Inhibition of tubulin-dependent ATPase activity in microtubule proteins from porcine brain by S100 protein

Neurochem. Int. Vol. 13, No. 4, pp. 509-516, 1988 Printed in Great Britain. All rights reserved

0197-0186/88 $3.00+0.00 Copyright © 1988PergamonPress pie

INHIBITION OF TUBULIN-DEPENDENT ATPase ACTIVITY IN MICROTUBULE PROTEINS FROM PORCINE BRAIN BY S100 PROTEIN HARUO ASAI, YUKIHIRO MIYASAKA,YOSHIYUKIKONDO and TOSHIHIROFUJII* Department of Functional Polymer Science, Faculty of Textile Science and Technology, Shinshu University, Ueda 386, Japan (Received 30 April 1988; accepted 14 June 1988)

Abatraet--Microtubule-associated proteins (MAPs) of brain microtubules exhibit an ATPase activity which is markedly enhanced by tubulin and Ca2+. Addition of S100 protein decreased the tubulindependent Ca2+-ATPase activity by about 85%, but did not affect the activity without tubulin. The inhibition by S100 protein was concentration-dependent and the apparent /(ffi value for ATP was not altered. A large amount of tubulin restored the inhibition, indicating that Sl00 protein acts through the binding to the tubulin molecule. Zn2+, which can bind both microtubule proteins and S100 protein, had little effect on the inhibitory action of Sl00 protein. The ATPase inhibition by Sl00 protein was partially restored by chlorpromazine or vinblastine. Sl00a is more effective than Sl00b on the inhibitory effect of tubulin-dependent ATPase activity. The results suggest that Sl00 protein may function as a regulatory factor of ATPase in brain microtubules. Microtubules play a role in cellular movement including the separation of chromosomes on the spindle formed during mitosis, axonal transport and hormonal secretion. These phenomena need ATPdependent energy generation as observed in cilia and flagella in which dynein has been reported to serve as a motor protein (Gibbons, 1981). Mammalian brain microtubule proteins exhibit an ATPase activity. When microtubule proteins were separated into tubulin and microtubule-associated proteins (MAPs) fractions by gel filtration, ammonium sulfate precipitation or phosphocellulose column chromatography, ATPase activity remained in the MAPs fraction (Gelfand et al., 1978; Larsson et al., 1979; Webb, 1979; White et al., 1980). Membrane ATPase inhibitors did not affect the ATPase activity in microtubule proteins (Gelfand et al., 1978). We have reported further that the addition of tubulin stimulates the MAPs ATPase activity (Ihara et al., 1979; Fujii et al., 1982, 1983), indicating that an ATPase is specifically associated with brain microtubules.

Recently, a microtubule-based motor protein has been isolated from squid giant axon, and termed as kinesin (Vale et al., 1985a). Video-enhanced differential interface contrast microscopy has indicated that this protein in the presence of ATP induces the movement of axoplasmic orgauelles along microtubules and also of latex beads on microtubules /n vitro. T h e purification of kinesin was carded out by forming a high affinity complex with microtubules in the presence of a non-hydrolyzable ATP analog adenylyl-imidodiphosphate (AMP-PNP) and its release by ATP (Vale et al., 1985a; Scholey et al., 1985). Kinesin has been also purified from chick brain (Brady, 1985), mammalian neuronal tissues (Vale et al., 1985a; Kuznetsov and Gvlfand, 1986) and sea urchin eggs (Scholey et al., 1985), and has been identified by immunoblot analysis in a variety of mammalian tissue culture cell lines, including 3T3 and PC12 cells (Vale et al., 1986). However, purified kinesin has a low ATPase activity. Kuznetsov and Gelfand (1986) have shown that kinesin, isolated from bovine brain using tripolyphosphate (PPPi) instead of AMP-PNP, shows relatively high Mg2+-ATPase activity and the ATPase activity is remarkably stimulated in the presence of taxolpolymerized tubulin. This evidence suggests that neuronal kinesin in itself may be an energy generator for fast axonal transport. S100 protein is an addle protein of central nervous

