THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc

Vol. 262, No. 10, Issue of April 5, pp. 4602-4609.1987 Printed in U. S. A.

A Novel Ca2+-dependent Protein Kinase from Paramecium tetraurelia”

(Received for publication, September 15, 1986)

Robert E. Gundersen and David L. Nelson From the Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison. Wisconsin 53706

The ciliated protozoan Paramecium tetraurelia con- tained two protein kinase activities that were depend- ent on Ca2+. We purified one of the enzymes to homo- geneity by Ca2+-dependent affinity chromatography on phenyl-Sepharose and ion exchange chromatography. The purified enzyme contained polypeptides of 50 and 55 kDa, with the 50-kDa species predominant. From its Stokes radius (32 A) and sedimentation coefficient (3.9 S), we calculated a native molecular weight of 51,000, suggesting that the active form is a monomer. Its specific activity was 65-130 nmol*min”-mg” and the K,,, for ATP was 17-35 p ~ , depending on the exogenous substrate used. Kinase activity was com- pletely dependent upon Ca2+; half-maximal activation occurred at approximately 1 p~ free Ca2+ at pH 7.2. Phosphatidylserine and diacylglycerol did not stimu- late activity, nor did the addition of purified Parame- cium calmodulin. The enzyme phosphorylated casein and histones, forming primarily phosphoserine and phosphothreonine, respectively. It also catalyzed its own phosphorylation in a Ca2+-dependent reaction; the half-maximal rate of autophosphorylation occurred at approximately 1-1.5 p~ free Ca2+, and both the 50- and 55-kDa species were autophosphorylated. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation in situ, the 50- kDa protein retained its Ca2+-dependent ability to phosphorylate casein, suggesting that Ca2+ interacts directly with this polypeptide. This was confirmed by direct binding studies; when the enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electro- phoresis transferred to nitrocellulose, and renatured, there was “‘Ca2+-binding in situ to both the 50- and 55-kDa polypeptides. The Paramecium enzyme ap- pears to be a new and unique type of Ca2+-dependent protein kinase.

the surface membrane, inducing the voltage-gated Ca2+ chan- nels in the ciliary membrane to open and allowing influx of Ca2+ into the ciliary compartment (8-10). The rise in free intraciliary Ca2+ produces, by unknown mechanisms, altera- tions in the orientation of the ciliary beat (11,12), and perhaps in its frequency as well (13, 14). The resulting changes in swimming speed and direction are transient, in part because the entry of Ca2+ causes the Ca2+ channels to close and the K+ channels to open, with consequent repolarization of the cell (9, 15, 16). Cyclic nucleotides are also involved in the regulation of the ciliary beat (17-20) and in the regulation of flagellar motion in sperm (21-23). The mechanisms by which Ca2+ and cyclic nucleotides act as second messengers in ciliary regulation are unknown. It seems likely that the cyclic nu- cleotides act by stimulating the phosphorylation of proteins in the ciliary membrane (ion channels) and/or the axoneme. Such cyclic nucleotide-dependent phosphorylations have been demonstrated in cilia, ciliary membranes, and axonemes of Paramecium (24-26). We have also detected Ca2+-dependent protein phosphorylation in permeabilized cells and have found that Ca2+ and CAMP can act antagonistically in modulating swimming speed.’ Ca2+-dependent phosphorylations have also been reported in the flagella of Chlamydomonas and may be involved in the light-directed motion of these cells (27). It is therefore possible that some or all of the effects of Ca2+ on ciliary motility are mediated through Ca2+-dependent protein phosphorylation.

The exocytotic release of trichocysts, secretory organelles that lie beside each cilium in the cortex of Paramecium, is also a Ca2+-triggered process (28, 29), and there is evidence that the exocytosis and recovery involve a cycle of protein dephosphorylation and rephosphorylation (30,31), which may be regulated by Ca2+ levels. Exocytosis triggered by picric acid (30) or by aminoethyl dextran (31) results in rapid dephos- phorylation (within 1 s) of a 65-kDa protein (30, 31) and in its rephosphorylation after a lag of about 5 s (31), on the same time scale as the recovery of exocytotic membrane into intra-

Ca2+ acts as a second messenger regulating a wide variety cellular vesicles. Mutants defective in exocytosis do not show

Paramecium, at least two well-studied processes, ciliary mo- sion of Ca2+ from the medium (30) or blockage of Ca2+

known to influence ciliary and flagellar motion in a variety of phorylation cycle. It therefore appears that either the dephos-

and in ciliated cells of metazoans (6, 7). In Paramecium, sensitive to intracellular Ca2+ concentration, implying the physical, chemical, or thermal stimuli cause depolarization of presence Of Ca2+-dependent protein phosphatase Or Ca2C-

* This research was supported by National Institutes of Health Two classes of Ca2+-dePendent Protein kinases have been Grants GM 32514, GM 22714, and GM 34906; National Research described in higher animal systems. In the first class are the Service Award Grant F32 NS07590; and a grant from the Graduate Ca2+/calmodulin-dependent protein kinases, of which there

this article were defrayed in part by the payment of page charges. School of the University of Wisconsin. The costs of publication of appears to be a wide variety (32-38). These kinases are

This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. N. M. Bonini and D. L. Nelson, manuscript in preparation.

of cellular processes in eukaryotes. In the ciliated protozoan the dephosphorylation Or rephosphorylation, and the exclu-

tion and exocytosis, are sensitive to ca2+ levels. ca2+ ion is channels (31) also prevents the dephosphorylation/rephos-

protozoans ( 1 4 ) , in vertebrate and invertebrate sperm (4, 5), phorylation Or the rephosphorylation, or perhaps both, are

dependent protein kinase.

