Crltlcxmon: Culmatiulin; Chcniicul madificatiun;

1. INTRODUCTION Caldesman is a thin filament-associated protein which has been implicated in the regulation of smooth muscle contraction 111,The isolated protein is capable of interaction in vitro with actin [2], myosin [3], tropomyosin [4] and calmodulin [2]. The calmodulin interaction is Ca’“-dependent and has been suggested to bc important in regulating the ability of caldesmon to inhibit smooth muscle actin-activated myosin MgATPase by dissociating caldesmon from actin [S], Chicken gizzard cafdestnon, cDNA clones have been sequenced in two laboratories [6,7]. The deduced amino acid SCquences probably represent the two isoforms of chicken gizzard caldesmon [g]. The dopmain structure of caldesmon has been extensively studied [9,10]. The myosin-binding domain is located in the N-terminal region of caldesmon, whereas the actin-, tropomyosinand calmodulin-binding domains are located near the opposite end of the molecule. Calmodulin-binding sites have been identified in several known cafmodulinbinding proteins and generally consist of - 18 amino acids having a basic amphilic u-helical structure [ 111. Examination of the amino acid sequence of calmodulinbinding fragments of cafdesmon reveals one such sequence: Arg-593-His-610 [6,12]. However, Bartegi et al. [ 131 recently concluded that this does not represent the calmodulin-binding site since a 10 kDa peptide corCowespondence address: M,P. Walsh, Biochemistry, University of Calgary, 3330 Calgary, Alberta T2N 4N 1, Canada

Published

by Elsevier Science Publishers

Dept. Hospital

B. V.

of Medical Drive N.W,,

Smuath muaclr

responding to the C-terminus of caldesmon (Trp-659Pro-756) binds calmodulin but does not contain this sequence. CNBr peptides corresponding to the remainder of the caldesmon molecule did not bind to immobilized calmodulin. We have pursued this surprising conclusion by examining the effects of specific chemical modification of His-610 located at the C-terminal end of the basic amphiphilic a-helix and conclude that this indeed is not the calmodufin-binding site. 2. MATERIALS AND METHODS [y.‘“P]ATI’ (20-40 Ci/mmol) was purchased from Amersham (Oakville, Ontario, Canada) and diethylpyrocarbonate and hydroxylaminc from Sigma (St, Louis, MO), Dithiothreitol was purchased from Woehringer Mannhcim (Dorval, Quebec, Canada) and electrophoresis reagents from Bio.Had (Mississauya, Ontario, Canada). Genera! laboratory reagents used were of analytical grade or better and were purchased from Fisher Scientific (Calgary, Alberta, Canada), Proteins were purified by methods published earlier: chicken gizzard caldesmon [Irl], tropomyosin [14] and actin [IS], rabbit skeletal muscle actin 116) and myosin [17], and bovine brain calmodulin [18]. Calmodulin and tropomyosin were coupled to CNBr-aciivated Sepharose 413 (Pharmacia, Bait d’Urfe, Quebec, Canada) according to the manufacturer’s insrructions. 2.2. CarbeUtoxylutiott of caldcsttton The sole imidazole function of caldesmon was carbethoxylated with diethylpyrocarbonate using the method described by Miles [IQ). Caldesmon (1 .O mg/ml) was incubated at 22°C in 0.1 M sodium phosphate buffer (pH 6.0) in the sample and reference cuvettes of a Pye Unicam PU8800 UV/visible double-beam spectrophotometer. The reaction was initiated by the addition to the sample cell of a if&& concentrated solution of diethylpyrocarbonate in ethanol to give a final reagent concentration of 50 PM (a 4.4.fold molar excess over

81

TIME (min)

Smooth m11rc1c nctin (9 &vl) and untreated or carbcthoxyletcd caldcrmons (0,57, l,l4, I .72, 2.26, 2.85, J,31 and 5.75&l) were in. cubn~cd for 30 min al 25’C in 30 mM Tris.HCI (pti 7.5), 2 mM MgCIz, I mM EGTA, 1 mM dithiothreitol, I mM ATP. Samples (0.2 ml) were thc?nccntrKu@cd nt IOOOWxg in a Bcckmnn TLIOO ten. trifuge for I Ii nr 2*C in order to sediment F-actin arid bind caldcsmon. Pcllcts and supernornnrs were subjcctcd to SDS-PAGE. The distribution of raldcsman bcbvecn the pcllcts and supernatants war qunntificd by dsnaitometric scnnning of the Coomassis bluestained gels using on LKB model 2202 Ultroscan laser densitometer equipped with a Hewlett-Packard model 3390A integrator. The & values of nctin for control and cnrbcthoxy-caldcsmons were crlculntcd by Scatchnrd analysis of the sedimentation

detn.

