Purification and characterization of a protein kinase in Tetrahymena cilia

Biochimica et Biophysica Acta, 327 (1973) 354-364 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s

BBA

67081

P U R I F I C A T I O N AND C H A R A C T E R I Z A T I O N OF A P R O T E I N K I N A S E IN T E T R A H Y M E N A CILIA

H1ROMU MUROFUSHI

Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bongo, Bunkyoku, Tokyo, (Japan) (Received J u l y 9th, 1973)

SUMMARY

A protein kinase was detected in T e t r a h y m e n a cilia and partially purified by hydroxyapatite column chromatography and Sephadex G-2oo gel filtration. This enzyme preferred casein to histone and protamine as snbstrate protein. The apparent Km for casein was about 0.6 mg/ml and that for ATP was about 25 #M when casein was used as substrate protein. The p H optimum for casein phosphorylation was about 8.5. IO mM Mg 2+ was required for its maximal activity. In the absence of Mg ~÷, other divalent cations such as Mn 2÷ and Co S+ could partially substitute for Mg 2+. The molecular weight of the enzyme was estimated to be about 53 ooo. The ciliary protein kinase was found to catalyze the phosphorylation of tubulin prepared from ciliary axonemes. Furthermore, adenosine 3',5'-monophosphate showed little effect on the phosphorylation of casein, histone, protamine and tubulin.

INTRODUCTION

It has been demonstrated recently that a mammalian brain tubulin fraction prepared by the method of Weisenberg et al. 1 shows protein kinase activity. Goodman et al. 2 have shown that the brain tubulin fraction contains a protein kinase which phosphorylates tubulin and that this activity is enhanced by the addition of adenosine 3',5'-monophosphate (cyclic AMP). This was confirmed later by Lagnado et al. s. In addition, evidence has been presented that a protein kinase, which was purified after Miyamoto et al. 4, catalyzes the phosphorylation of tubulin 2& On the other hand, Soifer et al. 6 and Soifer 7 have observed in a brain tubulin fraction another type of protein kinase which catalyzes the phosphorylation of casein and lysine-rich histone but cannot phosphorylate tubulin. In addition to the data obtained from in vitro studies, there is evidence that tubulin is actually phosphorylated in vivo. According to Eipper*, brain tubulin contained 0.8 mole phosphate per mole of tubulin dimer, and incubation of brain slices with orthola2P]phosphate resulted in phosphorylation of only the fl-subunit of the tubulin dimer.

CILIARY P R O T E I N K I N A S E

355

Besides brain kinases, cyclic AMP-dependent protein kinases have been partially purified from the sonicates of spermatozoa of the oxg, 1° and the sea urchin n. As the sonication caused tail disintegration on all spermatozoa with their heads remaining intact, those authors proposed a possibility that the protein kinases take part in the flagellar motile system. The present results, described below, demonstrate that a protein kinase can be extracted from the axoneme fraction of Tetrahymena cilia, and does catalyze phosphorylation of ciliary tubulin in vitro. MATERIALS AND METHODS

