Resolution of three Ca2+-dependent protein kinases and endogenous substrate proteins from bitter gourd seeds

ELSEVIER

Plant

Science 105 (1995) 31-44

Resolution of three Ca2’-dependent protein kinases and endogenous substrate proteins from bitter gourd seeds Alan Chang, Gregory M. Neumann, Gideon M. Polya* Department of Biochemistry, LA Trobe University, Bundoora, Victoria 3083. Australia

Received

I August

1994; revision

received 7 November

1994; accepted

22 November

1994

Abstract A protein that is phosphorylated by plant Cat+ -dependent protein kinase (CDPK) was isolated from seeds of bitter gourd (Momordica charantiu). This bitter gourd protein (referred to as BGP) was purified by a procedure involving batchwise elution from carboxymethylcellulose (CM-52), gel filtration, cation exchange HPLC and reversed phase HPLC. BGP preparations exhibit three bands (11 kDa, 7 kDa and 4 kDa, respectively) on SDS-PAGE, of which the 4-kDa material copurifies on SDS-PAGE with [32P]phosphoBGP phosphorylated by wheat CDPK or CDPKs isolated from M. charuntiu seeds. The 4-kDa [32P]phosphoBGP can be resolved from the other components after phosphorylation using [Y-~‘P]ATP and CDPK and subsequent reversed phase HPLC. Electrospray ionization mass spectrometry (ESMS) of BGP revealed a major component with an average molecular mass of 11450 Da, minor 11287 Da and 11 563 Da components and other minor components corresponding to K+ adducts of the major component. Reversed phase HPLC of BGP after treatment with 3 M guanidine HCl and 25% (v/v) 2-mercaptoethanol resolves three very similar small proteins (BGSl, BGS2 and BGS3) having average molecular masses of 3443 Da, 3606 Da and 3720 Da, respectively, and a larger protein (BGL) having an average molecular mass of 7850 Da. The 11287-Da, 11 450-Da and 11 563-Da BGP components correspond within experimental error to 1:l complexes of the large subunit (BGL) with a small subunit (BGSl, BGS2 or BGS3, respectively), with all complexes involving approximately three disulphide linkages (calculated masses 11287 & 2 Da, 11 450 f 1 Da and 11 563 f 2 Da, respectively). The 3606-Da BGS2 is a poor CDPK substrate in comparison with BGSl and BGS3. Three CDPKs (CDPKs I, II and III) were resolved from M. charuntiu. All three CDPKs are absolutely dependent on millimolar Mg2+ and about lo-‘- 10e6 M free Ca2+ for maximal activity and phosphorylate BGP, histone III-S, bovine serum albumin, casein and myosin light-chain derived synthetic peptide (MLCP) (KKRAARATSNVFA-NH,). CDPKs I and II phosphorylate the synthetic peptide kemptide (LRRASLG) better than CDPK III. CDPKs I, II and III are inhibited by poly+arginine (IC,, values 52, 70 and 43 nM, respectively) and the calmodulin antagonist calmidazolium (IC,, values about 30 PM). Keywordr: Momordica charantia; Phosphoprotein;

Protein kinase; Ca2+

Abbreviations: BGL, bitter gourd protein large subunit; BGP, bitter gourd protein kinase substrate protein; BGS, bitter gourd EGTA, ethyleneprotein small subunit; BSA, bovine serum albumin; CDPK, Ca *+-dependent protein kinase; DTT, dithiothreitol: bis(aminoethylether)N,N,N’,N’-tetraacetic acid; ESMS, electrospray ionization mass spectrometry; HPLC, high performance liquid chromatography; MES, 2-(N-morpholino)ethanesulfonate; MLCP, myosin light chain-based synthetic peptide KKRAARATSNVFA-NH,; PKA, cyclic AMP-dependent protein kinase; PMSF. phenyl-methylsulfonylfluoride; PTH, phenylthiohydantoin; QMA, quatemary methylamine; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TPCK. L-(I-tosylamido-2-phenyl) ethyl chloromethyl-ketone; W-7, N-(6-aminohexyl)-5-chloro-2-naphthalenesulfonamide. l Corresponding author.

Ol68-9452/95/$09.50 0 1995 Elsevier Science Ireland SSDI 0168-9452(94)04039-J

Ltd. All rights reserved

32

A. Chang et al. /Plant Science 105 (1995) 31-44

1. Introduction Ca2+ acts as an intracellular second messenger for external signals in plant cells [l-4] as in other eukaryotes [5]. Signal-induced transient elevation of cytosolic free Ca2+ concentration results in biochemical responses through the regulation of cellular processes by the Ca2+-binding protein calmodulin and by Ca*+-dependent protein kinases (CDPKs) [l-5]. Clearly resolution of CDPKmediated signalling pathways will require characterization of the CDPKs involved and definition of the sites and functional consequences of CDPKcatalysed protein phosphorylation. Soluble and membrane-located CDPKs have been resolved from a variety of plant sources [2,4] and several have been cloned and sequenced [6,7]. Histone Hl is one of the best CDPK substrates found [2,4,8] and the histone Hl-rich histone III-S preparation is phosphorylated by all plant CDPKs yet resolved [2,4]. However, other proteins phosphorylated by plant CDPKs include H+-ATPase [9], nodulin 26 [ 10,111, radish calmodulin antagonists [ 121, lipid transfer proteins [13,14] and soybean BowmanBirk trypsin inhibitor BBI-1 [15]. Plant CDPKs phosphorylate various synthetic peptides having a basic-X-X-Ser(Thr) motif [4,8,10] or a Ser(Thr)-X-basic motif [16]. Indeed substrate protein CDPK-catalysed phosphorylation sites having Ser(Thr)-X-basic [8,15], Ser(Thr)X-X-basic [ 11,151 or basic-X-X-Ser-X-X-basic [ 1l] motifs have been determined as well as sites having no nearby N-terminal or C-terminal basic residues [12,14]. The basic-basic-X-Ser(Thr) sequence is recognized by animal cyclic AMPdependent protein kinase (PKA) [ 17,181 and thus the synthetic peptide kemptide (LRRASLG) is a very good substrate for PKA [19]. However, kemptide is either not phosphorylated by resolved plant CDPKs or is a very poor substrate for plant CDPKs [2,4,8,20-221. Nevertheless, certain plant protein kinases have substrate specificities overlapping that of PKA. Thus plant phosphoenolpyruvate carboxylase is phosphorylated on its key regulatory site by animal PKA [23] and a multiplicity of plant substrates for PKA have been detected [24], A Ca2+-independent protein kinase resolved from Petunia phosphorylates kemptide