4602

Ca2+-dependent Protein Kinase from P. tetraurelia 4603

activated by interaction with a Ca2+/calmodulin complex; it is calmodulin which binds Ca2+ and senses the changes in cellular Ca2+. The second class is the Ca2+/phospholipid, diacylglycerol-dependent protein kinase, known as protein kinase C. Protein kinase C activation involves a complicated series of events; Ca2+ and phospholipids are required for activation but diacylglycerol appears to be the principal reg- ulator of kinase activity (39).

Very little information is available on Ca2+-dependent pro- tein kinases in the protozoans. In light of the various impor- tant functions Ca2+ performs in the protozoans, an effort was made to identify Ca2+-dependent protein kinases in Parame- cium. We report here the presence of two Ca2+-dependent protein kinase activities in Paramecium and describe the purification and properties of one of these enzymes, a unique Ca2+-dependent protein kinase that is activated directly by Ca2+, independent of calmodulin or lipids.

EXPERIMENTAL PROCEDURES

M~terials-[y-~~P]ATP and 'TaC12 were purchased from Amer- sham Corp. Mixed histones (H-IIAS), lysine-rich histones (H-IIIS), casein, N-a-p-tosyl-L-arginine methyl ester, phenylmethylsulfonyl

EDTA, EGTA,2 sucrose (Grade l), phenyl-Sepharose CL-4B, 0- fluoride, leupeptin, phosphatidyl-L-serine, 1,3-diolein, 1,2-diolein,

phospho-L-serine, 0-phospho-DL-threonine, 0-phospho-L-tyrosine, 2-mercaptoethanol, and SDS-PAGE molecular weight standards were all purchased from Sigma. Prestained SDS-PAGE molecular weight standards, acrylamide, bisacrylamide ((N,N'-methylene)-bis-acryl- amide), and SDS were purchased from Bethesda Research Labora- tories. Nitrocellulose sheets (0.1 pm pore size) were purchased from Schleicher & Schuell. Cellulose thin layer plates (100 pm) were obtained from EM Reagents. Diaflo ultrafiltration membranes and Centricon 10 microconcentrators were purchased from Amicon. All other compounds used were reagent grade or better.

Cells and Culture Conditions-Paramecium tetraurelia, wild-type stock 51s (supplied by Dr. Ching Kung, University of Wisconsin), was grown axenically at 28 "C to late logarithmic growth phase in Soldo's crude medium, modified by the addition of phosphatidyleth- anolamine to 50 mg/l and by the reduction of phosphatidylcholine to 125 mg/l (40).

Preparation of Crude Extract-Paramecia (10-20 1) were harvested by continuous-flow centrifugation and washed three times with Dryl's solution (41); 1 mM NaH2P04, 2 mM sodium citrate, 1.5 mM CaC12, pH 6.8. If cilia were to be isolated, the cells were deciliated according to Adoutte et al. (42). Preparation of whole cells or deciliated cell bodies was adapted from the procedure of Walsh et al. (43). All of the following procedures were performed at 4 "C. Cell pellets were resus- pended in 10 volumes of 40 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 2 mM EDTA, and 2 mM EGTA for a final wash. The cells were resuspended in approximately 2 volumes of Buffer H (40 mM Tris- HC1, pH 7.5,0.25 M sucrose, 2 mM EDTA, 10 mM EGTA, and 1 mM DTT) plus 0.3 mM N-a-p-tosyl-L-arginine methyl ester, 0.3 mM phenylmethylsulfonyl fluoride, and 5 pg/ml leupeptin (protease in- hibitors) and broken by repeatedly expelling the suspension from a syringe held tightly against the bottom of a beaker. The broken-cell preparation was centrifuged at 25,000 X g for 20 min, and the supernatant solution was centrifuged at 120,000 X g for 60 min. The high-speed supernatants were stored at -20 "C until 25-50 1 of cell culture had been processed (approximately 2-5 g of cell protein).

Phenyl-Sepharose Chromatography-Ca2+-dependent adsorption to phenyl-Sepharose CL-4B was performed according to Walsh et al. (43). The high-speed supernatant fraction (50-100 ml) was brought to 75% saturation with ammonium sulfate (0.476 g/ml) and allowed to mix for 60 min. The protein precipitate was pelleted at 25,000 X g for 30 min and resuspended in 50-100 ml of Buffer D (20 mM Tris- HCl, pH 7.5,l mM EDTA, 1 mM EGTA, 0.5 mM DTT) plus protease inhibitors (leupeptin was 0.2 pg/ml) and dialyzed against 3 1 of the same buffer with one change for 18-24 h. Following dialysis, MgC12

The abbreviations used are: EGTA, [ethylenebis (oxyethyleneni- tri1o)ltetraacetic acid; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; 1, liter; MES, 2-(N- morpho1ino)ethanesulfonic acid; HEPES, 4-(2-hydroxyethyl)-l-pi- perazineethanesulfonic acid.

and CaC12 were added to the dialyzed material to a concentration of 2 mM each, and the solution was clarified by centrifugation at 25,000 X g for 20 min. The cell extract was loaded onto a phenyl-Sepharose CL-4B column (25 X 1.5 cm) equilibrated with Buffer A (20 mM Tris- HCl, pH 7.5, 0.1 mM CaC12, 0.5 mM DTT). Protein not bound in the presence of Ca2+ was washed from the column with Buffer A and Buffer B (Buffer A plus 1 M NaCl). Ca2+-dependent protein kinase activity was eluted with Buffer C (20 mM Tris-HC1, pH 7.5, 1 mM EGTA, 1 mM DTT).