Tetrahymena pyriformis W was grown in a culture medium containing 2% (w/v) polypeptone, 0.5% (w/v) yeast extract and 0.85% (w/v) glucose with constant aeration. Cells in the stationary phase were used as experimental materials. Cilia were isolated by the method of Gibbons TM with some modifications as follows; the CaC12 concentration in the ethanol-calcium procedure was increased to 20 mM and a slight contamination due to the cell bodies was removed by passing the solution through a glass filter after the routine procedure in which the cilia were separated from the cell bodies by a brief centrifugation. Axonemes were prepared by the method of Stephens and Levine 1~. FV-32PIATP was prepared following the method described by Glynn and ChappelP ~. OrthoI32Plphosphate was purchased from The Radiochemical Centre, Great Britain, ATP from Kojin, Japan, and cyclic AMP from Daiichi, Japan. Proteins were obtained from commercial sources, as follows; casein, Merck; calf thymus histone (type II-A), herring protamine (grade III), soybean trypsin inhibitor and ovalbumin from Sigma; and bovine serum albumin and bovine Vglobulin from Armour. Hydroxyapatite was prepared according to the method of Tiselius et al. 15. Protein kinase assay A standard assay mixture, unless otherwise indicated, contained 5 #moles of Tris-HC1 (pH 8.5), I #mole of MgC12, o.i #mole of dithiothreitol, 0.2 #g of casein, o.oi ~umole of E3~P]ATP (4' 10520' lO5 cpm) and enzyme, in a total volume of o.I ml. The reaction was started by the addition of enzyme and allowed to proceed for 5 rain at 25 °C. It was found that the reaction was linear for at least io min. After incubation, 0.04 ml of the reaction mixtures was pipetted onto Whatman No. I paper discs and washed once with lO% trichloroacetic acid and three times with 5% triehloroacetic acid. After washing finally with ethanol and ether, the radioactivities were counted in a toluene-based scintillator containing PPO (4 g/l) and dimethylP O P O P (0.2 g/l) with an Aloka liquid scintillation system (Model LSC-65I ). When protamine was used as substrate protein, 0.6 mg of bovine serum albumin was added to the assay mixture before pipetting onto paper discs and washing was performed four times with lO% trichloroacetic acid. In the case of histone, 18% trichloroacetic acid was applied. All the trichloroacetic acid solutions mentioned above contained I % sodium pyrophosphate. One unit of enzyme activity was defined as the amount of enzyme which catalyzes the transfer of I nmole of 32p from ATP to casein per I rain in the standard assay system at 25 °C.

356

H. MUROFUSHI

Polyacrylamide gel electrophoresis Sodium dodecylsulfate polyacrylamide gel electrophoresis was performed according to the method of Weber and Osborn 16 with some modifications 17. The gels (0.6 cm × 6.5 cm) were made of 7.5% acrylamide-o.2% methylenebisacrylamide. In order to identify protein components which incorporated radioactive phosphate, gels were sliced into 37 equal pieces with a hand-made gel slicer and digested in 0.3 ml of 30% H202 (ref. 18) in counting bials. To the solubilized gels, were added 0.3 ml of I M HC1 and Io ml of toluene-based scintillator containing 2.8 g/1 of PPO, o.14 g/1 of dimethyl-POPOP and 30% (v/v) of Nonions NS-2IO (nonylphenolpolyethoxyether, Nippon Oil and Fats Co. Ltd) 19 and the radioactivities were measured as described above.

Protein determination The amount of protein was determined by the method of Lowry et al. 2° using bovine serum albumin as a standard. RESULTS

(I) Enzyme purification Solubilization of enzyme. About 5 ml of packed wet cilia was routinely obtained from a 9-i culture, from which axonemes were prepared using 1% (v/v) Triton X-Ioo (ref. 13). To the purified axonemes, about 20 ml of 0.6 M KC1 containing 0.5 mM EDTA, o.I mM ATP, o.I mM dithiothreitol and I mM Tris-HC1 (pH 8.2) were added and homogenized vigorously with a teflon homogenizer. The homogenate was dialyzed against 500 ml of the same buffer for 5 h and centrifuged at 5° ooo × g for 20 rain. The pellet was re-extracted with about 20 ml of the same KC1 solution. Both the supernatants were combined. Most of the protein kinase activity (more than 95%) was detected in the supernatant by this extaction procedure. Hydroxyapatite column chromatography. When the KC1 extract was chromatographed on hydroxyapatite, three peaks of protein kinase activity (Peaks A, B and C) were consistently eluted as shown in Fig. I. The fractions of the main peak, C (tubes 53-65) were combined. (NH4)2SO 4 fractionation. Solid (NH4)2SO4 (o.35g/ml) was added to the hydroxyapatite fraction. The precipitates formed were collected by centrifugation, dissolved and dialyzed against IO mM Tris-HC1 (pH 8.4) containing 0.6 M KC1, o.I mM ATP and o.I mM dithiothreitol. This fraction was called the "(NH4)2SO 4 fraction". Sephadex G-2oo gel filtration. For further purification, the (NHa)~SO a fraction was gel filtered through a Sephadex G-2oo column. The protein kinase activity was eluted as a single peak as is shown in Fig. 2. The active fractions (tubes 36-46) were combined. From the results summarized in Table I, the apparent specific activity of the Sephadex G-2oo fraction was found to be about 6o-fold higher than that of the KC1 extract of the axoneme. The enzyme in the Sephadex G-2oo fraction was fairly unstable, about 50% of the activity was lost in one week at o °C. The enzyme involved in the (NH4)2SO 4 fraction was more stable, little loss of the activity was detected when