[25]. However, while a number of potential elements of a cyclic nucleotide regulatory system are present in plants, no cyclic nucleotide-dependent protein kinases have been resolved from plants 1251. The present paper describes the resolution of three bitter gourd (Momordica charantia) CDPKs of which two phosphorylate kemptide at substantial rates relative to histone III-S phosphorylation. These CDPKs are also shown to phosphorylate endogenous bitter gourd proteins (BGPs) isolated from M. charantia seeds. These CDPK substrate proteins were initially detected through the screening of plants for good CDPK substrates in order to define targets of CDPK-mediated signalling. The BGPs and the constituent subunits have been purified and properties of the endogenous bitter gourd CDPKs examined. 2. Materials and methods 2.1. Materials Seeds of bitter gourd (Momordica charantia L.) were obtained locally. [T-~~P]ATP (4000 Cii mmol) was obtained from Bresa (Adelaide, Australia). Nucleotides, dephosphorylated casein, histone III-S, bovine serum albumin, dithiothreitol (DTT), phenylmethylsulfonylfluoride (PMSF), trifluoperazine, N-(6-aminohexyl)-5-chloro-2-naphthalenesulfonamide (W-7), calmidazolium, poly-Llysine, poly+ornithine and poly+arginine were obtained from Sigma Chemical Co. MLCP (KKRAARATSNVFA-NH2) and kemptide (LRRASLG) were obtained from Auspep (Melbourne, Australia). An Aquapore RP300 (C8) reversed phase HPLC guard column (4.6 mm x 30 mm; 7 Frn particle size) was obtained from Brownlee Laboratories and a Bakerbond C8 column (4.6 mm x 25 cm; 5 pm particle size) was obtained from J.T. Baker Research. QMA anion exchanger and an SP-5PW cation exchange column (4.6 mm x 75 mm) were obtained from Waters. 2.2. Purification of bitter gourd CDPK substrate protein (BGP) Eighty grams of fresh seed was used as the starting material. All operations were performed at 4°C

A. Chang et al. /Plant

unless stated otherwise. The seeds were ground in a mortar and pestle after freezing with liquid N,. A 320-ml quantity of buffer A (10 mM phosphate (Na+, pH 6.5), 10 mM 2-mercaptoethanol) containing 0.1 mM EGTA, 0.5 mM phenylmethylsulfonylfluoride (PMSF) and 0.25% (v/v) ethanol was added to the ground seed before blending in a Sorvall blender at maximum speed for 1 min. The resultant homogenate was filtered through two layers of cheese cloth and centrifuged at 15 000 x g for 30 min. The supcrnatant fraction was filtered through one layer of Miracloth and applied to 15 g of CM-cellulose (CM-52) that had been equilibrated with buffer A and the eluate (containing CDPK) was retained. The CM-52 was washed with 500 ml of buffer A followed by elution with 250 ml of 50 mM Tris (Cl-, pH 8.0), 10 mM 2mercaptoethanol, 1.0 M NaCl. This fraction, which contains the bitter gourd CDPK substrate (BGP), was concentrated by pressure filtration (Amicon YM3) to about 3 ml and applied to a Sephacryl S-ZOOHR gel filtration column (2 cm2 x 60 cm) which was eluted with 50 mM Tris (Cl-, pH 8.0), 10 mM 2-mercaptoethanol, 50 mM NaCl. Fractions of 4 ml were collected and assayed for activity as substrates for wheat CDPK. Peak activity fractions were pooled, concentrated by pressure filtration, desalted in 50 mM 2-(Nmorpholino)ethanesulfonate (MES) (Na+, pH 6.5), reconcentrated to about 4 ml and subjected to cation exchange HPLC. The desalted protein solution containing BGP was applied to an SP-5PW column (4.6 mm x 75 mm) that had been equilibrated in 50 mM MES (Na+, pH 6.0) at 25°C. The column was eluted with a linear gradient of increasing concentration of NaCl from 0 to 500 mM in 50 min at a flow rate of 1 ml/min. Fractions were assayed for CDPK substrate activity using the wheat CDPK. The peak activity fraction was subjected to further purification by reversed phase HPLC. The protein solution was brought to 0.2% TFA, filtered (0.2 pm filter) and applied to a C8 Bakerbond column (4.6 mm x 2.50 mm) equilibrated in O.l”/;,TFA. The column was eluted at room temperature with a linear gradient of increasing CHsCN concentration in 0.1% TFA from 0% to 60% in 60 min. BGP eluted at about 33% CH$N in 0.1% TFA.