Zon Exchange Chromatography-The Ca2+-dependent protein ki- nase activity peak from phenyl-Sepharose was pooled and concen- trated by filtration with a Diaflo (YM-5) ultrafiltration membrane and injected onto an anion exchange column (Mono Q HR 5/5, Pharmacia) equilibrated at 1 ml/min with 20 mM bis-Tris propane- HCl, pH 6.7, 0.1 M sucrose, 0.1 mM EDTA, and 1 mM DTT. Two Ca2+-dependent kinase activities were eluted with a NaCl gradient (Fig. 1B). The first (CaPK-1) eluted with the start of the salt gradient and the second (CaPK-2) eluted at approximately 0.13 M NaC1. The protein elution profile was monitored at A280 with a Waters model 420 variable wavelength detector. The NaCl gradient was created with a Waters model 720 System Controller and two Waters 501 pumps, and the theoretical salt gradient is plotted in Fig. 1B for simplicity. Actual salt gradients were routinely monitored in mock gradient runs with a YSI Scientific Model 35 conductance meter and were found to closely follow the theoretical gradient but with a delay of approximately 2 ml, a difference which has been incorporated into Fig. 1B.

Gel Filtration-CaPK-2 fractions from Mono Q were pooled and concentrated by centrifugation in a Centricon 10 microconcentrator. The concentrated sample (0.5-0.6 ml) was passed over a Sephacryl S-300 column (27 X 1.5 cm) equilibrated with 20 mM Tris-HC1, pH 7.5, 0.1 M sucrose, 0.1 M NaCl, 0.1 mM EDTA, and 1 mM DTT.

Sucrose Density Gradients-CaPK-2 (0.5 ml) concentrated as for gel filtration was loaded onto a 12.5-ml linear sucrose gradient (5- 12%) in 20 D M Tris-HC1, pH 7.5,l mM EGTA, and 1 mM DTT and centrifuged in a Beckman SW40Ti rotor a t 40,000 rpm for 15 h. Fractions of 0.37 ml were collected from the bottom of the tubes.

Caz+-dependent Protein Kinase Assay-Ca2+-dependent protein ki- nase assays were performed in plastic Eppendorf tubes at 30°C in either of two buffer systems. One was 20 mM MES-NaOH, pH 6.3, 10 mM magnesium acetate, 0.5 mM EGTA, k0.75 mM CaC12, 1 mM DTT, 100 pg of mixed histones, and 50 pM [y-32P]ATP (8-12 X 10' cpm/nmol) in a final volume of 0.1 ml. The other buffer system was 20 mM HEPES-NaOH, pH 7.2, 5 mM magnesium acetate, 0.5 mM EGTA, k0.6 mM CaC12, 1 mM DTT, 100 pg of casein, and 50 pM [y- 32P]ATP (8-12 X lo4 cpm/nmol) also in a volume of 0.1 ml. Reactions were initiated by the addition of ATP and terminated by spotting 90 pl of the reaction mixture onto a Whatman GF/C 2.4-mm filter disc and immersing it in 10% trichloroacetic acid (10 ml/filter) at 4 'C. The filters were washed several times with 10% trichloroacetic acid over a period of 5-15 h to reduce background. Phosphate incorpora- tion was quantified by counting Cerenkov radiation from the filters in 5 ml of water.

Free Ca2+ concentrations were calculated using the COMICS pro- gram (45) translated to BASIC by Stephen Tindall in our laboratory. This program calculates free ion concentrations in mixtures of metals (Ca2+ and M e ) and chelators (EGTA and ATP). Stability constants were taken from Martell and Smith (46) and Smith and Martell (47). To determine more accurately free Ca2+ in the Ca2+ activation of CaPK-2 (Fig. 5), free Ca2+ concentrations in the assay mixtures (less enzyme and histones and plus 0.1 M NaCl) were measured with a Ca2+-sensitive electrode, calibrated according to the method of Tsien and Rink (48) with 0.1 M NaCl.

SDS Gel Electrophoresis-SDS-PAGE was performed by the method of Laemmli (49) in 1.5-mm slab gels. The protein sample was solubilized in SDS sample buffer (62.5 mM Tris, 10% glycerol, 4% 8- mercaptoethanol, 1.5% SDS, pH 6.8). The stacking gel was 4% acrylamide and 0.1% bisacrylamide, and the resolving gels were either 10% acrylamide, 0.27% bisacrylamide, or an 8-12% acrylamide, 0.21- 0.32% bisacrylamide gradient. Gels were stained in 0.1% Coomassie Brilliant Blue R-250 dissolved in ethanol/acetic acid/H20 (992) and destained in 7% acetic acid and 5% ethanol, or silver stained according to the method of Poehling and Neuhoff (50). Molecular weight markers were myosin (205,000), /+galactosidase (116,000), phospho- rylase b (97,400), bovine serum albumin (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhy- drase (29,000), trypsinogen (24,000), soybean trypsin inhibitor (20,100), and a-lactalbumin (14,200). SDS-polyacrylamide gels for

4604 Ca2+-dependent Protein Kinase from P. tetraurelia the kinase assay in situ (see below) contained 1 mg/ml casein and could not be stained with Coomassie Brilliant Blue. To determine the molecular weight of the kinase, prestained molecular weight standards from Bethesda Research Laboratories were used. They were myosin (205,000), phosphorylase b (97,400), bovine serum albumin (66,000), ovalbumin (45,000), a-chymotrypsinogen (25,700), fl-lactoglobulin (18,400), and lysozyme (14,300).

Detection of Kinuse Activity in SDS-Polyacrylamide Gels-Detec- tion of kinase activity in samples run on SDS-polyacrylamide gels was performed according to Geahlen et al. (51), with modifications to the kinase assay in situ. The gel kinase assay was carried out in 20 ml of 20 mM HEPES-NaOH, pH 7.4, 5 mM magnesium acetate, 0.5 mM EGTA, 1 mM D m , and 50-100 pCi of [Y-~’P]ATP k 0.75 mM CaCl,.

‘Ta” Binding”%a’+ binding to nitrocellulose transfers was per- formed as described by Maruyama et al. (52).