357

CILIARY PROTEIN KINASE

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Fig. i. H y d r o x y a p a t i t e c o l u m n c h r o m a t o g r a p h y of t h e KC1 e x t r a c t . 4 ° m l of t h e KC1 e x t r a c t were l o a d e d on t h e h y d r o x y a p a t i t e c o l u m n (2 cm × 12.5 cm) e q u i l i b r a t e d w i t h o . i M p o t a s s i u m p h o s p h a t e buffer (pH 7-5) c o n t a i n i n g o.i mM A T P a n d o . i mM d i t h i o t h r e i t o l . A f t e r t h e c o l u m n was w a s h e d w i t h a b o u t 60 ml of t h e s a m e solution, a d s o r b e d m a t e r i a l s were e l u t e d w i t h a l i n e a r g r a d i e n t of p h o s p h a t e buffer c o n t a i n i n g o.i mM A T P a n d o.i mM d i t h i o t h r e i t o l (from o.I - 0.6 M, t o t a l 500 ml), c o l l e c t i n g f r a c t i o n s of 4 ml. A l i q u o t s of 5 °/*1 from e a c h f r a c t i o n were a s s a y e d using t h e s t a n d a r d a s s a y m i x t u r e .

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Fig. 2. Gel f i l t r a t i o n of t h e (NH4)2SO 4 f r a c t i o n on S e p b a d e x G-2oo. The (NH4)2SO 4 f r a c t i o n w a s l o a d e d on S e p h a d e x G-2oo c o l u m n (2 cm × 47 cm) e q u i l i b r a t e d w i t h IO mM Tris-HC1 (pH 8.4) c o n t a i n i n g 0.6 M KC1, o.I mM A T P a n d o.i mM d i t h i o t h r e i t o l , a n d e l u t e d w i t h t h e s a m e buffer solution, c o l l e c t i n g f r a c t i o n s of 2.5 ml. Th e n z y m e a c t i v i t y of e a c h f r a c t i o n was a s s a y e d w i t h a l i q u o t of 5 ° ffl u s i n g t h e s t a n d a r d a s s a y s y s t e m .

stored for a month at o °C. To determine the characteristics of this ciliary protein kinase, the (NHa)2SO 4 fraction was used as enzyme source.

(II) General properties of ciliary protein kinase The pH optimum of the enzyme activity was about 8.5. A second peak was detected at around p H 7 (Fig. 3). The apparent Km for ATP was about 25 ffM. Fig. 4 illustrates the divalent cation requirement for ciliary protein kinase. Mg 2+ was most effective among all the cations investigated, and revealed the optimum

358

H. M U R O F U S H I

TABLE I PURIFICATION STEPS OF A PROTEIN KINASE FROM Tetrahymena CILIA The a c t i v i t y was d e t e r m i n e d w i t h t h e s t a n d a r d a s s a y s y s t e m .

Fraction

Protein

Cilia Axoneme KC1 e x t r a c t Hydroxyapatite fraction (NH4)~SO~ fraction S e p h a d e x G-2oo fraction

(rag)

Total activity (units)

654 338 lO 9

65.8 144 2Ol

9.6

122

8.o 0.60

122 64.1

Specific activity (units/rng) o. i o 0.43 1.85 12. 7 15. 3 lO 7

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Fig. 3. Effect of p H on t h e r a t e of casein p h o s p h o r y l a t i o n . The r e a c t i o n r a t e w a s d e t e r m i n e d w i t h t h e s t a n d a r d a s s a y s y s t e m e x c e p t for t h e buffers i n d i c a t e d . The c o n c e n t r a t i o n of e a c h buffer w a s 5 ° mM. o.o43 u n i t of t h e e n z y m e ((NH4)2SO 4 fraction) w a s used p e r i n c u b a t i o n .