Science 105 (1995) 31-44

33

BGP was further purified after pre-phosphorylating the protein using [y-32P]ATP and wheat CDPK. Concentrated and desalted BGP, purified by cation exchange HPLC, was phosphorylated by wheat CDPK in the standard CDPK assay (see below) but with a l-ml reaction mixture volume. After 60 min incubation the reaction mixture was brought to 0.1% TFA in a 3-ml volume and applied to a Sep-Pak Cl8 cartridge pre-washed with 5 ml of CH$N and 5 ml of 10 mM ATP-0. 1% TFA. The cartridge was then washed with 30 ml of 10 mM ATP-0.1% TFA followed by 30 ml of 0.1% TFA before elution of phosphorylated protein with 5 ml of 0.1% TFAdO% CH$ZN in 1 ml fractions. The fractions were counted and those having the most radioactivity were concentrated using a Spe.edVac concentrator, pooled and reconcentrated to about 500 gl. The solution was brought to 3 M guanidine HCl in 1 ml volume, heated at 100°C for 5 min, acidified with TFA to O.l%, centrifuged at 11 000 x g for 10 min and subjected to reversed phase HPLC on a Bakerbond C8 column as previously described. 2.3. Protein phosphorylation Wheat germ CDPK (specific activity 0.014 ~mol/min/mg protein with 1.O mg/ml histone III-S as substrate), was partially purified as described previously [26] by an extensive procedure involving successive chromatography on DEAEcellulose (Whatman DE-52) phenyl Sepharose CL-4B (involving elution in the absence of Ca’+), Cibacron F3GASepharose 4B and on Ultrogel AcA44 (yielding a single peak of CDPK). CDPK was assayed at 30°C as described previously [8], the standard reaction medium (100 ~1) containing 62.5 mM Tris (Cl-, pH 8.0), 10 mM MgC12, 10 mM dithiothreitol, 2.5 mM 2-mercaptoethanol, 0.25 mM EGTA, 1.0 mM CaC12, 25 PM ATP (specific activity of [Y-~~P]ATP about 30 Ci/mol), protein substrate and CDPK. In the case of assays to examine the effect of CaM antagonists, the inhibitors were dissolved in DMSO and added to the assay with a final DMSO concentration of 20% (v/v). To measure the phosphorylation of proteins other than histone III-S, appropriate substrates or protein extracts were added instead of histone IIIS. Assays conducted in the presence of Ca2’ con-

34

A. Chang et al. /Plant

tained 0.25 mM EGTA and 1 mM Ca2+, whereas assays in the absence of Ca2+ contained only 0.25 mM EGTA. 2.4. Partial purification of CDP& I, IIand IIIfrom bitter gourd seed

The seed extract fraction that did not bind to CM-cellulose in the BGP isolation protocol described above and which contains the majority of CDPK activity was adjusted to a pH of about 8.0 with 3 M Tris base and applied to 15 g of DEAE-cellulose (Whatman DE-52) that had been equilibrated with buffer B (50 mM Tris (Cl-, pH 8.0), 10 mM 2-mercaptoethanol). The DE-52 was washed with 11 of buffer B before elution with 250 ml of buffer B containing 0.8 M NaCl, 0.25 mM PMSF and 0.125% (v/v) ethanol. CaC12 was added to this fraction to a final concentration of 2 mM and the solution was applied to 50 ml bed volume of Phenyl-Sepharose CL4B that had been equilibrated with buffer B, 0.5 mM CaC12. The matrix was washed with 1 1 of the same buffer before elution of Ca2+-dependent binding proteins with 300 ml of buffer B-2 mM EGTA. This fraction was concentrated by pressure filtration (Amicon Y M 10 membrane) to about 4 ml and was further purified by either gel filtration on an Ultrogel AcA 44 column (7 cm2 x 55 cm) in 50 mM Tris (Cl-, pH 8.0), 10 mM 2-mercaptoethanol, 0.2 mM EGTA or by anion exchange chromatography on a QMA (quaternary methylamine) column as described below. The fraction binding to Phenyl-Sepharose CL4B and eluted in buffer B-2 mM EGTA was concentrated to about 4 ml, diluted 2-fold with water and subjected to anion exchange column chromatography on a QMA (quaternary methylamine) column at 4°C. The protein solution was applied to a QMA column (bed volume 15 ml) that had been equilibrated in buffer C (25 mM Tris (Cl-, pH 7.5) 1 mM EGTA). The column was washed with 50 ml of buffer C at a flow rate of 1 ml/min and then eluted with a gradient of increasing NaCl concentration in buffer C (from 0 to 1 M over a period of 6 h at a flow rate of 0.6 mumin). This gradient elution was achieved using the BioRad Econo system. This procedure resolved three peaks of CDPK activity referred to as CDPKs 1,

Science 105 (1995) 31-44

II and III, respectively. Protein was determined by the method of Sedmak and Grossberg [27]. 2.5. SDS-PAGE and phosphoamino acid analysis SDS-PAGE was conducted using a Bio-Rad Minigel apparatus employing a 16% polyacrylamide gel and following the procedure of Schagger and Jagow [28]. Proteins were visualised by staining the gels with Coomassie Blue. For autoradiography the gels were dried between two pieces of cellophane and then exposed to Kodak AR film at -70°C using a cassette with an intensifier screen. Phosphoamino acid analysis of [ 32P]phosphoBGP was conducted essentially as described previously [ 151. High voltage electrophoresis (3 kV) was conducted for 3 h at pH 2.6 (running buffer containing 0.2% (v/v) pyridine and 10% (v/v) glacial acetic acid). The key phosphoamino acid migration positions were determined by inclusion of 600 nmol each of phospho+tyrosine, phospho-L-threonine and phospho-L-serine and detection by ninhydrin as described previously [29]. 2.6. Amino acid sequencing and electrospray ionization mass spectrometry (ESMS)

Amino acid sequencing by sequential Edman degradation was conducted using an Applied Biosystems 470A gas phase peptide sequenator and an applied Biosystems 13OAseparation system for automatic on-line analysis of PTH amino acids. Proteins (100-1000 pmol in 10 ~1 of MeOH/H20/acetic acid, 49.5:49.5:1) were introduced at a flow rate of 3 kl/rnin into a VG BIOQ electrospray mass spectrometer (VG Biotech, Cheshire, UK). The first quadrupole of the mass spectrometer was repeatedly scanned from m/z 500 to m/z 1500 (10 s per scan) over several minutes to obtain signal-averaged spectra at approximately unit mass resolution. The mass scale was calibrated using the multiply-charged ions from the separate introduction of myoglobin. 3. Results 3.1. Purification of bitter gourd CDPK substrate protein (BGP)

A bitter gourd protein fraction (BGP) that contains CDPK substrates was extensively purified by

