Cell Motility and the Cytoskeleton 20:95-108 (1991)

Epitope Mapping of Monoclonal Antibodies Against Caldesmon and Their Effects on the Binding of Caldesmon to Ca+ +/Calmodulin and to Actin or Actin-Tropomyosin Filaments Jim Jung-Ching Lin, Elizabeth J. Davis-Nanthakumar, Jian-Ping Jin, David Lourim, Robert E. Novy, and Jenny Li-Chun Lin Department of Biology, University of Iowa, Iowa City The effects of monoclonal anti-caldesmon antibodies, C2, C9, C18, C21, and C23, on the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca+ +/calmodulin were examined in an in vitro reconstitution system. In addition, the antibody epitopes were mapped by Western blot analysis of NTCB (2-nitro5-thiocyanobenzoic acid) and CNBr (cyanogen bromide) fragments of caldesmon. Both C9 and C18 recognize an amino terminal fragment composed of amino acid residues 19 to 153. The C23 epitope lies within a fragment ranging from residues 230 to 386. Included in this region is a 13-residue repeat sequence. Interestingly this repetitive sequence shares sequence similarity with a sequence found in nuclear lamin A, a protein which is also recognized by C23 antibody. Therefore, it is likely that the C23 epitope corresponds to this 13-residue repeat sequence. A carboxyl-terminal 10K fragment contains the epitopes for antibodies C2 and C21. Among these antibodies, only C21 drastically inhibits the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca+ +/calmodulin. When the molar ratio of monoclonal antibody C21 to caldesmon reached 1.O, a maximal inhibition (90%) on the binding of caldesmon to F-actin filaments was observed. However, it required double amounts of C21 antibody to exhibit a maximal inhibition of 70% on the binding of caldesmon to F-actin-tropomyosin filaments. These results suggest that the presence of tropomyosin in F-actin enhances caldesmon’s binding. Furthermore, C21 antibody also effectively inhibits the caldesmon binding to Ca+ +/calmodulin. The kinetics of C21 inhibition on caldesmon’s binding to Ca+ +/calmodulin is very similar to the inhibition obtained by preincubation of caldesmon with free Ca+ +/calmodulin. This result suggests that there is only one Ca+ +/calmodulin binding domain on caldesmon and this domain appears to be very close to the C21 epitope. Apparently, the Ca+ +/calmodulin-binding domain and the actin-binding domain are very close to each other and may interfere with each other. In an accompanying paper, we have further demonstrated that microinjection of C21 antibody into living chicken embryo fibroblasts inhibit intracellular granule movement, suggesting an in vivo interference with the functional domains [Hegmann et al., 1991: Cell Motil. Cytoskeleton 20:109-1201. Key words: actomyosin, smooth muscle contraction, nonmuscle cell motility, microinjection

INTRODUCTION

In vertebrate striated muscle, the thin filamentbased regulatory system (tropomyosin and troponin complex) plays a vital role in the regulation of muscle contraction [Ebashi et al., 1969; Smillie, 19791. In smooth 0 1991 Wiley-Liss, Inc.

Received January 3, 1991; accepted June 7, 1991. Address reprint requests to Dr. Jim Jung-Ching Lin, Department of Biology, University of Iowa, Iowa City, Iowa 52242.

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muscle and nonmuscle cells, the regulatory function of tropomyosin for actin-myosin interaction may exist in a different way, since no troponin proteins have so far been isolated [Cote, 19831 and since another type of regulation via myosin light chain phosphorylation has been found in these cells [Adelstein and Eisenberg, 1980; Kendrick-Jones and Scholey, 19811. Moreover, an actin- and Cat calmoddin-binding protein called caldesmon was discovered in chicken gizzard [Sobue et al., 19811 and has subsequently been identified in various smooth muscle and nonmuscle cells [Bretscher, 1986; Marston and Smith, 1985; Sobue et al., 19881. in vitro reconstitution studies reveal that caldesmon binds F-actin in a Ca+ +/calmodulin-inhibitable manner [Bretscher, 1986; Chalovich, 1988; Marston and Smith, 1985; Sobue et al., 19881. The binding of caldesmon to F-actin-tropomyosin strongly inhibits the interaction of myosin with actin and decreases myosin ATPase activity [Chalovich, 1988; Smith et al., 1987; Sobue et al., 19821. This inhibition is released upon the addition of Ca+ +/calmodulin. Several lines of evidence have demonstrated a direct interaction between caldesmon and tropomyosin [Fujii et al., 1988; Graceffa, 1987; Horiuchi and Chacko, 1988; Smith et al., 19871. Therefore, tropomyosin, caldesmon, and calmodulin may participate in a thin filament-based regulatory system in smooth muscle and nonmuscle cells. The intracellular localization of caldesmon demonstrated by immunofluorescence is consistent with this suggestion [Bretscher and Lynch, 1985; Dingus et al., 1986; Owada et al., 19841. In cultured nonrnuscle cells, caldesmon is distributed along stress fibers in a periodic pattern similar to the distribution of myosin, tropomyosin, and complementary to the distribution of cx-actinin. In addition, caldesmon can be also detected in the membrane ruffles of nonmuscle cells. However, a regulatory role of caldesmon in in vivo motility has not yet been demonstrated. Microinjection of monoclonal antibodies into living cells has shown great promise as an approach to probe the in vivo function of specific antigen molecules [Blose et al., 1984; Feramisco et al., 1985; Hegmann et al., 1989; Honer et al., 1988; Klymkowsky, 1981; Klymkowsky et al., 1983; Lin and Feramisco, 1981; Mercer et al., 1982; Sinard and Pollard, 19891. Recently, we have developed five monoclonal antibodies (C2, C9, C18, C21, and C23) against chicken gizzard caldesmon [Lin et al., 19881. In this study, we mapped their epitopes and characterized their in vitro effects on the binding of caldesmon to F-actin/F-actin-tropomyosin and to Ca+ +/calrnodulin. We have shown that the C21 antibody is a very potent inhibitor of caldesmon’s binding in each case. In the accompanying paper [Hegmann et al., 19911, we have microinjected these monoclonal antibodies into chicken embryo fibroblasts and provided

evidence for the involvement of caldesmon in the intracellular granule movements of nonmuscle cells. MATERIALS AND METHODS Purification of Muscle Proteins

Smooth muscle tropomyosin and caldesmon were purified by the method of Bretscher [ 19841. F-actin was purified from acetone powder of rabbit skeletal muscle as described previously [Lin et al., 1985bl. Monoclonal and Polyclonal Antibody Production

