containing 62.5 mM TrisHC1 (pH 8.0), 25 /~M ATP (specific activity of [3,-32p]ATP in the assay, 10~50 mCi/mmol), 10 mM MgC12, 0.22 mM EGTA, 2.5 mM 2-mercaptoethanol, protein kinase, 1 mg/ml protein substrate and 0.75 mM CaC12. Protein was determined using Coomassie blue [17] with crystalline bovine serum albumin as a standard. Electrophoresis of 32p-labelled phosphorylated proteins, autoradiography of electropherograms, acid hydrolysis of 32p-labelled proteins and analysis of acid hydro-

70 lysates were conducted as described previously [14]. CTPase was assayed at 30°C by determining phosphate release [18] in a medium comprising 1 mM CTP/62.5 mM Tris-HC1 (pH 8.0)/10 mM MGC12/0.22 mM E G T A / 2 . 5 mM 2-mercaptoethanol. Materials Raw wheat germ was purchased from Heidelberg Health Foods, Melbourne. [y-32p]ATP (3 Ci/mmol) was obtained from Amersham International, Amersham, U.K. Calmodulin was purified from wheat germ by Ca2+-dependent chromatography on phenyl-Sepharose CL-4B [19] as described previously [15]. Fluphenazine was obtained from Squibb (Australia) Melbourne. Dephosphorylated casein, calf thymus histones (catalogue specification: type II-AS), phosvitin, thiol reagents, nucleotides, chlorpromazine, phenyl-Sepharose 4B, hemin and enzymes for column calibration were obtained from Sigma, St. Louis, MO, U.S.A. Sephacryl S-200 was obtained from Pharmacia. The Ca2+-dependent protein kinase from wheat-germ chromatin was partially-purified as described previously [14]. The major wheat-germ cytokinin-binding protein was purified by affinity chromatography [20]. Casein-Sepharose 4B was prepared by coupling casein to CNBr-activated Sepharose 4B [20]. Results and Discussion

We will refer below to the previously described Ca2+-dependent protein kinase derived from wheat-germ chromatin [12,14] as protein kinase I

and to the second wheat-germ Ca2+-dependent protein kinase that is the subject of the present paper as protein kinase II. The overall purification of protein kinase II (see Materials and Methods) 17500-fold (Table I). However, note that measureable protein kinase is greatly increased after the DEAE-cellulose step (Table I). With respect to the protein kinase retained by DEAE-cellulose, the overall yield is only 0.07% (Table I).The specific activity of the final preparations is only approx. 2 n m o l / m i n per mg protein (cf. Ref. 21). The final gel filtration on Sephacryl S-200 yields one major peak of Ca2+-de pendent protein kinase activity; peaks of protein kinase activity determined in assays containing casein or histones as substrates were coincident (data not shown). The apparent molecular weight of protein kinase II as determined from gel filtration is 86000 + 9000 (mean + S.D. from five determinations of elution volume). The protein kinase 1I preparations lose 50% of activity on storage in buffer C at 4°C for 5 days. In contrast, a preparation of protein kinase I lost only 8% of activity on storage in buffer C at 4°C for 2 months. The pH- and cation-dependence of protein kinase II is very similar to that of protein kinase I [14]. Protein kinase II exhibits a broad pH-activity profile when assayed with either casein or histones as substrates (Fig. 1). The protein kinase is largely dependent upon added Ca 2+ at pH values above 6.0 when assayed in the presence of EGTA as a Ca 2+ buffer (Fig. 1). Note that EGTA has a decreased affinity for Ca 2+ at lower pH values (see Ref. 22). Casein phosphorylation is markedly activated by micromolar free Ca 2+ concentrations

TABLE I PARTIAL PURIFICATION OF THE PROTEIN KINASE 500 g wheat germ was the starting material for the purificationprocedure. Protein kinase was assayed in the standard assay conditions with 0.5 mM free Ca2+ present and 1 mg/ml dephosphorylatedcasein as substrate. Purificationstep

Protein (mg)