A. Chang et al. /Plant

1 l5 z 2 10

T m :

-5 CL

35

Science IO5 (1995) 31-44

a procedure successively involving batchwise chromatography on CM-cellulose, gel filtration on Sephacryl S-200 HR, cation exchange HPLC on an SP-5PW column and reversed phase HPLC

a

kD 97.4 66.0 45.0 29.0 20.1 14.3’7.8 3.4 -

b

El&on

time

12

3

1 2

3

kD

(mln)

Fig. 1. Purification of bitter gourd CDPK substrate protein (BGP). (a) Gel filtration of BGP on Sephacryl S-ZOOHRin 50 mM Tris (Cl-, pH 8.0), 50 mM NaCI. 0, protein concentration; n, wheat CDPK activity determined using 20 ~1 of the indicated fraction as substrate (relative protein kinase activity given as counts/mm incorporated into phosphoprotein). (b) Cation exchange of BGP on a SP-SPW column eluted with a gradient of increasing NaCl concentration in 50 mM MES (Na+, pH 6.5). Continuous trace, A,,,; histogram height indicates CDPK activity (counts/min incorporated into phosphoprotein) with 20 pl of the indicated fraction as substrate; the straight line indicates NaCl concentration. (c) Puritication of BGP by reversed phase HPLC. BGP purified by cation exchange HPLC (Fig. lb) was subjected to reversed phase HPLC on a C8 column eluted with a gradient of increasing CHsCN concentration in 0.1% TFA. Continuous trace, Also; the straight line indicates CHsCN concentration.

Fig. 2. SDS-PAGE and autoradiography of BGP. (a) BGP by cation exchange HPLC by reversed phase HPLC I, position of M, standards; lane 2, Coomassie Blue-stained gel; 3, autoradiograph of lane 2 electropherogram.

36

A. Chang et al. /Plant

Science 105 (199s)

(Fig. 1). One peak of CDPK substrate activity containing BGP is resolved by gel filtration (Fig. la). Subsequent purification of the peak activity fractions by cation exchange HPLC yields two major protein peaks of which the second contains the majority of CDPK substrate activity (Fig. lb). This preparation shows multiple bands (4 kDa, 7 kDa and 11 kDa, respectively) on SDS-PAGE analysis (Fig. 2a). Reversed phase HPLC of this material on a C8 column resolves a single protein peak eluting at around 33% CHjCN (Fig. lc). However, this preparation still contains three

,

Elution

b

time

31-44

polypeptides (4 kDa, 7 kDa and 11 kDa, respectively) as resolved by SDS-PAGE (Fig. 2b), suggesting that all three entities derive from an II-kDa complex (see below). Of these three entities, the CkDa material is clearly phosphorylated by wheat germ CDPK (Fig. 2b). With 1 mg/ml of BGP preparation (isolated by cation exchange HPLC) as protein substrate, wheat embryo CDPK activity is 73% of the rate with 1 mg/ml histone IIIS, noting that histone III-S is one of the best substrates for wheat embryo CDPK [8]. The K,,, for BGP was not determined because of the heter-

C

100

kD

d

(min)

25

0

20

30

Elution

Fig. 3. Purification

time

40

(min)

50

1.5

25

35

Elutlon

time

45

55

(mln)

of [32P]phosphoBGP by reversed phase HPLC. Cation-exchange HPLC-purified BGP (Fig. lb) was phosphorylated using [Y-~‘P]ATP and wheat CDPK and subjected to reversed phase HPLC on a C8 column eluted with a gradient of increasing CH,CN concentration in 0.1% TFA as described in Section 2. (a) Initial reversed phase HPLC. Continuous trace, A2s0; histogram height, counts/min from Cerenkov counting of fractions. (b) Final purification of [32P]phosphoBGP. Continuous trace, A,,,; histogram height, counts/min. (c) SDS-PAGE and autoradiography of purified [32P]phosphoBGP from (b). Lane 1, position of M, standards; lane 2, Coomassie Blue-stained gel; lane 3, autoradiograph of lane 2 electropherogram. (d) Final puritication of BGSs 1, 2 and 3 by reversed phase HPLC on a Cl8 column of cation exchange-purified BGP (Fig. lb) after treatment at 100°C for 5 min in the presence of 0.25% (v/v) 2-mercaptoethanol and 3 M guanidine HCI. The fractions were concentrated 3-fold using a SpeedVat vacuum concentrator and 25 ~1 aliquots were assayed for CDPK substrate activity in the standard assay conditions. Relative CDPK activity is presented as histograms (counts/min); continuous trace, A,,,; inclined straight line, CHsCN concentration.

A. Chang et al. /Plant

ogeneity of the preparation. Phosphoamino acid analysis of BGP (isolated by cation exchange HPLC) and phosphorylated by wheat embryo CDPK was conducted as described in Section 2 and showed that the amino acid phosphorylated is serine. No peaks corresponding to phosphothreonine or phosphotyrosine were detected in this procedure. Electrospray ionization mass spectrometry (ESMS) applied to the BGP preparation purified up to and including the cation exchange step (Fig. lb) revealed a major component with an average molecular mass (*95% confidence limits) of 11 450.4 f 0.9 Da, as well as two lesser components with average molecular masses of 11 563.0 i 1.7 Da and 11 287.3 f 2.4 Da. Minor components corresponding to dipotassium adducts of these three proteins were also observed in some spectra. The three 1I-kDa proteins clearly correspond to the 11-kDa component seen on SDS-PAGE (Fig. 2a); however, the 4-kDa and 7kDa components observed on SDS-PAGE were not evident in ESMS spectra of intact BGP, confirming that they derive from 1 1-kDa BGP. Final purification of 4-kDa material from BGP preparations was accomplished by a procedure involving preparation of [ 32P]phosphoBGP using [T-~~P]ATP and wheat CDPK in the standard reaction conditions described in Section 2. It should be particularly noted that these reaction conditions include the presence at 10 mM concentration of the reductant dithiothreitol (DTT). Subsequent reversed phase HPLC of this material as described in Section 2 resolved three major protein peaks of which the earliest eluting peak elutes just prior to a major peak of radioactivity (Fig. 3a). Further purification of the [32P]phosphoprotein peak material near the earliest eluting protein peak yielded coincident zones of A,,, and radioactivity (Fig. 3b). SDS-PAGE and autoradiography of this material revealed a single radioactive band of 4 kDa (Fig. 3c), but there was too little of the actual protein for detection of protein in the gel. The stoichiometry of phosphorylation of this material is about 1.3 mol/mol 4-kDa protein (assuming A,,, of 20 corresponding to 1 mg/ml and an M, of 4000). We conclude that this procedure has resolved near-stoichiometrically phosphorylated 4-kDa

