THE JOURNAL OF BIOLOGICAL CHEMISTRY (0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

VOl. 266, No . 32, Issue of ‘November 15, pp ,21784-21790,1991 Printed in U.S.A.

Stimulation of the Interaction between Actin and Myosin by Physarum Caldesmon-like Protein and Smooth Muscle Caldesmon”

(Received for publication, April 29, 1991)

Ryoki IshikawaS, Tsuyoshi Okagakij, Sugie Higashi-FujimeT, and Kazuhiro KohamaS From the $Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371, the $Physical Science Laboratories, Nihon University at Narashino, Funabashi, Chiba 274, and the 7lDepartment of Molecular Biology, Faculty of Science, Nagoya University, Nagoya, Aichi 464, Japan

We have purified an actin-binding protein from the plasmodia of a lower eukaryote, Physarum polyce- phalum, with an apparent molecular mass of 210,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein bound to actin filaments with a stoichiometry of 1:7-8 in a Ca2+-calmodulin- dependent manner. Antibody raised against caldesmon from smooth muscle cross-reacted with the 210-kDa protein. In vitro motility assay revealed that the 210- kDa protein increased the sliding velocity of actin filaments on Physarum myosin. The 210-kDa protein more than doubled the actin-activated ATPase activity of Physarum myosin under comparative conditions of in vitro motility assay. Further increases in the con- centration of the 210-kDa protein decreased its stim- ulatory effects. Ca2+-calmodulin prevented the stimu- latory effects of the 210-kDa protein. Unexpectedly, smooth muscle caldesmon also increased the sliding velocity of actin filaments on smooth muscle myosin at lower concentrations. The well-known inhibitory ef- fect of smooth muscle caldesmon on the actin-myosin interaction was observed with this motility assay when the concentration of the caldesmon was increased fur- ther. The stimulatory and inhibitory effects were con- firmed by measurements of actin-activated ATPase activity of smooth muscle myosin. From estimations of the intracellular concentrations of the 210-kDa pro- tein and smooth muscle caldesmon in vivo, it appears that effects of the former and the latter on actin-myosin interactions in vivo are stimulatory and inhibitory, respectively.

The contractile system of the plasmodia of Physarum po- lycephnlum is uniquely down-regulated by Ca2+ (see Refs. l and 2 for reviews). Biochemical studies have shown that most of this inhibition by Ca2+ is mediated by myosin. The direct binding of Ca2+ to one of the light chains of myosin inhibits the actin-activated ATPase activity of myosin (3-5). In ad- dition to this myosin-linked regulation, there is evidence to suggest that actin-linked regulation is also involved in the Ca2+-inhibition (see Ref. 1 for review). While extensive studies on the myosin-linked regulation have been reported, the actin-

* This work was supported by grants from the Japan Research Foundation for Clinical Pharmacology, the Life Science Foundation of Japan, the Naito Foundation, the Uehara Memorial Foundation, and by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

linked regulation has received less attention and is poorly understood.

In smooth muscle, the caldesmon/calmodulin system has been well characterized as an actin-linked regulatory system (see Ref. 6 for review). The actin-activated ATPase activity of smooth muscle is inhibited by caldesmon. The inhibition is released by calmodulin in the presence of Ca2+ (7-12). Recently, the inhibitory effect of caldesmon was confirmed in an in uitro motility assay, in which ATP-dependent movement of actin filaments is measured on a coverslip coated with smooth muscle myosin (13).

In this report, we described purification of Physarum 210- kDa protein, the biochemical properties of which are quite similar to those of smooth muscle caldesmon. Furthermore, we performed the in uitro motility assay and measurements of ATPase activity using Physarum myosin, to study the effects of the 810-kDa protein/calmodulin system on inter- actions between actin and myosin in Physarum. From the present study it appears that the 210-kDa protein stimulates the interaction, an effect that is quite opposite of the well- known effect of vertebrate caldesmon (7-12). Under certain conditions, smooth muscle caldesmon also functions to stim- ulate the interaction. These results are discussed in terms of the possible role of the 210-kDa protein and caldesmon on the interactions between actin and myosin in Physarum plas- modia and other nonmuscle cells.

MATERIALS AND METHODS

Proteins-Physarum 210-kDa protein was purified by a modified version of the methods of Yamashiro-Matsumura et al. (14). 300 g of plasmodia was washed twice with 20 mM KC1 and 15 mM Na phos- phate buffer (pH 6.5), and homogenized with a Polytron (30 s X 2 at maximal speed) in 2 volumes of 0.5 M NaCl, 10 mM EGTA,’ 10 mM 2-mercaptoethanol, and 0.1 mM phenylmethylsulfonyl fluoride. After adjustment of the pH to 8.0 with NaOH, the homogenate was centri- fuged at 100,000 X g for 1 h. The supernatant was used as high-salt extract (HSE). The HSE was heated in a boiling water bath for 15 min, cooled on ice for 20 min, and centrifuged at 16,000 x g. The supernatant was used as the heat-stable HSE of plasmodia. Solid ammonium sulfate was mixed with the heat-stable HSE to 45% saturation. The mixture was centrifuged at 12,000 X g for 20 min. Solid ammonium sulfate was gradually added to the supernatant to 55% saturation. The precipitate after centrifugation at 12,000 X g for 20 min was dialyzed with three changes against 2 liters of buffer A, which contained 10 mM 2-mercaptoethanol, 0.1 mM phenylmethyl- sulfonyl fluoride, and 20 mM Tris-HCI (pH 7.6). The solution was clarified by centrifugation at 16,000 X g for 10 min and applied to a column of DEAE-Toyopearl 650 M (3 cm i.d. X 20 cm; Toyo Soda, Tokyo, Japan), which was incorporated into an HPLC system (655A type; Hitachi, Tokyo, Japan) and equilibrated with buffer A. The