Other Procedures-Samples for phosphoamino acid analysis were prepared by precipitating samples with 10% trichloroacetic acid for 30 min on ice. Protein pellets were dissolved in 6 M HC1 (constant boiling) and hydrolyzed under vacuum at 110 ‘C for 120 min. Follow- ing hydrolysis, HCl was removed under reduced pressure, and the samples were redissolved in 0.5 ml of H20 and lyophilized. Samples were dissolved in a small volume of HZ0 containing phosphoserine, phosphothreonine, and phosphotyrosine at 1 mg/ml and separated by electrophoresis on cellulose thin layer plates (100 pm) by the procedure of Hunter and Sefton (53) using glacial acetic acid/pyri- dine/water (505:945), pH 3.5. Following autoradiography, phospho- amino acid standards were detected with ninhydrin. Autoradiograms were obtained with Kodak X A R B or XRP film. Densitometry of the x-ray films was performed on a Zeineh Soft Laser Scanning Densi- tometer Model SL-504-X4 (Biomed Instruments, Inc.). Protein esti- mates were performed on samples precipitated with 0.02% deoxycho- late and 10% trichloroacetic acid according to Lowry et al. (54), with bovine serum albumin as the protein standard.

RESULTS

Purification-Crude homogenates of Paramecium revealed only minimal Ca2+-dependent protein kinase activity, but removal of the particulate fraction to produce a 120,000 X g supernatant allowed the detection of significant Ca2+-depend- ent protein kinase activity. Ammonium sulfate precipitation and dialysis increased the total Ca2+-dependent activity slightly, yet a high background of Ca2+-independent protein kinase activity persisted (Table I).

Pseudoaffinity chromatography on phenyl-Sepharose CL- 4B separated a peak of Ca2+-dependent protein kinase activity from the Ca2+-independent kinase activity, which passed through the column along with the bulk of the protein (Fig. 1A). The Ca2+-dependent protein kinase activity was retained on the column in the presence of 0.1 mM CaC12, but could be eluted with 0.1-0.3% of the applied protein by buffer contain- ing EGTA to chelate Ca2+. This peak of kinase activity was

highly dependent on the presence of Ca2+, having barely measurable to no activity when Ca2+ was omitted from the assay (Fig. lA and Table I).

Ion exchange high performance liquid chromatography re- solved two protein kinase activities in the peak from phenyl- Sepharose. One (CaPK-1) eluted at low ionic strength and the other (CaPK-2) came off the anion exchange column at about 0.13 M NaCl (Fig. 1B). Both peaks were entirely de- pendent upon Ca2+ for activity ( d a t a for CaPK-2 shown in Table I). When all column fractions were assayed in the presence of added Paramecium calmodulin, or in the presence of phosphatidylserine and diacylglycerol, no new peaks were detected, and no stimulation of CaPK-1 or CaPK-2 was seen above the level with Ca2+ alone (data not shown).

Gel electrophoresis in SDS revealed many polypeptide spe- cies in CaPK-1 (Fig. 2), whereas CaPK-2 contained predom- inantly two detectable peptides of 50 and 55 kDa (Fig. 2). In five repetitions of this purification, CaPK-2 always contained these two species, with the 50-kDa species predominating. In some preparations minor bands of approximately 200 and 15 kDa ran into the CaPK-2 peak from adjacent Mono Q frac- tions, but these could be effectively removed with a gel filtra- tion step.

When subjected to gel permeation chromatography, CaPK- 2 migrated as a single, symmetrical peak (Fig. 3A), and by comparison with markers of known molecular weights, we estimated a Stokes radius of 32 A. The sedimentation coeffi- cient of CaPK-2, measured in sucrose gradients by compari- son with markers (Fig. 3B), was about 3.9 S. From these two measurements, using the method of Seigel and Monty (55) and assuming a partial specific volume of 0.725, we calculated a molecular weight of 51,000 and a frictional ratio (f/fo) of 1.3 for the active and presumably native enzyme.

Catalytic Properties-The pH activity profile for purified CaPK-2 depended upon the protein provided as substrate for phosphorylation. With casein, the enzyme showed a broad optimum between pH 6.5 and 7.5. Kinase activity was optimal at pH 6.2-6.4 with mixed histones as the substrate, but rather poor a t pH 7.2; at the higher pH, lysine-rich histones were a slightly better substrate than mixed histones. When each substrate was phosphorylated at its pH optimum, casein was a better substrate than mixed histones, accepting 2-3 times more phosphate per milligram.

During the 5-min assays, protein kinase activity was linear with respect to time and to enzyme concentration up to 0.2 pg/assay (for freshly prepared kinase). Optimal activity re- quired 5-10 mM M$+. Substitution of Ca2+ATP for M$+ATP

TABLE I Purification summary

Data are drawn from two purifications. Ca*+-dependent protein kinase assays were performed at pH 6.3 as described under “ExDerimental Procedures.”

Total Kinase activity Ca2+-dependent Ca*+/EGTA T&..&e Yield Purification protein -caz+ +caz+ activity ratio

mg nml . mg-’. min” 1. Cell homogenate 4520 0.05 0.06 2. 120,000 X g superna- 1540 0.28 0.72