Science IO5 (1995)

31-44

37

protein. Purification of substrate proteins without prior phosphorylation involved treatment of the cation exchange-purified BGP at 100” for 5 min in the presence of 0.25% (v/v) 2-mercaptoethanol and 3 M guanidine HCl followed by reversed phase HPLC on a Cl8 column eluted with a linear gradient of increasing CH3CN concentration in 0.1% TFA (Fig. 3d). This procedure resolves three fractions having activity as substrates of wheat CDPK of which fractions 1 and 3 are the most active (Fig. 3d). Electrospray ionization mass spectrometry of fractions 1, 2 and 3 revealed in each case a single major entity with average molecular masses (&95% confidence limits) of 3442.9 f 1.3 Da, 3606.3 f 0.7 Da and 3719.5 f 1.2 Da, respectivkly. We have denoted these three major components BGSl, BGS2 and BGS3, respectively. Each fraction also contained a minor entity corresponding in each case to the dipotassium adduct of the major component. The average molecular mass differences between BGPs 1 and 2 and between BGPs 2 and 3 (Fig. 3d) are 163.4 Da and 113.2 Da, respectively. These differences do not correspond to single amino acid substitutions. However, these differences could correspond simply to loss of Y (difference, 163.1 Da) and loss of L or I (difference, 113.1 Da), respectively. This would be consistent with the order of elution of BGSs l-3 from a Cl8 column on reversed phase chromatography (Fig. 3d), i.e. 1 (-Y and -I/L), 2 (-I/L) and then 3 (the most hydrophobic of the three). The lack of absorbance at 280 nm of 1 but not of 2 and 3 (Fig. 3d) is also consistent with a lack of Y in 1 but not in 2 and 3. N-terminal sequencing of BGS 1, BGS2 and BGS3 was unsuccessful, indicative of Nterminal blockage. The major protein component resolved by reversed phase HPLC of reduced BGP and eluting at about 45% CH3CN in 0.1% TFA (Fig. 3a,d) was analysed by ESMS and shown to have an average molecular mass ( f 95% confidence limits) of 7850.1 f 0.9 Da. This corresponds to the approximately 7-kDa component seen on SDSPAGE of reduced BGP (Fig. 2a,b). We have denoted this entity as BGL. As observed with the BGS polypeptides, N-terminal sequencing of 500 pmol of purified BGL was unsuccessful, indicating N-terminal blockage.

A. Chang et al. /Plant Science 105 (1995) 31-44

38

a complex with BGL (7850.1 f 0.9 Da) the resultant complex would have a mass of 11 456.4 f 1.6 Da, 6 Da greater than that of the major BGP component (11 450.4 f 0.9 Da), consistent with three disulphide linkages in the oxidized BGSZBGL complex. Similarly BGS 1-BGL and BGS3-BGL complexes involving three disulphide bonds would have average molecular masses of 11 287.0 f 2.2 Da and 11 563.6 f 2.1 Da, as compared with the observed masses of BGP components of 11 287.3 f 2.4 Da and 11 563.0 f 1.7 Da, respectively. We conclude that the major 11 450 Da component and the minor 11 287 Da and 11 563 Da components of the BGP preparations are 1:1 complexes of a large subunit (BGL) with a small subunit (BGS2, BGSl and BGS3, respectively), all involving approximately three disulphide linkages.

0

10

20

30 Froctlan

40

50

60

70

number

Fig. 4. Resolution of bitter gourd CDPKs by gel filtration and anion exchange chromatography. The CDPK fraction eluted from Phenyl-Sepharose CL4B was applied (a) to an Ultrogel AcA44 column eluted with 50 mM Tris (Cl-, pH 8.0), IO mM 2-mercaptoethanol, 0.2 mM EGTA or (b) a QMA column eluted with a gradient of increasing NaCl concentration in 25 mM Tris (Cl-, pH 8.0), I mM EGTA. Protein kinase was assayed in the standard assay conditions with I mg/ml histone III-S as substrate. Cl, protein concentration; m, CDPK activity; straight line, NaCl concentration.

The occurrence of 4-kDa, ‘I-kDa and 11-kDa components in the BGP preparation as revealed by SDS-PAGE of cation exchange- or HPLC-purified BGP (Fig. 2a), suggested the possibility that the 1I-kDa component might be a complex of a small 4-kDa subunit and a large 7-kDa subunit. Very precise support for this is provided by analysis of the ESMS-derived average molecular masses of the three 11-kDa BGP components and of the large (BGL) and small (BGSl, BGS2 and BGS3) components resolved by reversed phase HPLC of reduced BGP (Fig. 3d). Thus if the major BGS component (BGS2, mass 3606.3 f 0.7 Da) forms