Preparation and characterization of anti-caldesmon monoclonal antibodies (C2, C9, C18, C21, and C23) have been described previously [Lin et al., 19881. All these antibodies are IgG, class. Thus, antibodies were purified from ascites fluids by protein A-sepharose column chromatography as described previously [Lin et al., 1985al. Polyclonal antiserum against chicken gizzard caldesmon (R 19) was prepared by immunizing a rabbit with purified caldesmon following the procedure and schedule described previously [Matsurnura et al., 19831. The preimmune serum and postimmunization antiserum were tested by enzyme-linked immunosorbant assay (ELISA) with purified caldesmon (0.5 pg/well) as coated antigen. The preimmune serum showed negative reactivity in ELISA, whereas the antiserum R19 showed strong positive reactivity. The specificity of R19 antiserum was further tested by indirect immunofluorescence and by Western blots [Lin et al., 19881. Immunofluorescence staining of cultured chicken gizzard cells and several nonmuscle cells from different species showed strong stress fiber and membrane ruffle region staining. This staining was abolished by preabsorption of the antiserum with purified caldesmon prior to immunofluorescence staining. Autoradiograms of Western blots showed that R19 antiserum reacted specifically with caldesmon in total cell lysates of chicken gizzard and a variety of cultured nonmuscle cells, identical to that obtained by monoclonal antibody C21 as reported previously [Lin et al., 19881. Chemical Cleavages of Caldesmon

Cleavage of caldesmon at cysteine residues was carried out according to the method of Jacobsen et al. [ 19731. Briefly, lyophilized caldesmon was reduced in NTCB reaction buffer containing 8 M urea, 0.2 M sodium borate, pH 9.0, and 0. l mM dithiothreiotol (DTT) for 2 hours at 37°C. A 0.1 M DTNB [ 5 , 5’-dithiobis (2-nitrobenzoic acid)] stock solution was added to a final concentration of 0.5 mM. After 15 min at room temperature, KCN was added in a I0-fold excess over the DTNB. The solution was allowed to stand for 2 hours at

Caldesmon Domains Defined by Monoclonal Antibodies

room temperature and then dialyzed against DE52 buffer (30 mM NaCl, 10 mM immidazole buffer, pH 7.0, 0.1 mM DTT, and 0.1 mM EGTA). The identification and orientation of the cleaved fragments was performed according to Riseman et al. [1989]. These fragments (called NTCB fragments) contained two partially cleaved polypeptides (108K and 105K), as well as three completely cleaved polypeptides (a 28K amino-terminal fragment, an 80K central fragment, and a 25K at the carboxyl-terminal fragment). The 25K fragment was purified from flow-through fractions after passing the mixture through a DE52 column previously equilibrated with DE52 buffer. The bound fragments and intact caldesmon were further eluted with a linear salt gradient (0-0.3 M NaCl in DE52 buffer) to yield partially purified fragments. The 28K and 80K fragments were further purified by preparative SDS PAGE as described previously [Lin et al., 1985al. Cleavage of 25K, 80K, and 28K fragments at methionine residues was performed with cyanogen bromide (CNBr). The lyophilized polypeptides (about 5 nmoles) were dissolved in 70% formic acid. Two mg of CNBr was added, mixed, and incubated at room temperature overnight. The reaction was stopped by diluting the solution ten times with water and then lyophilized. A 10K fragment was purified from the CNBr digestion of the 25K fragment by either HPLC (C18 reverse phase column) or Biogel P-30 column chromatography. An aliquot of 10K fragment was hydrolyzed in a sealed, evacuated tube containing 200 pl of 1% phenol in 6 N HC1 solution at 110°C for 24 hours. The hydrolysates were evaporated to dryness and derivatized for amino acid composition analysis. For actin binding assay, a 10 K fragment was prepared from the CNBr digestion of caldesmon according to the method of Bartegi et al. [1990]. Binding of Caldesmon to Ca+ +/Calmodulin

Caldesmon binding to Ca+ +/calmodulin was assayed by a modified ELISA [Tijssen, 19851. Bovine brain calmodulin (Sigma Chemical Co., St. Louis, MO) was dissolved in 0.5 M carbonate buffer, pH 9.6, 0.2 mM CaC1, at 5 pg/ml; 100 pl of the calmodulin solution was added to the wells of Immulon I1 microtiter plates and incubated at 4°C overnight. After removing the excessive amounts of Ca+ +/calmodulin solution, the wells were blocked for 30 min at room temperature with 3% bovine serum albumin (BSA) in PBS-T-Ca+ (10 mM phosphate buffer, pH 8.2, 0.15 M NaCl, 0.05% Tween 20, and 0.2 mM CaC1,). After washing three times with PBS-T-Ca+ , an increasing amount of caldesmon in 100 pl of PBS-T-Caf+ was added to each well and incubated for 1 hour at room temperature. Caldesmon bound after three washes was detected by incubation +

+

97

with rabbit polyclonal R19 antiserum against caldesmon for 1 hour. The wells were incubated for an additional hour with horseradish peroxidase conjugated goat antirabbit IgG (Sigma Chemical Co., St. Louis, MO; 1,000fold dilution). The enzyme activity in each well was estimated from the color intensity developed by incubation for 10 min with 100 pl of the chromogenic substrate containing 1 mM 2,2'-azinobis-(3-ethylbenzthiazoline sulfonic acid) in 0.1 M citrate buffer, pH 4.2, and 0.03% H,O,. The color reaction was stopped by the addition of 50 p l per well of 5% SDS. The plate was read photometrically at 405 nm by an ELISA reader. PBS-T-Ca+ was used both for washing plates between each incubation step and for all dilutions, when the binding was tested in the presence of C a t + . Otherwise, 0.2 mM EGTA was replaced by the Cat in the PBS-T buffer for washings and for dilution. For testing the effects of anti-caldesmon monoclonal antibodies on the binding of caldesmon to Ca+ + / calmodulin, a constant concentration (0.2 pM) of purified caldesmon was preincubated with increasing amounts of various monoclonal antibodies or Ca+ + / calmodulin (used as a positive inhibition control) for 30 min at room temperature. The caldesmon-antibody or caldesmon-Ca+ +/calmodulin complex was used in the ELISA assay as described above. The absorbance at 405 nm with no inhibitor added (Ao) and the absorbance with various tested concentration of inhibitor added (Ax) were used to calculate the percent inhibition by the following equation: +

+

% inhibition =

[Ao;oAxl

x 100%.

L

A

Actin Binding Assay

The binding of caldesmon to F-actin or F-actintropomyosin was performed as described previously [Lin et al., 1985bI using conditions of 10 mM Tris, pH 8.0, 100 mM KC1, and 10 mM MgCI,. In order to determine the effect of various monoclonal antibodies on the binding of caldesmon to either F-actin or F-actin-tropomyosin, increasing amounts of monoclonal antibody were added to the mixture of F-actin (1.1 mg/ml) and caldesmon (0.15 mg/ml) in the absence or presence (0.18 mg/ ml) of tropomyosin. After incubation for 30 min at room temperature , samples were centrifuged using a Beckman airfuge at 26 psi for 20 min. Aliquots of the resulting supernatants and pellets of each sample were analyzed on SDS PAGE. Gels were stained with Coomassie blue and scanned with a densitometer. The area under the caldesmon peak was calculated in the pellet and supernatant of each sample. The percent caldesmon bound was calculated and plotted against antibody concentration.