3. Phenyl-Sepharose 1010 0.44 1.42

4. Phenyl-Sepharose 590 0.58 0.58

5. Phenyl-Sepharose 1.91 0.22 83.6

6. Mono Q (CaPK-2) 0.123 0.48 292 7. Sephacryl S-300 0.024 0.14 324

tant

load

pass-through

EGTA eluate

nmol. mg“. min” 0.01 0.44

0.98

0

83

292 324

nmllrnin % -fold 1.2 63 2.6 680

3.2 990 100 I

1.0 0

380 160 16 85

610 36 4 300 2300 8 1 330

Ca2+-dependent Protein Kinase from P. tetraurelia 4605

60 0.3

50 - Q

40 0.2 - L3 a

30

20 0.1

10

0 0

6o IEl 1 Y

CoM

50 - ? I- -

CoPK-2 f

f I

I 4 0 -

CoPK-I I -0.1

0 5 IO I5 20 25 30 F R A C T I O N NUMBER

FIG. 1. Cas+-dependent protein kinase purification by phenyl-Sepharose and anion exchange chromatography. A, phenyl-Sepharose chromatography was performed as described under "Experimental Procedures." The A, of the column pass-through and beginning of the Buffer A wash are omitted for clarity. A, B, and C under the arrows refer to the start of the corresponding buffer. For Buffers A and B, fractions were 9 ml; for Buffer C, fractions were 5 ml. The break in the x axis at fraction 30 is the omission of an additional 150 ml of Buffer A. Aliquots of 10 pl were assayed for kinase activity with (0) and without (0) added Ca2+ at pH 6.3. B, ion exchange chromatography on Mono Q HR 5/5 was performed as detailed under "Experimental Procedures." Fractions were 1 ml, and 10-pl aliquots were assayed for kinase activity at pH 7.2 (0). Protein was monitored at A, (. . . .), and the NaCl gradient is also shown (- - -). The position of calmodulin (Cam elution was determined by a phosphodiesterase stimulation assay (44).

resulted in minimal kinase activity. The dependence of initial velocity upon ATP concentration was uncomplicated. From double-reciprocal plots we obtained values of approximately 35 p~ and 130 nmol.min".mg" for K , and Vmer, respec- tively, with casein as the substrate at pH 7.2, and values of approximately 17 pM and 65 nmol. min" . mg", respectively, with histones at pH 6.3 (data not shown). The Vmax in both cases was less than optimal due to the gradual decay of enzyme activity during storage at -20°C. Kinetic values using histones at pH 6.3 and freshly isolated enzyme yielded an essentially identical K , for ATP (20 p ~ ) and an optimal V,, (470 nmol. min".mg"). Neither GTP (up to 200 p ~ ) nor Pi (up to 1 mM) inhibited the reaction with ATP over a concentration range of 5-100 p ~ , but ADP was a competitive inhibitor with a Ki of 140 p ~ . ADP inhibition was not due to a reduction in the free Ca2+ concentration, since even at the highest ADP concentration tested, 0.5 mM, the reduction in free Ca2+ was calculated to be only 6 p~ from a level of 260 p~ in the absence of ADP. The enzyme was found to label serine residues in casein, whereas histones were labeled on threonine residues (data not shown).

During the course of purifying CaPK-2, we discovered that the enzyme was considerably more stable in extracts when

DTT was present at 1 mM, and we therefore included DTT in all buffers; during storage at -20 "C, CaPK-2 activity decreased approximately 50% over 2-4 weeks. CaPK-2 was very sensitive to inhibition by the sulilydryl reagent N- ethylmaleimide (Fig. 4); it seems possible that the enzyme requires one or more cysteine-SH groups for its activity or stability.

Regulatory Properties: Dependence on Calcium-Purified CaPK-2 was completely dependent upon added Ca2+ when assayed in the presence of 0.5 mM EGTA; at pH 7.2, this stimulation was dependent on Caz+ concentration, and half- maximal activation occurred at approximately 1 pM free Ca2+ (Fig, 5). At pH 6.3 the dependence on Ca2+ was also complete; a half-maximal rate required approximately 100 pM Caz+ (Fig. 5). The stimulation by CazC was also completely reversible by the addition of excess EGTA, although a 30-45-s delay in inactivation was seen (data not shown). No other divalent ions tested could substitute for Ca2+; they included M P , Mn2+, Ba2+, Sr", Co2+, Ni", and ZnZ+ (each metal was 0.6 mM in the presence of 0.5 mM EGTA).

The activation of CaPK-2 by micromolar Ca2+ suggested the possibility that calmodulin might mediate the effect of Ca2+ on the protein kinase activity. In the ion exchange chromatography step used to purify CaPK-2, calmodulin was well separated from the two peaks of kinase activity (see Fig. 1B), and SDS-PAGE did not reveal the presence of calmod- ulin in molar amounts equivalent to the 50- or 55-kDa com- ponents (see Fig. 2), suggesting that CaPK-2 was relatively free of calmodulin (see "Discussion"). The addition of purified Paramecium calmodulin (0.3 p ~ ) failed to increase kinase activity at either pH 6.3 or 7.2 above the level obtained with Ca2+ alone (Table 11). The addition of phosphatidylserine and diacylglycerol at levels known to stimulate the Caz+-depend- ent activity of protein kinase C (43, 56) did not increase activity of CaPK-2. At pH 6.3 the lipids were somewhat inhibitory, perhaps due to the binding of Ca2+ by phosphati- dylserine. Likewise, no stimulation resulted from the addition of 10 p~ CAMP (Table 11).

Since CaPK-2 appeared to be regulated solely by the addi- tion of Ca2+, we sought to determine whether the polypeptides of the kinase bound Ca2+. When CaPK-2 was subjected to SDS-PAGE, transferred to nitrocellulose, renatured in situ, and incubated with radiolabeled calcium, preferential binding to the 50- and 55-kDa species was detected by autoradiogra- phy (Fig. 6). 45Ca2+ bound also to the control proteins (molec- ular weight markers) to a small extent but a comparison of the ratio of 45Ca2+ binding to Coomassie staining demonstrates a preferential binding of the 45Ca2+ to the 50- and 55-kDa bands of CaPK-2 (Table 111). Ca2+ binding directly to the 50- kDa species was also suggested by a Ca2+-induced shift in electrophoretic mobility; in the presence of Ca2+ the 50-kDa protein moved faster than when EGTA was added to the sample before electrophoresis (not shown). The 55-kDa pro- tein does not show such a Ca2+ shift. In CaPK-1, a 48-kDa protein was also found to undergo a Ca2+-dependent mobility shift in SDS gels.