3.2. Resolution of CDPKS I, II and IIIfrom bitter gourd seeds Ca*+-dependent protein kinase from the seed of bitter gourd was extensively purified by a protocol employing anion exchange chromatography and Ca*+-dependent hydrophobic chromatography, the latter process being a key step in the procedure. The fraction binding to Phenyl-Sepharose CL4B in a Ca*+-dependent fashion and eluted by 2 mM EGTA contains protein kinase activity that is essentially Ca *+-dependent. Only one peak of protein kinase activity was resolved when this fraction was chromatographed on an Ultrogel AcA 44 gel filtration column (Fig. 4a). The apparent molecular mass of the CDPK is about 55 000 as determined by calibration of the gel filtration column using standards of known M,. However, anion exchange chromatography of the CDPK from the Phenyl-Sepharose CL4B step on QMA in the presence of 1 mM EGTA resolved three peaks of CDPK activity (Fig. 4b). C&-dependent protein kinases eluting at around 0.2,0.25 and 0.3 M NaCl are denoted here as CDPK I, CDPK II and CDPK III, respectively. The specific activities of CDPK I, II and III were 9.5, 10.3 and 14.2 nmol/mg protein/mm, respectively, representing purifications of 500-800-fold (Table 1). An attempt was made to see if reversed phase HPLC using a CHsCN gradient in 0.1% TFA

A. Chang et al. /Plant

Science IO5 (199.5) 31-44

39

Table 1 Partial purification of CDPKs from the seed of bitter gourd Step

Total protein (mg)

Purification (fold)

Protein kinase activity

Total activity (nmol/min)

Specific activity (nmol/mg proteimmin)

High speed supematant DE-52

Phenyl-Sepharose

1554

25

437

232

0.53

33

69

5.1

317

8.9 10.0 13.4

556 623 838

13.6

0.016

QMA CDPK I CDPK II CDPK II

0.21 0.29 0.26

I.9 2.9 3.5

The CDPKs were purified from 80 g of seed. Protein kinases were assayed in duplicate in the standard CDPK assay with I mg/ml histone III-S as substrate. The activities represent the difference between assays conducted in the presence of Ca2+ (0.25 mM EGTA and I.0 mM CaCI, in the assay) or the absence of Ca2+ (0.25 mM EGTA in the assay).

could be used to resolve a CDPK polypeptide that could be partially renatured by subsequent removal of CH,CN in vacua and addition of 10 mM Tris (Cl-, pH 8.0). After the initial chromatographic separation a peak of CDPK activity was obtained after renaturation in material

eluting at 60% CH$N in 0.1% TFA. However, after subsequent rechromatography of this material a single peak of material absorbing at 220 nm was obtained but the yield was very low (estimated to be about 0.5 pg) and no CDPK activity was detected after renaturation. 3.3. Characterization of bitter gourd CDPKs The activities of three bitter gourd CDPKs resolved by QMA column chromatography are highly dependent on Ca 2+. There is very little or no detectable protein kinase activity in the absence

.

I

. . 09

,-----

6

p&2+

12

18

_1 24

.

. .

[Mg*‘] CmM)

Fig. 5. Dependence on Ca2+ and Mgz+ concentration for the activity of bitter gourd CDPKs. (a) Protein kinase was assayed in the standard conditions but with I mg/ml bovine serum albumin (BSA) as substrate, and with 0.45 mM EGTA present. CaCI, was added to give the indicated free Ca*+ concentrations [291.(b) Protein kinase was assayed in the standard assay conditions (i.e. with I .O mM CaC12 and 0.25 mM EGTA present) but with I mdml BSA as substrate and at the indicated Mg2+ concentrations. W, CDPK I; 0, CDPK II; A, CDPK III.

Fig. 6. Dependence on pH of bitter gourd CDPK. The bitter gourd CDPK fraction from gel filtration (see Fig. 5a) was assayed in the standard conditions with I mg/ml histone III-S histone as substrate but with the reactions buffered to give the indicated pH.

40

A. Chang et al. /Plant

Table 2 Substrate specificity of bitter gourd CDPKs Substrate

Histone III-S BSA Casein MLCP Kemptide

Protein kinase activity (% control) CDPK I

CDPK II

CDPK III

100 46 18 55 6

100 11 6 28 4

100 12 6 26 0.6

Science 105 (1995)

0 k g

0

160

c 120 > t Z 80

8 $

40

0

L Y

o

Protein kinases were assayed in the standard CDPK assay conditions with I mg/ml histone III-S, BSA or casein as substrate or with 25 pM MLCP or 250 PM kemptide as substrate. Ca*+dependent protein kinase activity (which represents the difference between results of assays conducted in the presence of Ca*+) (0.25 mM EGTA and I mM CaC12 in the assay) or in the absence of Ca*+ (0.25 mM EGTA in the assay) is expressed as a percentage of the activity obtained with histone III-S as substrate for each CDPK preparation: 0.40.0.84 and 0.53 nmol incorporated per mitt/ml for CDPK preparations I, II and III, respectively.

of added Ca*+, but phosphorylation is markedly activated by free Ca*+ concentrations as low as 0.1 PM for all three CDPKs (Fig. 5a). In addition to sub-micromolar Ca*+, a millimolar concentration of Mg*+ is required for maximal activity of all three CDPKs (Fig. 5b). The pH optimum of the combined bitter gourd CDPK activity resolved on gel filtration is about pH 8.0 (Fig. 6). The best substrate found for the bitter gourd CDPKs is the histone III-S preparation (Table 2). The K,,, for histone III-S determined using the combined CDPK preparation from gel filtration (see Fig. 4a) is 0.25 f 0.03 mg/ml as compared with 0.6 f 0.3 mg/ml with the wheat embryo CDPK [8]. Other substrates found include the exogenous substrates bovine serum albumin, dephosphorylated casein and the synthetic peptide MLCP (KKRAARATSNVFA-NH$. All of these polypeptides are also substrates for wheat embryo CDPK (81. It is notable that CDPKs I and II phosphorylate kemptide (LRRASLG) and do so relatively much more effectively than CDPK III (Table 2). However, the K, values for the phosphorylation of kemptide are 2.5 f 0.4 mM for CDPK I and 1.8 f 0.3 mM for CDPK II and thus kemptide is not a good substrate for either CDPK

31-44

0.0

0.2

0.0

0.2

0.4 0.6 ~rifluoperazin~

0.4

0.6

b-4

2 > ;; 0

0.8 1.0 (mM)

1.2

0.8

1.2

1.0

(mW

60 40

0.0

0.2

0.4

0.6

[Calmidazollum]

0.8

I.0

(mM)

Fig. 7. Effect of calmodulin antagonists on bitter gourd CDPKs. Protein kinase was assayed in the standard assay conditions with 1 mg/mI histone III-S as substrate and with the inclusion of increasing concentrations of inhibitors. Inhibitors were added dissolved in DMSO to give a final DMSO concentration of 20% (v/v). Protein kinase activity is expressed as % of control (no added inhibitor): 0.06, 0.15 and 0.09 nmol/min/mI for CDPK preparations 1, II and III, respectively. (a) Trifluoperazine; (b) W-7; (c) calmidazolium. n, CDPK I; 0, CDPK II; A, CDPK III.

