Role of non-kinase activity of myosin light-chain kinase in regulating smooth muscle contraction, a review dedicated to Dr. Setsuro Ebashi

Myosin light-chain kinase (MLCK) of smooth muscle consists of an actin-binding domain at the N-terminal, the catalytic domain in the central portion, and the myosin-binding domain at the C-terminal. The kinase activity is mediated by the catalytic domain that phosphorylates the myosin light-chain of 20 kDa (MLC20), activating smooth muscle myosin to interact with actin. Although the regulatory role of the kinase activity is well established, the role of non-kinase activity derived from actin-binding and myosin-binding domains remains unknown. This review is dedicated to Dr. Setsuro Ebashi, who devoted himself to elucidating the non-kinase activity of MLCK after establishing calcium regulation through troponin in skeletal and cardiac muscles. He proposed that the actin-myosin interaction of smooth muscle could be activated by the non-kinase activity of MLCK, a mechanism that is quite independent of MLC20 phosphorylation. The authors will extend his proposal for the role of non-kinase activity. In this review, we express MLCK and its fragments as recombinant proteins to examine their effects on the actin-myosin interaction in vitro. We also down-regulate MLCK in the cultured smooth muscle cells, and propose that MLC20 phosphorylation is not obligatory for the smooth muscle to contract.     

平滑肌肌球蛋白轻链激酶及ATP结合位点突变体对ATP酶活性的调节作用

目的：探寻MLCK的非激酶活性区域对MLCK活性的影响,进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制。方法：利用编码MLCK全长的pColdI表达载体对其ATP结合位点进行定点突变,获得无激酶活性的MLCK突变体;应用Glycerol—PAGE鉴定肌球蛋白磷酸化水平;应用孔雀绿方法检测重组MLCK对肌球蛋白ATP酶活性的影响。结果：MLCK／△ATP（突变型）失去磷酸化肌球蛋白轻链的激酶活性;重组MLCK（野生型）和MLCK／AATP（突变型）均可以在非钙条件下激活非磷酸化肌球蛋白Mg2＋-ATP酶活性,抑制磷酸化肌球蛋白的Mg2＋．ATP酶活性,而且激活与抑制作用均随着MLCK浓度的增加而增大,但二者对肌球蛋白的ATP酶活性的作用没有显著差异（P〉0．05）。结论：平滑肌肌球蛋白轻链激酶及ATP结合位点突变体具有激活非磷酸化肌球蛋白ATP酶活性的作用。     

Inhibiting myosin light chain kinase induces apoptosis in vitro and in vivo

Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis.     

Diphosphorylation of platelet myosin by myosin light chain kinase.

Recently, one of the authors (K.I.) and other investigators reported that myosin light chain (MLC) of smooth muscle (gizzard, arterial and tracheal) was diphosphorylated by myosin light chain kinase (MLCK) and that diphosphorylated myosin showed a marked increase in the actin-activated myosin ATPase activity in vitro and ex vivo. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated diphosphorylation of MLC of platelet by MLCK in vitro. Our results are as follows. (1) Platelet MLC was diphosphorylated by a relatively high concentration (greater than 20 micrograms/ml) of MLCK in vitro. As a result of diphosphorylation, the actin-activated myosin ATPase activity was increased 3 to 4-fold as compared to the monophosphorylation. (2) Both di- and monophosphorylation reactions showed similar Ca2+, KCl, MgCl2-dependence. Maximal reaction was seen at [Ca2+] greater than 10(-6) M, 60 mM KCl and 2 mM MgCl2. This condition was physiological in activated platelets. (3) Di- and monophosphorylated myosin showed similar Ca2+, KCl-dependence of ATPase activity but distinct MgCl2-dependence. Diphosphorylated myosin showed maximal ATPase activity at 2 mM MgCl2 and monophosphorylated myosin showed a maximum at 10 mM MgCl2. (4) The addition of tropomyosin stimulated actin-activated ATPase activity in both di- and monophosphorylated myosin to the same degree. (5) ML-9, a relatively specific inhibitor of MLCK, inhibited the aggregation of human platelets induced by thrombin ex vivo in a dose-dependent manner. Moreover, this drug also partially inhibited both di- and monophosphorylation reactions and actin-activated ATPase activity. On the other hand, H-7, a synthetic inhibitor of protein kinase C, had little effect on the aggregation of human platelets induced by thrombin ex vivo. From these results, we conclude that diphosphorylation of platelet myosin by MLCK may play an important role in activated platelets in vivo.     

