INTRODUCTION

Myosin light chain kinase (MLCK)¹ has an important regulatory role in smooth muscle contraction (see Ref. 1 for a review). MLCK phosphorylates the 20-kDa light chain of smooth muscle myosin together with calmodulin in the presence of Ca²⁺ ions (Ca²⁺/CaM), thereby activating the myosin, which can then interact with actin filaments to induce contraction.

In addition to this kinase activity, MLCK can act as an actin-binding protein; MLCK is present in association with the sarcomeric I-band (2), and it binds to actin filaments with a high affinity (3-5). We have shown that MLCK can inhibit the ATP-dependent interaction between actin and myosin by binding to actin filaments. This inhibition can be relieved by Ca²⁺/CaM, as was demonstrated by avoiding the complication derived from its kinase activity (6-8), although the demonstration was limited to in vitro only.

Such an inhibition, however, is not peculiar to MLCK. Caldesmon (see Ref. 9 for a review) and calponin (10) are known to inhibit the actin-myosin interaction by binding to actin, an inhibition that is relieved by Ca²⁺/CaM. Twenty years ago, Hartshorne et al. (11) obtained an inhibitory fraction from chicken gizzard in a preliminary form. The activity of the 130-kDa component of their fraction relates to the actin-binding property of MLCK. By measuring the content of MLCK, caldesmon, and calponin in the myofibril of smooth muscle (12), we have shown previously how the actin-binding property of MLCK participates in regulating smooth muscle contraction.

The relationship between the structure of MLCK and the function of the kinase activity in phosphorylating the myosin light chain and how Ca²⁺/CaM modulates the activity has been well established (see Ref. 13 for a review). However, there has been little study of the structure-function relationship of the actin binding activity except for the purification of an actin-binding fragment from MLCK (14).

In this study, we have investigated which sequence of MLCK is responsible for the actin binding activity, which sequence inhibits the actin-myosin interaction, and which sequence binds CaM to relieve the inhibition. Our approach has been to cleave MLCK to prepare native fragments containing the actin binding activity (14), to design MLCK cDNA to express the recombinant fragments of actin binding activity in Escherichia coli, and then to analyze them biochemically. A few peptides have been synthesized to confirm these analyses. We have shown that: (i) MLCK has two actin-binding sites on the N-terminal side away from the central kinase domain, (ii) the binding site responsible for the inhibitory effect is at the Met¹-Pro⁴¹ sequence, and (iii) CaM interacts with MLCK at the Pro²⁶-Pro⁴¹ sequence.

MATERIALS AND METHODS

All procedures were carried out at 0-4 °C. The purity of proteins was routinely monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see below) so that they were >95% pure. The purified proteins, except actin and myosin (see below), were divided into aliquots and stored at 80 °C until they were used. All columns (see below) were incorporated into a high performance liquid chromatography system (model L-6200; Hitachi, Tokyo, Japan). MLCK was prepared by the method of Adelstein and Klee (15) with slight modification (16). In short, MLCK was extracted from the smooth muscle of chicken gizzard and was subjected to ammonium sulfate fractionation. After fractionation, MLCK was purified using column chromatography in DEAE-Toyopearl 650M columns and subsequently in SP-Toyopearl 650M (Tosoh, Tokyo, Japan) columns; this MLCK was used in all experiments unless otherwise specified. We also purified MLCK from bovine stomach smooth muscle by a modification (6) of the method of Kuwayama et al. (17) and used this for a few experiments, in which it is specified as bovine stomach MLCK.

MLCK was cleaved by 2-nitro-5-thiocyanatobenzoic acid (NTCB) complex at a Cys site, and an actin-binding fragment was purified as described by Kanoh et al. (14). The fragment consisted of the Met¹-Lys¹¹⁴ sequence (14, 18) and is referred to as the NTCB fragment.

Skeletal muscle actin was purified from an acetone powder of chicken breast muscle (19) and used as actin filaments after polymerization. The concentrations of actin filaments were expressed in terms of monomericactin. Myosin was purified from chicken gizzard as described by Nakamura and Nonomura (20), who modified the method of Ebashi (21) to remove phosphatase activities. The myosin was phosphorylated by incubation with MLCK in the presence of Ca²⁺/CaM for 10 min at 25 °C as described by Okagaki et al. (22) and used as myosin in our experiments. CaM from bovine brain, glucose oxidase, and catalase were purchased from Sigma.