A. Chang et

al./ Planr Science IOS (1995) 31-44

I or CDPK II. It should be noted that kinetic data were obtained by assaying CDPK in standard assay conditions at a fixed ATP concentration (25 PM) and by varying the concentration of protein or peptide substrates. Kinetic parameters were determined by fitting this data to the MichaelisMenten equation employing a least squares curvefitting program. The three CDPKs phosphorylate BGP in a Ca*+-dependent manner as shown by SDS-PAGE and autoradiography (data not shown). [ 32P]PhosphoBGP was prepared using BGP (isolated by cation exchange HPLC) and bitter gourd CDPK (prepared by gel filtration). After high voltage electrophoresis of the acid hydrolysates of [32P]phosphoBGP thus prepared the only major peak of radioactivity corresponded to phospho+serine. No radioactivity was detected in the phospho-L-tyrosine zone and radioactivity in the phospho-L-threonine zone was only 3.0% of that in the phospho-L-serine zone. 3.4. Inhibitors of the bitter gourd CDPKs While the calmodulin antagonists trifluoperazine [30] and W-7 [30] are either not inhibitory or relatively poor inhibitors of CDPKs I, II and III, the calmodulin antagonist calmidazolium [30] is a good inhibitor of all three enzymes (ICso value for each about 30 PM) (Fig. 7). Poly-L-arginine (aver-

3

140

0, E

120

8

100

E 1 r_ 5

8o

8 m 5 c hi

20

60 40

0 0

5

10

15

poly-L-Arglnln~

Fig. 8. Inhibition

of bitter gourd

20

30

25

35

(/.~g/ml)

CDPKs

by poly-L-arginine.

Protein kinase was assayed in the standard CDPK assay with 1mglml histone III-S as substrate and with the inclusion of increasing concentrations of poly-L-arginine (average M, 115 000). Protein kinase activity is expressed as a percentage of control (no added poly-r-arginine): 0.10, 0.18 and 0.08 nmol/min/mI for CDPK preparations I, II, and III, respectively. n, CDPK I; 0, CDPK II; A, CDPK III.

41

age h4,, 115 000) is a potent inhibitor of CDPKs I, II and III (I& values 6, 8 and 5 &ml i.e. 52, 70 and 43 nM, respectively) (Fig. 8). However, poly-L-lysine (average M,, 173 000) is a poorer inhibitor of CDPKs I, II and III (activities with 32 &ml poly-L-lysine present 65%, 60% and 51%, respectively, of control activity with no added inhibitor) as is poly-L-ornithine (average M,, 19 000) (CDPK I, II and III activities with 32 pg/ml poly-L-ornithine present are 58%, 41% and 79% of control activity with no added inhibitor). The effects of various salts, cations and nucleotides on the activity of the ‘combined’ CDPK preparation from gel filtration (see Fig. 4a) was examined. High ionic strength inhibits the CDPK activity. Thus activity is 80%, 84% and 79%, respectively, of control activity (no added inhibitor) when 100 mM KCl, NaCl or Na acetate is included and 9%, 11% and 10% of control, respectively, when these salts are included at 500 mM in the reaction. Activity with inclusion of (NH&SO4 at 100 mM and 500 mM is 14% and 3% of control, respectively, and inclusion of 100 mM KH2P04 causes 97% inhibition. While Ca*+, Zn*+, Ba*+ and Mn*+ at 0.2 mM or 2 mM cause no inhibition of the CDPK, activity in the presence of 0.2 mM and 2.0 mM LaC13 is 68% and 3% of control, respectively. Similarly, CDPK activity in the presence of 0.2 mM or 2 mM FeC13 is 93% and 7% of control, respectively. While inclusion of additional unlabelled ATP at 0.1 mM and 1 mM in the standard radiochemical CDPK assay decreases (through isotope dilution) phosphoproteinincorporated countslmin to 37% and 6% of control, respectively, the inclusion of 0.1 mM or 1.O mM CTP, GTP, UTP or ITP has no inhibitory effect. This suggests an ATP-specificity of the bitter gourd CDPK activity. Interestingly, while inclusion of 1 mM 3’,5’-cyclic AMP causes slight CDPK inhibition (to 77% of control), addition of 0.1 mM 3 ‘,5 ‘-cyclic AMP causes a modest apparent activation of CDPK (to 182% of the control activity). 4. Discussion This paper demonstrates that seeds of bitter gourd (Momordica charantia) contain three very

