Enzymatic activity in tubulin preparations

I82

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 36139 E N Z Y M A T I C A C T I V I T Y IN T U B U L I N P R E P A R A T I O N S I. I N T R I N S I C P R O T E I N K I N A S E A C T I V I T Y IN L Y O P H I L I Z E D P R E P A R A T I O N S OE T U B U L I N F R O M P O R C I N E B R A I N

DAVID SO1FER, ARMIN H. LASZL() AND JOSEPH M. SCOTT() Department of Neurochenlistry, N . Y . S . Institute for Research in Mental Retardation, 5tah~z lslamt, N . Y . ~o3~4 ( U . S . A . )

(Received December i3th, 197 l) (Revised version received March ~4th, 1972)

SUMMARY Lyophilized tubulin which has been isolated from pig brain has the ability to catalyze the transfer of a2p from [y-a~P1ATP to casein or to lysine-rich histone, A m o n g the properties of this kinase activity are : (a) linear dependence on a m o u n t of tubulin present over a range of 5 to 2oo/~g of tubulin in Ioo #1 incubation volume ; (b) linear rate of casein phosphorylation from I to 2o min; (c) variation of activity with pH, m a x i m u m activity is at p H of 6.6. Activity is inhibited by Nat" concentrations in excess of IO raM. Neither colchicine (io nM IOO/~M) nor vinblastine (too nM-2 mM) have a n y effect on activity. A T P is only slightly more effective a substrate than is GTP, The apparent Km for A T P is 2- 4 #M; t h a t for G T P is IO ~2o #M.

INTRODUCTION Microtubules function in undetermined manners in such diverse cell activities as pigment granule m o v e m e n t 1, hormone secretion ~,a and the regulation of metabolism b y insulin 4. The first two of these processes are stimulated b y 3',5'-cY clic-AMPS-7 ; the last involves a series of metabolic actions in which the effects of insulin are opposite to those of cyclic AMP. It is generally considered t h a t a primary, if not the sole site of action of cyclic AMP in regulating cell function is at the level of a class of enzymes called protein kinases, which are activated b y the cyclic nucleotide 8 10,32 Thus, it has seemed reasonable to several investigators to look for an association between microtubules and protein kinase activity. G o o d m a n et al. al investigated the ability of microtubule protein (tubulin) front bovine brain to serve as a substrate for protein kinase. In the course of their studies t h e y found t h a t tubulin itself appears to have intrinsic protein kinase activity and t h a t this activity can be stimulated b y cyclic AMP. L a g n a d o et al. 12 also reported t h a t tubulin possesses an intrinsic "cyclic AMP activated phosphokinase" activity. Biochim. Biophys. Acta, 271 (1972) t82-I92

ENZYMATIC ACTIVITY IN TUBULIN PREPARATIONS

I83

In neither of these reports, were any features of this activity described except for its sensitivity to stimulation by cyclic AMP. As a part of a series of investigations on the biology of microtubules, we have examined the intrinsic protein kinase activity of tubulin in detail. This report describes some of the general characteristics of this enzyme activity in preparations of lyophilized tubulin from porcine brain. MATERIALS AND METHODS

Chemicals

[y-3*p~ATP and Ey-a*pIGTP were purchased from ICN Corporation. Nucleotides were purchased from Boeringer-Mannheim. Histone fraction HfI was purchased from Worthington. Vinblastine was a gift from Eli Lilly and Company. Preparation of tubulin

Fresh pig brains were obtained from a local slaughter-house and transported to the laboratory on ice. Tubulin was prepared by the method of Weisenberg et al. 13 and stored after lyophilization of 2-3-mg aliquots. In order to minimize aggregation of the tubulin, the conditions suggested by Weisenberg and Timasheffla were applied to these preparations prior to lyophilization. The protein concentration was maintained at about io mg/ml and Mg~+ was removed by dialysis against three changes of IOO volumes each of lO-4 M GTP in lO-3 M sodium phosphate buffer (pH 6.5). The samples were stored in the vials in which they were lyophilized, below o °C. Electrophoresis

Purity of tubulin preparations was monitored by acrylamide gel electrophoresis in a Canalco apparatus. Tubulin was reduced by making it o.12 M in mercaptoethanol and adding urea to 6 M and sodium dodecyl sulphate to 1%. After heating to 95 °C for IO min, sucrose and bromphenol blue were added and the reduced, denatured protein was then layered over running gel from which persulphate had previously been removed by electrophoresis at 5 mA per gel for I h. Gels were 6-7 cm long by 0. 5 cm in diameter. The gels were normally 7% acrylamide, made up with running buffer which was Tris-glycine (o.I M, pH 8.5) containing 6 M urea and o.1% sodium dodecyl sulphate. Electrophoresis was started at I mA per gel until the tracking dye had run entirely into the gel (usually about 30 min). The current was then turned up to 5 mA per gel and the electrophoresis was allowed to proceed until the tracking dye reached the bottom of the gel. Gels were fixed in 7% trichloroacetic acid and stained with coomassie blue. Protein kinase assay