For the expression of recombinant MLCK fragments, the pET system (Novagen, Madison, WI) was used according to the manufacturer's instructions. Series of cDNA fragments (see Fig. 6 for their topology) encoding various domains of bovine stomach MLCK (23) were amplified by the polymerase chain reaction. The amplified fragments were subcloned into a pET21 vector and verified by DNA sequencing. The recombinant MLCK fragment was overproduced in E. coli BL21(DE3). The cells were grown to an absorbance at 600 nm of 0.5-0.6 in 2 liters of Luria broth medium containing ampicillin (50 µg ml¹) and then were induced with 1 mM isopropyl-1-thio--D-galactopyranoside. All of the fragments were soluble in low salt. Therefore, cells expressing the fragments were collected by centrifugation, and then were suspended in 40 ml of a solution of 14 mM 2-mercaptoethanol and 20 mM Tris-HCl (pH 7.5). The resulting supernatant was fractionated with ammonium sulfate. The fragments were purified from the fraction by column chromatography using a combination of DEAE-Toyopearl 650M and CM-Toyopearl 650M columns (Tosoh, Tokyo, Japan). When any minor contaminants were found in SDS-PAGE, they were removed by gel filtration with Superose 12HR (Pharmacia Biotech Inc.). The MLCK fragments were: NN-, NC-NN/25, and NN/41 fragments, which were composed of Met¹-Lys³³⁷, Pro³¹⁹-Val⁷²¹, Pro²⁶-Lys³³⁷, and Lys⁴²-Lys³³⁷ of bovine stomach MLCK (23), respectively. The amino acid sequences of these fragments were confirmed by sequencing about 7 residues from their N termini using an Applied Biosystems model 477A analyzer.

Peptides of the MLCK sequences (18) of Met¹-Pro⁴¹, Lys⁴²-Ala⁸⁰, Met¹-Gly²⁵, Ser⁷⁸⁷-Ser⁸¹⁵, Ala⁷⁹⁶-Ser⁸³⁴, and Pro²⁶-Pro⁴¹ were synthesized using a MilliGen 9050 peptide synthesizer and then purified according to the manufacturer's instructions for Fmoc (N-(9-fluorenyl)methyloxycarbonyl) chemistry. The sequences of the peptides were routine ly confirmed by amino acid sequencing using an Applied Biosystems model 477A analyzer.

Actin filaments at a concentration of 12 µM were mixed with specified amounts of MLCK, its fragments, and/or CaM in 50 mM KCl, 20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, and 0.1 mM EGTA or 0.1 mM Ca²⁺ unless otherwise specified in the figure legends. The mixture was incubated for 0.5 h at 25 °C. The mixture after incubation was centrifuged at 100,000 × g for 45 min in a Beckman Airfuge at 25 °C. The amount of MLCK both in the supernatants and the pellets was determined by SDS-PAGE followed by densitometry as described above, and then the amount of MLCK bound to actin filaments was calculated (24). The assay for the binding activity of the fragments of MLCK was done in the same way. The results were analyzed on Scatchard plots.

The binding interaction between CaM and MLCK or its fragments was also measured by surface plasmon resonance with the IAsys Cubette System (Fissons, Cambridge, United Kingdom). CaM was immobilized to the cubette of carboxymethylated dextran matrix via N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide according to the manufacturer's protocol. MLCK and its recombinant fragments, after dissolving in 50 mM KCl, 5 mM MgCl₂, 20 mM Tris-HCl (pH 7.5), and 0.1 mM Ca²⁺ or 0.1 mM EGTA, were added to the cubette. Then the resultant resonance at 25 °C was recorded. In some experiments, the binding of MLCK or its fragments was monitored in the presence of various concentrations of synthetic peptides.

The effect of MLCK and its fragments on the ATP-dependent interaction between actin and myosin was analyzed by means of an in vitro motility assay on a myosin-coated surface (see Ref. 25 for the method), as modified for smooth muscle myosin by Okagaki et al. (22). In short, 3 nM actin filaments labeled with rhodamine-phalloidin (Molecular Probes, Eugene, OR) were mounted on a coverslip coated with myosin together with various concentrations of MLCK in 50 mM KCl, 30 mM Tris-HCl (pH 7.5), 4 mM MgCl₂, 1 mM ATP, 25 mM dithiothreitol, and 0.1 mM EGTA. To see the effect of Ca²⁺/CaM, EGTA was replaced by 0.1 mM Ca²⁺ and CaM at the same molar concentration as MLCK. Glucose oxidase (0.2 mg ml¹, Sigma), catalase (0.04 mg ml¹, Sigma), and glucose (4.5 mg ml¹) were added to the above solution to prevent photobleaching of rhodamine. The movement of actin filaments was recorded under a fluorescence microscope equipped with a videocamera and a recorder. The average velocity was measured using an image analyzer (Image Sigma III, Nippon Avionics, Tokyo, Japan).