ATPasc activities were measured as previously described [2 I ] under the following conditions: 25 mM Tris.HCI (pH 7.5), 50 mM KC1, 3.5 mM MgCh, I mM dithiothrcitol, 0.2 mM EGTA, I mM [y-“P)ATP (-9OO0 cprn/nnrol), 3.6brM skeletal ectin, 0.57 PM skeletal myosin, in the absence or presence of conlrol or carbelliol;y-cnldesrnon (I ,2 and Z.OpM), in reaction volumes of 0.3 ml at 30°C. Reactions were started by addition of ATP, Samples (SO/rl) of reaction mixtures were withdrawn at 0.5.min intervals up to 2.5 min forquantificarion of ‘*Pi release (21). Rates of ATP hydrolysis werecalculated by linear regrcssion analysis of the linear time-course data,

I, Chcmic;rl mocliricntian of cnldamon with diclhylpyrouarbtrnats. Caldcrmon WRJ incub:tbxl with clicrhylpyrecarbollnrs Ww NW follewcd bsr tlcxeribed in 5cction 2. hr.snrbcthuxyloiion uontinuc)us rccordlny of A~.Q,,,,,~ The rtoiehlumctry 0r nroclifi~mian WIX cnlrulatcd from the nbrorbance vnlrrca, Fig.

tyrosinc rcsiducs has occurred. The spectra in Fig. 2B itlustrate the time-dependent increase in AzJ~,,,,,(histidine modification) with no significant change in A27Rt~m, indicative of no chemical modification of ryrosine residues. Treatment of caldesmon with a4.4.fold molar excess of diethylpyrocarbonate therefore results in specific and stoichiomctric modification of the sole histidine residue. Catbethoxylation of a 1: 1 molar mixture of caldcsmon and calmodulin in the presence of Ca*’ resulted in the incorporation of 0.92 mol Ncarbethoxyhistidine/mol protein (Fig. 3), Calmodulin, like caldcsmon, contains one histidine residue [28]

2.5. Oilier procedures Protein concentrations were determined by spectrophotometric measurements in a Deckman DU-813 UV/visible spcctrophotomctcr using the following extinction coefficients: calmodulin, &$,,, = I .9 [22]; caldesmon, L$&,= 3,3 [23]; tropomyosin, E%& = 2.9 1241; actin, &&,.=6.5 [25]; and myosin, EJ!~,,~,= 5.3 [26]. SDS-PAGE was performed in O,l% SDS/lO% acrylamide mini-slab gels to analyse or in full-size 0.1% SDS/7.5-20% column elution profiles, polyacrylamide gradient slab gels with a 50/, acrylnmide stacking gel to analyse catdesmon binding to actin. In each case the Laemmli [27J buffer system was used.

3.

RESULTS AND DISCUIjSION

The time course of carbethoxylation of caldesrnon with diethylpyrocarbonate is illustrated in Fig. 1. At the plateau, - 1 mol N-carbethoxyhistidine was detected/ mol caldesmon. This modification was completely reversed by incubation of carbethoxylated caldesmon overnight with 0.1 M hydroxylamine at pH 7 and 22’C, indicating that modification of lysyl or sulfhydryl residues did not occur (data not shown) [19]. The spectra in Fig. 2A demonstrate very little change in A278,,,~ following incubation of caldesmon with diethylpyrocarbonate, indicating that little or no modification of

0 220

WAVELENGThI

Enm)

260

WAVELENGTH

300

(nm)

Fig. 2. Spectral analysis of the carbethoxylation reaction. A. Spectra were recorded before (-) and after (-- -) the reaction shown in Fig? 1 with reaction buffer in the reference cell of the Pye Unicam spectrophotomcter. B. During the course of a reaction identical to that shown in Fig. I, spectra were recorded at selected times (10, 15, 17, 20, 25, 30, 40 and 50 min). The reference cell contained an identical mixture except for the omission of diethylpyrocarbonate which was replaced by an equal volume of vehicle (ethanol).