*Address correspondence to: Dr Toshihiro Fujii, Department of Functional Polymer Science, Faculty of Textile Science and Technology, Shinshu University, Ueda 386, Japan. Abbreviations: AMP-PNP, adenylyl-imidodiphosphate; MAPs, microtubule-associated proteins; MES, 2-(Nmorpholino)ethansulfonic acid; PPPi, tripolyphosphate; SDS, sodium dodecyl sulfate; NEM, N-ethylmaleimide. 509

ll~R~o A.s.~l ct a/

510

system with Ca 2+-binding d o m a i n s h o m o l o g o u s to calmodulin a n d several o t h e r Ca: +-binding proteins (Moore, 1965; Calissano et al., 1969; Szebenyi et al., 1981). This protein m a y play roles in m i c r o t u b u l e p o l y m e r i z a t i o n ~ l e p o l y m e r i z a t i o n and p h o s p h o r y lation o f a brain 73 k D a protein (Baudier et aL, 1982b; E n d o a n d Hidaka, 1983; D o n a t o , 1983, 1986; Fujii et aL, 1986a; Patel a n d M a r a n g o s , 1982). Ca :~ is essential for these effects. However, the precise biological function of Si00 protein is u n k n o w n . We have reported recently that SI00 protein inhibits C a : " - A T P a s e activity in m i c r o t u b u l e proteins from porcine brain (Fujii et al., 1987). We report here t h a t t u b u l i n - d e p e n d e n t A T P a s e inhibition by S100 protein is observed only when tubulin a n d Ca "~ are present. This inhibitory effect is described further with respect to its m e c h a n i s m and pharmacological properties. T u b u l i n - d e p e n d e n t A T P a s e is c o m p a r e d with kinesin a n d m i c r o t u b u l e activated ATPase. EXPERIMENTAL PROCEDURES

Materials ATP and 2-(N-morpholino)ethansulfonic acid (MES) were purchased from Boehringer; chlorpromazine, W-7. trifluoperazine and AMP-PNP from Sigma; DEAE Sephadex A-50, Sephadex G-75 and Phenyl-Sepharose CL-4B from Pharmacia; [~,-32P]ATP from ICN Radiochemicals; PPPi from Nakarai Chemicals; vinblastine was a gift from Shionogi Co. Ltd: pepstatin A from Peptide Institute Inc. Other general laboratory reagents were of the highest grade. Preparation o f proteins S100 protein was purified from fresh porcine brains by ammonium sulfate fractionation followed by column chromatography on DEAE-Sephadex A-50 and Sephadex G-75 (Isobe et al., 1977). S100a and Sl00b were prepared according to Baudier et al. (1982a) as follows: the crude SI00 protein was dialyzed against buffer A (50 mM Tris-HCl, pH 7.5, 2 mM 2-mercaptoethanol). ZnSO4 was added to the dialyzed solution to a concentration of 1 mM and immediately applied to a Phenyl-Sepharose 4B column (2 × 15 cm) which had been equilibrated with buffer A. The column was washed with buffer A containing 0.3 M NaCI and 0.25 mM ZnSO4, and the bound protein was eluted with buffer A containing 2 m M EDTA. Sl00b, a highly hydrophobic protein, was selectively bound in the presence of Zn > , whereas Sl00a was not bound. The fractions were analyzed by electrophoresis on 12.5% SDS-polyacrytamide gels (Laemmli, 1970). Gels were stained with 0.1% Coomassie Brilliant Blue R-250 in ethanol-acetic acid-water (5:1:4) and destained by diffusion in 10% acetic acid. Microtubule proteins were prepared from porcine brains by three cycles of temperature-dependent polymerization and depolymerization with some modifications (Ihara et al., 1979). Brains were homogenized in buffer B (100mM MES-KOH, pH6.6, 0.5mM Mg(CH~COO):, 1 mM EGTA) containing 2#g/ml pepstatin and centrifuged at 70,000g for 50 min at 4°C. To the supernatant was added l m M ATP, 0.2mM GTP and 15% glycerol in a final