The abbreviations used are: EGTA, [ethylenebis(oxyethyl- enenitri1o)ltetraacetic acid; DTT, dithiothreitol; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HSE, high salt extract; HPLC, high performance liquid chromatography.

21784

Physarum Caldesmon-like Protpin 21 785 column was washed with two column volumes of huffer A and devel- oped with a linear gradient of NaCI (0-400 mM) in huffer A. Column fractions were examined hy SDS-PAGE and Western hlotting with monoclonal antihody against caldesmon from smooth muscle of chicken gizzard designated as SM12 in Ref. 15. One of the major hands that reacted with the antilmly was that of a protein of 210 kDa, which was eluted by approximately I50 mM NaCl (see Fig. 1). The fractions containing the 210-kDa protein were pooled and then diluted with an equal volume of water to reduce the ionic strength. After addition of (:aCI? to a final concentration of 1 mM, the fractions were directly applied to a column of calmodulin-agarose (1 cm i.d. X 5 cm: Sigma), equilibrated with huffer 13, which contained 1 mM CaCI?, 3 mM MgVl,, 10 mM 2"mercaptoethanol. 0.1 mM PMSF, and 50 mM imidazole-HCI (pH 7.0). The column was washed with two column volumes of buffer H and eluted with huffer H that contained 150 mM NaCI. The fractions shown hy SDS-PAGE to contain the 210-kDa protein were concentrated hy ultrafiltration with Ultracent 10 (Tosoh, Tokyo, .Japan), and dialyzed against 100 mM KC1 and 20 mM imidazole-HCI (pH 7.0). The protrin was used as I'hysnrum 210- kDa protein or raldesmon-like protein.

Smooth muscle caldesmon was purified from chicken gizzard by the method descrihed hy Rretscher ( I f ) . I'hvsarurn myosin was pu- rified in the phosphorylated form as descrihed previously (4). Smooth muscle myosin (17) and myosin light-chain kinasr (18) were purified from chicken gizzard as reported elsewhere. Smooth muscle myosin was phosphorylated with myosin light chain kinase and hrain cal- modulin (18). and med for ATPase measure and in vitro motility assay. Actin was purified from chicken breast muscle in glohulous forms descrihed elsewhere (19). Glohnlous actin was polymerized by adding KC1 to the final concentration of 100 mM. The polymerized, filamentous actin was centrifuged at 140,000 X ,q for 1 h. The precip- itate was suspended in 100 mM KC1 and 20 mM imidazole-HCI (pH 7.0) and used as actin filaments. Calmodulin. glucose oxidase, and catalase were purchased from Sigma. The molecular masses used for calculations of molarities of solutions of actin, I ' h y a r u m 210-kr)a protein, smooth muscle caldesmon, and calmodulin were 42, 210, 87, and 17 kDa, respectively.

Assay of Actin Rindin,q-(i) Actin hindings of the 210-kDa protein under the high concentrations, namely, more than 0.35 PM of the 210-kDa protein. were determined hv the following methods. Varying Concentrations of the 210-kDn protein (0.:15-:1.6 p ~ , final concentra- tions) were incubated with 12 pM (final concentration) of actin filaments at room temperature for 1 h in 50 mM KCI, 1 mM DT'I', a n d 20 mM imidazole-HCI (pH 7.0). In addition, calmodulin and CaCI? or EGTA were added to the reaction mixtures in some experi- ments. Samples were centrifuged at 140,000 X p for 30 min in an Airfuge (Heckman). The amounts of the 210-kDa protein and actin in hoth supernatants and pellrts were determined quantitatively hy densitometry, after SDS-PAGE, as descrihed previously (20). ( i i ) T h e amounts bound to actin filaments of the 210-kDa protein or smooth muscle caldesmon under the low concentrations, namely less than 0.18 pM of the 210-kDa protein or 0.74 p M of smooth muscle caldes- mon. were determined as follows. Cnldesmon and the 210-kDa protein