42

A. Chang et al. /Plant Science IO5 (1995) 31-44

similar small proteins (BGSl , BGS2 and BGS3), of which two (BGSI, BGS3) can be phosphorylated by wheat CDPK. These small polypeptides evidently form 1:1 complexes with a larger subunit (BGL) involving disulphide linkages. The small polypeptide components of BGP are also phosphorylated (on Ser residues) by three endogenous CDPKs of bitter gourd seeds. Three CDPKs can be extensively purified from iki. charantia by a procedure that critically involves Ca*+-dependent chromatography on a hydrophobic matrix (Phenyl Sepharose CL4B). Ca*+-dependent hydrophobic chromatography has previously been used in the purification of a variety of soluble [2,4,20,22,26] and solubilized CDPKs [21] and indeed of calmodulin [29,31]. This behaviour is consistent with the possible presence of Ca *+-binding calmodulin-like domains on such CDPKs as found for several plant CDPKs that have been cloned and sequenced [6,7]. The inhibition of the bitter gourd CDPKs by the calmodulin antagonist cahnidazolium but not by the structurally different calmodulin antagonists trifluoperazine and W-7 (Fig. 7) is consistent with calmidazolium interaction with a calmodulin-like domain on the CDPKs that is nevertheless quite different from calmodulin, a protein that shows a high degree of conservation over a wide range of eukaryotes [32] and which binds all three inhibitors tightly (301. W-7, calmidazolium and trifluoperazine variously inhibit other plant CDPKs [2,4,20-22,33,34] but the concentrations required for inhibition of CDPK are higher than those required for inhibition of calmodulin-dependent enzymes [4]. Calmidazolium is a much more potent inhibitor of the bitter gourd CDPKs (IC, values about 30 PM) than of oat leaf CDPK (I(&, 2-5 mM) [22]. Such differences in sensitivity of plant CDPKs to calmodulin antagonists may reflect differences in calmodulinlike domains on these enzymes. The bitter gourd CDPKs are similar to other plant CDPKs in relation to inhibition by La3+ [20,22,33] and by poly+arginine [35]. The I& values for poly+arginine for CDPKs I, II and III (52, 70 and 43 nM, respectively) are similar to the very low ICsOvalues found for such polycations in relation to other plant CDPKs [35]. The lack of inhibition of radiochemical assays of the bitter

gourd CDPKs involving [Y-“P]ATP by inclusion of nucleoside triphosphates other than ATP suggest that these CDPKs preferentially use ATP as a substrate. The bitter gourd CDPKs phosphorylate histone III-S, casein and bovine serum albumin as do a variety of other soluble [2,4,8,20,22] and membranederived [21] plant CDPKs. The synthetic peptide MLCP (KKRAARATSNVFA-NH,) is a substrate for the bitter gourd CDPKs and for wheat embryo CDPK [8]. It is notable that kemptide (LRRASLG) is a much better substrate for CDPK I and CDPK II than for CDPK III but is nevertheless a poor substrate in comparison with histone III-S (Table 2). Kemptide, while a very poor substrate for silver beet leaf CDPKs relative to histone III-S, is a much better substrate for silver beet CDPK I than for silver beet CDPK II [20]. Bitter gourd CDPK III is much more like other plant CDPKs for which kemptide is either not a substrate or is a very poor substrate [8,20,22]. We have previously resolved a Cat+-independent protein kinase from Petunia petals which also phosphorylates kemptide [25]. Thus while a plant equivalent of animal and fungal 3 ’ ,5 ‘-cyclic AMP dependent protein kinase (PKA) has not yet been found [25], there are plant protein kinases (of which bitter gourd CDPKs I and II are examples) that in a qualitative sense have substrate specificities overlapping that of PKA. Other examples include the Petunia petal kemptide-phosphorylating protein kinase [25] and phosphoenolpyruvate carboxylase kinase [23]. There is a considerable homology existing between the catalytic domains of eukaryote Ser/Thr-specific protein kinases [ 181. Further, it is estimated that any eukaryote genome may code for as many as 1000 different protein kinases [36]. It is therefore possible that there are plant protein kinases with much higher affinities for good PKA substrates such as kemptide than exhibited by bitter gourd CDPKs I and II. The definition of CDPK-mediated signalling pathways in any plant tissue will require the characterization of the CDPKs and substrate proteins involved. The present work has resolved a multiplicity of CDPKs from seeds of Momordica charantia and isolated endogenous protein substrates. Application of ESMS to analysis of

A. Chang et al. /Plant

BGP has revealed that the phosphorylated proteins are small 4-kDa subunits that form 1:1 complexes with a larger 7-kDa subunit involving an estimated three disulphides linkages. It is notable that small, 4.5kDa subunits of radish seed napins are phosphorylated by plant CDPK [IZ] and form l:l, disulphide-linked complexes with a larger lokDa subunit [37]. The radish seed napins and other napins have KCl-sensitive antifungal properties connected with permeabilisation of fungal membranes [37-391. The propensity toward K+adduct formation by BGP and its small subunits observed by ESMS could be connected with a possible function of BGP, e.g. as a defensive protein. The BGP sequences, the sites of phosphorylation on these substrate proteins and the functional consequences of such phosphorylation remain to be determined. Acknowledgements This work was supported by a grant from the Australian Research Council to G.M. Polya. We are grateful to Mrs Rosemary Condron for amino acid sequencing and to Ian Thomas for electrospray ionization mass spectrometry. References 111P.K. Hepler and R.O. Wayne, Calcium and plant development. Annu. Rev. Plant Physiol., 36 (1985) 397-439. VI G.M. Polya and S. Chandra, Ca2+-dependent protein phosphorylation in plants: regulation, protein substrate specificity and product dephosphorylation. Curr. Topics Plant B&hem. Physiol., 9 (1990) 164-180. [31 A. Trewavas and S. Gilroy, Signal transduction in plant cells. Trends Genet., 7 (1991) 356-361. 141 D.M. Roberts and A.C. Harmon, Calcium-modulated proteins: targets of intracellular calcium signals in higher plants. Annu. Rev. Plant Physiol. Mol. Biol., 43 (1992) 375-414. 151 P.J. Blackshear, A.C. Nairn. J.F. Kuo, Protein kinases 1988: a current perspective. FASEB J., 2 (1988) 2957-2969. 161 J.F. Harper, M.R. Sussman, GE. Schaller, C. PutnamEvans, H. Charbonneau and A. Harmon, A calciumdependent protein kinase with a regulatory domain similar to calmodulin. Science, 252 (1991) 951-954. [71 K.-L. Suen and J.H. Choi, Isolation and sequence analysis of a cDNA clone for a carrot calcium-dependent protein kinase: homology to calcium/calmodulin-dependent

Science 10.5 (1995) 31-44

43

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