concentration. ]'he mixture was incubated tbr 30 mm ,tL 37 C. and then centrifuged at 70,000g for 45 min at 2YC through a 25% glycerol cushion. The microtubule pellet was dispersed in buffer B at 0°C, followed by centrifugation at 70,000g for 50min at 4~C. The polymerization and depolymerization cycle was repeated twice except that ATP was omitted in polymerization step. MAPs and tubulin were separated from the depolymerized microtubule proteins by column chromatography on phosphocellulose (Fuiii ~'~ al.. 1982). .4 TPase activity The standard reaction mixture contained 60raM MES-KOH (pH6.5), 5mM CaCl2, 2 m M 2-mercaptoethanol, 12.5% (vol/vol) glycerol, 0.5mM [~,>:P]ATP (4.5/~Ci/#mol), 13yg MAPs and 18/tg tubulin in a final volume of 50y1. After incubation for 20min at 37 (?, the reaction was stopped by 0.4 ml of cold 20 mM silicotungstic acid in 0.02N H2SO 4. The 32p in the each sample was assayed by a liquid scintillation spectrometer (Aloka, LSC-1000) to determine the amount of Pi released during the reaction. Protein concentration Protein concentration was determined according to the method of Lowry et al. (1951) using bovine serum albumin as the standard.

RESULTS

S lO0 protein inhibits A T P a s e activity

tubulin-dependent

MAPs

The M A P s A T P a s e was activated by the addition of tubulin a n d Ca 2+. The effect o f SI00 protein on A T P hydrolysis by M A P s in the presence or absence of tubulin is presented in Fig. 1. SI00 protein in-

14 12

C

4 O

O.

, ± ÷ , 0.2 & 0,6 0.8 SIO0 Protein (mglml)

0

0 1.0

Fig. l. Effect of SI00 protein on Ca2 ~-ATPase activity in MAPs, tubulin, MAPs-tubulin mixture. ATPase activity was measured as described in Experimental Procedures, in media containing 60mM MES-KOH (pH6.5), 5mM CaCI 2, 2 mM 2-mercaptoethanol, 12.5% (vol/vol) glycerol and 0.5 mM [Z-32P]ATP, made up to contain the concentration of S100 protein indicated. The concentration of MAPs and tubulin were 0.26 and 0.36 mg/ml, respectively. O , MAPs-tubulin mixture: C), MAPs; A, tubulin.

SI00 protein inhibits microtubular ATPase hibited tubulin-dependent ATPase activity of MAPs, but had little effect on the activity in the absence of tubulin. The hydrolysis was inhibited by S100 protein about to 15% of the original level over 1.0mg/ml SI00 protein added. The concentration of S100 protein required to obtain a 50% inhibition was 0.15mg/ml. SI00 protein could not alter Mg'+-ATPase activity of MAPs in the presence or absence of tubulin. When calmodulin was used in the place of S100 protein, the Ca2+-ATPase activity was unaffected (data not shown). The MAPs ATPase as a function of tubulin is illustrated in Fig. 2(a). The stimulation of MAPs ATPase activity was observed whenever tubulin was included in the reaction mixture. As determined by double-reciprocal plots, the concentration of tubulin

511

O~ O4

0.2

-O01

OO1

0 IIATP (pM)"(~

Fig. 3. Tubulin-depcndent ATPase activity of MAPs in the presence or absence of SI00 protein as a function of ATP concentration. Tubulin-dependent Ca'+-ATPase activity in the presence (0, 0.45mg/ml) or absence (O) of Sl00 protein was measured at 37°C for 10rain in standard conditions modified the concentration of A T P indicated.

16 14

~ ~ ~ - ' - ' ~

!: lo

8

2

•

0.2

(14

Tubulin

(16

0.8

(mglml)

1.0

(a)

1.01 / !o,/

7/"

I/.,,," 0

2

4

6

llTubulin (b)

8 10 12 14 (mglml) "4

Fig. 2. Dependence on tubulin concentration of MAPs ATPase activity. (a) Ca2+-ATPase activity of MAPs in the presence (&, 0.23 mg/ml; O, 0.45 mg/ml) or absence (C)) of SI00 protein was measured in standard conditions modified to contain the concentrations of tubulin indicated. (b) The double reciprocal plot.

required for half-maximal activation of MAPs ATPase was 0.09 nmol in the absence of S100 protein [Fig. 2(b)], while the concentrations were 0.45 and 0.59 nmol in the presence of 0.23 and 0.45 mg/ml S100 protein, respectively. Addition of excess tubulin was able to overcome completely the inhibition by Sl00 protein, suggesting that S100 protein binds to tubulin and cancels out the effect of tubulin enhancement on MAPs ATPase activity. The kinetic parameters of ATP hydrolysis by MAPs ATPase were also studied. The ATPase activity was assayed in the presence of 0-500/~ M ATP with or without Sl00 protein,-and the results were shown in the double-reciprocal plots (Fig. 3). The Km of tubulin-dependent MAPs ATPase for ATP was indicated to be 100 #M irrespective of S100 protein.