1 2 3 4

220K . ' -210K

- -13OK -11OK

94K

67K

43K

FIG. 1. The plnsmodia of Physarum contain the 210-kDn and the 180-kDn proteins which cross-react with n cnldes- mon-specific nntihody. Heat-stable, high-salt extract (20 pg) of plasmodia (lancs I :Ind 3 ) and 210-kDa protein (0.6 p g ) partially digested by <r-chymotr,ypsin (lnncs 2 and 4 ) were subjected to SDS- PAGE and blotted. Imnes I and 2. staining with amidohlack; lnnrs 3 a n d 4 . the corresponding immunohlots prepared using a monoclonal antibody (SM12) against smooth muscle caldesmon. Note that the 130-kDa protein appears to hr proteolytic product of the 210-kIh protein.

were laheled with ""'I using Rolton-Hunter J h g r n t ( 1 ) ~ I'ont-Nwv England Nuclrar). ~'"'1-1,aheIed 2IO-kI>a protein fo ,rl~I5-~l . lx p ~ ) or '.''I-caldesmon (0.074-0.74 pM) was mixed with 1.2 p v o f actin f i l : ~ . ments in 50 mM KCI, 1 mM D'T'I', and 'LO mM imidazolr-Il( ' l ( p f l 7.0) for 1 h. The mixtures werr crntrifugrrl at 1 . ~ 0 . 0 0 ( ~ X c for :<f) nlin in an Airfugr. The radioactivity in both sclprrnatant.; and pc,llrts wrre determined with a y-counter (Auto \Vrll (;amma Sy.;trm ;!It('- 3 0 0 , Aloka, Tokyo, Japan), and the amorlnts hound t o actin filrlmrnts was calculated.

I n Vitro Motility Assay-Actin filaments Inhrlrrl with rhotl:lminr- phalloidin were allowed t o move on I'hy.wrrrm mynsin nr smooth muscle myosin which had hren fixrd to a nitrocrllulosr-coatrrl gla.;.; surface as descrihrrl hy 'I'oyoshima et o l . (21 1 , with minor rnodific.n- tions in the procedure used for coating with I'hysrtrum myosin (221 and smooth musclr myosin (18). 'I'hr movrments n f the actin fib. ments were monitored under a flnoreswncr mirrwcopr rquipprd with a videocamera (C2400; Hamamatsu Photonics. Hnm:~matsrr. .Inpan). Actin filaments with the fluorescent lalwl (6 n\qI wrrr inruhatrd with 0.9 nM of the 210-kI)a protein at room trmprratrrrr. o f 1 h in a solution of :X0 mM KCI, 2 mM ATP, 1 mM \Ig('l:, 25 m v II'T'r, .I.:) mg/ml glucose, 0.22 mg/ml glucose oxidnsr. 11.0: lG mg/ml cntnlnsr, and 20 mM imidazolr-HCI (pH 7 . 0 ) , in t h r presrncc. o f 0 . 1 m\f ( 'n( ' l or 0.1 mM E(;TA. and then srrhjectetl t o t h r 111 [ , ; f ro motility ns.;:~y with Physnrum myosin (22). The vrlority o f actin filnnlents wrrr mrasured only whrn they smoothly moved. Thr rflrct o f smooth muscle caldesmon o n the motility with smooth musrlr myosin \vas

examined in the same hrrffer as that drsrrihrd nlmvr rxcrpt t h n t concentrations of KC1 and MgCI, werr 50 mw nnd et mw, rrsprrtivvly.

Assay of A7'I'n.w Actiuity-ATPase activity wn.; tletrrminc~tl t)y t h r malachite green method rlrsrriherl hy Kodnn1:l ~t a(. ( 2 : { ) . Artin in its filamentous form (1.2 p ~ ) was mixrd with varying cnncrntrntions o f the 2lO-kIh protein (0-0.18 p ~ ) or smnnth muscle ralrlr.;mon ( 0

0.74 p ~ ) . Each mixture was incubated in a solrrtion of . I f ) o r l 1 1 i ) m u KCI, 50 p M MgCI:, and 2 0 mM imitlazole-H('l ( p l l 7 . 0 ) in thr. prrsr*ncv or ahsence of 0.1 mM Carl: and 5.5 p M cnlmodrrlin. A T I ' l i l t a finnl concentration of 50 phi) was sub.iertrd t o hydrolysis I)? myosin in t h r presence of the each mixture for 5 t o 15 min at 25 '(', nnt l thc. hydrolysis was stopped by mixing o f the eqrlal vnlumr o f 0.1; M perchloric acid. 'The mixtures wrrr crntrifugrrl at l'L,f)OO x fnr I O min. The amount o f I', in each supernatant after rentrifr~grttion w:~.; determined hy the use of malachite grrrn. A'l'l ' hydrnlysis w11s in linear rangr during our assay periods.

Ihfimation of the Concrntrntions of the ( 'nntrnrt i lo I'r<ttvln.y in HSb,'-The protein concentration in the HSI? o f plasmodia wn.; determined (see below) and 45 pglwell of HSK was suhjrrtccl t o SI)S- PAGE (see helow). Purified actin and I'hy.wrurn mynsin (O,O75-:I pg/ well) were also loaded on the same gel as stnntlard.;. T h r grl w;~.; stained with Coomnssie Hrillinnt lilue, and the amounts o f nctin wa.; drterminerl hv densitometry (sre helow).