The basic assay was similar to those described by Reimann et al. 1~ and by Miyamoto et al. 16. Casein, when used as substrate, was prepared according to Reimann et al. 1~. Incubations normally contained ethyleneglycol-bis(aminoethyl)-tetraacetic acid (0.03 #mole), MgC12 or magnesium acetate (2/~moles), potassium phosphate or glycerophosphate buffer (5/~moles (pH 6.5) except as stated) and bovine serum albumin (IOO#g) in an incubation volume of o.i ml. [~,-32p]ATP or Ey-32plGTP were Biochim. Biophys. •cta, 271 (1972) 182-192

184

o. SOIFER et al.

normally IO6 cpm per incubation. Other additions were made as indicated. In early experiments (Method i) incubations were terminated by addition of o. 5 nfl of io°:,, trichloroacetic acid. Precipitates were resuspended in o.I ml I M NaOH and reprecipitated by addition of 0.5 ml i o % triehloroacetic acid. After 4 5 reprecipitations, the trichloroacetic acid precipitates were dissolved in Soluene (Packard TM) and counted in toluene-(CHa)2POPOP-PPO with a Packard liquid scintillation spectrometer. More recently (Method 2), we have used the procedure of Reimann ctal)" terminating our reactions by pipetting 5° #1 of reaction mixture on 2 cm / 2 cm squares of Whatman ET3I filter paper and washing the squares in to".{, and then 5% triehlor()acetic acid in the cold, and 5% trichloroacetic acid for 4 washes at room tenlperaturc. After washes in ethanol and ether, the papers were placed in counting vials and counted as above, but without Soluene. Data presented is in terms of total pmoles of phosphate transferred to trichloroacetic acid precipitable protein, normalized to tubulin concentration, time or both.

Protein determination Protein was measured by the method of Lowry et al3 7 using bovine serum albmnin as a standard. RESULTS

PuriO, of tubulin Each preparation of tubulin was examined by eleetrophoresis on acrylamide gels in the sodimn dodecyl sulfate urea system to provide an indication of the degree of purity of the protein. Typical patterns are included in Fig. I. The pair of bands is characteristic of brain tubulin is 2o. Preparations of tubulin were used for studies of intrinsic protein kinase activity only if no other bands could be resolved on these gels when the gels were overloaded to a point where the tubulin bands became distorted or smeared.

Fig. i. A c r y l a m i d e gel electrophoresis of typical t u b u l i n p r e p a r a t i o n used for protein kinase exp e r i m e n t s . Conditions as in Methods. A ,B, (', I) : 5-, to-, 2o-, 5o-/tl a p p l i c a t i o n s of a p p r o x , i m g / m l p r o t e i n samples.

lliochim. Biophys. Acla, 271 (I97 z) ~82 ~92

ENZYMATIC ACTIVITY IN TUBULIN PREPARATIONS PROTEIN KINASE ACTIVITY OF AS A

185

TUBULIN

FUNCTION OF TIME

1200 z

I000

D

800

~;

600

#,

400 200

2

4

;

8

lO

I'2

'4

I'6

"8

20

INCUBATION TIME

Fig. 2. P h o s p h o r y l a t i o n of casein b y tubulin as a function of time. E a c h incubation included 6o0 leg casein, 5 ° # g tubulin, 2 nmoles ATP, i #mole NaF, ioo nmoles caffeine. O t h e r conditions as detailed in Methods. [~2P]Protein collected, washed and p r e p a r e d for c o u n t i n g b y Method I. The line represents a least-squares fit of the d a t a to a straight line. Each point represents a separate incubation.

PROTEIN KINASE ACTIVITY OF OF

TUBULIN

TUBULIN CONCENTRATION IN

°t

AS A

FUNCTION

ASSAY

It

n,IaJ Z rr" ~-

5

LtJ ~ a

4

(D 0

x

m.

i UME

•

I O 0 sJI.

3

J

2'0 4o

60

do

ioo ,~o ,4o ,;o ,8o z;o

,ug TUBULIN

IN ASSAY

Fig. 3. P h o s p h o r y l a t i o n of casein b y t u b u l i n as a function of the a m o u n t of t u b u l i n in the assay. Same conditions as Fig. 2 except each incubation contained I nmole ATP. I n c u b a t i o n time: io rain. Least squares fit to a s t r a i g h t line. E a c h point represents a separate incubation.