Further evidence that CaPK-2 requires only the addition of Ca2+ for activity was obtained by performing a kinase assay on CaPK-2 that had been subjected to SDS-PAGE and then renatured in the gel (Fig. 7). Casein incorporated into the gel acted as the substrate and it was phosphorylated in the region of the 50-kDa band when Ca2+ was added to the assay mixture but not when Ca2+ was omitted. Only one band of label was detected, suggesting that the 55-kDa protein does not possess kinase activity. However, the predominance of the 50-kDa species and the consequent heavy labeling in this region may

4606 ea2+-dependent Protein Kinase from P. tetraurelia

FIG. 2. Purification of CaPK-2 as followed by SDS-PAGE. Fractions from each purification step of CaPK-2 were subjected to SDS-PAGE (8-12% acrylamide) and stained with Coomassie Brilliant Blue as described under “Ex- perimental Procedures.” A, lune 1, cell homogenate (87 pg); lune 2, 120,000 X g supernatant (66 pg); lune 3, phenyl- Sepharose load (58 pg); lune 4, phenyl- Sepharose EGTA pool (12.5 pg); lune 5, Mono Q CaPK-2 (3.5 pg); lune 6, Se- phacryl S-300 CaPK-2 (4.6 pg). B, Mono Q, CaPK-1 (4 pg).

A

2 0 5 .

116. 97.4.

66-

4 5 -

36 -

29- 24-

20. I -

14.2-

1 2 3 4 5 6

+55 I- 50

B

- 205

-I I6

- 97.4

-66 z - 0 (D

S - -45

-36

-29 * -24

T

(D -.

X

-20.1 6 I

w -14.2

mask activity at the 55-kDa band. When purified CaPK-2 was incubated with [32P]ATP in

the absence of protein substrates, it catalyzed autophospho- rylation of both the 50- and 55-kDa polypeptides, approxi- mately in proportion to their mass contributions. This auto- phosphorylation was also completely dependent upon Ca2+; half-maximal labeling of the 50-kDa species occurred a t 1.5 PM free Ca2+ (Fig. 8). Half-maximal labeling of the 55-kDa species occurred at approximately 1 PM free Ca2+. Autophos- phorylation did not change the Ca2+-dependent mobility of these CaPK-2 peptides in SDS gels; the 50-kDa peptide demonstrated such a shift, whereas the 55-kDa peptide did not (not shown).

DISCUSSION

The protein kinase we describe here appears to be unlike any previously described kinase; it depends upon Ca2+ for maximum activity, but is neither a Ca”/calmodulin-type nor a protein kinase C. The homogeneous enzyme is half-maxi- mally activated by Ca2+ in the physiological range (1 PM) but it does not contain calmodulin in equimolar amounts with the 50- and 55-kDa peptides that comprise the kinase. Calmodulin is not seen on SDS-PAGE (Fig. 2), and the amount of cal- modulin detected in the purified enzyme by a very sensitive enzyme-linked immunosorbent assay using monoclonal anti- body against Paramecium calmodulin was a t most 1% of the 50-kDa protein? The addition of purified Paramecium cal- modulin to the purified CaPK-2 did not stimulate it above the level seen with Ca2+ alone. The purified CaPK-2 did not bind radioiodinated calmodulin from Paramecium in a gel overlay assay that does detect other calmodulin-binding pro- teins in Paramecium with high sensitivity: Perhaps the most convincing evidence that the enzyme requires no calmodulin (or lipid) for its activity is the assay for protein kinase in situ

A. Burgess-Cassler, personal communication. ‘ T. C. Evans and D. L. Nelson, manuscript in preparation.

after SDS-PAGE; only the addition of Ca2+ was required for kinase activation in the 50-kDa polypeptide.

In addition to the gel assay in situ, there are several other lines of evidence that the Paramecium enzyme is not protein kinase C. We saw no activation, at either pH 6.3 or 7.2, by a combination of phosphatidylserine and diacylglyceride, which characteristically activates protein kinase C. Protein kinase C is known to be stereospecific for 1,2-diacylglycerol(39), and activation of protein kinase C by 1,3-diacylglycerol is due to an isomerization to the 1,2-isomer during sonication in vesicle preparation. The experiment in Table I1 used 1,3-diolein, which failed to stimulate kinase activity. To exclude the possibility that 1,3-diolein did not isomerize in our vesicle preparation, we repeated the experiment with 1,2-diolein, which also failed to alter kinase activity. Protein kinase C from several sources has a catalytic subunit of about 80 kDa, substantially larger than the 50- and 55-kDa peptides in the Paramecium enzyme.

Could the enzyme we isolated be a proteolytic fragment of one of the Ca2+-dependent protein kinases known in other organisms? We consider this unlikely; Ca2+ dependence is retained by CaPK-2, whereas proteolytic cleavage of protein kinase C is known to produce an active but Ca2+-independent kinase (56). We know of no reports of proteolytic activations of Ca2+/calmodulin kinases that yield Ca2+-dependent, cal- modulin-independent forms. The Ca’+/calmodulin protein ki- nase of Aplysia, which is bound to a cytoskeletal-membrane complex, has been reported to release into the cytoplasm a Ca2+/calmodulin-independent protein kinase under appropri- ate conditions (37). Nor is CaPK-2 activity the result of Ca2+- dependent proteolysis, since activation was completely re- versible by the addition of excess EGTA. The Ca2+/calmod- ulin kinases are not known to bind Ca2+ directly, unlike CaPK-2, which appears to bind Ca2+ preferentially as deter- mined by radiolabeled calcium binding on nitrocellulose blots of SDS-PAGE, and as reflected in its Ca2+-dependent mobility