Part of t h r H S E was boiled at 100 " C for I O min nnrl thrn crwtri- fuged at 12,000 x E for 1 0 min. The supernatant ($1 p g o f protrin! wrll) together with thr purifird 210-klh protein (O.:jS 2.:{ pg ( 1 1

protein/well) as a standards was sul)jertrd t o SI)S-I'A(;F:. 'l'hr amount of the 2IO-kIh protrin in the heated wprrnntnnt w : ~ s dr te r - mined hv densitometry (see helow). To rstimatp thr rrcnvrry I ) I ' th r 210-kDa protein from HSK after hoiling, wr mixrd thr 0 . 1 1 p~ o f "'I-labeled 210-kIh protein t o HSIC. The mixturr wa.; hoilrd fur 1 1 ) min and centrifuged at 12,000 X ,q for I O min. Thr rntio o f radionctiv. itv of snprrnatant to pellet was W 1 8 . suRgesting thnt X?'"; o f t hr 2111. kI)a protein in H S E was rrcovrrerl in thr srqwrnatant.

Part of HSE was dialyzed against 9 volumrs ofroltl w:ltrr t o rrdocv the concentration of KC1 to 50 rnsq and. therrhy, t o prrcipitrltr the, actomyosin. Dialysate was centrifugrtl at l 2 , ~ l f ~ O X for 'LO min rind the pellet was used as crudr actomyosin, Thr rat i o o f t hr amount of' actin in the pellet to that in the supernatant waq drtrrminrd n f t r r SIX-PAGE and snhsequent densi tometn. \Ve usrd thi.; v a l ~ ~ r 11.; t h c s

ratio of filamentow- to glohulous-actin in r,ic.o (:I>:(;:'; ) on 1111.

assumption that actin in the filamentorrs fnrm in t h r llSI< W:I\

recovered as actomyosin. Other / 'rorcdfrrcs--~estern hlots wrrr prrformrrl nrcorcling t o t hr

methods o f Towhin c t a/ . (2.1). I'rotrin crlncrntratinns wrrt' ( I r t r r - mined by t he met hod of liradftrrd (25) or 1,owry c t nl. f2Gl with h v i n t . serum albumin asa standard. SI)S-I'A(;E was c n r r i r r l o ~ ~ t 11s tlrsrrihrrl elsewhere ( 2 0 ) . Densitometry was cnrrird o u t with a nlicrotlm.;ito. meter (Chromosrnn I l l , Vickers Instrument Inc.. \1:1lrIrn, \1,.!1

2 1786 Physarum Caldesmon-like Protein

RESULTS

Purification of the 210-kDa Protein from Physarum- Monoclonal antibody raised against smooth muscle caldes- mon (15) cross-reacted with 210- and 130-kDa bands in the heat-stable HSE (Fig. 1, lanes I and 3 ) . We attempted to purify these proteins by the conventional methods used for purification of smooth muscle caldesmon. As shown in Fig. 2, the 210-kDa protein was purified to homogeneity, as judged

The heavy chain of Physarum myosin could not be sepa- rated from the 210-kDa protein by SDS-PAGE under the conditions described under "Materials and Methods." How- ever, a monoclonal antibody raised against heavy chain of Physarum myosin (27) did not cross-react with the 210-kDa protein (data not shown). It is likely that myosin in the HSE can be removed from HSE by boiling. We obtained 1.2 mg of the 210-kDa protein from 300 g of plasmodia.

Partial digestion of the 210-kDa protein by a-chymotrypsin produced two peptides, with apparent molecular masses of 130 and 110 kDa, respectively (Fig. 1, lane 2 ) . Caldesmon- specific antibody cross-reacted to the 130-kDa peptide (Fig. 1, lane 4 ) . These results suggested that the 130-kDa protein in the heat-stable HSE that cross-reacted to caldesmon- specific antibody may be a proteolytic product of the 210-kDa protein.

Binding of the 210-kDa Protein to Actin and Reversal of Binding by Ca"-Calmodulin-We examined the actin-binding activity of the 210-kDa protein by changing its concentrations in the wide range. The amount of the 210-kDa protein bound to actin filaments was increased with increase in the 210-kDa protein mixed with actin (Fig. 3). The saturation was achieved at an approximate molar ratio of one 210-kDa protein to seven to eight actin monomers. The dissociation constant

All these values were calculated on the assumption that the molecular mass of the 210-kDa protein was actually 210,000 daltons. The actual molecular mass of smooth muscle caldes- mon from chicken gizzard is 86,974 daltons which is derived from the nucleotide sequencing of its cDNA (28). However, the apparent molecular mass on SDS-PAGE is about 140,000 daltons (7,16). Therefore, the true molecular mass of the 210- kDa protein could be much lower than 210,000 daltons.

We examined the effects of calmodulin on the actin-binding activity of the 210-kDa protein in the presence of Ca2+ (Fig. 4). Only 31% of the 210-kDa protein bound to actin filaments in the presence of Ca'+ (lane 4 ) , while 99% of 210-kDa protein

by SDS-PAGE.

(&) Was 0.6 X M.