Biochim. Biophys. Acta, 271 (z972) 182-192

1~. soH:v:It et al.

186 PROTEIN KINASE ACTIVITY OF TUBULIN AS A

FUNCTION OF pH

I

BATION • It

~0"

MINUTES

,60"

40"

20" 00"

80"

40 20

pH F i g . 4. P h o s p h o r y l a t i o n o f c a s e i n b y t u b u l i n a s a f u n c t i o n o f p H . C o n d i t i o n s a s in l;ig. 2. E a c h point represents the mean of at least three separate incubations. Vertical bars represent standard deviations.

General properties The intrinsic protein kinase activity of tubulin was proportional to the reaction time for at least 20 min (Fig, 2) and was proportional to the amount of tubulin in the reaction in the range 0.05 to 2 mg/ml (Fig. 3). The pH optimum was about 6.6 (Fig. 4).

TABLE

I

EFFECT OF ~ [*M 3',5'-C3"CLIC A M P ON PROTEIN KINASE ACTIVITY OF DIFFERENT PREPARATIONS OF TUBULIN D a t a i n c l u d e d in "Fable I is f o r f i v e d i f f e r e n t p r e p a r a t i o n s o f t u b u l i n . E a c h v a l u e r e p r e s e n t s t h e m e a n o f a t l e a s t t h r e e s e p a r a t e i n c u b a t i o n s , :L i s t a n d a r d d e v i a t i o n . I n c u b a t i o n t i m e , i o m i n ; A T P concentration, io/~M. Each incubation included 6oo #g casein and IOO/,moles of caffeine; other c o n d i t i o n s a s i n M e t h o d s . R e a c t i o n s t e r m i n a t e d a n d p r o t e i n p r e p a r e d f o r c o u n t i n g a s in M e t h o d 1.

pmoles of phosphate transferred from A T P per rain per mg tubulin --3",5'-cyclic A M P +3",5'-cyclic A M P 33 46 35 43 51

k _!:L ~_~

IO 2 io 12 I5

2S 74 3° 52 57

~ ~2. 2~_ J-

4 3 5 iI 2I

Biochim. Biophys. Acta, 2 7 I ( I 9 7 2 ) I 8 2 - I 9 2

187

ENZYMATIC ACTIVITY IN TUBULIN PREPARATIONS

Effect of cyclic nucleotides 3',5'-cyclic AMP had no significant effect on the protein kinase activity of these preparations (Table I). In the presence of cyclic AMP, neither the properties described in Figs 2-4 nor the apparent relations between velocity and substrate were affected. 3',5'-cylic GMP was also without discernable effect.

Effect of NaF NaF is a potent inhibitor of many phosphatases and phosphotransferases. It has been included in most protein kinase assays15,~6,z5 to block enzymatic removal of the phosphorylated protein produced by the kinase reaction and to maintain ATP concentration in the face of possible ATPase contamination. NaF inhibits the intrinsic protein kinase activity of tubulin at concentrations in excess of IO mM (Fig. 5). PROTEIN KINASE ACTIVITY OF TUBULIN AS A FUNCTION OF NoF CONCENTRATION

IO MIN. INCUBATION IN/VIAL

C

i0-4

i0-3

i0-2

i0-I

MOLAR CONCENTRATION OF NoF

Fig. 5. Effect of N a F on intrinsic protein kinase activity of tubulin. Conditions as in Fig. 2, except no caffeine was included, and 300/~g of casein and 60/~g of t u b u l i n were used for each incubation. E a c h point represents m e a n of three incubations; vertical bars represent s t a n d a r d deviation.

Effect of tubulin-binding alkaloids Colchicine and vinblastine are known to bind to specific sites on tubulin13,14, 21-z~. To determine whether these sites themselves might be involved in the protein kinase activity or whether the presence of these alkaloids might cause rearrangement of the tubulin with consequent effects on the intrinsic kinase activity, the effects of colchicine and vinblastine on the intrinsic protein kinase activity were measured (Figs 6 and 7). Neither alkaloid inhibited the kinase activity.

Substrate specificity Both casein and a lysine-rich fraction (HfI) of histone were effective phosphate acceptors, the histone being more effective on a gram basis as a substrate for the Biochim. Biophys. Acta, 271 (1972) I82-192

1). SOH:E~ et al.