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Fig. 4- D i v a l e n t c a t i o n r e q u i r e m e n t . The r e a c t i o n r a t e w a s m e a s u r e d w i t h t h e s t a n d a r d a s s a y m i x t u r e e x c e p t t h a t MgCI~ w a s s u b s t i t u t e d for v a r i o u s c o n c e n t r a t i o n s of d i v a l e n t c a t i o n s i n d i c a t e d . 0.040 u n i t of the e n z y m e ((NH4)2SO 4 fraction) w a s used p e r i n c u b a t i o n .

CILIARY PROTEIN KINASE

359

at around IO mM. Mn 2+ and Co2+ were less effective with their optima at o.2 and 2 raM, respectively. Each 2, 5 and IO mM of Ca 2+, Sr 2+, Ni 2+, Zn 2+ and Cd ~+ were completely ineffective. Among the proteins tested for substrate specificity, casein was the best substrate for the enzyme in the present assay condition. The apparent Krn for casein was about 0.6 mg/ml. Protamine and histone (8 mg/ml at the maximum) were poorer substrates than casein, revealing one-fifth and one-fourth of the rate of casein phosphorylation, respectively. IO-S-IO-4 M cyclic AMP showed little effect on the phosphorylation of casein, histone and protamine. The enzyme activity was depressed as the concentration of KC1 and NaC1 increased. The effects of both salts were quite similar. About half of the enzyme activity was depressed by the addition of 0.2 M KCI or NaCI. It has been established by Miyamoto et al. 4 and Guthrow et al. 21 that Ca 2+ inhibits cyclic AMP-dependent protein kinases in the presence of Mg2+. The same inhibitory effect of Ca 2+ was true with the present enzyme. CaC12 reduced the enzyme activity to half the original level, however, by increasing the concentration up to IO raM, the phosphorylation was largely diminished. The molecular weight of ciliary protein kinase was estimated with Sephadex G-2oo gel filtration (Fig. 5)- The apparent molecular weight was estimated to be about 53 ooo, which was close to that of tubulin.

(III) Tubulin phosphorylation catalyzed by ciliary protein kinase Identification of tubulin as the major component of Peak I fraction from hydroxyapatite chromatography. Tubulin is a main component of the axoneme and it is known 1.0

0.8

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Moleculor weight (10-4) Fig. 5. Molecular w e i g h t e s t i m a t i o n of c i l i a r y p r o t e i n k i n a s e w i t h S e p h a d e x O-2oo gel fi l t ra t i on. As p r o t e i n m a r k e r s , s o y b e a n t r y p s i n i n h i b i t o r s , o v a l b u m i n , b o v i n e s e r u m a l b u m i n a n d ~ - g l o b u l i n were so lubilized s e p a r a t e l y in I ml of i o mM Tris-HC1 (pH 8.4)-0.6 M KC1 c o n t a i n i n g B l ue D e x t r a n a n d sucrose. As source, i m l of t h e (NH4)2SO t f r a c t i o n w a s m i x e d w i t h B l ue D e x t r a n as well. E a c h s a m p l e s w a s r u n s e p a r a t e l y . The S e p h a d e x G-2oo c o l u m n (2 c m × 47 cm) w a s e q u i l i b r a t e d w i t h T r i s - H C l a n d t h e e l u t i o n was p e r f o r m e d w i t h t h e s a m e buffer. 2 ml of f r a c t i o n s were collected. M a r k e r p r o t e i n s were d e t e c t e d b y t h e m e a s u r e m e n t of A 2s0 nm. The voi d a n d c o l u m n v o l u m e s were m e a s u r e d b y t h e aid of Blue D e x t r a n a n d sucrose, r e s p e c t i v e l y . The a s s a y c o n d i t i o n of p r o t e i n k i n a s e was t h e s a m e as s h o w n in Fig. 2.