Ca2+-dependent Protein Kinase from P. tetraurelia 4607 7 0 I

A ’p I 2 3 l 4 ’ I

I I I

6ol 50

40 t I \

I I I

i l l 0

FRACTION NUMBER FIG. 3. CaPK-2 gel filtration and sucrose density gradient

centrifugation. A, gel filtration on Sephacryl S-300 was performed according to procedures outlined under “Experimental Procedures.” Fractions of 1 ml were collected once the sample entered the column bed. Aliquots of 10 pl were assayed for kinase activity at pH 6.3. Standards were run separately. 1, thyroglobulin (Stokes radius = 85 A); 2, bovine serum albumin (35.5 A); 3, ovalbumin (30.5 A); 4, chymotrypsinogen A (20.9 A). B, sucrose gradients were prepared and run as detailed under “Experimental Procedures.” Fractions of 0.37 ml were collected starting from the bottom of the tube, and 10-pl aliquots were assayed for kinase activity at pH 7.2. Standards were run in separate, parallel gradients. a, thyroglobulin (19.2 S); b, ferritin (17 S); c, catalase (11.3 S); d, aldolase (7.35 S); e, bovine serum albumin (4.5 S).

shift on SDS-PAGE. (We are now attempting to measure Ca2+ binding by CaPK-2 using more direct methods.)

The relationship between the 50- and 55-kDa species in purified CaPK-2 is unknown; the native molecular weight of the enzyme is about 50,000, which would seem to preclude their being subunits of a larger holoenzyme. The 55-kDa species was always the minority species, and it is conceivable that it is a biosynthetic precursor of the 50-kDa chain. The two peptides might differ in the presence of a post-transla- tional modification (glycosylation, covalent lipid, etc.), or the 50-kDa species might, in principle, be a proteolytic fragment of the 55-kDa protein. It is also conceivable that the 55-kDa peptide is a contaminating peptide which has no kinase activ- ity but is a substrate for the kinase.

Autophosphorylation of CaPK-2 appears to lead to some degree of Ca2+-independent protein kinase activity similar to Ca2+/calmodulin-dependent protein kinase I1 (57-60). The Ca2+-independent activity produced by autophosphorylation may in part be responsible for the high degree of apparent cooperativity seen for CaPK-2 activity versus free [Ca”] at

R. E. Gundersen, unpublished observation.

I 1

[NEM] , M

FIG. 4. N-Ethylmaleimide inhibition of CaPK-2. Protein ki- nase assays were performed as described under “Experimental Pro- cedures” at pH 7.2 in the presence (+) or absence (-) of 1 mM DTT with various amounts of N-ethylmaleimide (NEM). Both assays contained 0.1 mM 2-mercaptoethanol, which was contributed by CaPK-2.

Free Ca2’, M

FIG. 5. Caa’ activation of CaPK-2. Kinase assays were per- formed as described under “Experimental Procedures,” except that the Ca2+ concentration was varied while keeping EGTA (0.5 mM) and pH (6.3 or 7.2) constant. Lysine-rich histones (100 pg) replaced casein as the substrate at pH 7.2. Curve pH 6.3, maximum activity was 111 nmol.min” .mg“; curve pH 7.2, maximum activity was 33 nmol. min”. mg”.

pH 7.2 (Fig. 5). Multiple Ca2+-binding sites on CaPK-2 may also account for the cooperativity. Further investigations into the effects of autophosphorylation and the number of Ca2+- binding sites of CaPK-2 may help in determining this en- zyme’s function.

What physiological role(s) do the Ca2+-dependent protein kinases of Paramecium play? We are currently attempting to obtain antibodies against purified CaPK-2, with which we hope to determine the intracellular location of the enzyme. Several events in the regulation of ciliary motility are known to be triggered by Caz+ in the micromolar range. Preliminary experiments have localized Ca2+-dependent protein kinase activity within isolated cilia, which suggests a role for the enzyme there. Ca2+-dependent phosphorylations have also been detected in detergent-permeabilized “models” of Para- mecium which occur at the same Ca2+ concentrations required to alter ciliary beating in such models.’

Segal and Luck (27) detected the Ca2+-dependent phospho- rylation of an identified axonemal protein in Chlamydomonas flagella. They tested several compounds known to alter the

4608 ea2+-dependent Protein Kinase from P. tetraurelia TABLE I1

Requirements for CaPK-2 activation Assays were performed as described under "Experimental Proce-

dures" with the following changes: at pH 6.3 and 7.2 added Ca" was 0.5 mM (pH 6.3,63 p M free Ca2+; pH 7.2, 24 p M free Ca2+) and at pH 7.2 the substrate was 100 pg of lysine-rich histones. Values are mean f S.D. of triplicates.

pH 6.3 pH 1.2

Addition Phosphate Percent of Phosphate Percent of incorporation Ca2+ only incorporation Ca2+ only

P m l % Pmol % None - 0.6 f 0.3 1 1.1 f 0.1 5 None + 1 3 2 f 3 100 21.2 f 0.4 100 Calmodulin" - 1.1 f 0.3 1 0.9 f 0.2 4 Calmodulin + 123 f 4 94 22.2 f 1.0 105 Lipidsb Lipids

- 1.5 f 0.3 1 4.2 f 2.9 20

cAMP + 74 f 2.0 56 20.9 -C 0.4 99 - 0.9 f 0.4 1 1.0 f 0.5 5

' Purified Paramecium calmodulin (0.5 pg). *Lipids were phosphatidylserine (10 pg) and 1,3-diolein (0.2 pg),

prepared by mixing stock solutions, evaporating the organic solvents under a stream of N2, and sonicating the lipids in buffer to form vesicles.

cAMP was 10 p ~ .