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Other Procedures SDS PAGE was carried out according to Laemmli [ 19701 with a low concentration of bisacrylamide (12.5%

acrylamide and 0.104% bisacrylamide) [Blattler et al., 19721. For separation of chemically cleaved caldesmon fragments, a Tricine-SDS PAGE was used as described by Schagger and von Jagow [1987]. Western immunoblotting was performed as described previously [Lin et al., 1985al. Protein concentrations were determined by the method of Lowry et al. [1951] with BSA as standard. Solid-phase radioimmunoassay was performed as described previously [Lin et al., 1985al. RESULTS Monoclonal Antibody Epitope Mapping

Partial chemical cleavage of gizzard caldesmon at cysteine residues gives rise to the intact molecule and five NTCB fragments with apparent molecular weights of 108,000, 105,000, 80,000, 28,000, and 25,000 (Fig. 1A). Riseman et al. [1989] have recently oriented these fragments into a linear map for the caldesmon molecule, as shown in Figure 1. The 80K polypeptide is located in the central portion of the molecule, whereas the 28K and 2SK are derived from the amino terminus and carboxyl terminus, respectively. This information allows us to roughly map the epitopes of monoclonal anti-caldesmon antibodies C2, C9, C18, C21, and C23 by Western immunoblotting on the partially purified NTCB fragments. As can be seen in Figure 1 , both C2 and C21 antibodies reacted with the carboxyl 25K fragment (lane 6 in Fig. 1B and E), whereas both C9 and C18 antibodies recognized the amino 28K polypeptide (lane 5 in Fig. 1C and D). Neither 25K nor 28K fragment was detected by C23 antibody. However, C23 antibody strongly reacted to the central fragment (80K) of the caldesmon (lane 4 in Fig. 1F). It should be noted that autoradiograms shown in Figure 1 are the experiment with the exposed time of 3 days for C9, C18, and C21, 1 day for C2, and 4 hours for C23 antibody. F-Actin Binding Domain It has been shown that the carboxyl 25K polypeptide of caldesmon is able to bind F-actin in a saturable manner [Riseman et al., 19891. Further cleavage of 25K fragment by cyanogen bromide gave rise to a carboxyl 10K fragment, which has an amino acid composition very similar to that calculated from the cDNA sequence of caldesmon reported by Bryan et al. [1989a] (see Table 1). Actin binding experiments showed that unlike the 2SK fragment (lane A in Fig. 2), carboxyl 10K fragment purified by HPLC or Biogel P-30 column chromatography of CNBr fragments of 25K was unable to bind F-

actin (data not shown). However, a 10K fragment prepared according to the method of Bartegi et al. [1990] retained strong binding activity to Ca+ +/calmodulin affinity column (data not shown) and weak binding activity to F-actin filaments (lane B in Fig. 2). These results are consistent with the report by Bartegi et al. [1990]. Thus, the F-actin binding domain of caldesmon appeared to be located in this 10K fragment from residue 658 to 756 (Fig. 3). Both 25K and 10K fragments were used in Western blots with C2 and C21 antibodies to determine their epitopes. As can be seen in Figure 4, the C2 antibody reacted strongly and equally well with the 10K and 2SK fragments of caldesmon. On the other hand, the C21 antibody recognized the 10K fragment weakly, although C21 reacted with the 25K fragment as well as C2 antibody did (Fig. 4).The differential reactivity of C2 and C21 antibodies to the 10K fragment was also observed in the radioimmunoassay (data not shown). The fact that cleavage of the 25K fragment by CNBr reduced the C21 reactivity might suggest the C21 epitope closer to the amino-terminus of the 10K polypeptide than the C2 epitope. This conclusion is further supported by the following evidence. Amino acid sequence comparison between chicken gizzard caldesmon [Bryan et al., 1989al and human low M, caldesmon (Novy and Lin, in preparation) revealed a 85% identity in this carboxyl-terminal 10K fragment, and the nonhomologous region was clustered in the extreme carboxyl-terminus (residues from 730 to 756) of chicken gizzard caldesmon. Moreover, we have previously shown that C21 antibody but not C2 antibody is able to crossreact with human low M, caldesmon [Lin et al., 19881. Thus, it is apparently that the C21 epitope is distinctive from the C2 epitope and locates closer to the amino terminus of the 10K fragment than the C2 epitope. As we will describe below, only C21 antibody but not C2 can competitively inhibit the binding of caldesmon to actin filaments, further supporting this epitope map. Monoclonal C21 Antibody Interferes With the Binding of Caldesmon to F-Actin and F-Actin-Tropomyosin Filaments The effect of monoclonal anti-caldesmon on the binding of caldesmon to F-actin or F-actin-tropomyosin filaments was examined by cosedimentation actinbinding assay. An increasing amount of each monoclonal antibody was incubated with a constant amount of caldesmon and then the mixture was added to a F-actin solution. Free and bound caldesmon were separated by airfuge and quantified by scanning SDS-PAGE gels as described in Materials and Methods. Figure 5 shows an example of such experiments. Most of the caldesmon was cosedimented with F-actin filaments in the absence of monoclonal antibody (lane 1 P). The presence of an

Caldesmon Domains Defined by Monoclonal Antibodies

N

28K

V

c,?, LlU

4s

80 K

25K

y 4

C23

cys

99

C

c2 c21

P

F - a c t i n and calmodul i n binding d o m a i n

I A \Arnido black

(BJ c 2

123456

fcl c9

123456

1 2 3 4 5 6

{El C21 123456

IF} C 2 3

28 k25k-

(DI C18

1234S6

Fig. I . Western blot analysis of monoclonal antibodies binding to gizzard caldesmon and its fragments obtained from the chemical cleavage of caldesmon at cysteine residues. Cleavage was performed according to the presence of Jacobsen et al. [1973] using the reagent NTCB (2-nitro-5-thiocyanobenzoicacid). The partially purified, cleavage products were separated on 12.5% SDS PAGE. After electrophoresis, proteins were transferred to nitrocellulose papers and either stained with amido black (A) or reacted with C2 (B), C9 (C), C l 8 (D), C21 (E),or C23 (F) monoclonal anti-caldesmon antibody, fol-

123456

lowed by 1251-labeledgoat anti-mouse IgG. Bound antibody was detected by autoradiography. The exposed times for radiogram B, C, D, E, and F were 1 day, 3 day, 3 day, 3 day, and 4 hours, respectively. The orientation of the cleaved fragments was arranged according to data obtained by Riseman et al. [I9891 using C14-labeled KCN in the reaction. The schematic diagram is shown here with the locations of epitopes recognized by monoclonal antibodies, as determined by these Western blots.

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TABLE 1. Amino Acid Compositions of 10 K CNBr Fragment Purified From Caldesmon Amino acid

10 K

Theoretical"

Asx Glx Ser glY His Arg Thr Ala Pro TYr Val Met Ile Leu Phe LYS TrP CYs

8.3 11.3 11.8 13.4 0 4.2 7.7 6.8 10.0 0.6 6.1 0 1.8

8.2 10.2 12.2 11.2 0 4.1 8.2 5.1 9.2 0 6.1 0 1.0 6.1 2.0 13.3 3.1 0

4.8 2.0 11.3 n.d. n.d.