The effect of recombinant MLCK fragments on the actin-myosin interaction was also examined by measuring ATP hydrolyzed at 25 °C for 10 min with 0.05 µM phosphorylated myosin in the presence of various concentrations of MLCK and its fragments under the conditions of 2 µM actin filaments, 0.5 mM ATP, 60 mM KCl, 5 mM MgCl₂, 0.1 mM EGTA, and 20 mM Tris-HCl (pH 7.5) unless otherwise specified. The liberated phosphate was quantified in duplicate or triplicate by the malachite green method of Kodama et al. (26). We ensured that the rate of ATP hydrolysis was constant and that the specific activity of the ATPase did not differ from our previously reported values. Therefore, we expressed the activity as a normalized ATPase activity (27).

SDS-PAGE was carried out using the method of Laemmli (28) with a slight modification (24) so that we could check the purity (>95%) of MLCK and its fragments. Protein concentrations were determined using the methods of Bradford (29) and Lowry et al. (30) with bovine serum albumin as the standard.

The molecular masses used for calculating concentrations (M) of proteins and peptides were obtained from their amino acid sequences as follows: myosin, 440 kDa; actin, 41.8 kDa; CaM, 16.7 kDa; MLCK, 107.5 kDa; bovine stomach MLCK, 128.8 kDa; NTCB fragment, 12.4 kDa; NN fragment, 35.3 kDa; NC fragment, 44.4 kDa; NN/41 fragment, 30.7 kDa; NN/25 fragment, 33.3 kDa; 787-815 peptide, 3.4 kDa; 796-834 peptide, 4.1 kDa; 1-25 peptide, 3.0 kDa; 26-41 peptide, 1.7 kDa; 1-41 peptide, 4.8 kDa; 42-80 peptide, 4.0 kDa.

Homology of the specified amino acid sequences of MLCK with the sequences of other proteins was examined using MacDNASIS Pro 020-00 (Hitachi Software Engineering, San Bruno, CA) to search the GenBank/NBRF-PIR/SWISS-PROT data bases. Isoelectric points of the synthetic peptides were calculated according to Skoog and Wichman (31).

RESULTS

We designed a fragment so that it consisted of Met¹-Lys³³⁷ of bovine stomach MLCK (see Fig. 6 for its topology in MLCK), and subjected this NN fragment to an actin-binding assay. As shown in Fig. 1a by open circles, the amount of the NN fragment bound to actin filaments increased with increasing concentration of the NN fragment. The K_a of the binding of the NN fragment to actin filaments was 3.72 ± 0.20 × 10⁵ M¹ (mean ± S.E., n = 3). The binding was abolished totally by Ca²⁺/CaM (Fig. 1b, open circles). Thus, we deduce that the 1-337 sequence contains a site for Ca²⁺/CaM-sensitive actin binding.

[View Larger Version of this Image (18K GIF file)]

Previously, we investigated the importance of the sequence between Lys⁴² and Ala⁸⁰ to the inhibitory effect of MLCK by searching for a part of MLCK that is homologous to the actin-binding domain of caldesmon (6). We synthesized a peptide consisting of Lys⁴²-Ala⁸⁰ of MLCK and used it to compete against the actin binding activity of the NN fragment. However, as shown in Fig. 1c by the open circles, the binding was not at all affected.

We obtained another peptide, this time of the Met¹-Pro⁴¹ sequence, and found its inhibitory effect on the actin binding of the NN fragment as shown by the filled circles in Fig. 1c. Thus, the sequence responsible for the binding of the NN fragment to actin filaments, i.e. the Ca²⁺/CaM-sensitive site of MLCK, must be located at the N terminus of the MLCK molecule. We divided peptide Met¹-Pro⁴¹ into peptide Met¹-Gly²⁵ and peptide Phe²⁶-Pro⁴¹. Both of them failed to inhibit the actin binding (open and filled triangles in Fig. 1c), suggesting that there is a critical region for actin binding within the 1-41 residues.