360

H. MUROFUSHI

that a considerable amount of ciliary tubulin can be extracted with o.6 M KC122. In the present work, the KC1 extract of the axoneme was separated into three peaks of Protein I, I I and I I I (see Fig. i). Fig. 6A represents the electrophoretic patterns of the protein components in Peaks I, I I and I I I . The run-off fraction (Peak I) exhibited the presence of a major component. From its mobility, it was supposed that the protein might be tubulin. Therefore, the major component of Peak 1 was further analyzed by electrophoresis on sodium dodecylsulfate polyacrylamide gels (Fig. 6B) with a marker of ciliary tubulin derived from outer fibers 2a. Since the outer fiber consists mainly of tubulin, the major band of solubilized outer fiber (Fig. 6B-c) was identified as tubulin. The RF value of the main band of Peak I (Fig. 6B-a) was coincident with that of the major band of solubilized outer fiber (Fig. 6B-c), and the molecular weight was estimated to be 54 ooo, which was close to that obtained by Renaud et al32. Moreover, the RF value of the main band was identical with that of porcine brain tubulin prepared by the method of Weisenberg et al. 1. These facts support the idea that the major component of Peak I was ciliary tubulin.

Fig. 6. (A) Sodium dodecylsulfate polyacrylamide gel electrophoresis of c o m p o n e n t s in Peak I, I I and I I I . Several main fractions of each Peak I, I I and I I I were combined a n d concentrated. After treated w i t h 8 IV[ u r e a - I % sodium d o d e e y l s u l f a t e - I % 2 - m e r c a p t o e t h a n o l - 5 mM E D T A 2o mM Tris-HC1 (pH 8.5), a b o u t 5 ° / i g of each p r o t e i n fraction was applied on sodium dodecylsulfate polyacrylamide gels. a, P e a k I; b, Peak II, and c, P e a k I I I . (B) Sodium dodecylsulfate polyacrylamide gel electrophoresis of c o m p o n e n t s in Peak I and solubilized outer fibers as a m a r k e r of tubulin. Several m a i n fractions of Peak I of h y d r o x y a p a t i t e column c h r o m a t o g r a p h y were combined and dialyzed a g a i n s t the u r e a - s o d i u m dodecylsulfate 2 - m e r c a p t o e t h a n o l E D T A - T r i s buffer. Outer fibers were directly solubilized in the same solution and dialyzed as well. A b o u t 5 °/~g of Peak I protein a n d solubilized outer fibers were applied to Gels a and c, respectively. A b o u t 2 5/~g of b o t h fractions were mixed and applied, b.

361

CILIARY P R O T E I N K I N A S E

Time course of tubulin phosphorylation catalyzed by ciliary protein kinase. The a b i l i t y of this as a s u b s t r a t e of ciliary t u b u l in was f u r t h e r i n v est i g at ed . In order to obtain a c o n c e n t r a t e d t u b u l i n fraction for assay, t h e P e a k I fraction was f u r t h er f r a c t i o n a t e d b y (NH4)2SO 4 precipitation. This fraction is, hereafter, called the "axonemal tubulin fraction". A l t h o u g h m o s t of protein kinase was s e p a r a t e d from ciliary t u b u l i n by the h y d r o x y a p a t i t e column c h r o m a t o g r a p h y , the P e a k I fraction still contained a d e t e c t a b l e a m o u n t of p r o te in kinase (see Fig. i). A f t er it was c o n c e n t r a t e d b y (NH4)2SO 4 f r a c t i o n a t i o n to o b t a i n the a x o n e m a l t u b u l i n fraction as substrate, a fairly high c o n t a m i n a t i n g p r o t e i n kinase a c t i v i t y was d e t e c t e d w i t h o u t addition of exogenous enzyme. W h e n purified protein kinase was added, a higher rate of protein p h o s p h o r y l a t i o n was d e m o n s t r a t e d (Fig. 7A). A