"Theoretical value calculated from cDNA sequence data of caldesmon [Bryan et al., 1989aJ. n.d., not determined.

A

B

S P S P

42OOk -116.5k -94k

Act in,

430k 25K*

421k increasing amount of C2 1 antibody gradually inhibited the binding of caldesmon to F-actin filaments (lanes 2, 3, and 4 in Fig. 5 ) . On the contrary, the addition of C18 antibody did not show significant reduction on the binding of caldesmon to F-actin filaments (lanes 5 and 6 in Fig. 5). It should be noted that the C18 antibody was readily cosedimented with the actin-caldesmon filaments in the pellet fractions (lanes 5P and 6P in Fig. 5). This was also true for C2, C9, and C23 but not for C21 antibody. These results suggest that most of these antibodies are able to bind to the caldesmon-decorative filaments. The control experiment with either antibody and caldesmon in the absence of F-actin showed all of the caldesmon remaining in the supernatant fraction (data not shown), suggesting that cosedimentation of caldesmon with the F-actin fraction was not due to the crosslinking and precipitation of the antigen by monoclonal antibody. Similarly, there appeared to be no effect on the caldesmon binding, when C2, C9, or C23 antibody was added to the assay. Identical results were also obtained in experiments in which antibody was added to preformed filaments of F-actin-caldesmon. It has been reported that smooth muscle tropomyosin can enhance the binding of caldesmon to F-actin filaments [Horiuchi and Chacko, 1988; Smith et al., 19871. Thus, the effect of these monoclonal antibodies on the binding of caldesmon to F-actin-tropomyosin filaments was also examined. Again, only C21 antibody showed a significant interference on the binding of caldesmon to F-actin-tropomyosin filaments. Figure 6 shows a quantitative summary of these binding assays. In

10KP

-14.3k

Fig. 2. Tests for the actin-binding ability of the carboxyl-terminal 25K and 10K fragments of caldesmon. Purified fragments (9.0 pg each) were mixed with 100 pg of F-actin in 100 p1 of a buffer containing 10 mM Tris, pH 7.5, 10 mM MgCI,, and 100 mM KCI. After incubation for 30 min at room temperature, the samples were centrifuged for 20 min in a Beckman airfuge at 26 psi. Aliquots of the resulting supernatants (S) and pellets (P) were analyzed on SDS PAGE. A, actin plus the 25K fragment; B, actin plus the 10K fragment. Under this condition, neither the 25K nor the 10K fragment was pelleted (data not shown).

the presence of 0.5 p M C21 antibody, the extent of binding of caldesmon to F-actin was only 30% of that found in the control, whereas 60% of the control binding could still be observed when preformed F-actin-tropomyosin filaments were used. At a very high concentration (2.75 pM) of C21 antibody, the caldesmon bound to F-actin filaments and to F-actin-tropomyosin was reduced to about 15% and 35%, respectively, of the control binding. These results suggest that the presence of tropomyosin in the F-actin filaments may protect the caldesmon from being stripped off by the C21 antibody. This is consistent with the idea that tropomyosin may strengthen the binding of caldesmon to F-actin filaments.

Caldesmon Domains Defined by Monoclonal Antibodies 1

153

yet

N":

101

cYS

v

A met

TCOOH A

19

met

met

A

met

h

h met

230

386

446

548

h met

h met 606 658

h

Cad15

I

A

-

A

A

25K

28K

10K

Fig. 3. A linearized representation of the caldesmon molecule and the epitopes of monoclonal anti-caldesmon antibodies mapped in this study. The positions of met and cys residues were derived from the predicted amino acid sequence of cDNA clones by Bryan et al. [ 1989al. Chymotryptic fragment (CT40) and CNBr fragment (CB40) have been purified and partially sequenced [Leszyk et al., 1989bI. Each fragment contains one cysteine residue. Both fragments bind to a Ca' +/calmodulin affinity column [Fuji et al., 1987; Szpacenko and Dabrowska, 1986; Wang et al., 1989; Yazawa et al., 19871, but only CT40 contains a F-actin binding domain. Cad,, corresponds to the region Ser,,, to Phe,,,, which is not directly involved in actin binding but may participate in strengthening the actin binding [Leszyk et al.,

The Inhibition on the Caldesmon Binding to F-Actin-Tropomyosin Filaments by C21 Antibody Is a Competitive Inhibition

A

1989a; Mornet et al., 19881. In addition, Cad,, contains residues Glu,,, to Lys,,,, a region homologous to the tropomyosin binding region of troponin T [Bryan et al., 1989a; Leszyk et al., 1989al. The NTCB fragments (28K, 80K, and 2SK) of caldesmon were further cleaved at Met residues by CNBr. The resulting fragments were immunoblotted to determine the epitope maps of the monoclonal antibodies. Both C9 and C18 antibodies recognize a fragment composed of residues 19 to 153 near the amino terminus of the molecule. The epitope of C23 antibody lies within the region of residues 230 to 446. The C21 epitope appears to be closer to the amino-terminus of the 10K fragment than the C2 epitope.

Amido Black

1 2 3

-c2

1 2 3

c9 --

123

c21 i -

1 2 3

To examine whether the C21 inhibition on the 200k4aD kcaldesmon's binding to F-actin-tropomyosin filaments 116.5 94kwas of a purely competitive mode, two inhibition curves 68kwere generated at two different concentration of caldes45 kmon in the binding assays. Dixon analysis [Dixon, 19531 of these data (Fig. 7) shows that C21 antibody is a competitive inhibitor of caldesmon binding to F-actin-tropomyosin filaments with a Ki of 0.48 pM. Therefore, the 2lk25K K, for caldesmon binding to F-actin-tropomyosin, cal14.3kculated from this analysis, is 2-2.6 X lop6 M. This -10K result suggests that the C21 epitope is located within the actin binding domain of caldesmon and that the binding of C21 antibody to caldesmon is strong enough to dis- Fig. 4. Western blot analysis of monoclonal antibodies (C2, C9, and place the binding of caldesmon to actin filaments. C21) binding to purified 10K, 2SK, and intact caldesmon. Protein The Binding of Caldesmon to Calmodulin Is Ca+ +/Dependent In order to examine the effect of monoclonal antibodies on the interaction between caldesmon and calmodulin, we have developed a modified ELISA method to demonstrate the binding of caldesmon to calmodulin.

fragments were separated on SDS PAGE and transferred to nitrocellulose papers. The blots were either stained with amido black, or reacted with C2, C9, or C21 antibody, followed by 'Zsl-labeled goat antimouse IgC. Bound antibody was detected by autoradiography . The exposed time for these immunoblots was 2 days. C9 immunoblot was used as a control. Lane 1, intact caldesmon; lane 2, the 25K fragment; lane 3, the 10K fragment. The reactivity of C2 to the 10K fragment was much stronger than that of C21 antibody. CaD, caldesmon .

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Lin et al.