Protein kinase (nmol/min)

Filtered homogenate DEAE-cellulose Phenyl-SepharoseCL-4B Casein-Sepharose4B SephacrylS-200

143800 2306 8.5 7.6 0.072

15.1 213.8 7.3 1.0 0.139

Protein kinase (pmol/min per mg protein) 0.11 93 85 132 1 930

71 0 20

i

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015 E 010

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4

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6

7

8

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Fig. 2. Dependence of casein and histone phosphorylation on free Ca 2+ concentration. Protein kinase was assayed in triplicate in the standard assay at pH 8.0 containing 1 m g / m l histone (e) or 1 m g / m l casein (zx, O). The open symbols represent the normalized results of two consecutive experiments conducted under identical conditions. All assays contained 0.22 m M EGTA and free Ca 2+ concentrations at a variety of total Ca 2+ concentrations were calculated using a value of K a p p (pH 8.0) for Ca2+-binding to EGTA (in the presence of 10 m M MgCI2) of 3.8-107 M -1 derived from the data of Potter and Gergely [22]. Reagent Ca 2+ concentration was determined by titration against an EGTA solution.

3 • 10 -6 M free Ca 2+ (Fig. 2). In contrast, histone phosphorylation is activated at much higher free Ca 2+ concentrations, half-maximal activation being obtained at 6 . 1 0 5 M and maximal activation at 5 - 1 0 - 4 (Fig. 2). Increasing the free Ca 2÷ concentration further to approx. 5 mM inhibits Ca 2+dependent phosphorylation (Fig. 2). As found for protein kinase I, protein kinase II requires a divalent cation (Mg 2+ or Mn 2+) in addition to Ca 2+ for maximal casein or histone phosphorylation rates (Figs. 3 and 4). The protein phosphorylation rate is largely Ca2+-independent at millimolar Mn 2+ (Figs. 3B, 4B). A variety of metal ions inhibit the CaZ+-dependent igrotein kinase. 1 mM Co 2+, Hg 2+, Cd 2+, Cu 2+, Zn 2+ and La 3+ inhibit by 74, 100, 97, 99, 99 and 89%, respectively. The enzyme is markedly inhibited at elevated salt con-

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(Fig. 2) that are commensurate with the cytosolic free Ca 2+ concentrations in excited plant cells [4]. Casein phosphorylation is markedly activated by free Ca 2+ concentrations above approx. 10 - 7 M , half-maximal activation being obtained at 3 • 10 7 M free Ca z+ and maximal activation at approx.

E

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Fig. 2. Dependence of casein and histone phosphorylation on free Ca 2+ concentration. Protein kinase was assayed in triplicate in the standard assay at pH 8.0 containing 1 m g / m l histone (o) or 1 m g / m l casein (zx, ©). The open symbols represent the normalized results of two consecutive experiments conducted under identical conditions. All assays contained 0.22 m M EGTA and free Ca 2+ concentrations at a variety of total Ca 2+ concentrations were calculated using a value of Kapp (pH 8.0) for Ca2+-binding to EGTA (in the presence of 10 m M MgC12) of 3.8-107 M -1 derived from the data of Potter and Gergely [22]. Reagent Ca 2+ concentration was determined by titration against an EGTA solution.

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Fig. 3. Cation dependence of casein phosphorylation catalyzed by the Ca2+-dependent protein kinase. Protein kinase was determined in triplicate in the standard assay with 1 m g / m l casein as protein substrate, at various Mg 2÷ or Mn 2+ concentrations in the presence (closed symbols) or absence (open symbols) of 0.5 m M free Ca 2÷. (A) Mg2+-dependence: O, without Ca2+; e, with Ca 2+, (B) Mn2 +-dependence: zx, without Ca2+; ,~, with Ca 2+. Fig. 4. Cation dependence of histone phosphorylation catalyzed by the Ca2+-dependent protein kinase. Protein kinase was determined in triplicate in the standard assay with 1 m g / m l histones as protein substrate, at various Mg 2+ or Mn 2+ concentrations in the presence (closed symbols) or absence (open symbols) of 1.0 m M free Ca 2+. (A) MgE+-dependence: O, without Ca2+; e, with Ca 2+. (B) Mn2+-dependence: zx, without Ca2+; A, with Ca 2+.