B

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Fig. 7. Time-cource of phosphorylation of the axonemal tubulin fraction (A), and of the ureatreated tubulin fraction (B), with and without exogenous protein kinase. The Peak I fraction of hydroxyapatite chromatography (Fig. i, tubus 7-18) were combined and proteins were precipitated with (NH4)zSO4 (o.31 g/ml), dissolved in 3° mM Tris-HC1 (pH 8.3) and dialyzed against the same buffer to obtain the axonemal tubulin fraction. As for the preparation of the urea-treated tubulin fraction, the axonemal tubulin fraction was dialyzed against 5 M urea containing 3° mM TrisHC1 (pH 8.3), 20 mM 2-mercaptoethanol and 0. 5 mM EDTA for I h at o °C. Urea was then removed by dialysis against 3° mY[ Tris-HC1 (pH 8.3). 0.28 mg of the axonemal tubulin fraction or the urea-treated tubulin fraction was used per incubation as substrate. The time-course of the reactions were measured with the standard assay mixture except for the substrate proteins. o.o3o unit of exogenous protein kinase ((NH4),SO 4 fraction) was added when indicated. (A) a, (+)axonemal tubulin fraction, (+)exogenous enzyme; b, (+)axonemal tubulin fraction, (--)exogenous enzyme; c, (--)axonemal tubulin fraction, (+)exogenous enzyme. (B) a, (+)urea-treated tubulin fraction, (+)exogenous enzyme; b, (+)urea-treated tubulin fraction, (--)exogenous enzyme; c, (--)urea-treated tubulin fraction, (+)exogenous enzyme. I t was difficult to s e p a r a t e the c o n t a m i n a t i n g p r o t ei n kinase from the t u b u l i n fraction. Therefore, an a t t e m p t was m a d e to reduce the e n z y m e a c t i v i t y in t h e a x o n e m a l t u b u l i n fraction. W h e n the fraction was t r e a t e d with 5 M urea (see legend of Fig. 7), t h e e n z y m e a c t i v i t y decreased to less t h a n 15% of the original level. This fraction was d e s i g n a t e d as " u r e a - t r e a t e d t u b u l in f r a c t o n " . As shown in Fig. 7 B, the u r e a - t r e a t e d t u b u l i n fraction served as a s u b s t r a t e for ciliary p r o t ei n kinase. T h e rate of p h o s p h o r y l a t i o n in this fraction was a b o u t half of t h a t of casein at the same p r o te in concentration. F r o m these data, it was strongly

362

H. M U R O F U S H I

suggested that the axonemal tubulin molecule could actually serve as a phosphate acceptor for this enzyme. In order to obtain evidence an experiment was designed to analyze phosphorylated proteins by electrophoresis.

Sodium dodecvlsulfate polyacrylamide gel electrophoresis of phosphorvlated proteins. Phosphorylated proteins in the urea-treated tubulin fraction were electrophoresed and the radioactivity of the sliced gels was counted (Fig. 8). A peak of the radioactivity emerged coincident with the major band of protein. It was concluded, therefore, that tubulin actually served as a substrate for ciliary protein kinase.

3000]

+

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O O -o 1 0 0 0 0 13::

5

10

15

20

25

30

35

S l i c e No, Fig. 8. Identification as t u b u l i n of p h o s p h o r y l a t e d protein b y sodium sodium dodecylsulfate polyacrylamide gel electrophoresis, i ml of an assay m i x t u r e containing 2.8 mg of urea-treated t u b u l i n fraction was incubated at 2 5 °C for I h. The concentration of the reagents were the same as those in the s t a n d a r d assay mixture, o.3o u n i t of the enzyme ((NH4)2SO 4 fraction) was used. E q u a l volume of 2o% trichloroacetic acid containing 2% sodium p y r o p h o s p h a t e was added to stop the reaction. The precipitates were t h e n washed five times with 5% trichloroacetic a c i d - 1 % sodium p y r o p h o s p h a t e and were dissolved in o. 5 ml of the u r e a - s o d i u m dodecylsulfate-2-mercaptoe t h a n o l - E D T A - T r i s buffer (see legend of Fig. 6), followed b y dialysis against the same solution. A b o u t 5° t,g of the protein was electrophoresed. After staining, the gels were sliced and t h e i r radioactivities were counted as in Materials and Methods.