1 -

-

2 -

C

3 )

r

4 C

5

6

-

c2 c9 C18 C23

200k w

-CaD

116.5k w 94kw 68kw

45kw

4 A c t in

jC2(

30kw

SlLC

1

21kw

I

0.5

I 1.0

lmAbl

14.3kw

Fig. 5 . Effects of monoclonal antibodies CIS and C21 on the binding of caldesmon to F-actin filaments. Increasing amounts of monoclonal C21 or CIS antibody were added to the mixture of F-actin (1.1 mg/ml) and caldesmon (0.15 mg/ml) in a buffer containing 10 mM Tris, pH 7.5, 10 mM MgCl,, and 100 mM KCI. The samples were centrifuged after incubation for 30 min, at room temperature. The aliquots of the resulting pellets (P) and supernatants (S) were analyzed on 12.5% SDS PAGE. Sample 1, no antibody added; samples 2,3, and 4 contained 0.2, 0.4, and 0.6 mgiml, respectively, of C21 antibody. Samples 5 and 6 contained 0.2 and 0.4 mglml, respectively, of CIS antibody. Monoclonal C21 antibody effectively inhibits the binding of caldesmon to F-actin, whereas C18 antibody may increase slightly the actinbinding ability of caldesmon.

As can be seen in Figure 8, caldesmon appeared to bind to calmodulin in the presence of 0.2 mM CaCl,. However, when the assay was performed in the presence of EGTA, the caldesmon binding was abolished. When bovine serum albumin was used instead of calmodulin in the assay, there was negative reactivity in both the presence and absence of Cat (data not shown). Moreover, as the concentration of caldesmon was increased the reactivity was shown to plateau, thus indicating a saturable binding of caldesmon to Ca+ +/calmodulin (Fig. 8). +

Monoclonal C21 Antibody Inhibits the Binding of Caldesmon to Ca+ +/Calmodulin The binding of caldesmon to calmodulin appeared to be specific in our modified ELlSA assay. Incubation of caldesmon with Ca+ +/calmodulin before the assay abolished the caldesmon binding to the coated Cat + / calmodulin (Fig. 9). When monoclonal anti-caldesmon antibodies were used as inhibitor in this assay, we have

I

I

I

1.5

2.0

2.5

I

3.0

pM

Fig. 6 . Effects of monoclonal antibodies, C2, C9, C18, C21, and C23 on the binding of caldesmon to F-actin or F-actin-tropomyosin filaments. A cosedimentation assay was used to examine the effects of increasing amounts of monoclonal antibody on the actin-binding ability of caldesmon. Caldesmons in bound fractions (pellets) or free fractions (supernatants) were quantified by densitometric scanning of coomassie blue-stained gels. The percent bound obtained from three separate experiments were plotted against increasing amounts of monoclonal antibody (mAb). The vertical bars at each point indicate the standard deviations of the means. A-CaD and A-TM-CaD represent the binding of caldesmon to F-actin and F-actin-tropomyosin filaments, respectively. The concentration of actin, tropomyosin, and caldesmon used in this experiment were 1 mg/ml, 0.15 mg/ml, and 0.15 mgiml, respectively. It appears that only the C21 antibody inhibits the binding of caldesmon to both F-actin and F-actin-tropomyosin filaments. It requires 0.4 pm C21 antibody to inhibit 50% of the caldesmon binding to F-actin filaments, whereas about double amounts (0.8 pM) of C21 antibody are needed in order to inhibit 50% of the caldesmon binding to the F-actin-tropomyosin filaments.

observed that only C21 antibody showed a significant inhibition and the rest of the antibodies (C2, C9, C18, and C23) did not have any effect on the binding of caldesmon to Ca+ +/calmodulin (Fig. 9). The C21 antibody gave a maximum percent inhibition (85%) close to that (90%) obtained with free Ca+ /calmodulin. These results suggest a proximity of the C21 epitope to the Ca +/calmodulin binding site of caldesmon. +

Monoclonal C23 Antibody Recognizes Both Gizzard Caldesmon and Nuclear Lamin A To further map the epitopes for C9, C18, and C23 antibodies, we have purified the 28K and 80K NTCB caldesmon fragments by preparative gel electrophoresis. The purified polypeptides were subjected to CNBr cleavage at methionine residues. The resulting CNBr frag-

Caldesmon Domains Defined by Monoclonal Antibodies

20 C

3 0

m

15

-1.5 -1.0 -0.5

0.0

I

l

l

1

1

0.5

1.0

1.5

2.0

2.5

3.0

[rnAb] uM Fig. 7. Dixon analysis of the inhibition by C21 antibody on the binding of caldesmon to F-actin-tropomyosin filaments. Two actin-binding assays similar to the one shown in Figure 6, with caldesmon concentration at 0.1 mg/ml (open circle) or 0.125 mg/ml (closed circle). Data are plotted according to Dixon 119531. The intersection point does not meet at a point on the base line, indicating that the inhibition is a competitive. The K, calculated from this plot is 0.48 p M (indicated by arrowhead).

103

but also nuclear lamin A in muscle and nonmuscle cells from a variety of species (Lin et al., 1988). Since the cDNA sequences of both gizzard caldesmon and nuclear lamin A have been reported [Bryan et al., 1989a; Fisher et al., 1986; McKeon et al., 19861, a sequence comparison between these two protein sequences was performed. As can be seen in Figure 11, five different regions, spanning 13 to 16 amino acid residues, of human nuclear lamin A and caldesmon have greater than 50% sequence homology. Both regions I and I1 are located within the fragment (residues 230-446) recognized by C23 antibody. Moreover, region I in caldesmon contains a sequence repeated eight times. These repeated sequences have homology ranging from 38.5% to 61.5% with human nuclear lamin A [Fig. 11 (I)]. At the present time, we still do not know which of these regions (I or 11) is the correct epitope for C23 antibody. Furthermore, it is known from cDNA cloning studies [Bryan et al., 1989b; Novy and Lin, in preparation] that nonmuscle caldesmon appears to lack the central repetitive sequence (region I) and region I1 as compared to chicken gizzard caldesmon. We have also shown that the C23 antibody does not react with the nonmuscle isoform of caldesmon [Lin et al., 19881. These results are consistent with the conclusion that we made for the C23 epitope. However, in the same experimental condition, the C23 antibody gives a stronger signal to caldesmon on Western blots (Fig. 1) relative to that obtained with the other antibodies (C2, C9, C18, and C21), suggesting that the C23 epitope may correlate with this repeated sequence.