平滑肌肌球蛋白轻链激酶的非激酶作用及对ATP 酶活性的调节

肌球蛋白轻链激酶（myosin light chain kinase, MLCK）具有激酶活性和非激酶活性,在平滑肌收缩过程中起着关键酶调控的作用.为探寻MLCK的非激酶活性区域对MLCK活性的影响,以进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制.采用PCR技术构建MLCK部分氨基酸缺失的重组表达载体pGEX-F6-5/D,经大肠杆菌表达得到可溶性GST融合蛋白,利用SDS-PAGE及Western 印迹鉴定表达的MLCK在细胞中的分布,结果还显示,提取液的上清和沉淀中均有MLCK片段的表达.运用亲和层析技术分离并纯化删除前、后表达的MLCK片段（F6.5和F6-5/D）,经谷胱甘肽琼脂糖凝胶 4B 纯化,SDS-PAGE鉴定显示为单一表达条带.应用EnzChek磷分析试剂盒和孔雀绿两种方法分别测定不同浓度的MLCK对非磷酸化肌球蛋白Mg²⁺-ATP酶活性的影响.两种MLCK的片段均具有激活ATP酶活性的作用,并随MLCK浓度的增加,酶的活性增加.比较删除前后不同MLCK片段对ATP酶活性的影响结果显示,删除MLCK片段1002位丙氨酸至1019位亮氨酸后,对ATP酶的激活作用较删除前明显降低,表明删除的部分氨基酸序列为MLCK非激酶活性所必需的区域.利用电镜技术观察到MLCK片段（F6.5）使非磷酸化肌球蛋白构象发生明显的变化.加入MLCK片段后肌球蛋白的构象由非活性型转化为活性型,并且MLCK片段还具有促进肌球蛋白单体形成肌丝的作用.     

Inhibition of the ATP-dependent interaction of actin and myosin by the catalytic domain of the myosin light chain kinase of smooth muscle: possible involvement in smooth muscle relaxation

Myosin light chain kinase (MLCK) phosphorylates the light chain of smooth muscle myosin enabling its interaction with actin. This interaction initiates smooth muscle contraction. MLCK has another role that is not attributable to its phosphorylating activity, i.e., it inhibits the ATP-dependent movement of actin filaments on a glass surface coated with phosphorylated myosin. To analyze the inhibitory effect of MLCK, the catalytic domain of MLCK was obtained with or without the regulatory sequence adjacent to the C-terminal of the domain, and the inhibitory effect of the domain was examined by the movement of actin filaments. All the domains work so as to inhibit actin filament movement whether or not the regulatory sequence is included. When the domain includes the regulatory sequence, calmodulin in the presence of calcium abolishes the inhibition. Since the phosphorylation reaction is not involved in regulating the movement by MLCK, and a catalytic fragment that shows no kinase activity also inhibits movement, the kinase activity is not related to inhibition. Higher concentrations of MLCK inhibit the binding of actin filaments to myosin-coated surfaces as well as their movement. We discuss the dual roles of the domain, the phosphorylation of myosin that allows myosin to cross-bridge with actin and a novel function that breaks cross-bridging.     

Stable transfectants of smooth muscle cell line lacking the expression of myosin light chain kinase and their characterization with respect to the actomyosin system

We constructed a plasmid vector having a 1.4-kilobase pair insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells, producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to platelet-derived growth factor-BB, which was supported by lamellipodia. However, the transfectants showed neither chemotaxic motility nor developed lamellipodia, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by a few tests including the rescue experiment. Despite this importance of MLCK, platelet-derived growth factor-BB failed to induce MLC20 phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to the novel property of MLCK that stimulates the ATPase activity of smooth muscle myosin without phosphorylating its light chain (Ye, L.-H., Kishi, H., Nakamura, A., Okagaki, T., Tanaka, T., Oiwa, K., and Kohama, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6666-6671).     

Inhibitory effect of the catalytic domain of myosin light chain kinase on actin-myosin interaction: insight into the mode of inhibition.