Effect of several factors on the inhibition by SIO0 protein Both Sl00 protein and microtubule proteins bind Zn 2+ (Baudier and G6rard, 1983; Lee et al., 1983) and microtubule assembly is suppressed by S100 protein in the presence of Zn 2+ (Deinum et al., 1983; Fujii et al., 1986b). The effect of Zn ~+ on the inhibition of tubulin-dependent ATPase activity by Sl00 protein is shown in Table 1. In the presence of Ca 2+, addition of Zn 2+ potently inhibited the ATPase activity with or without Sl00 protein. In the absence of Ca 2+ under which the ATPase activity was relatively low, Zn 2+ slightly inhibited the ATPase activity and Sl00 protein had little effect on the ATPase activity. These results suggest that Zn z+ has no influence on Sl00 protein-dependent ATPase inhibition.

,512

HARt() ASAi ~'t" U[,

trifluoperazine, calmodulin antagonists, affected the ATPase inhibition by S100 protein (data not shown). Dynein ATPase was specifically inhibited by low concentration ( ~ I 0 p M ) of vanadate, whereas tubulin-dependent MAPs ATPase remained about 85% of the original level by 10pM vanadate (Table 3). N-ethylmaleimide (NEM) (0.SmM), AMP-PNP (lmM) and PPPi ( l m M ) inhibited tubulin-dependent ATPase activity by 74, 67 and 50%, respectively. The inhibition was also observed in the presence of SI00 protein but the extent was low.

Table I. Effect of Zn 2+ on tubulin-dependent ATPase activity of MAPs in the presence or absence of St00 protein Pi released (nmol mg/min) (-)SI00 ( ÷)SI00

Zn 2. (mM) ( + ) C a -'+

0 0.~ 0.2 0.4 0 0,1 0.2 0.4

( - )Ca 2+

13.8 5.31 1.69 0.77 3.46 2.85 2.70 2.40

2.85 1.62 0.85 {L69 3.46 3.00 3.08 2.31

Experimental conditions were the same as for Fig. l except that 5 mM CaCI 2 was replaced by 1 mM EGTA in (--)Ca 2 ÷ condition. The concentration of SI00 protein was 0.45 mg/ml.

Eff~,ct of SlOOa and SlOOb Si00 protein is known to be a mixture of two principal components SI00a and S100b, with a subunit compositions ~fl and fl/~ (Isobe and Okuyama, 1978, 1981). Electrophoretic analysis in t2.5% polyacrylamide gel shows that S100a and Sl00b were highly purified by Zn2+-dependent affinity chromatography [Fig. 4(a)]. The effects of Si00a and Sl00b on the tubulin-dependent MAPs ATPase activity are shown in Fig. 4(b). Both S100a and S100b suppressed ATPase activity of MAPs in a

Chiorpromazine restored the inhibition of tubulindependent ATPase activity by S100 protein to some extent (Table 2), and the reagent had little effect on the activity in the absence of S100 protein under the experimental condition employed. Antimitotic drug vinblastine, which is capable of activating tubulindependent ATPase activity (Fujii et al., 1982), also restored the ATPase inhibition by SI00 protein. However, neither N-(6-aminohexyl)-5-chloro-lnaphthalenesulfonamide hydrochloride (W-7) nor

Table 2. Effect of calmodulin antagonists on tubulin-dependent Ca2*-ATPase activity of MAPs in the presence or absence of S100 protein Pi released (nmot/mg/min) Cone, (#M)

(-)SI00

(+)SI00

Inhibition (%)

30 60 120 30 60 120

13.5 12.4 11.5 11,2 20.5 19.2 24.4

3.24 3.91 5.15 5.87 14.8 14.6 14.5

76.0 68.5 55.2 47.6 27.8 24.0 40.6

Control Chlorpromazine

Vinblastine

Enzyme activity was measured in standard conditions modified to contain the concentrations of drugs indicated. The concentration of SI00 protein was 0.45 mg/ml. Table 3. Effect of various reagents on the tubulin-dependent ATPase activity of MAPs in the presence or absence of SI00 protein Pi released (nmol/mg/min) Cone, Control Vanadate