A

I -

2 - 3- 4 - 5 - 6- 7 -

1 2 1 2 FIG. 6. "Ca2+ binding to CaPK-2. Samples were subjected to

SDS-PAGE, transferred to nitrocellulose, incubated with "Ca2+, and rinsed with H20 for 5 min. Following autoradiography (B) the nitro- cellulose sheet was stained with Coomassie Brilliant Blue (A) . Lane 1, molecular weight standards (each 4 pg, see Table 111 for identifi- cation); lane 2, CaPK-2 (2.5 pg).

activities of various kinases in an attempt to identify the kinase responsible for the Ca2+-dependent phosphorylation. None of the compounds noticeably altered the degree of Caz+- dependent phosphorylation. By these experiments, they elim- inated cyclic nucleotide-dependent protein kinases, calmod- ulin-dependent protein kinases, protein kinase C, and myosin light chain kinase as possible candidates. Although these results do not definitively exclude these kinases, they do suggest the possible existence of another, uncharacterized Ca2+-dependent protein kinase, possibly the same Ca2+-de- pendent protein kinase we have described here in Parame- cium.

Although there is general agreement that Ca2+ concentra- tion regulates at least the orientation of the ciliary beat (1, 12, 14, 18, 61) and perhaps the ciliary beat frequency (14),l the direct target(s) of intraciliary Ca2+ action are not known

1 2 3

205 - m ' 97.4 0 -

Ca2+ + - FIG. 7. Gel kinase assay in situ. CaPK-2 (2 pg) was subjected

to SDS-PAGE and renatured in the gel. The kinase assay in situ was performed as outlined under "Experimental Procedures." Lane 1 was incubated in the presence of Ca2+ and lane 3 in the absence of Ca2+. The gel was run with prestained molecular weight standards (lane 2) for estimating the molecular weight of kinase activity.

TABLE 111 Evaluation of the specificity of "Ca2+ binding

Protein 'Ta'+/Coomassie ratio"

CaPK-2 50 kDa 6.6 55 kDa (>3.5)b

1. Bovine serum albumin (66 kDa) 0.1 2. Ovalbumin (45 kDa) 0.3 3. Glyceraldehyde-3-phosphate dehydro- 0.8

4. Carbonic anhydrase (29 kDa) 0.1 5 . Trypsinogen (24 kDa) 0.1 6. Soybean trypsin inhibitor (20.1 kDa) 0.6 7. a-Lactalbumin (14.2 kDa) 1.6

Molecular weight standards

genase (36 kDa)

a This value is the ratio of the peak areas from densitometer scans of the '%a2+ autoradiogram and from the negative of the Coomassie Brilliant Blue-stained nitrocellulose sheet.

The 55-kDa Coomassie Brilliant Blue-stained band was not de- tected by the scannirlg densitometer. A minimum value was assumed.

with certainty. Calmodulin is present within Paramecium cilia (62, 63), and some investigators have reported inhibition by calmodulin antagonists of ciliary reorientation in living cells (64, 65, 71). In permeable models of Paramecium trifluoper- azine blocks ciliary reversal (65), but in Tetrahymena models there appears to be no effect of this same antagonist on swimming direction (66), leaving uncertain the involvement of calmodulin in ciliary reversal. There is similar uncertainty about the target for Ca2+ in trichocyst discharge. We (67) and others (72) initially reported the presence of calmodulin in trichocysts, but it now appears that this association is an artifact of isolation (68).6 Calmodulin antagonists were re- ported to inhibit exocytosis in Paramecium (69), but such

S. H. Tindall, L. D. DeVito, and D. L. Nelson, manuscript in preparation.

Ca2+-dependent Protein Kinase from P. tetraurelia 4609 8. Naitoh, Y., Eckert, R., and Friedman, K. (1972) J. Exp. Biol. 66,667-681 9. Eckert, R., Naltoh, Y., and Machemer, H. (1976) Symp. SOC. Exp. Bml. 30,

233-255

B

5 t e i - / I ‘ 1 1 50 i

/- L-,-HH I 1 1 I I I I I

0 10-7 10-6 10-5 10-4

Free Ca2+, M FIG. 8. Effect of Ca2+ concentration on CaPK-2 autophos-

phorylation. A kinase assay was performed at pH 7.2 without substrate using CaPK-2 and various free Ca2+ concentrations. This reaction was stopped after 2 min by precipitating the protein with 0.01% deoxycholate and 1 ml of 10% trichloroacetic acid. After 30 min on ice, the precipitate was pelleted and washed with 1 ml of 10% trichloroacetic acid to remove excess [3ZP]ATP. Acid pellets were subjected to SDS-PAGE (10%) as described under “Experimental Procedures.” A, autoradiogram of CaPK-2 autophosphorylation. Ap- proximate free Ca2+ levels were: lane 1, no added Ca2+ (shown as 0 on the x axis in B); lane 2, 0.2 pM; lane 3, 1.7 pM; lane 4, 8.5 pM; lane 5, 25 p ~ ; lane 6,44 PM; lane 7,125 pM; lane 8,270 pM. B, autoradiogram band density (50 kDa) plotted against free Ca2+ concentration.

antagonists are known to block Ca2+ channels in Paramecium (70) and may therefore act indirectly on exocytosis. We tested two calmodulin antagonists (trifluoperazine and calmidazo- lium) for inhibition of the Ca2+-dependent protein kinases in the pooled phenyl-Sepharose fractions and found no inhibi- tion by either in the micromolar range;5 the enzymes we describe here are therefore potential Ca2+ targets not sensitive to calmodulin antagonists. We intend to explore the role, if any, of CaPK-2 in ciliary reversal and exocytosis by charac- terizing the enzyme from mutants defective in each process and by microinjection of inhibitory antibodies against CaPK- 2.

Acknowledgments-We would like to thank Brook Soltvedt for her contribution of purified Paramecium calmodulin and for her help in preparing this manuscript, Tom Evans for performing the calmodulin overlay assay, Dr. Anthony Burgess-Cassler for the enzyme-linked immunosorbent assay, Dr. Grant Nicol for his assistance with the calcium electrode, Dr. Stephen Tindall for providing the computer program to calculate free Caz+ concentrations, and Dr. Paul Ludden for the use of his scanning densitometer.

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