72

centrations: 0.5 M K2HPO 4, ( N H 4 ) 2 8 0 4 , NaC1 and KC1 inhibit by 100, 98, 91 and 91%, respectively (cf. Ref. 14). The partially-purified preparations of protein kinase II are absolutely dependent upon added substrate protein for activity. With 1 mg/ml protein substrate present, the rates of phosphorylation of histones and phosvitin are 20% and 7%, respectively, of the rate with dephosphorylated casein as substrate. The major wheat-germ cytokinin-binding protein [20] is not a substrate for protein kinase II. Calmodulin at micromolar concentrations markedly activates phosphorylation of histones but not of casein or phosvitin (cf. Ref. 14). Calmodulin (7.3 ~M) activates histone phosphorylation by 100% or more at histone concentrations between 0.2 and 2 m g / m l (Fig. 5). As found for protein kinase I [14], calmodulin inhibits histone phosphorylation at lower histone concentrations (below 0.2 mg/ml) and does not substantially activate at high histone concentrations (above 5 mg/ml) (Fig. 5). Calmodulin activates the protein kinase at micromolar concentrations but maximum activation is not obtained at concentrations up to 14.5 /~M wheat-germ calmodulin (which gives 6.5-fold activation). Calmodulin is not a substrate for the protein kinase as determined from electrophoresis/autoradiography experiments and direct analysis in the standard protein kinase assay conditions. Electrophoresis of 32p_ labelled casein and subsequent autoradiography revealed major 32p-labelled casein polypeptides with molecular weights of 24000, 26000 and

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Fig. 6. Inhibition of the Ca2+-dependent protein kinase by

phenothiazine derivatives. Protein kinase was assayed as described in the legend to Table I in the presence of various concentrations of fluphenazine ( O ) or chlorpromazine (e). Protein kinase activity is expressed as percentage of control activity (no added phenothiazine derivative). The error bars indicate standard deviations from triplicate determinations.

30000. From a similar analysis, the major 32p_ labelled histone polypeptides have molecular weights of 16000 and 15000 and calmodulin increases the labelling of both of these polypeptides in the standard reaction conditions. The histone

TABLE II D I F F E R E N T I A L I N H I B I T I O N OF TH E W H E A T - G E R M CALCIUM-DEPENDENT PROTEIN K I N A S E S BY NUCLEOTIDES

Ca2+-dependent protein kinases I and II were assayed in the standard reaction medium containing 1 m g / m l casein and 0.5 mM free C a 2+ in the presence or absence of the indicated compound. Addition

Protein kinase (% control) Protein kinase 1

Protein kinase lI

0.1 mM

1.0 mM

0.1 mM

1.0 mM

inhibitor

inhibitor

inhibitor

inhibitor

None

100

100

100

100

2'AMP 3'AMP 5'AMP

102 99 29

97 88 11

99 99 100

99 94 79

ADP CDP UDP XDP

22 34 29 24

11 6 5 5

69 108 113 101

18 105 103 101

G TP CTP UTP ITP ATP

24 23 19 21 24

7 4 3 2 5

87 107 93 97 30

64 96 90 90 5

(rng/rnl)

Fig. 5. Dependence of calmodulin activation on histone con-

centration. Protein kinase was assayed in triplicate in the standard assay containing the indicated histone concentrations and 0.8 mM free Ca 2+ in the presence (O) or absence ( O ) of 7.3 /~M wheat embryo calmodulin.