To confirm this idea by another method, we produced the NN/41 fragment, which is devoid of the Met¹-Pro⁴¹ sequence of MLCK, and the NN/25 fragment which is devoid of the Met¹-Gly²⁵ sequence of MLCK (see Fig. 6 for their topology in MLCK), and then assayed for their actin binding activity. As shown by filled triangles in Fig. 1a, the binding of the NN/41 fragment to actin filaments remained at a basal level. Although the NN/25 fragment definitely bound to actin filament with K_a = 1.01 ± 0.53 × 10⁵ M¹ (mean ± S.E.,n = 3), the affinity was much lower than that of the NN fragment. The length of the Pro²⁶-Pro⁴¹ sequence when added to the NN/41 fragment (i.e. the NN/25 fragment) is probably too short to endow it with an actin-binding ability as high as that of the NN fragment, confirming the importance of the 1-41 sequence as an actin-binding site.

We also produced the NC fragment, which comprises the Pro³¹⁹-Val⁷²¹ sequence of bovine stomach MLCK (see Fig. 6 for its topology in MLCK). We assayed the NC fragment for its actin-binding ability. As shown in Fig. 1a by filled circles, it shows a definite actin binding activity with a K_a = 1.83 ± 0.47 × 10⁵ M¹ (mean ± S.E., n = 3). We used CaM in the presence of Ca²⁺ to compete against the actin binding of the NC fragment, but failed to observe any effect of Ca²⁺/CaM (see Fig. 1b, filled circles). Considering the Ca²⁺/CaM-sensitive, actin binding activity of the NN fragment, parent MLCK would be expected to bind to actin filaments in both Ca²⁺/CaM-sensitive and insensitive ways, which will be shown later and discussed (see Fig. 8a).

We immobilized CaM on an IAsys cubette surface, and allowed MLCK to interact with the surface. In the presence of Ca²⁺, the surface plasmon resonance of IAsys increased with the increase in the concentration of MLCK. However, in the presence of EGTA, the increase is only slight (data not shown). Such a difference indicates that the IAsys system could be reliably used to detect binding activity of MLCK and its fragments to CaM.

As shown in Fig. 2a by open (MLCK) and filled (bovine stomach MLCK) circles, the difference in the resonance between Ca²⁺ and EGTA at the specified concentrations was readily increased at low concentrations of both MLCKs and approached saturation at their higher concentrations. This Ca²⁺-dependent change in the resonance of the NN fragment shown in Fig. 2a by filled squares is quite different from that of parent MLCK; it increased gradually. However, it was never saturated, indicating the low affinity of the fragments for CaM.

[View Larger Version of this Image (18K GIF file)]

We assayed the NN/41 fragment with IAsys using the same surface of the cubette. As shown in Fig. 2a, the Ca²⁺-dependent changes in the resonance were not detected with this fragment (filled triangles), indicating that the CaM-binding site is not located in the C-terminal portion away from Lys⁴². The failure to detect these changes suggests that the site is probably within the sequence of Met¹-Pro⁴¹, and so we allowed the Met¹-Pro⁴¹ peptide to compete against the CaM binding activity of the NN fragment. As shown by the triangles in Fig. 2b, the Ca²⁺-dependent changes in the resonance of the NN fragment reduced with the increase in the concentration of the peptide. The molecular mass of the peptide itself was too low to produce a resonance (data not shown), so the reduction in the resonance is an indication of the binding of the 1-41 sequence of MLCK to CaM.

The NC fragment was also constructed to include a Ca²⁺/CaM-insensitive, actin-binding site, which was confirmed by the actin-binding assay (see Fig. 1). Accordingly, the NC fragment did not cause any Ca²⁺-dependent change in the resonance (Fig. 2a, open triangles).

To identify the position of the CaM-binding site in the 1-41 sequence, we synthesized peptides of Met¹-Gly²⁵ and Pro²⁶-Pro⁴¹. Because of their low molecular mass, their interaction with CaM could not be detected directly by resonance (data not shown). Therefore, their interaction with CaM was detected by observing the interaction of the NN fragment with the CaM-coated surface of an IAsys cubette in the presence of various concentrations of the peptides (Fig. 2b). The peptide that competed with the NN fragment was Pro²⁶-Pro⁴¹.

We also tested the NN/25 fragment for CaM-binding ability. As shown by asterisks in Fig. 2a, its binding ability is comparable to that of the NN fragment. Taken together with the competing abilities of the peptides, we conclude that the boundary of the CaM-binding sequence of MLCK is at Pro²⁶.