Effect of cyclic A M P on the phosphorylation of tubulin, As described in Section II, cyclic AMP did not significantly affect the phosphorylation of casein, histone and protamine. The same held true with the effect of varying the concentration of cyclic AMP (lO-1°-1o -4 M) in tubulin phosphorylation. In addition, the phosphorylation of tubulin in the axonemal tubulin fraction catalyzed by the contaminating enzyme was not influenced by cyclic AMP. DISCUSSION

It has been established that Tetrahymena cilia contains a protein kinase which catalyzes the phosphorylation of ciliary tubulin. In the present paper, the extraction

CILIARY PROTEIN KINASE

363

of the enzyme was usually carried out with the axoneme. When the outer-fiber fraction was obtained according to Gibbons ~3, most of the protein kinase activity (about 70~o) remained in the fraction, from which the same enzyme fraction was extracted and purified by hydroxyapatite column chromatography. This indicates that the protein kinase is tightly bound to the outer fiber. Even when the outer-fiber suspension was incubated with L32PIATP, a considerable amount of the radioactivity was detected in the acid precipitate. This m a y confirm that the true substrate of the protein kinase is tubulin. The phosphorylation in the outer-fiber fraction was not also affected by cyclic AMP. It has been reported so far that tubulin phosphorylation is cyclic AMP dependent in vitro 2,a. It is noteworthy, in the case of the present ciliary kinase, that the Triton X-Ioo treatment and the KC1 extraction induced a three times increase in the total activity of protein kinase (see Table I). Therefore, a possibility was considered that the R-subunit 23-25 might be removed during these steps. However, the possibility m a y be ruled out, because the protein kinase activity of the whole axoneme fraction was hardly affected by IO-S-IO-4 M cyclic AMP. Furthermore, the incorporation of a2p from ATP into the outer-fiber fraction was also cyclic AMP independent. A possibility still remained, however, that the R-subunit might be removed by Triton X-Ioo used in the procedure of the axoneme preparation. But it is reasonable to decide that ciliary kinase contains no R-subunit in si/u, judging from the fact that brain protein kinases which are cyclic AMP dependent could be prepared after Triton treatment 26. Further experiments are required to demonstrate the absence of cyclic AMP-binding protein in cilia. As inhibitor other than R-subunit, on the other hand, might be removed with the KC1 extraction, or ciliary protein kinase might be partially masked in the axonemal structure and be activated in this procedure. In the treatment of tubulin with urea to reduce the contaminating protein kinase activity, care should be taken due to the following possibility: Proteins which have little substrate activity might become good substrates after urea treatment. It is true that the urea treatment slightly enhanced the activities of ovalbumin, bovine serum albumin and y-globulin, as substrate, however, these activities were Io-fold lower than that of the urea-treated tubulin fraction in the present work. The activity of urea-treated bovine serum albumin was maximally only about one-seventh of that of the same concentration of the urea-treated tubulin fraction. Furthermore, the activity of casein was not affected by the urea treatment. For further analysis of tubulin phosphorylation in vi/ro, it is necessary to prepare pure native tubulin which is flee of protein kinase activity. ACKNOWLEDGEMENTS

The author wishes to express his thanks to Dr H. Sakai for his valuable suggestions and discussions. In the course of the purification of ciliary protein kinase, the author was greatly indebted to Mr K. Kaji for his valuable suggestions and for the fractionation of axonemal proteins by hydroxyapatite column chromatography which was originally developed by him (K. Kaji, Ph.D. thesis in preparation). Author's thanks are also due to Mr T. Kobayashi, Mr M. Hoshino, Miss R. Kuriyama and Mr T. Shimizu for their valuable discussions in the course of the present work

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and their kind supply of the preparations of outer fibers and brain tubulin. This work was in part supported by a graduate fellowship of the California Foundation of Biochemical Research and research grant from Takeda Science Foundation given to Dr H. Sakai.

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