1 2 3 4 5 6 220k- - - - - - 2 1 0 ~ r - -Caldnmon

9 4 k r

em- ;

43kr bL - P -

30k-

ZOkr

FIG. 2. Purification of the 210-kDa protein from Physa- rum. SDS-PAGE. Iane I, HSE of plasmodia; lane 2, heat-stable fraction of HSE; lane 3, fraction precipitated by 45-55% saturation with ammonium sulfate; lane 4,210-kDa protein fraction after column chromatography on DEAE; lane 5 , purified 210-kDa protein from Physarum after chromatography on a calmodulin-agarose column; lane 6, purified caldesmon from chicken gizzard. Asterisks in lane 1 indicate the heavy chain of myosin and actin.

2 1 0 K ( p M )

FIG. 3. Binding to actin of the 210-kDa protem from Phy- sarum. Actin (12 p M ) was incubated with varying Concentrations of 210-kDa protein (0.3.5-3.6 JIM) in 50 mM KCI, 0.5 mM DTT, and 20 mM imidazole-HCI (pH 7.0). After a I-h incubation, samples were centrifuged in an Airfuge (140,000 X g, 30 min). Supernatants and pellets were dissolved separately in equivalent volumes of SDS- sample buffer and subjected to SDS-PACE. The amounts of 210-kI)a protein and actin were determined by densitometry.

1 2 3 4

0 0 - *no*

-0lrndutln

FIG. 4. Binding of the 210-kDa protein from Physarum to actin is regulated by Ca'+-calmodulin. A solution of 0.71 p~ 210- kDa protein was incubated with 12 p~ actin and 20 JIM calmodulin in the presence of EGTA or CaCI2. After high-speed centrifugation in an Airfuge, each supernatant and pellet were analyzed hv SDS- PAGE. h n e s I and 2, supernatant and precipitate, respectively. ohsewed with 1 mM EGTA; lancs 3 and 4. supernatant and precipi- tate, respectively. observed with 1 mM CaCI,.

bound to actin filaments in the presence of EGTA (lane 2). These results indicate that Cay+-calmodulin inhibits the bind- ing activity of the 210-kDa protein. Similar effecb of Ca"- calmodulin have been reported for vertebrate caldesmons (7, 15, 16).

Increases in the Sliding Velocity of Actin Filaments on Physarum Myosin Caused by the 210-kDa Protein-We ex- amined the effects of the 210-kDa protein on the sliding velocity of actin filaments (Fig. 5), and the effects are sum- marized in Table I. Actin filaments moved at a velocity of between 0.68 and 2.40 pm/s, with 1.39 pmls as the average velocity, in the absence of the 210-kDa protein. In the pres- ence of 0.9 nM of the 210-kDa protein, the velocity increased and showed a wide distribution, from 0.85 to 3.40 pm/s, with 1.95 pm/s as the average velocity.

These experiments were carried out in the absence of Ca". In the presence of Ca2+, the average velocity of sliding was reduced from 1.39 to 0.94 pm/s, a reduction that confirms our previous report that Cay+ inhibits the velocity of actin fila- ments on Physarurn myosin (22). The 210-kDa protein in- creased the average sliding velocity of actin filaments from 0.94 to 1.26 pm/s. When calmodulin at 14 nM was mixed with the 210-kDa protein, the average velocity was reduced from 1.26 to 1.08 pm/s. Although the difference was not statistically significant, ATPase measurement (see below) confirmed the

Physarum Caldesmon-like Protein 21 787

FIG. 5. Translocation o f fluorcscrncc~-l;thrlc.d actin fila- ments by Phyrrnrcrrn myosin. Movrlnent 01' actin filaments was ohserved as drscril)rtl untlcr "Mtlteritlls tlntl Methods" in the presence of EGTA. Photogrnphs were tnken at 0 time ( A , 1 ) ) and 10 s later ( H , E ) . C and F a r e photographs from a 10 s exposure from 0 time to 10 s. A-C, control actin filaments in the ahsence of210-kI)a protein; 1)- F , in the presence of 0.45 nM 210-kl)a protein. ArrouJs in A and I ) indicate the direction of sliding. Arrowheads in H-F indicate the endpoints of sliding a t 10 s. Note that frarm with arrowheads in F are longer than those in ('.

TABLE I Sliding orhritirs o f actin lilaments on coucr slips coated with

Physarum myosin Velocitv (n = 30)

pm/s

Control (0.1 mM ECTA) 1.39 f 0.49 +I'h.vsarum 210-kh protein (0.1 mM ECTA) 1.95 f 0.66" Control (0.1 mM CaCI:) 0.94 2 0.42 +l'hysarum 210-kI)a protein (0.1 mM CaCI,) 1.26 f +Ph.vsarum 210-kDn protein + calmoddin 1.08 2 0.50'

(0.1 mM CRCI.,)

" Hy Student's f test, p < 0.01 when compared with the control

" p < 0.05 when compared with the control value (0.94 pm/s). ' Not significant at 0.05 level when compared with the value in the

value (1.39 pm/s).

presence of Cay* and caltlesmon (1.26 pm/s).

effect of calmodulin in the presence of Ca'+. Therefore, we believe that calmodulin removes the effect of 210-kDa protein.