The catalytic domain of myosin light chain kinase (MLCK) not only exerts kinase activity to phosphorylate the 20 kDa light chain but also inhibits the actin-myosin interaction. The site of action of this novel role of the domain has been suggested to be myosin [Okagaki et al. (1999) J. Biochem. 125, 619-626]. In this study, we have analyzed the amino acid sequences of MLCK and myosin that are involved in the inhibition. The ATP-binding peptide of Gly526-Lys548 of chicken gizzard MLCK exerted the inhibitory effect on the movement of actin filaments on a myosin-coated glass surface. However, the peptide that neighbors the sequence failed to inhibit the movement. The inhibition of the ATP-binding peptide was confirmed by measuring ATPase activities of the myosin. The inhibition by parent MLCK of the movement was relieved by the 20 kDa light chain, but not by the 17 kDa myosin light chain. The peptide of the 20 kDa light chain sequence of Ser1-Glu29 also relieved the inhibition. Thus, the interaction of the ATP-binding sequence with the 20 kDa light chain sequence should cause the inhibition of the actin-myosin interaction. Concerning the regulation of the inhibition, calmodulin relieved the inhibitory effect of MLCK on the movement of actin filaments. The calmodulin-binding peptide (Ala796 Ser815) prevented the relief, suggesting the involvement of this sequence. Thus, the mode of regulation by Ca2+ and calmodulin of the novel role of the catalytic domain is similar, but not identical, to the mode of regulation of the kinase activity of the domain.     

Maximal stimulation-induced in situ myosin light chain kinase activity is upregulated in fetal compared with adult ovine carotid arteries

Postnatal decreases in vascular reactivity involve decreases in the thick filament component of myofilament calcium sensitivity, which is measured as the relationship between cytosolic calcium concentration and myosin light chain (MLC20) phosphorylation. The present study tests the hypothesis that downregulation of thick filament reactivity is due to downregulation of myosin light chain kinase (MLCK) activity in adult compared with fetal arteries. Total MLCK activity, calculated as %MLC20 phosphorylated per second in intact arteries during optimal inhibition of myosin light chain phosphatase activity, was significantly less in adult (6.56+/-0.29%) than in fetal preparations (7.39+/-0.53%). In situ MLC20 concentrations (microM) in adult (198+/-28) and fetal arteries (236+/-44) did not differ significantly. In situ MLCK concentrations (microM), however, were significantly greater in adult (8.21+/-0.59) than in fetal arteries (1.83+/-0.13). In situ MLCK activities (ng MLC20 phosphorylated.s(-1).ng MLCK(-1)) were significantly less in adult (0.26+/-0.01) than in fetal arteries (1.52+/-0.11). In contrast, MLCK activities in adult (15.8+/-1.5) and fetal artery homogenates (17.3+/-1.3) were not significantly different. When in situ fractional activation was calculated, adult values (1.72+/-0.17%) were significantly less than fetal values (9.08+/-0.83%). Together, these results indicate that decreased thick filament reactivity in adult compared with fetal ovine carotid arteries is due at least in part to greater MLCK activity in fetal arteries, which in turn cannot be explained by differences in MLCK, MLC20, or calmodulin concentrations. Instead, this difference appears to involve age-related differences in fractional activation of the MLCK enzyme.     

Role of myosin light chain kinase and myosin light chain phosphatase in the resistance arterial myogenic response to intravascular pressure

The intrinsic ability of vascular smooth muscle cells (VSMCs) within arterial resistance vessels to respectively contract and relax in response to elevation and reduction of intravascular pressure is essential for appropriate blood flow autoregulation. This fundamental mechanism, referred to as the myogenic response, is dependent on apposite control of myosin regulatory light chain (LC₂₀) phosphorylation, a prerequisite for force generation, through the coordinated activity of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Here, we highlight the molecular basis of the smooth muscle contractile mechanism and review the regulatory pathways demonstrated to participate in the control of LC₂₀ phosphorylation in the myogenic response, with a focus on the Ca²⁺-dependent and Rho-associated kinase (ROK)-mediated regulation of MLCK and MLCP, respectively.     

The 2-arachidonoylglycerol effect on myosin light chain phosphorylation in human platelets

In human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) stimulates some important pathways leading to thromboxane B₂ formation, calcium intracellular elevation, ATP secretion and actin polymerisation. The aim of the present study was to examine the 2-AG effect on myosin light chain (MLC) phosphorylation and to investigate the mechanisms involved. We demonstrated that 2-AG induced a rapid MLC phosphorylation, stimulating both the RhoA kinase (ROCK) and MLC kinase (MLCK) in a dose and time-dependent manner. In addition MLC phosphorylation was strengthened through the MLC phosphatase inhibition. MLC phosphatase inhibition was accomplished through the RhoA/ROCK and protein kinase C mediated phosphorylation of MLC phosphatase inhibiting subunits MYPT1 and CPI-17. The presence of CB1 receptor in human platelets and the involvement of CB1 receptor in MLC phosphorylation and MLC phosphatase inhibition was shown.     