ments were immunoblotted with C9, C18, and C23 antibodies (Fig. 10). Based on the cDNA sequence of caldesmon [Bryan et al., 1989a1, the 28K polypeptide will give raise to a very small fragment with only 17 amino acid residues, which cannot be resolved in our gel, and a second CNBr fragment, which is slightly smaller in size than 28K. Both C9 and C18 antibodies DISCUSSION recognized the intact 28K and this second CNBr fragment. The 80K fragment contains four methionine resiIn this study, we have mapped the epitopes for five dues at positions 230, 386,446, and 548 of the predicted monoclonal anti-caldesmon antibodies (C2, C9, C 18, sequence derived from cDNA sequence [Bryan et al., C2 1, and C23) by Western immunoblotting of the NTCB 1989al. CNBr cleavage gives rise to five completely and CNBr fragments of gizzard caldesmon. The results cleaved fragments, nine partially cleaved fragments, and are summarized in the schematic model shown in Figure an intact 80K fragment. As can be seen in lane 2 of 3. The epitopes for C9 and C18 antibodies lie in the Figure 10, the top five amido black-stained bands can be residues from 19 to 153 near the amino terminus of the identified as intact 80K (residues 153-548), 70K (resi- molecule. The C23 antibody may recognize either a 13dues 153-148), 60K (residues 230-SO), 55K (residues residue sequence repeated within the fragment from 230 153-446 and residues 230-548), and 45K (residues to 386 or a 16-residue sequence (396-411) within the 153-386 and residues 230-446), based on their apparent fragment from 387 to 446. Both of these putative C23 size. These bands were recognized by the C23 mono- epitopes are also present in the nuclear lamin A and clonal antibody (Fig. 10). Therefore, the C23 epitope absence in the nonmuscle isoform of caldesmon. This is may be located in the region from residues 230 to 446 of consistent with our previous results, showing that C23 gizzard caldesmon. Further cleavage of this region into antibody crossreacts with both gizzard caldesmon and fragments containing residues 230-386 and 387-446 re- nuclear lamin A [Lin et al., 19881. From the extent of sulted in the loss of C23 reactivity, since no fragment reactivity to caldesmon among these five monoclonal smaller than 45K was recognized in the C23 immuno- antibodies, we would like to further suggest that the C23 blotting (Fig. 10). epitope is located in the repeated sequence of the caldesWe have previously demonstrated that the C23 an- mon. Both C2 and C21 epitopes are located in the 10K tibody not only recognizes chicken gizzard caldesmon fragment near the carboxyl terminus of the molecule.

104

Lin et al.

-

0

+Ca++

L. v) O ..

0 +EGTA 0.4 I

P

a

0.2-

”

I

4

-

I

I

8

12

”

” A

v

A

U

I

16

10

A

Q

I

I

28

24

I

32

pmoles CaD addedlwell Fig. 8. Test of the binding of caldesmon to Ca++/calmodulin by enzyme-linked immunoabsorbant assay (ELISA). Bovine brain calmodulin (0.31 FM) was coated onto microtiter plates in 100 pl of 0.5M carbonate buffer, pH 9.6, containing 0.2 mM CaCI, at 4°C overnight. Caldesmon was added in an increasing amount to the coated plate, which had been blocked with 3% BSA, in the presence of either 0.2 mM CaCl, or 0.2 mM EGTA. After incubation for 1 hour at room temperature followed by three washes, bound caldesmon was detected by incubation with a rabbit polyclonal antibody against caldesmon for 1 hour at room temperature. After three washes, the plate was incubated for an additional hour with HRP-conjugated goat

anti-rabbit IgC (1:1,000 dilution), and enzyme activity was estimated from color intensity developed by incubation with 1 mM 2,2’-azino-di (3-ethyl benzothiazoline)-6-sulfonate(ABTS) in 0.1 M citrate buffer pH 4.2 and 0.03% H,O,. The plate was read spectrophotometrically at 405 nm by a microtiter plate reader. PBS-Tween-CA++ (10 mM phosphate buffer, pH 8.2, 0.15 M NaCI, 0.05% Tween 20, and 0.2 mM CaCl,) was used for washing plates and for all dilutions, when the binding was tested in the presence of C a + + . Otherwise, 0.2 mM EGTA replaced the Ca+ for washes and dilutions. The binding of caldesmon to coated Ca’. +/calmodulin appears to be Ca+ + dependent.

Among these monoclonal antibodies, only C2 1 has previously been shown to react specifically to chicken gizzard caldesmon and to crossreact with several nonmuscle caldesmons from a variety of species [Lin et al., 19881. The C21 epitope appears to be conserved across species, suggesting that this portion of the caldesmon may be very important for its function. In the present study we have shown that the C21 antibody interferes with caldesmon binding to Ca+ /calmodulin nearly as effectively as free Cai +/calmodulin. In addition, the C2 1 antibody also competitively inhibits the binding of caldesmon to F-actin or F-actin-tropomyosin filaments. These results suggest that the F-actin-binding and Ca+ + / calmodulin-binding domains appear to be in close proximity and very close to the C21 epitope itself. Other investigators have previously shown that both F-actin-binding and Ca+ calmoddin-binding domains lie at the extreme carboxyl terminus of caldesmon [Bryan et al., 1989a; Fuji et al., 1987; Makuch et al.,

1989; Riseman et al., 1989; Szpacenko and Dabrowska, 1986; Yazawa et al., 19871. After chymotrypsin digestion of gizzard caldesmon, a 37-40 kDa fragment (called CT40, see Fig. 3) has been found to retain Ca++/calmodulin- and F-actin-binding abilities [Fujii et al., 1987; Leszyk et al., 1989b; Szpacenko and Dabrowska, 1986; Wang et al., 1989; Yazawa et al., 19871. Furthermore, cleavage of caldesmon at cysteine residues gives rise to a carboxyl-terminal 25K NTCB fragment, which has previously been shown to contain both domains also [Riseman et al., 19891. Recently, Mornet et al. [1988] have identified and characterized a 15 kDa fragment (called Cad,,, see Fig. 3), which can be chemically crosslinked to actin molecules. By itself, Cad,, cannot bind to F-actin. Amino acid sequencing of the Cad,, polypeptide places this fragment in the region from Ser,,, to Phe,,, [Leszyk et al., 1989aj. Although Cad,, is not directly involved in binding to F-actin, Mornet et al. have suggested that it may play a role in twisting the

+

+

Caldesmon Domains Defined by Monoclonal Antibodies

105

0 Ca++-CaM

0 c21

0

C2,C9,C18

pmoles Inhibitor addedlwell Fig. 9. Effects of monoclonal antibodies C2, C9, C18, and C21 or Cai +/calmodulin on the binding of caldesmon to the coated Ca+ + / calmodulin. The ELISA method as described in Figure 7 legend was used for examining the effects. Caldesmon (0.2 mM) was preincubated with an increasing amount of monoclonal antibody or Ca+ + / calmodulin for 30 min at room temperature and then added to the

coated plates. The effects were expressed as % inhibition and plotted against the concentrations of monoclonal antibody or Ca+ +/calmedulin added. Note that C21 antibody at 1 pM is able to inhibit about 82% of caldesmon binding to Ca+ +/calmodulin, whereas the addition of Ca+ +/calmodulin at 1 pM effectively inhibits about 90% of the binding.