I88 EFFECT

OF COLCHICINE ON PROTEIN

KINASE

ACTIVITY

OF

TUBUUN

b xlO" X

~8

8

•

10-8

10-7

•

:

•

.

|

:

,1

~s _>

b-

ga ¢I

z

COLCHIC|NE

i0-6

CONCENTRATION

10-5

IN

ASSAY

10-4

(MOLAR)

Fig. 6. Effect of colchicinc on p r o t e i n k i n a s e a c t i v i t y of t u b u l i n . C o n d i t i o n s as for Fig. 5. E a c h point represents a separate incubation.

protein kinase of tubulin (Table II). Maximal activity could be achieved of ttfI histone while 6oo # g of casein were required to reach the same level Bovine serum albumin was completely ineffective as a substrate. Tubulin is normally prepared in the presence of I o o / , M GTP and w a y ~a. In order to estimate effective A T P concentrations and nucleotide

EFFECT

~2

OF

VINBLASTINE ACTIVITY

OF

ON

PROTEIN

with 5 ° # g of activity. stored that specificity,

KINASE

TUBULIN

'o

:

x

:

• :

b

(.lO0 ~g TUBULIN) [9 X LO'flM ATP) ~600 ~1 CASEIN) (PREINCUBATE WITH VINBLASTtNE- 15 MIN, ot 4eC} (10 MtN. tNCUBATtON)

f 10`7

io-~

,b-s

~o-,

~o-~

VLNBLASTINE CONCENTRATION {MOLAR) Fig. 7. Effect of v i n b l a s t i n e on p r o t e i n k i n a s e a c t i v i t y of t u b u l i n . C o n d i t i o n s as for Fig. 2 e x c e p t e a c h i n c u b a t i o n c o n t a i n e d 9 n m o t e s A T P a n d i o o / z g of t u b u l i n . Caffeine w a s not i nc l ude d. E a c h p o i n t r e p r e s e n t s a s e p a r a t e i n c u b a t i o n . T u b u l i n w a s a d d e d t o r e a c t i o n m i x t u r e w h i c h i n c l u d e d all e l e m e n t s e x c e p t ATP. A f t e r 15 rain i n c u b a t i o n a t 4 °C, t h e k i n a s e r e a c t i o n s were s t a r t e d b y a dd i t i o n of 9 n m o l e s A T P i n c l u d i n g IOe c p m [y-a2P]ATP.

Biochim. Biophys. Acta, 27t (I972) I 8 2 - t g z

189

ENZYMATIC ACTIVITY IN TUBULIN PREPARATIONS T A B L E II A COMPARISON OF THE RELATIVE TORS FOR THE INTRINSIC

PROTEIN

EFFECTIVENESS

OF CASEIN AND HISTONE

AS P H O S P H A T E

ACCEP-

K I N A S E OF T U B U L I N

Assay conditions as in Methods. E a c h incubation was initiated b y addition of 15 ° / 2 g of tubulin. A T P concentration, 5.5 FM; incubation time, 15 min. Values are m e a n s of 5 separate incubations, - - s t a n d a r d deviation.

Substrate

#g used in incubation

pmoles phosphate transferred per mg tubulin

Casein

15 ° 3 °0 600

55.i ± 6.8 74.7 -4- 4.7 lO8.2 ± IO.I

Histone H f I

5° IOO 200 400

99.5 117.o lO8.O 115.1

~ 9.8 i 14.3 ~ 6.8 ! 13.1

lyophilized preparations of tubulin were suspended in water and dialyzed against three changes of ioo vol. of each of I mM phosphate buffer (pH 6.5) in order to remove unbound GTP. These preparations retained their protein kinase activity. The apparent Km for ATP in several preparations was in the range 2- 4/~M (Fig. 8 shows typical experiment). Although the Km remained nearly constant from one preparation to the next, the V varied from as low as 15 pmoles/mg per min to more than ioo pmoles/mg per rain with different preparations of tubulin.

7ooo 6ooo PHOSPHATE DONOR" ATP

X

5000

ACCEPTOR " CASEIN (300 Ug)

4000

ENZYME • TUaULIN D98 -DIALYZI:D E GTP {EO.Eeg VIAL)

~ 3000 2000,

10OO

// -soo - ~

/

j

Km • 3.7 X I(~EM ATP

m

400 800 '2o0 ,6~0 2~0 2~,oo 2;00 ~00 3'6oo gooo I,//S (x ~05 )

Fig. 8. Double reciprocal plot illustrating the effect of A T P concentration on the rate of p h o s p h a t e t r a n s f e r from A T P to casein b y intrinsic protein kinase of tubulin. V is pmoles p h o s p h a t e t r a n s ferred per m g t u b u l i n per rain. S is pmoles of A T P ir~cluded in the incubation. Assay conditions as in Methods. E a c h incubation included 3oo/*g casein and was initiated b y addition of 60.6 Fg tubulin. I n c u b a t i o n s were t e r m i n a t e d and protein p r e p a r e d for c o u n t i n g b y Method 2. Each point represents a separate incubation. Line is a least-squares fit of the data. A p p a r e n t K m = 3.7 FM ATP.