Thus, the results in Table I suggest that the 210-kDa protein controls the interaction between actin and myosin by activating the interaction, and that the activation may he abolished by Ca"-calmodulin.

Physarum 210-kDa Protein Stimulates the Actin-activated Mg-ATPase Activity of Myosin-To confirm the results in the in vitro motility assay, we examined the effects of the 210- kDa protein on the actin-activated Mg-ATPase activity of Physarum myosin. The 210-kDa protein a t 0.07 PM stimulated the ATPase activity 2.3-fold (Fig. 6A) . The 210-kDa protein bound to actin at a level equivalent to 10% saturation under similar conditions (Fig. GB). When we increased the amount of the 210-kDa protein to the 20% saturation level, the enhancement of ATPase activity was reduced to only 1.7-fold. When the concentration of the 210-kDa protein was further increased to 1.7 PM, the effect of the 210-kDa protein became inhibitory, i.e. the ATPase activity fell to 80% of the activity observed in the absence of the 210-kDa protein (data not shown).

Next, we examined the way in which Ca2+ and calmodulin

0 "0.. "1" " 9 . . , /.I ,!. 1.

2 1 0 K (,,MI Z I O K ~ r h t l

FIG. 6. Effects of the 210-kDa protein on the actin-acti- va ted ATPase ac t iv i ty o f Physarum myosin ( A ) and the ac t in- binding of the 210-kDa protein (R). A , each assay rontained varying amount of the 210-klh protein (0-0.18 pMJ. 1.2 pM artin. and 0.024 p~ myosin in a solution of 50 mM KCI. 50 p M MgCI,, 50 p~ ATP, and 20 mM imidazole-HCI (pH 7.0). H , ""I-laheled 'L10-kl)a protein and actin were incuhated and then centrifuged. The amounts of the 210-kDa protein in hoth supernatants and pellets werr drter- mined as descrihed under "Materials and Mrthods." The ATPase activity of myosin in the ahsence of the 210-kDa protein was 186 nmol rnin"mg" myosin.

T

0.1

Z I O K ( p M )

2

FIG. 7. Effects of Ca2+ and calmodulin on the s t imulat ion of the act in-act ivated ATPase act ivi ty of Physarum myo- sin. The activities were measured as described in the Irgrnd to Fig. 6 with the exception that 0.1 mM CaCI, and 5.5 p~ of calmodulin were present. 0, assayed in the ahsence of calmodulin; 0. assayed in the presence of calmodulin. The ATPase activity in the. ahsence of the 210-kDa protein was 87 nmol min-lmg" myosin. Note that Cn"- calmodulin completely negated the stimulatory effects of the 210-klh protein.

modify the effects of the 210-kDa protein on the actin- activated ATPase activity of Physarum myosin. In the ah- sence of Ca", the activity was 186 nmol min-lmg-'. Ca" (0.1 mM) reduced the activity to 87 nmol min-'mg", a result that confirms previous observations (4, 22). As shown in Fig. 7 . the 210-kDa protein effectively stimulated the activity of the myosin in the presence of Ca2+, a result that confirms the results shown in Fig. 6A. However, when calmodulin at 5.5 PM was mixed with the 210-kDa protein, the activity did not increase, i.e. the stimulatory effect of the 210-kDa protein was completely negated (Fig. 7). Thus, results from measure- ments of ATPase activity were consistent with those obtained from the in vitro motility assay as shown in Table I.

Estimation of the Amount of thp 210-kDa Protpin in HSR- To understand the physiological role of the 210-kDa protein

21788 Physarum Caldesmon-like Protein

in vivo, we estimated the amount of the 210-kDa protein in HSE. Plasmodial cells contain too much slime to determine the amount of the 210-kDa protein by analyzing the total cell lysate on SDS-PAGE. Therefore, we used HSE as an approx- imation of the living plasmodial cell. As shown in Table 11, 0.12% of total protein in HSE was the 210-kDa protein. The concentration of the 210-kDa protein in plasmodia was cal- culated to be 0.13 p ~ . However, the actin that interacts with myosin is well-known to be in the filamentous form. The HSE of Physarum contains various actin-modulating pro- teins, such as fragmin (29) and profilin (30), which reduce the level of filamentous actin. Therefore, we can expect that only part of the actin is in the filamentous form in vivo. We estimated the filamentous actin accounted for 35% of the total actin. Thus, the molar ratio of the 210-kDa protein and actin filaments in HSE was calculated to be 1:70. Because the concentration of the 210-kDa protein in HSE (0.13 p ~ ) is much lower than the Kd of the 210-kDa protein (0.6 p ~ ) , only part of the 210-kDa protein bind to actin filaments, suggesting that actin binding of the 210-kDa protein in vivo is less than 10% saturation. The 210-kDa protein i n vivo may stimulate the interaction between actin and myosin.