Azaa(--)

0

I

I

10

1

I

20

o,04

1

I

40

TIMECri$ 0.02

salmotlulin.

0

0

10

15

2g

Fraction number which can be carbcthoxylatcd in the presence or absence of Ca’” [29]. Removal of Ca’* with EGTA, causing the dissociation of calmodulin and ealdesmon, did not result in any spectroscopic change. We can conclude therefore that His-610 of caldesmon is accessible to when complcxed with diethylpyrocarbonate calmodulin, suggesting that this residue is not part of the calmodulin-binding site. In support of this conclusion, carbechoxylated caldesmon, like untreated caldesmon, bound to a calmodulin-Scpharose affinity column in the presence of Ca”+ and was elut,ed with EGTA (data not shown). The affinities of smooth muscle actin for control and carbethoxylated caldesmons were compared using a sedimentation assay as described in section 2. The Kd for control caldesmon was determined to be 0.88 x 10” M and for carbethoxylated caldesmon, 1.29x 10V6M. Histidine modification therefore caused a slight, possibly insignificant, reduction in the affinity of caldesmon for actin. Consistent with this observation, carbethoxylated caldesmon was as effective as the unmodified protein in inhibition of the actin-activated MgATPase activity of skeletal myosin (Table I). Table I Comparison of ihe effects of control and carbethoxylatcd caldesmons on the skeletal actin-activated myosin MgATPase Caldesmon None Control, 1.2 ,LLM Control, 2.0 gM Carbethoxylated, 1.2 PM Carbethoxylated, 2.0 kM

ATPase rate (nmol Pl/min*mg myosin) 6939% 14.7 371,6~tll.l 387,8rn 4.7 383,7ri1*5 332,1~11.2

(n=2) (n=4) (n=2) (n=4) 01=2)

Fig. 4. Uindina of ctirbcrhex~caldesnlon to immabilizcd trapumyonin. Cnrbcthuxylatcd ealdcsmon (3 mg; see Fil. 1) WI dialyzed va 20 mM Trir-HCI (pl-I 7.9, I 111blEGTA, 1 r&l dithiothrcital and applied, al a flow rate of IO ml/h, to a column (1 X ia cm) of fropomyasilr-Spkurese 40, The column was washed with the same buffer and bound pratcin wns elutcd with this buCfer containing 0.4 M NnCI, Fractions of 2,5 ml were collected. The inrc~ Shows SDS-PACE analysis of the unbound material (peak A) and bound protein (peak B). The positions of M, markers arc sJ~owo.

Cacbcthoxylated caldesmon, like the untreated protein, was found to bind to a tropomyosin-Sephzrose affinity column at low ionic strength and was eluted with a buffer containing 0.4 M NaCl (Fig. 4). The flowthrough peak from this column contained polypeptides of M, 110 and 90 kDa which probably represent proteolytic fragments of caldesmon (see gel inset of Fig. 4). Specific modification of His-610 of caldesmon did not affect its interaction with calmodulin suggesting that the basic amphiphilic a-helical sequence (Arg-59%His-610) does not represent the calmodulinbinding site. These results support the conclusion of Bartegi et al. [13] that this site is located in a different part of the molecule (towards the C-terminus). Carbethoxylation of His-610 of caldesmon had little, if any, effect on its affinity for actin or ability to inhibit the actin-activated MgATPase activity of skeletal muscle myosin suggesting that this residue does not play an important role in the interaction of caldesmon with acbound to carbethoxy-saldesmon tin. Finally, tropomyosin-Sepharose at low ionic strength and could be eluted with NaCl, as for the untreated protein [301. On the basis of sequence homologies with the tropomyosin-binding domains of skeletal muscle troponin T, two regions within the caldesmon molecule 83