Biochim. Biophys. Acta, 271 (1972) 182-192

I). SOIFt,;R C[ a[.

I9O

/

J

5000

/

4000

0

×

3000,

% 2000

I000

-

100

/

PHOSPHATE

ENZYME = TUBULIN TO REMOVE

../ i&

Km = l i b

300

28o

DONOR- GTP

ACCEPTOR - CASEIN

400

(300 ~Jq)

D98" GYP

DIALYZED (88~g//VIAL)

X 10"6M GTP

580

~60

1/S [x 1o~) Vig. 9. Double reciprocal plot illustrating the effect of ( ; T P concentration on the rate of phosp h a t e transfer from G T P to casein by intrinsic protein kinase of tubulin. Conditions, concentrations, units as in Fig. S except each incubation was initiated with 88 ]~g tubulin. A p p a r e n t K,,, I 1.8 t~M GTP.

GTP was almost as effective a substrate as ATP: the apparent Km for GTP being between IO #M and 2o/~M (Iqg. 9). DISCUSSION

These observations show that lyophilized porcine brain tubulin preparations have intrinsic protein kinase activity and define some properties of that activity. As in the case with the ox-brain protein kinase described by Rodnight and Lavin 24, G T P is almost as effective a phosphate donor as ATP for the phosphorylation of histone or casein by the intrinsic protein kinase of tubulin. However, unlike the protein kinase that they studied, which was first purified by Rabinowitz and Lipmann 25, the p H optimum for the protein kinase of tubulin is about pH 6.6. Rabinowitz and Lipmann found a broad p H curve with an optimum between p H 7 and pH 8 and significant activity at pH values near 9. At p H values greater than 7.5, the kinase activity of lyophilized tubulin is minimal. The failure of colchicine and vinblastine to interfere with activity indicates that the site of kinase activity is independent of the binding sites for these alkaloids. Furthermore, any conformational changes that colchicine or vinblastine induce in the tubulin do not affect the kinase activity. At vinblastine concentrations in excess of io mM, there was some indication of increased kinase activity. At these concentraBiochirn. Biophys. Acts, 271 (1972 ) 182-192

ENZYMATIC ACTIVITY IN TUBULIN PREPARATIONS

191

tions, tubulin precipitates ~°,23. The apparent stimulation of kinase activity may be real or may be due to sampling errors consequent to precipitation of the tubulin in the incubation vial. There is considerable evidence that cyclic nucleotides regulate many protein kinases through a mechanism by which binding of the cyclic nucleotide to a regulatory peptide causes that peptide to partly or completely dissociate from the catalytic unit of the enzyme ~6-3°. Thus, the enzyme appears fully active either in the presence of cyclic nucleotides or under conditions where the regulatory unit has been modified or displaced. The lack of effect of cyclic nucleotides on the intrinsic protein kinase of tubulin appears to be a consequence of the tubulin-kinase being already fully active. In another series of experiments (to be described in a separate publication) cyclic AMP and cyclic GMP have been shown to regulate the intrinsic protein kinase activity of non-lyophilized tubulin but the regulatability of the enzyme activity has proved to be exceedingly labile. The non-regulatable protein kinase activity described in this report may represent a fully or partially active enzyme whose regulatory elements have been inactivated or damaged as a consequence of lyophilization. The biological role of a microtubule associated protein kinase can only be guessed at on the basis of the evidence in hand. If secretory mechanisms, including those governing neurotransmitter release, function along the lines proposed by Rasmussen 31, the site for cyclic AMP activation of the secretory process may well be the intrinsic protein kinase of tubulin. An explanation of the role of microtubules in the expression of the directive effect of insulin on adipoyte metabolism 4 may be attempted here as well: when tubulin is assembled into microtubules, the intrinsic protein kinase activity of the tubulin can no longer continue to maintain the activities of the lipolytic and glycogenolytic enzyme systems; the kinase is available for regulation of these systems only when not incorporated into microtubules. The presence of protein kinase activity in tubulin preparations provides a point of attack for the investigation of microtubule function in a variety of systems. ACKNOWLEDGMENTS

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192 II I2

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Biochim. Biophys. Acta, 271 (i972) ~82 I0-'