Mutation analysis of the non-muscle myosin light chain kinase (MLCK) deletion constructs on CV1 fibroblast contractile activity and proliferation

Smooth muscle myosin light chain kinase (MLCK) is a multifunctional molecule composed of an N-terminal actin binding domain, a central kinase domain, and C-terminal calmodulin- and myosin-binding domains. We previously cloned and characterized a novel MLCK isoform from endothelial cells (EC MLCK) consisting of 1,914 amino acids displaying a higher molecular weight (210 kDa) and a novel-amino-terminal stretch of 922 amino acids not shared by the smooth muscle isoform (smMLCK, 150 kDa). To further define the role of specific EC MLCK motifs in endothelial and non-muscle cells, we constructed two epitope-tagged EC MLCK deletion mutants in mammalian expression vectors lacking either the C-terminal auto-inhibitory and calmodulin-binding domain (EC MLCK1745) or the ATP-binding site (EC MLCKATPdel). Expression of EC MLCK1745 in CV1 fibroblasts showed increased basal actin stress fiber formation, which was markedly enhanced after tumor necrosis factor (TNF-alpha) or thrombin treatment. Distribution of EC MLCK1745 was largely confined to stress fibers, cortical actin filaments, and focal adhesion contacts, and co-localized with myosin light chains (MLCs) diphosphorylated on Ser(19) and Thr(18). In contrast, immunofluorescence staining demonstrated that EC MLCKATPdel abolished thrombin- and TNFalpha-induced stress fiber formation and MLC phosphorylation, suggesting this kinase-dead mutant functions as a dominant-negative MLCK construct, thereby confirming the role of EC MLCK in stress fiber formation. Finally, we compared the serum-stimulated growth rate of mutant MLCK-transfected fibroblasts to sham controls, and found EC MLCK1745 to augment thymidine incorporation whereas EC MLCKATPdel reduced CV1 growth rates. These data demonstrate the necessary role for MLCK in driving the contractile apparatus via MLC phosphorylation, which can alter fibroblast growth and contractility.     

Decrease in myosin light chain kinase activity of rabbit fast muscle by chronic stimulation

Analysis of myosin light chain kinase (MLCK) activity in tibialis anterior muscles of the rabbit revealed that chronic stimulation at a frequency of 10 Hz for 24 h per day reduced the enzyme activity in a timedependent manner. Since fast twitch muscle contains significantly more myosin light chain kinase than slow twitch muscle, the observed reductions are consistent with the type of fast-to-slow transformation observed for other type-specific muscle characteristics. The present data also indicate that the stimulation-induced decrease in MLCK activity precedes the fast-to-slow conversion of the myosin molecule as judged by pyrophosphate-polyacrylamide gel electrophoresis.     

Biochemistry of smooth muscle myosin light chain kinase

The smooth muscle isoform of myosin light chain kinase (MLCK) is a Ca²⁺-calmodulin-activated kinase that is found in many tissues. It is particularly important for regulating smooth muscle contraction by phosphorylation of myosin. This review summarizes selected aspects of recent biochemical work on MLCK that pertains to its function in smooth muscle. In general, the focus of the review is on new findings, unresolved issues, and areas with the potential for high physiological significance that need further study. The review includes a concise summary of the structure, substrates, and enzyme activity, followed by a discussion of the factors that may limit the effective activity of MLCK in the muscle. The interactions of each of the many domains of MLCK with the proteins of the contractile apparatus, and the multi-domain interactions of MLCK that may control its behaviors in the cell are summarized. Finally, new in vitro approaches to studying the mechanism of phosphorylation of myosin are introduced.     

肌球蛋白轻链激酶活性片段对肌球蛋白轻链的非钙依赖性磷酸化作用特征

肌球蛋白轻链激酶 (MLCK)的活性片段 (MLCKF)能比完整的MLCK更有效地、以非钙依赖性的方式磷酸化肌球蛋白轻链 (MLC2 0 )。该片段是用胰蛋白酶水解MLCK ,再经DEAE 5 2柱层析分离而获得的 ,分子量约为 6 1kD。Western印迹已证实该MLCKF与完整的MLCK同源。MLCKF对肌球蛋白轻链的磷酸化作用及其作用特征通过甘油电泳及ScoinImage扫描软件检测 ,肌球蛋白ATP酶活性通过分光光度法检测。实验结果证实 ,MLCKF催化的MLC2 0 非钙依赖性磷酸化 (CIPM)比MLCK催化的CIPM效力高、耗能多 ,但比MLCK催化的MLC2 0 钙依赖性磷酸化 (CDPM)效力低、耗能少 ;MLCKF催化的CIPM与MLCK催化的CIPM均较MLCK催化的CDPM稳定 ,不易受温育温度、温育时间及离子浓度等变化的影响 ,且对MLCK抑制剂ML 9敏感性低。     

Functional role of the C-terminal domain of smooth muscle myosin light chain kinase on the phosphorylation of smooth muscle myosin