As shown in Fig. 3a by filled circles, the motility of actin filaments on a myosin-coated glass surface was inhibited with the increase in the concentration of the NN fragment. Half-maximal inhibition was observed in the presence of 41.1 ± 5.5 nM (mean ± S.E., n = 3) of the NN fragment. The inhibition was relieved by Ca²⁺/CaM (Fig. 3a, open circles). Actin-activated ATPase activities of myosin (Fig. 3b, filled circles) were reduced with the increase in the concentration of the NN fragment. Half-maximal inhibition was observed in the presence of 0.276 ± 0.028 µM (mean ± S.E., n = 3). Ca²⁺/CaM effectively relieved the inhibition; the inhibition caused by 1 µM NN fragment was abolished by an 8-10-fold molar excess of CaM over the NN fragment (Fig. 3c, filled circles).

[View Larger Version of this Image (20K GIF file)]

It must be noted that the concentration required for inhibiting the activity half-maximally was about 6.7-fold higher than the concentration required for inhibiting the motility. Such a discrepancy has been noted by Sato et al. (8) by using aortic MLCK. The difference in the concentrations of actin filaments used for the motility and ATPase assays may be one possible explanation.

The regulatory activity of the NN/41 fragment was also tested both with motility and ATPase assays. As expected from the absence of actin binding activity (Fig. 1a), we failed to find any effect, irrespective of the presence or absence of Ca²⁺/CaM (Fig. 3, a-c, squares). Taking the absence of the actin binding activity of the NN/41 fragment (Fig. 1a) into consideration, the absence of any effect indicates that Met¹-Pro⁴¹ is the sequence of MLCK that is responsible for the inhibition of myosin through actin binding activity. The NN/25 fragment, which showed a weak actin binding activity (Fig. 1a), failed to inhibit the effect in the motility and ATPase assays (Fig. 3, a and b, asterisks), suggesting that the residues on both N- and C-terminal sides of Gly²⁵ are required for inhibiting the actin-myosin interaction.

The NC fragment, on the other hand, bound to actin filaments in a Ca²⁺/CaM-insensitive manner (Fig. 1b). As shown in Fig. 3 (a and b) by triangles, the NC fragment did not exert any regulatory activity as monitored both by the motility and ATPase assays. The absence of activity was confirmed in the presence of various concentrations of Ca²⁺/CaM (Fig. 3c, triangles). Thus, we conclude that the Ca²⁺/CaM-insensitive binding site of MLCK has no regulatory activity.

We allowed parent MLCK to bind to actin filaments in a similar way to its recombinant fragments. In the presence of 1 mM EGTA, MLCK bound to actin filaments at its high affinity site at 0.15 ± 0.02 mol/mol actin (mean ± S.E., n = 3) with an affinity constant K_a = 2.16 ± 0.65 × 10⁶ M¹ (mean ± S.E., n = 3). At its low affinity site, MLCK bound to actin filaments with a K_a = 4.67 ± 0.17 × 10⁵ M¹ (mean ± S.E., n = 3). In the presence of 1 mM Ca²⁺ and CaM at an 8-fold molar excess over MLCK, binding of MLCK to the actin filaments had only a single K_a of 3.1 × 10⁵ M¹. Because this value is closer to the K_a of the low affinity site, we conclude that the actin binding at the high affinity site is Ca²⁺/CaM-sensitive.

As we describe below (see Fig. 8a), the Ca²⁺/CaM-insensitive binding is confirmed by the persistence of actin binding activity in MLCK when the concentration of CaM was increased. Sellers and Pato (4) showed Ca²⁺/CaM-sensitive binding of MLCK to actin filaments. However, we conclude that parent MLCK binds to actin filaments in both Ca²⁺/CaM-sensitive and -insensitive ways, confirming the Ca²⁺/CaM-sensitive and -insensitive binding of the NN- and NC fragments, respectively.

We produced the NTCB fragment from MLCK by chemical cleavage. In the presence of 1 mM EGTA, the NTCB fragment showed actin binding activity, confirming the results of Kanoh et al. (14). Scatchard plots of the data showed that the NTCB fragment bound to actin filaments maximally at 0.2 mol/mol actin with a single K_a of 4.8 × 10⁵ M¹. In the presence of 1 mM Ca²⁺ and CaM at a 9-fold molar excess over MLCK, we detected no binding activity of the NTCB fragment to actin filaments. We consider that the actin binding of the fragment is very similar to that of the NN fragment, i.e. Ca²⁺/CaM-sensitive, although we are aware that the K_a of the NTCB fragment is lower than that of the Ca²⁺/CaM-sensitive site of parent MLCK (K_a = 2.16 × 10⁶ M¹). The difference is discussed in more detail under "Discussion."