(Glu=rTOB=Lys-565and Lyti4Xl’l-Arg-Bill) hnvr bm inv plieetcd a$ rro9orrryarrin=bindirry regions [31). The 1attcr ecquenct:eonrrina Hts-btt) but e8wbcrhoxylarion elenrly dtd net prcvenr the binding of caldermon 10 tragamyosin, Either this rwidrre is nac invnlved in tropamyastn binding or ir ptays w reinlively ~ntnor role in the interacrion of cnldesmon with trapemyoain,

K. 1%reclglcnt al R Fellawahip lram M.R.C.C, nnd M.P.W. ir RII M.H,C,C. Scicnrist w.t Albcrfr &rltage Paunttmion t’ar htedicat Research Scholiw. The wrhors we fgnlrful IoGcrry Cirwnclr for ward procenln~, RBP;EREiNCES [I] Walsh, M-P. (1990) Prog. Clin, tdiol. Res. 327. 127-140, 121 Sobue, K., Murfimo~o, Y., Fujiw Cf. nnd Kekiuchi, S. (1981) Proc. NatI. Acad, Sci. USA 78, 5652-5655. 13) tkebe, M, and Rio&n, S. (1988) J. Biol, Chcm. 263. 3055-3058, 141 eracerra, P, (1987) FEUS LCIU, 218, 139-w. (51 Sobuc, K., Morimoto, K,, tnui, M*, Ksnda, K. and Kakiuchi, S. (lP82) Biomed, Rcr. 3, 188-196. [6] Bryan, J,, tmai, M., Lee, R,, Moore, P., Cook, R.G. nrd tin, W.-G, (1989) J. Biol. Chcm. 264. 13873-13879. [I] Huynshi, K., Kanda, K., Klmizuka, F., KPIO, I. and Sobue, K. (1989) Riochcm. Biophys. Rcs. Common. 164, 503-511. (81 Riremnn, V.M., Lynch, W-P., N&sky, 8. and Brewher, A. (1989) J. Biol. Chcm. 264, 2869-2875. [9] Szpaccnko. A. snd Dabrowska, R. (1966) FEBS Lat. 202, 182-186. [IO] Fujii, T., Imai, M., Rorenfcld, G.C. and Bryan, J, (1987) J. Biol. Chem. 262, 2757-2763.

ll6] %01, tf,G. and Porter, J.U, (1981) Prep. Biashem, II, 381-395. 1171 Perrrchini, A. nnd Ruwc, A-1. (1984) 1. hlni. Bltrl, 1X&23-39, [IS) Walsh, M.P,, Vntcnrinc, K.A,, Ngai, P,K., Cnrrurhers, C,A. nntl Etrsttcnbery, M.D, (tPB4) Btachcm, J. 234, tl7-12’1. (191 Milts, E.W, (1977) Melhoda Ennymat. 47, 431-442, [2(lj Ovilrli, J., Libor, S, untl EIBdi, P. (1967) Biochim. Blophyx. Aclr 2, 455”4s. [Zll Ikcbc, M, ontl Harlsharnc, t1.J. (1985) Biochcmlwlry 24, 2380-23137. (22) KICC, C.B. (1977) Ukchcmialry 16, IOtt-1024. (23) C;rncefh, P,, W’nng, C.&.A. end Slitft’ortl, W.Fd (1988) J. Riot, Chcm. 263, 14196-14202, (24) Eisenberg, E. and Kieltey, W,.Wv, (1974) J. Biol, Chcm, 249, 4742-4148. 125). Recr, M,R, and Young, M. (1967) J. t3iul. Chem. 242, 4449-4358. I261 Margosinn, S,S, and Lowry, S. (t9tt2) Mcfhodx Enrymol. 84, 55-71. Lawnmti, U.K. (1970) Nruurc 227, 680-685, 1:;; Watterron, D.M.. Sharicf, F. and Vnnamen, f.C, (1980) J, Dial. Chcm. 2$S, 962-975. 16, 1291Wnlsh, hl, and Srevenr, F.C. (1977) Biochemiswy 2742-2749. [301 Fujii, T., Oznwa, J., Ogoma, Y. and Kondo, Y. (1988) J. Uiol. Chrm. (Tokyo) 104, 734-737, 1311 Hnyaski, K., Yamadn, S., Knnda, K., Kimiruka, F., Kn~o, I. and Sobue, K. (1989) Biochcm. Diophys, Rcs. Comnrun. 161, 38-4s.