Smooth Muscle Caldesmon Enhances the Movement of Actin Filaments on Smooth Muscle Myosin-We examined whether or not smooth muscle caldesmon could also stimulate the movement of actin filaments on smooth muscle myosin. With- out smooth muscle caldesmon, the average sliding velocity of actin filaments was 0.69 pm/s (Fig. a), confirming our recently reported data (13). The velocity increased to 0.98 pm/s at 0.98 nM smooth muscle caldesmon (Fig. 8B). How- ever, when the concentration was increased to 4.1 nM, the velocity was hardly greater than in controls. Finally, with the increase in concentration to 11 nM, the effect was reversed from activation to inhibition of movement. The sliding veloc- ity was decreased to 0.23 pm/s (Fig. 8 E ) , which was only 33% of the control velocity of actin filaments in the absence of smooth muscle caldesmon. Thus, as in the case of the 210- kDa protein, regulation by smooth muscle caldesmon was stimulatory with respect to the actin-myosin interaction at lower concentrations and inhibitory at higher concentrations.

Smooth Muscle Caldesmon at L o w Concentrations Stimu- lates Actin-activated Mg-ATPase Activity of Myosin-As shown in Fig. 9, smooth muscle caldesmon stimulated the actin-activated ATPase activity of smooth muscle myosin from 1.5- to 2-fold, and maximum stimulation was obtained when it was present at 0.07 p~ in 50 pM KC1 (Fig. 9A), or at 0.27 p~ in 100 mM KC1 (Fig. 9B). Under the same conditions, the actin-binding of smooth muscle caldesmon was equivalent to about 10% saturation (see Fig. 9, dotted lines), which values

TABLE I1 Estimated abundance of actin, myosin, and the 210-kDa protein in

H S E of Physarum plasmodia

Protein Mass ratio“ Protein con- Molar centration ratio

% totalproteins* P M

Actin 4.7 f 0.7 26 100 Filamentous actin 1.6 & 0.2 9.1 35 Myosin heavy chain 1.0 f 0.2 0.52 2 210-kDa protein‘ 0.12 f 0.03 0.13 0.5

a Estimated from scans after SDS-PAGE of the high salt extract (HSE) and heat-treated HSE, after staining with Coomassie Brilliant Blue ( n = 4).

* The total amount protein in Physarum plasmodia was 24 mg/ml. This value was determined on the assumption that the recovery

rate of the 210-kDa protein in HSE from boiling was 82%. The recovery rate was derived by boiling the mixture of 12sI-labeled 210- kDa protein and HSE, as described under “Materials and Methods.”

10

5

10

5

10

5

10

5

10

5

A

1 .o 2 . 0

Sliding Velocity (pm/s)

FIG. 8. Stimulatory and inhibitory effects of smooth muscle caldesmon on the sliding velocity of actin filaments on smooth muscle myosin. Fluorescence-labeled actin filaments (6 nM) were incubated with varying concentrations of smooth muscle caldesmon (0-11 nM) for 1 h at room temperature, then they were subjected to the i n vitro motility assay. A , histogram of the sliding velocity of actin filaments in the absence of smooth muscle caldesmon (mean velocity k S.D. was 0.69 f 0.17 pm/s); B, histogram of the sliding velocity in the presence of 0.98 nM smooth muscle caldesmon (0.98 f 0.35 pmls); C, in the presence of 2.0 nM smooth muscle caldesmon (0.97 & 0.23 @m/s); D , in the presence of 4.1 nM smooth muscle caldesmon (0.72 f 0.16 pm/s); E , in the presence of 11 nM smooth muscle caldesmon (0.23 f 0.06 pm/s).

are compatible with that of the 210-kDa protein. When we increased the concentration of smooth muscle caldesmon above these values, the stimulatory effects were gradually eliminated (Fig. 9). Finally, the smooth muscle caldesmon inhibited the ATPase activity, as reported by many authors (7-12). Such profiles were very similar to those obtained into the 210-kDa protein. We also examined the effects of calmod- ulin on the stimulation of the ATPase activity by smooth muscle caldesmon, and found that Ca2+-calmodulin com- pletely negated the stimulatory effect of smooth muscle cal- desmon (data not shown).

DISCUSSION

We purified and characterized a 210-kDa protein from the plasmodia of the lower eukaryote Physarum. In terms of the following four properties, the 210-kDa protein was similar to previously described vertebrate caldesmons: (i) the ability to bind to actin filaments in a Ca2+-calmodulin-dependent man- ner (Fig. 4); (ii) the ability to bind to calmodulin in a Ca2+- dependent manner (Fig. 2); (iii) the ability to cross-react with an antibody raised against smooth muscle caldesmon (Fig. 1); (iv) stability to heating (Fig. 2). From these results, we have designated this protein the Physarum caldesmon-like protein.