Fig. 4 shows the competition experiment for synthetic peptides against the actin binding of the NTCB fragment. Resembling the result of the NN fragment (Fig. 1c), neither peptide Met¹-Gly²⁵ nor peptide Pro²⁶-Pro⁴¹ was able to abolish the actin binding of the NTCB fragment. The abolition was brought about only by peptide Met¹-Pro⁴¹, confirming the idea that the boundary of the Ca²⁺/CaM-sensitive actin-binding site is probably around Pro⁴¹.

[View Larger Version of this Image (17K GIF file)]

The inhibitory effects of MLCK and the NTCB fragment on the actin-myosin interaction are shown in Fig. 5. MLCK effectively inhibited the motility of actin filaments in the presence of EGTA. However, there was no inhibition in the presence of Ca²⁺/CaM. Similar inhibition and its relief by Ca²⁺/CaM were observed for the NTCB fragment, confirming the results obtained with the NN fragment (Fig. 3). The higher concentration of the NTCB fragment compared with that of parent MLCK (Fig. 5, compare a and b) is attributable to their different affinities to actin filaments.

1

[View Larger Version of this Image (12K GIF file)]

Taken together with the CaM binding activity of the NTCB fragment (see Fig. 3, asterisks), the above data for the NTCB fragment confirm those for the NN fragment. We consider that the NN fragment is a recombinant form of the NTCB fragment and that it contains the whole sequence (Met¹-Lys¹¹⁴) of the NTCB fragment (see Fig. 6).

[View Larger Version of this Image (33K GIF file)]

DISCUSSION

This report analyzes two classes of actin-binding site of MLCK, i.e. Ca²⁺/CaM-sensitive and Ca²⁺/CaM-insensitive sites as shown in Table I. The former is involved in the inhibitory effect of MLCK on the actin-myosin interaction. The latter site has no regulatory activity.

Table I. Summary of binding to actin filaments of MLCK and its fragments and their inhibitory effects on the actin-myosin interaction MLCK and its fragments Actin binding Inhibitory effects Ca2+/CaM-sensitive Ca2+/CaM-insensitive MLCK (full-length, native) + + + NTCB fragment (native) +   + NN fragment (recombinant) +   + NC fragment (recombinant)   +   NN/41 fragment (recombinant)       NN/25 fragment (recombinant) ±

The inhibitory effect of MLCK on the actin-myosin interaction is brought about by its actin binding activity. This actin-linked nature was first demonstrated with a Nitella-based motility assay (see Ref. 25 for the method), where we allowed MLCK to bind to the actin cables that run on the inner surface of the plasma membrane of Nitella internodal cells (6). With the myosin-coated motility assay used in the present study, Sato et al. (8) suggested the actin-linked nature of the effect after observing that the inhibitory effect was obscured with the increase in the concentrations of actin filaments. The present study (Table I) demonstrates the actin-linked nature of the effect by using actin-binding fragments of MLCK which were produced (i) by purifying the NTCB fragment with conventional protein chemistry and (ii) by producing recombinant fragments with the pET expression system.

Fig. 6a depicts the domain structure of bovine stomach MLCK (23). The central kinase domain (see Ref. 1 for a review) and C-terminal telokin domain (32) are known to interact with myosin. Therefore, the present study analyzes the function of the remaining part of MLCK, i.e. the N-terminal portion of MLCK, with respect to its actin-linked regulatory role as follows. The Ca²⁺/CaM-sensitive, actin-binding site is localized at the extreme N terminus of MLCK, consisting of Met¹-Pro⁴¹ (Fig. 6, a and b). The localization was determined (i) by comparing actin binding activity of the recombinant NN fragment containing only the Ca²⁺/CaM-sensitive site with that of the NN/41 fragment, which is devoid of the 1-41 sequence of the NN fragment (Fig. 1a); and (ii) by using a synthetic peptide of Met¹-Pro⁴¹ to compete against the actin binding of the native NTCB fragment (Fig. 4) and its recombinant form, the NN fragment (Fig. 1c).