NEM

AMP-PNP

PPPi

5 ,uM 10 100 0.125 mM 0.25 0.5 0.25 mM 0.5 1.0 0.25 mM 0.5 1.0

(-)SI00 18.7 16.6 15.1 7.58 I 1.7 8.41 4.89 10.9 8.56 6.26 19.2 16.9 9.37

Inhibition (%) 11.2 19.3 59.5 37.4 55.0 73.9 41.7 54.2 66.5 9.6 49.9

(+)S100

Inhibition (%)

4.50 4.19 4.19 3.34 3.40 2.65 1.95 3.25 2.53 2.63 4.50 3.28 3.68

6.9 6.9 25.8 24,4 41.1 56.7 27.8 43,8 41.6 0 27.1 18.2

Enzyme activity was measured in standard conditions modified to contain the concentrations of reagents indicated, The concentration of Sl00 protein was 0.45 mg/ml.

S100 protein inhibits microtubular ATPase

o

513

concentration-dependent manner. However, S100a was more effective than Sl00b on the inhibition of ATPase activity. The concentration of SlOOa and Sl00b required for 50% inhibition of ATPase activity were 0.13 and 0.19 mg/ml, respectively.

o

M.W.

DISCUSSION

I - subunit ~- subunit

(a) 14 12

~1o f8 4 2 0 Protein

(mg/ml)

(b) Fig. 4. Effect o f Sl00a (a//) a n d Sl00b (p//) on tubulindependent M A P s ATPase activity. (a) SDS-polyacrylamide gel electrophoretic pattern of the components S100a (10 ~g) and Sl00b (10~g). The arrows indicate the positions o f molecular weight markers: carbonic anhydrase (30,000), soybean trypsin inhibitor (20,000) and ~,-lactalbumin (14,400). (b) Tubulin-dependent Ca2+-ATPase activity was measured made up to concentration of Sl00a ( 0 ) and Sl00b (C)) indicated. Experimental conditions were the same as for Fig. 1.

Recently, we have reported that ATPase activity contained in the preparation of microtubule proteins is considerably suppressed by $100 protein (Fujii et al., 1987). The data presented in this report show that S100 protein also exhibits a significant inhibition on tubulin-dependent Ca2+-ATPase activity of MAPs, while the MAPs ATPase activity in the absence of tubulin was not affected by S100 protein (Fig. 1). Half-maximal inhibition of ATPase activity was found at 2mol of SI00 protein per mol of tubulin. Recently, the similar ratio has been reported when S100 protein suppresses microtubule assembly to 50% in the presence of 0.2 mM CaC12 (Endo and Hidaka, 1983; Fujii et al., 1986a). In addition, the direct interaction between S100 protein and tubulin has been observed by the use of affinity chromatography. A large amount of tubulin cancels out the inhibitory action of S100 protein (Fig. 2), indicating that SI00 protein-tubulin complex, as compared to tubnlin alone, would be a less effective species on the activation of MAPs ATPase activity. However, we have no suitable explanation for the requirement of unphysiologically high Ca 2+ concentration for tubuiin-dependent ATPase activity of MAPs and its inhibition by S100 protein. Although the involvement of cytoplasmic elements in axonal transport is well established, the molecular mechanism is largely unknown (Allen et aL, 1985). The intracellular movement of organelles and several cytoplasmic matrix proteins in axonal cytoplasm is modulated by an ATP-dependent mechanism (Adams, 1982). Recently, Vale et al. (1985a) have shown that an energy generation protein, kinesin, is

Table 4. Comparison of mierotubule-related ATPases Dyneina Kinesinc Microtubule activated ATPasec Tubulin-dependent ATPase

NEM

Vanadate

AMP-PNP

0.3 mM activation 4 mM 20% inhibition 2 mM 88% inhibition 0.5 mM 74% inhibition

10/~M 90% inhibitionb 10/4 M 27% inhibition 100/JM no effect 10/~M 20% inhibition

1 mM 25% inhibition 1 mM 50% inhibitiond -I mM 67% inhibition

a--Shimizu and Kimura (1977); b--Gibbons et aL (1978); c--Kuznetsov and Gelfand (1986); d--Cohn et al, (1987); e---Collins and Vallee (1986).