F-actin filament and enhancing the actin-binding [Leszyk et al., 1989a; Mornet et al., 19881. Bartegi et al. [1990] have further shown that the extreme carboxyl 10K fragment derived from the CNBr cleavage of caldesmon retains its actin binding ability. In this study, we have confirmed this conclusion by showing that the binding of C21 antibody to its epitope near the amino terminus of the 10K fragment competitively inhibits the binding of caldesmon to F-actin or F-actin-tropomyosin. Therefore, the C21 epitope locates within the F-actin binding domain of caldesmon. The Ca +/calmodulin-binding domain of caldesmon has previously been located in the carboxyl-terminal CT40 [Fujii et al., 1987; Makuch et al., 1989; Szpacenko and Dabrowska, 1986; Yazawa et al., 19871, 25K NTCB fragments [Bryan et al., 1989a; Riseman et al., 19891, and 10K CNBr fragment [Bartegi et al., 19901. In this paper, we have shown that the C21 antibody, recognizing a carboxyl-terminal 10K fragment, can interfere with the binding of caldesmon to C a + + / calmodulin and that the interference by C21 antibody is as effectively as free Cat +/calmodulin. This result sug-

gests a close proximity between the C21 epitope and the Ca +/calmodulin-binding domain. The amino acid sequence of this 2% fragment has been deduced from protein sequencing [Leszyk et al., 1989b] and cDNA sequence analysis [Bryan et al., 1989al. Moreover, a structural framework containing basic, amphiphilic ahelix (Baa-helix) has been proposed to be the C a + + / calmodulin binding domain [James et al., 1988; O'Neil and DeGrado, 19901 for a number of calmodulin binding proteins. Although there is no direct proof yet, residues Arg,,,-His,,, may form such a Baa-helix which interacts with calmodulin [James et al., 1988; O'Neil and DeGrado, 1990; Leszyk et al., 1989bl. However, the results obtained by Bartegi et al. [ 19901 and us in this study seem to argue against this Baa-helix for the Ca++/calmodulin domain. In addition to this C-terminal Cat +/calmodulin binding domain, Wang and his colleagues have recently used crosslinking [Wang, 19881 and calmodulin-affinity column [Wang et al., 19891 to locate the other calmodulin-binding site on the amino-terminal CB40 fragment (see Fig. 3 ) . In the present study we have shown that the

+

+

106

Lin et al. SEOUENCE

L L

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-

C18

CaD

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123

(I)

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7/13 (53.8%)

K,,,Q~EEEKKAAEE,,,

5/13 (38.5%)

RZ,,AKAEEEKllAAEE,,,

6/13 (46.1%)

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7/13 (53.8%)

R,,,AKAEEERKAAEE,,,

7/13 (53.8%)

R,,,AKAEEERKAAEE,,,

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R,,,AKAEKERKAAEE,,,

8/13 (61.5%)

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** *

'=

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(11)

(111)

C21 antibody inhibits the binding of caldesmon to coated calmodulin as effectively as free Ca+ +/calmodulin (maximal inhibition about 85-90%). This result may imply that there is only one calmodulin-binding site on the caldesmon. However, we cannot rule out the possibilities i) that a second calmodulin-binding domain is inaccessible when calmodulin is coated on microtiter plates, or ii) that thts second domain binds caldesmon too weakly to be detected under our assay conditions. There has been a great deal of speculation that calmodulin-caldesmon-tropomyosin interaction may play a role in the regulation of smooth muscle contraction or nonmuscle cell motility. Recently, we have demonstrated that microinjection of the monoclonal antitropomyosin antibody CG1, which recognizes a functional domain on nonmuscle tropomyosin, into chicken embryo fibroblasts causes a specific inhibition of intracellular granule movement [Hegmann et al., 19891. This result provides the first in vivo evidence that nonmuscle tropomyosin plays a role in granule motility. In the accompanying paper [Hegmann et al., 19911, microinjection of anti-caldesmon antibodies into CEF cells was carried out. As expected, the C21 antibody, whose epitope is close to both the F-actin-binding and Caf + / calmodulin-binding domains of the protein, is a very

**

R,,,LIAEKEREMAEM,,,

CaD

9/16 (56.2%)

hL4

-6.2K

Fig. 10. Western blot analysis of monoclonal antibodies C9, C18, and C23 binding to caldesmon and to CNBr fragments of the 28K or 80K NTCB fragments. The gel-purified 28K and 80K NTCB fragments were further cleaved at met residues by CNBr. The resulting fragments (lanes I, 2) and intact caldesmon (lane 3) were separated on TricineSDS PAGE. After electrophoresis, proteins were transferred to nitrocellulose papers and either stained with amido black (the right-most blot) or reacted with C9, CIS, or C23 antibody, followed by '251labeled goat anti-mouse IgG. Bound antibody was detected by autoradiography. CaD, caldesmon; Std, molecular weight standard polypeptides.

IDENTITY

R,,,LKAEEEKKAAEE,,,

(IV)

(V)

CaD

A,,,FGRSNLKGAANAE,,,

hL4

AZ7,ERNSNLVGAAHEEZp1

*** *** *

*

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N,,,LKGAANAEAGSEK,,,

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*

* **

* ***

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*

** * ***

8/14 (57.1%)

8/14 (57.1%)

8/15 (53.3%)

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Fig. 11. Amino acid sequence homology between gizzard caldesmon and human nuclear lamin A. Amino acid sequences shown here are derived from the cDNA sequence of gizzard caldesmon [CaD, Bryan et al., 1989al and human lamin A [hLA, Fisher et al., 1986; McKeon et al., 1986). * indicates identical residues between these two proteins. There are five different regions of homology between caldesmon and nuclear lamin A, spanning 13 to 16 amino acid residues. These regions share between 38 to 61% homology. Region I in caldesmon correlates with an internal repeated sequence identified by Bryan et a]. [1989a].

potent inhibitor of intracellular granular movement. Although the C2 antibody recognizes the same fragment as C21 antibody does, there is no effect on granule movement in C2 injected cells. Therefore, the in vivo inhibition of granule motility by C21 antibody is very specific. These results further support the belief that a calmodulincaldesmon-tropomyosin system participates in the in vivo regulation of nonmuscle cell motility.

ACKNOWLEDGMENTS

This work was supported in part by grants HD18577, GM40580 from the National Institutes of Health, and by grants from the Muscular Dystrophy Association and the Pew Memorial Trust. Dr. J.J.-C. Lin is a recipient of a Pew Scholarship in Biomedical Sciences from the Pew Memorial Trust.