Calponin (10) and caldesmon (9) are actin-binding proteins in smooth muscle that inhibit the actin-myosin interaction in a similar way to MLCK. Recent biochemical studies have narrowed the length of amino acid sequence responsible for actin binding activity down to 37 amino acids for calponin (33), and 32 and 46 amino acids for caldesmon (34). In agreement with these values, a length of 41 amino acids was shown to be a Ca²⁺/CaM-sensitive, actin-binding site. This was demonstrated as follows. (i) When the peptide of Met¹-Pro⁴¹ was split into Met¹-Gly²⁵ and Pro²⁶-Pro⁴¹, both peptides lost the antagonism for the actin binding of the NTCB (Fig. 4) and NN fragments (Fig. 1c). (ii) The extension of fragment length from the NN/41 fragment to the NN/25 fragment failed to restore full actin binding activity (Fig. 1a). This weak actin-binding ability of the NN/25 fragment suggests the possibility that some residues within the 1-41 sequence may not be involved in the actin binding.

It must be noted that the calculated pI of the sequence of Met¹-Pro⁴¹ is 10.67. Because actin is a highly acidic protein, we wonder whether the alkaline property of the sequence could cause MLCK to bind to actin nonspecifically. As shown in Fig. 4, the peptide of the Lys⁴²-Ala⁸⁰ sequence did not affect the ability of the NTCB fragment to bind to actin filaments, although its pI was similarly alkaline (pI = 10.51). Therefore, the interaction between MLCK and actin filaments at the site of Met¹-Pro⁴¹ is not attributable to the nonspecific binding between the acidic and alkaline sequences. We searched for homology of Met¹-Pro⁴¹ with other actin-binding proteins and found sequences that show a 35% identity within the -actinin sequence, a 32.3% identity within the dystrophin sequence, a 30.0% identity within the villin sequence, and a 28.1% identity within the coronin sequence.

Unlike the Ca²⁺/CaM-sensitive, actin-binding site, the position of the Ca²⁺/CaM-insensitive, actin-binding site was not determined precisely (asterisk in Fig. 6a). What we can conclude is that the Ca²⁺/CaM-insensitive site is included in the NC fragment as shown in Fig. 6. The sequence of Pro³¹⁹-Lys³³⁷ in the NC fragment overlaps with the NN fragment and is obviously devoid of the Ca²⁺/CaM-insensitive site (compare the NC fragment with the NN fragment in Fig. 6b). Therefore, the Ca²⁺/CaM-insensitive site must be present somewhere in the Gly³³⁸-Val⁷²¹ sequence of bovine stomach MLCK.

Is there any other sequence in MLCK that binds to actin filaments in a Ca²⁺/CaM-sensitive manner? The NN/41 and NC fragments cover the N-terminal portion of MLCK except for the 1-41 sequence. Neither the NN/41 fragment nor the NC fragment showed Ca²⁺/CaM-sensitive binding to actin filaments (Table I, Fig. 1). We obtained the central kinase domain by proteolyzing MLCK,² and the C-terminal myosin-binding domain by purifying telokin (32). Because these domains failed to show actin binding activity,² the 1-41 sequence is the sole sequence responsible for the Ca²⁺/CaM-sensitive binding of MLCK that allows it to exert its regulatory role in the actin-myosin interaction.

Although parent MLCK shares the 1-41 sequence with the native NTCB fragment and the recombinant NN fragment, there is a difference in K_a values. When the Ca²⁺/CaM-dependent site was separated from parent MLCK (K_a = 2.16 × 10⁶ M¹) as the NTCB fragment, its K_a was reduced to 4.8 × 10⁵ M¹. The K_a of the NN fragment, a recombinant form of the NTCB fragment, is also low, i.e. 3.72 × 10⁵ M¹. Such a reduction in the affinity upon cleavage from a parent molecule has also been noticed for the actin binding activity of the -actinin family (35). On the other hand, affinity to actin filaments of the Ca²⁺/CaM-insensitive site is affected only slightly upon its separation from the parent molecule. The NC fragment, which carries only this site, binds to actin filaments with a K_a = 1.83 × 10⁵ M¹ (Fig. 1a), an affinity that is comparable with the K_avalues of the Ca²⁺/CaM-insensitive sites of MLCK (K_a = 4.67 × 10⁵ M¹ as measured in the absence of the Ca²⁺/CaM, and K_a = 3.1 × 10⁵ M¹ as measured in the presence of Ca²⁺/CaM).