Smooth muscle myosin light chain kinase (MLCK) is known to bind to thin filaments and myosin filaments. Telokin, an independently expressed protein with an identical amino acid sequence to that of the C-terminal domain of MLCK, has been shown to bind to unphosphorylated smooth muscle myosin. Thus, the functional significance of the C-terminal domain and the molecular morphology of MLCK were examined in detail. The C-terminal domain was removed from MLCK by alpha-chymotryptic digestion, and the activity of the digested MLCK was measured using myosin or the isolated 20-kDa light chain (LC20) as a substrate. The results showed that the digestion increased K(m) for myosin 3-fold whereas it did not change the value for LC20. In addition, telokin inhibited the phosphorylation of myosin by MLCK by increasing K(m) but only slightly increased K(m) for LC20. Electron microscopy indicated that MLCK was an elongated molecule but was flexible so as to form folded conformations. MLCK was crosslinked to unphosphorylated heavy meromyosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the absence of Ca(2+)/calmodulin (CaM), and electron microscopic observation of the products revealed that the MLCK molecule bound to the head-tail junction of heavy meromyosin. These results suggest that MLCK binds to the head-tail junction of unphosphorylated myosin through its C-terminal domain, where LC20 can be promptly phosphorylated through its catalytic domain following the Ca(2+)/CaM-dependent activation.     

The long myosin light chain kinase is differentially phosphorylated during interphase and mitosis

We have shown previously that the activity of the long myosin light chain kinase (MLCK) is cell cycle regulated with a decrease in specific activity during mitosis that can be restored following treatment with alkaline phosphatase. To better understand the role and significance of phosphorylation in regulating MLCK function during mitosis, we examined the phosphorylation state of in vivo derived MLCK. Phosphoamino acid analysis and phosphopeptide mapping demonstrate that the long MLCK is differentially phosphorylated on serine residues during interphase and mitosis with the majority of the phosphorylation sites located within the N-terminal IgG domain. Biochemical assays show that Aurora B binds and phosphorylates the IgG domain of the long MLCK. In addition, phosphopeptide maps of the endogenous full-length MLCK from mitotic cells and in vitro phosphorylated IgG domain demonstrate that Aurora B phosphorylates the same sites as those observed in vivo. Altogether, these studies suggest that the long MLCK may be a cellular target for Aurora B during mitosis.     

ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells.

A novel myosin light chain kinase (MLCK) cDNA was isolated from a HeLa cell cDNA library. The deduced amino acid sequence was identical to that of a zipper-interacting protein kinase (ZIPK) which mediates apoptosis [Kawai et al. (1998) Mol. Cell. Biol. 18, 1642-1651]. Here we found that HeLa ZIPK phosphorylated the regulatory light chain of myosin II (MRLC) at both serine 19 and threonine 18 in a Ca2+/calmodulin independent manner. Phosphorylation of myosin II by HeLa ZIPK resulted in activation of actin-activated MgATPase activity of myosin II. HeLa ZIPK is the first non-muscle MLCK that phosphorylates MRLC at two sites.     

A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability

Activation of Na(+)-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 +/- 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na(+)-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na(+)-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes.     

The interaction between the regulatory light chain domains on two heads is critical for regulation of smooth muscle myosin

Recent findings have suggested that the interaction between the two heads is critical for phosphorylation-dependent regulation of smooth muscle myosin. We hypothesized that the interaction between the two regulatory light chains on two heads of myosin dictates the regulation of myosin motor function. To evaluate this notion, we engineered and characterized smooth muscle heavy meromyosin (HMM), which is composed of one entire HMM heavy chain and one motor domain truncated heavy chain containing the S2 rod and regulatory light chain (RLC) binding site, as well as the bound RLC (SMDHMM). SMDHMM was inactive for both actin-translocating activity and actin-activated ATPase activity in the dephosphorylated state, demonstrating that the interaction between the two RLC domains on the two heads and/or a motor domain and a RLC domain in a distinct head is sufficient for the inhibition of smooth muscle myosin motor activity. When phosphorylated, SMDHMM was activated for both actin-translocating activity and actin-activated ATPase activity; however, these activities were lower than those of double-headed HMM, implying partial release of inhibition by phosphorylation in SMDHMM and/or cooperativity between the two heads of smooth muscle myosin. The present results indicate that the RLC domain is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity. On the other hand, similar to double-headed HMM, SMDHMM showed both "folded" and "extended" conformations, and the ratio of those conformations is dependent on ionic strength, suggesting that the RLC domain is sufficient to regulate the conformational transition in myosin.