Physarum Caldesmon-like Protein 2 1789

A ( P h y s a r u m )

0 7 0 . 0 6 0 8 0 7 0 . 01 0"

Cao ( w ) CaD (vM)

FIG. 9. A low concentration of smooth muscle caldesmon stimulated the actin-activated ATPase activity of smooth muscle myosin. Actin filaments (1.2 p ~ ) were incuhated with vary- ing concentrations of smooth muscle cddesmon (0-0.74 pM) in a solution of 50 mM ( A ) or 100 mM ( H ) KCI, 50 p~ MgC12, 20 mM imidazole-HCI (pH 7.0) for 1 h at room temperature, and then 0.024 pM smooth muscle myosin that had been phosphorylated hy myosin light-chain kinase was included in the mixture. After 10 min, ATP was added to a final concentration of 50 pM, and the mixtures were incuhated with 25 "C for 15 min to determine the amount of phos- phate hydrolyzed. Actin binding of smooth muscle caldesmon (dottrd linr) was determined under the same condition as described in the legend to Fig. 6.

We describe that novel property of Physarurn caldesmon-like protein and smooth muscle caldesmon, namely, its stimula- tory effect on the interaction between actin and myosin. The effect has been confirmed by two different methods, i.e. assay of actin-activated ATPase activity and in vitro motility assay. For both Physarurn caldesmon-like protein and smooth mus- cle caldesmon, the stimulatory effect is obvious when they are present a t lower concentrations which are equivalent to less than 10% saturation of actin-binding capacity (Fig. 6R and Fig. 9).

Bretscher (16) reported that the molar ratio in uiuo of actin:caldesmon in chicken gizzard smooth muscle was 100:2.4 using a molecular mass for caldesmon of 140 kDa (16). This ratio can be recalculated to 100:3.6 if the true molecular mass value of 87 kDa (from cDNA sequence (28)) is used. Our estimate of the actin concentration in chicken gizzard smooth muscle is 300 pM (data not shown). If our estimate is the case, the concentration of smooth muscle caldesmon in chicken gizzard is calculated to be 11 p ~ , which is high enough to inhibit the ATPase activity of myosin. Thus, the regulatory effect on the actin-myosin interaction in uiuo of smooth muscle caldesmon was inhibitory. As reported by many au- thors, smooth muscle caldesmon forms complex with actin a t the lower concentrations of Ca2+, and under these conditions the interaction between actin and myosin is inhibited (9-12, Fig. 10R). Furthermore, in the relaxed smooth muscle, myosin itself is inactive because the light chain of smooth muscle myosin remains unphosphorylated. When the concentration of Ca2+ is elevated, calmodulin hinds Cay+ to release the inhibition. At the same time, the myosin is phosphorylated by the myosin light-chain kinase/calmodulin system and be- comes active. Thus, the interaction between actin and myosin is enhanced through both the caldesmon/calmodulin system and myosin light-chain kinase/calmodulin system.

Whether the Physarurn caldesmon-like protein is inhibitory or stimulatory depends on the fraction of actin containing bound Physarurn caldesmon-like protein. While precise de- termination of this level of saturation, in uiuo, is very impor- tant, it is also subject to large errors. However, from our approximate estimation of the Physarurn caldesmon-like pro-

. .

Active lnnctlvc

B ( s m o o t h muscle)

. . - Innctlve Active

FIG. 10. Schematic illustrntion of the regulntion by caldcs- mon of the interaction between actin and myosin. Ahhrevint Inns used in this figure are: M , myosin; A, actin; ( ' I ) , caklesmnn-like protein or caldesmon; C M . calmodulin. Shaded moleculrs refer t o active forms.

tein in HSE, the effects of Physarurn caldesmon-like protein on the interaction hetween actin and myosin may be stimu- latory. It must be noted that the effect is not attributed to the intrinsical difference in the properties of actin and myosin between Physarurn and smooth muscle. Rather the effect depends on the concentrations of caldesmon-like protein as follows. At lower concentrations of Ca'+, actin forms a com- plex with caldesmon-like protein to interact actively with myosin even though calmodulin is present (Fig. 10A ), Fur- thermore, myosin itself is free of Ca" and is in an active form in lower concentration of Ca2+ (2). At higher concentrations of Ca'+, caldesmon-like protein is released from act.in fila- ments by the Ca"-calmodulin complex, with the resultant negation of the stimulatory effects of caldesmon-like protein. As reviewed previously, myosin itself is inactivated hy the direct binding of Cay+ in ATP-dependent actin-myosin inter- action (2). Thus, the interaction het.ween actin and myosin is effectively regulated by Ca" through the actin-linked and the myosin-linked regulatory systems.

In vertebrate nonmuscle cells, such as cultured fihrohlasts from the rat, the concentrations of actin and caldesmon are 27 pM and 0.097 p M , respectively.' Such a concentration of caldesmon is low enough to stimulate the actin-myosin inter- action. It will be interesting to determine whether Ca" ions bind to fibroblast myosin, as they do to I'hysarurn myosin, to inhibit its interaction with actin.

Acknowledgments-We wish to thank Drs. Fumio Mntsumura and Sigeko Yamashiro at Rutgers University, I'iscataway. N.J, for provid- ing us the caldesmon-specific antihodv and for helpful discussions. We also thank Yukie Iioppongi and Chichiro Hayashi for their excellent technical assistance.

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