73 concentration for half-maximal phosphorylation is approx. 0.05 m g / m l and the rate of histone phosphorylation decreases at very high histone concentration (Fig. 5). The Lineweaver-Burk plot of casein phosphorylation as a function of casein concentration is linear and the K m for casein is 1.3 mg/ml. The K m for ATP with casein as substrate is 16 /~M, as determined from Lineweaver-Burk analysis. The acid hydrolysate of 32p-labelled casein from a reaction catalyzed by protein kinase II was chromatographed on Dowex 50W-X8 as described previously [14]: 30% of applied radioactivity was associated with inorganic phosphate, 27% with phosphoserine and 4% with phosphothreonine. Protein kinase II is inhibited by relatively high concentrations of the phenothiazine-derived calmodulin antagonists fluphenazine and chlorpromazine (Fig. 6), as is protein kinase I [14]. These inhibitions may derive from hydrophobic interactions not necessarily involving calmodulin (for discussion see Refs. 6 and 7). Thus, the calmodulin antagonist trifluoperazine does not inhibit phosphorylase b kinase (containing tightly bound calmodulin subunits) in the absence of added calmodulin [23] whereas Ca2÷-dependent phospholipid-activated protein kinase (which does not contain a calmodulin subunit [24,25]) is inhibited by trifluoperazine [26], fluphenazine [26] and chloropromazine [26,27]. Hemin and adenosine, inhibitors of several Ca2+-independent wheat-germ protein kinases [20,21] and of protein kinase I [14], inhibit protein kinase II by 97% and 48%, respectively, at 1 mM concentration. Protein kinase II (freed of 2-mercaptoethanol by gel filtration) is activated 5.7-fold by addition of 10 mM dithiothreitol or 10 mM 2-mercaptoethanol and is inhibited 95% by 1 mM N-ethylmaleimide. The ratio of activity with histone to activity with casein (1 m g / m l substrates) is 0.2 for protein kinase II as compared to 1.0 for protein kinase I. Addition of 1 m g / m l histones to a reaction mixture containing 1 m g / m l casein and protein kinase I results in less than additive total protein phosphorylation. In contrast, addition of 1 m g / m l histones to a reaction mixture containing 1 m g / m l casein and protein kinase II results in an approx. 2-fold stimulation of protein phosphorylation.

Protein kinase I (from wheat germ chromatin) [12,14] is very similar to the present protein kinase II in many properties. However, the two enzymes differ markedly in sensitivity to a variety of nucleotides (Table II). In particular, protein kinase I is inhibited by nucleoside 5'-diphosphates and nucleoside 5'-triphosphates that have little or no effect on the activity of protein kinase II (Table II). The differential sensitivity of the two enzymes to nucleoside 5'-triphosphates was employed to confirm the difference in apparent molecular weight of protein kinase I (M r 90000) [14] and protein kinase II (M r 86000) as determined from gel filtration. On gel filtration of a mixture of the two enzymes, the CTP-sensitive protein kinase I elutes just prior to the CTP-insensitive protein kinase II. No CTPase activity was detectable in either protein kinase preparation. We conclude that protein kinases I and II represent two distinct (if possibly structurally related) wheat-germ Ca 2+ dependent protein kinases. The concentrations of phenothiazine-derived calmodulin antagonists required for 50% inhibition of protein kinases I and II are similar to the concentrations required for 50% inhibition of plant developmental responses to certain phytohormones and red light [6,7]. Ca 2+-dependent protein kinases of the kind described here are therefore conceivably - but not necessarily - involved in such developmental processes. The very low free Ca 2+ concentrations for basal (approx. 0.1 btM) and maximal (2-3 /~M) casein phosphorylating activity of protein kinase I [14] and protein kinase II (Fig. 2) are similar to the free cytosolic Ca 2+ concentrations found for unexcited (0.2 /~M) and electrically stimulated (6.7 ~tM) plant cells [4], respectively. Thus, these types of CaZ+-dependent plant protein kinase may be involved in plant cellular responses to elevation in cytosolic free Ca 2+ concentration induced electrically [4], by Ca2+-ionophore A23187 [13,28-30] or by red light via the phytochrome system [6,31].

Acknowledgement This work was supported by a grant from the Australian Research Grants Scheme.

74

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