related to axonal transport and succeeded to move latex beads along microtubules by the addition of purified kinesin. In addition, affinity chromatography using monoclonal antibody to kinesin suggests that kinesin might be associated with anterograde translocation (Vale et al., 1985b). Axonal transport occurs either from or to the cell body. Recent evidence has shown that MAPIC, a high molecular weight component of MAPs, will be related to retrograde transport (Paschal and Vallee, 1987). Bovine brain kinesin shows a low ATPase activity and the activity is strongly enhanced by high Ca 2~ concentration or microtubules polymerized by taxol. We have reported that tubulin-dependent Mg2~-ATPase activity of MAPs is stimulated by adding taxol under conditions to form microtubules (Fujii et al., 1983). Vanadate has been reported to be a specific inhibitor of dynein ATPase (Gibbons et al., 1978), while both tubulindependent ATPase and kinesin are less sensitive to vanadate (Kuznetsov and Gelfand, 1986) (Table 3). A significant ATPase inhibition by AMP-PNP or PPPi is observed in both kinesin and tubulin-dependent ATPase. Thus, there are many similarities in properties between kinesin and tubulin-dependent MAPs ATPase. However, the ATPase was associated with microtubules even when ATP was added at the first step of microtubule polymerization (see Experimental Procedures). On the other hand, the binding of kinesin to microtubules is released in the presence of ATP, but not by AMP-PNP (Vale et al., 1985a). Addition of NEM lowered the MAPs ATPase activity, while kinesin was not sensitive to this reagents (Kuznetsov and Gelfand, 1986) (Table 3). These findings indicate that tubulin-dependent ATPase is distinct from kinesin. Collins and Vallee (1986) have indicated that cytoplasmic extracts from unfertilized sea urchin eggs contain a prominent microtubule activated ATPase which is stimulated by polymerized tubulin, but not by tubulin dimer. The sea urchin egg ATPase activity was not affected by 100t~M vanadate (Collins and Vallee, 1986). Thus, tubulindependent MAPs ATPase seems to be distinct from cytoplasmic dynein, kinesin and sea urchin eggs microtubule activated ATPase concerning its biochemical and pharmacological properties. These data are summarized in Table 4. High-molecular-weight 4, a component of MAPs, isolated from bovine spinal nerve roots has been shown to have ATPase activity (Hollenbeck and Chapman, 1986). Its interaction with microtubules is ATP-sensitive and the activity is completely inhibited by 10-t00 # M vanadate. Many of the biochemical actions of the phenothiazine antipsychotic reagents can be explained by

the common mechanism of their binding t(~, and inhibiting, calmodulin and SI00 protein, lI has been reported that S100 protein does not compete with chlorpromazine for interacting with phosphocellulose-purified tubulin (Donato, 1984). Chlorpromazine led to a considerable restoration of SI00 protein-dependent ATPase inhibition in microtubule proteins (Fujii et aL, 1987), while the extent of restoration is less when tubulin-dependent ATPase was examined (Table 2). It has been reported that Sl00a and S100b are involved in the Ca2*-mediated control o! the polymerization-depolymerization of microtubule proteins in vitro (Baudier et al., 1982b; Endo and Hidaka, 1983; Donato, 1983). Donato et al. (1985) have indicated that microtubule polymerization in the presence of Ca 2+ is modulated by brain Sl00a0, Sl00a and SI00b with the similar degree. However, as indicated in Fig. 4, c~-subunit might be more effective than fl-subunit in inhibiting tubulindependent ATPase activity of MAPs. Kimura et al. (1984) have reported using immunoassay that SI00 protein is distributed in not only glial cells but also in various regions of brain including white matter and spinal cord with the various ratio of 7/~¢ subunit. These suggest that S100a and S100b might participate in different roles in cellular functions. Ca `'+- regulatory system of ATPase in microtubule proteins including tubulin-dependent ATPase, kinesin, MAP1C and microtubule activated ATPase is not known. Thus, S100 protein is one likely candidate for the regulatory protein on microtubule-mediated motility. Acknowledgement--The authors thank Dr A. Hachimori

for stimulating discussions and critical reading of the manuscript. REFERENCES

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