107

Caldesmon Domains Defined by Monoclonal Antibodies

of monoclonal antibodies against caldesmon. Cell Motil. Cytoskeleton 20: 109-120. Adelstein, R.S., and Eisenberg, E. (1980): Regulation and kinetics of Honer, B., Citi, S . , Kendrick-Jones, J., and Jockusch, B.M. (1988): the actin-myosin ATP interaction. Annu. Rev. Biochem. 49: Modulation of cellular morphology and locomotory activity by 921-956. antibodies against myosin. J. Cell Biol. 107:2181-2189. Bartegi, A,, Fattoum, A,, Derancourt, J., and Kassab, R. (1990): Horiuchi, K.Y., and Chacko, S . (1988): Interaction between caldesCharacterization of the carobxyl-terminal lOKDa cyanogen mon and tropomyosin in the presence and absence of smooth bromide fragment of caldesmon as an actin-calmodulin-binding muscle actin. Biochemistry 27:8388-8393. region. J. Biol. Chem. 265:15231-15238. Jacobsen, G.R., Schaffer, M.H., Stark, G.R. and Vanaman, T.C. Blattler, D.P., Garner, F., van Slyke, K., and Bradley, A. (1972): (1973): Specific chemical cleavage in high yield at the amino Quantitative electrophoresis in polyacrylamide gels of 2-40%. peptide bonds of cysteine and cystine residues. J. Biol. Chem. J. Chromatogr. 64:147-155. 24816583-6591, Blose, S.H., Meltzer, D.I., and Feramisco, J.R. (1984): 10-nm fila- James, P., Maeda, M., Fischer, R., Verma, A.K., Krebs, J., Pennisments are induced to collapse in living cells microinjected with ton, J.T., and Carafoli, E. (1988): Identification and primary monoclonal and polyclonal antibodies against tubulin. J. Cell structure of a calmodulin binding domain of the Ca+ pump of Biol. 98:847-858. human erythrocytes. J. Biol. Chem. 263:2905-2910. Bretscher, A. (1984): Smooth muscle caldesmon: rapid purification Kendrick-Jones, J., and Scholey, J.M. (1981): Myosin-linked reguand F-actin crosslinking properties. J. Biol. Chem. 259: latory systems. J . Muscl. Res. Cell Motil. 2:347-372. 12873-12880. Klymkowsky, M.W. (1981): Intermediate filaments in 3T3 cells colBretscher, A. (1986): Thin filament regulatory proteins of smootblapse after intracellular injection of a monoclonal anti-intermeand nonmuscle cells. Nature 321:726-727. diate filament antibody. Nature 291:249-251. Bretscher, A,, and Lynch, W. (1985): Identification and localization of immunoreactive forms of caldesmon in smooth and nonmus- Klymkowsky, M.W., Miller, R.H., and Lane, E.B. (1983): Morphology, behavior and interaction of culture epithelial cells after the cle cells: A comparison with the distributions of tropomyosin antibody-induced disruption of keratin filament organization. J. and a-actinin. J. Cell Biol. 100:1656-1663. Cell Biol. 96:494-509. Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R.G., and Lin, W.-G. (1989a): Cloning and expression of a smooth muscle caldes- Laemmli, U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680mon. J. Biol. Chem. 264:13873-13879. 685. Bryan, J., Lee, R., Chang, S.J., and Lin, W.-G. (1989b): Cloning and expression of a low molecular weight caldesmon. J. Cell Biol. Leszyk, J., Mornet, D., Audemard, E., and Collins, J.H. (1989a): Amino acid sequence of a 15 kilodalton actin-binding fragment 109:189a. of turkey gizzard caldesmon: Similarity with dystrophin, tropoChalovich, J.M. (1988): Caldesmon and thin-filament regulation of myosin and the tropomyosin-binding region of troponin T. Biomuscle contraction. Cell Biophys. 12:73-85. chem. Biophys. Res. Comm. 160:210-216. Cote, G.P. (1983): Structural and functional properties of the nonmusLeszyk, J., Mornet, D., Audemard, E., and Collins, J.H. (1989b): cle tropomyosins. Mol. Cell. Biochem. 57:127-146. Caldesmon structure and function: Sequence analysis of a 35 Dingus, J., Hwo, S . , and Bryan, J. (1986): Identification by monokilodalton actin- and calmodulin-binding fragment from the clonal antibodies and characterization of human platelet caldesC-terminus of the turkey gizzard protein. Biochem. Biophys. mon. J. Cell Biol. 102:1748-1757. Res. Commun. 160:1371-1378. Dixon, M. (1953): The determination of enzyme inhibitor constants. Lin, 3.J.X. and Feramisco, J.R. (1981): Disruption of the in vivo Biochem. J. 55:170-171. distribution of the intermediate filaments in fibroblasts through Ebashi, S . , Endo, M., and Ohtsuki, I. (1969): Control of muscle the microinjection of a specific monoclonal antibody. Cell 24: contraction. Quart. Rev. Biophys. 2:351-384. 185-193. Feramisco, J.R., Clark, R., Wong, G., Arnheim, N., Milley, R., and McCormick, F. (1985): Transient reversion of ras oncogene- Lin, J.J.-C., Chou, C.-S., and Lin, J.L.-C. (1985a): Monoclonal antibodies against chicken tropomyosin isoforms: Production, induced cell transformation by antibodies specific for amino characterization, and application. Hybridoma 4:223-242. acid 12 of ras protein. Nature 314:639-642. Fisher, D.Z., Chaudhary, N., and Blobel, G. (1986): cDNA sequenc- Lin, J.J.-C., Helfman, D.M., Hughes, S.H., and Chou, C.-S. (1985b): Tropomyosin isoforms in chicken embryo fibroblasts: ing of nuclear lamins A and C reveals primary and secondary Purification, characterization, and changes in Rous sarcoma structural homology to intermediate filament proteins. Proc. virus-transformed cells. J. Cell Biol. 100:692-703. Natl. Acad. Sci. U.S.A. 835450-6454. Fujii, T., Imai, M., Rosenfeld, G.C., and Bryan, J. (1987): Domain Lin, J.J.X., Lin, J L - C . , Davis-Nanthakumar, E.J., and Lourim, D. (1988): Monoclonal antibodies against caldesmon, a Ca+ + / mapping of chicken gizzard caldesmon. J. Biol. Chem. 262: calmodulin- and actin-binding protein of smooth muscle and 2757-2763. nonmuscle cells. Hybridoma 7:273-288. Fujii, T., Ozawa, J., Ogoma, Y., and Kondo, Y. (1988): Interaction between chicken gizzard caldesmon and tropomyosin. J. Bio- Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951): Protein measurements with Folin phenol reagent. J. chem. 104:734-737. Biol. Chem. 193:265-275. Graceffa, P. (1987): Evidence for interaction between smooth muscle Makuch, R., Walsh, M.P., and Dabrowska, R. (1989): Location of tropomyosin and caldesmon. FEBS Lett. 218: 139-142. the calmodulin- and actin-binding domains at the C-terminus of Hegmann, T.E., Lin, J.L.-C., and Lin, J.J.-C. (1989): Probing the caldesmon. FEBS Lett. 247:411-414. role of nonmuscle tropomyosin isoforms in intracellular granule movement by microinjection of monoclonal antibodies. J. Marston, S.B., and Smith, C.W.J. (1985): The thin filaments of Cell Biol. 109:1141-1 152. smooth muscles. J. Muscl. Res. Cell Motil. 6:669-708. Hegmann, T.E., Schulte, D.L., Lin, J.L.-C., and Lin, J.J.-C. (1991): Matsumura, F., Yamashiro-Matsumura, S , . and Lin, J.J.-C. (1983): Inhibition of intracellular granule movement by microinjection Isolation and characterization of tropomyosin-containing mi-

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