According to Dabrowska et al. (3), the concentrations of MLCK and actin filaments in chicken gizzard smooth muscle cells are estimated to be 3 µM and 830 µM, respectively, and MLCK in the cells is adequately absorbed by the actin filaments. Our estimation of affinity for actin of the Ca²⁺/CaM-sensitive site of MLCK explains their data, indicating that 0.36% of actin filaments in the cells are in the MLCK-bound form. The concentration of MLCK that gives half-maximal inhibition of the motility of actin filaments is 2.1 nM (Fig. 5a). Using a 3 nM concentration of actin filaments in the motility assay, we calculate that 0.42% of actin filaments would be in the MLCK-bound form. A comparable figure has also been calculated from the concentration of MLCK that gives half-maximal inhibition of the actin-activated ATPase (see Fig. 2 in Ref. 6). To establish the physiological role of the actin binding activity of MLCK (36), it remains to be demonstrated whether or not such a small percentage of actin filaments in smooth muscle cells could participate in regulating their contraction. Alternatively, the in vivo binding of MLCK to actin filaments may serve not to regulate the actin-myosin interaction per se but simply to localize the kinase activity to the vicinity of its substrate.

Actin binding of calponin (10) and caldesmon (9) is regulated by Ca²⁺/CaM in a similar way to MLCK (see Fig. 1). The sites that bind CaM in calponin (10) and caldesmon (9) are located close to the sites for their actin binding. Therefore, the CaM-binding site that regulates the actin binding of MLCK would be expected to be close to the actin-binding site. Accordingly, we demonstrated that the CaM-binding site is included in the actin-binding site, i.e. Met¹-Pro⁴¹ sequence, by showing (i) that the NN/41 fragment that is devoid of the Met¹-Pro⁴¹ sequence fails to bind CaM (Fig. 2a) and (ii) that the Met¹-Pro⁴¹ peptide competes against the NN fragment for CaM binding (Fig. 2b). Further, when we divided the 1-41 peptide into two peptides of Met¹-Gly²⁵ and Pro²⁶-Pro⁴¹, the sequence that competes for CaM binding is the latter (Fig. 2b). The calculated pI values of the former (pI = 10.25) and the latter (pI = 10.82) are similar. Therefore, the failure of the former peptide to bind to CaM suggests that the interaction between CaM and the latter peptide was not caused by the nonspecific binding of an alkaline peptide to an acidic protein such as CaM. As shown in Fig. 7, the IQ motif, a consensus sequence in CaM-binding proteins (37), can be assigned to the latter peptide, although the matching was not complete.

[View Larger Version of this Image (25K GIF file)]

MLCK has another CaM-binding site for regulating its kinase activity; the site is the Ala⁷⁹⁶-Ser⁸¹⁵ sequence for chicken gizzard MLCK (18) and the Ala¹⁰⁰²-Ser¹⁰²¹ sequence for bovine stomach MLCK (23) (see Fig. 6a for their topology). Does peptide Pro²⁶-Pro⁴¹ interact with the 796-815/1002-1021 sequence? We confirmed that this sequence is a CaM-binding site as follows. Peptide Ser⁷⁸⁷-Ser⁸¹⁵ and peptide Ala⁷⁹⁶-Ser⁸³⁴, both of which contain the 796-815/1002-1021 sequence, effectively abolished phosphorylation of myosin by MLCK in the presence of Ca²⁺/CaM (Fig. 8b, open circles and triangles). However, peptide Pro²⁶-Pro⁴¹ did not affect this kinase activity of MLCK (Fig. 8b, filled circles), demonstrating that the amino acid sequence of MLCK that binds to CaM regulate its kinase activity is totally different from the one that regulates its actin binding activity.

[View Larger Version of this Image (15K GIF file)]

Alternatively, we allowed Ca²⁺/CaM to antagonize the actin binding of parent MLCK in the absence and presence of peptide Pro²⁶-Pro⁴¹ (Fig. 8a). In its absence, the actin binding of parent MLCK was antagonized by Ca²⁺/CaM as shown by filled circles in Fig. 8a. When the peptide was present, such an antagonism by Ca²⁺/CaM was much weakened (Fig. 8a, open circles), confirming that the 26-41 sequence works as a CaM-binding site for regulating actin binding activity of MLCK. On the other hand, peptide Ser⁷⁸⁷-Ser⁸¹⁵ (Fig. 8a, filled triangles) exerted no effect. Thus, we concluded that parent MLCK has at least two classes of CaM-binding sites, which rules out the possibility that the CaM binding activity of the 26-41 sequence was produced upon fragmentation of MLCK.