REVIEW Circ J 2009; 73: 208 – 213

Phosphorylation of Regulatory Light Chain by Myosin Light Chain Kinase, and Muscle Contraction

Seiji Takashima, MD

Not only muscle contraction, but also most cell movements depend on myosin – interaction using ATP. Many components of the contraction machinery are involved in the efficient coupling of energy source and force devel- opment. Among these, I have focused on myosin light chain kinase (MLCK) in this review. MLCK phosphorylates myosin regulatory light chain and controls all 3 types of muscle contraction: skeletal muscle, smooth muscle, and cardiac muscle. However, each muscle has specific MLCK and the role of MLCK in each muscle is different. This difference explains the specific role of each muscle in vivo and contributes to the activity of various force development in different ways in each tissue. Therefore, I also review the differences in the connection between each MLCK and muscle contraction in the 3 muscle types. (Circ J 2009; 73: 208 – 213) Key Words: Muscle contraction; Myosin light chain kinase; Regulatory light chain

iochemical analysis of the involved in The role of skMLCK or smMLCK in skeletal muscle where muscle contraction has a long history, since 1950, works as the main [Ca2+]i sensor for contraction is B because many researchers have been interested in still controversial. Surprisingly, in 2007 a third MLCK was the physiologically dynamic output of muscle contraction. identified, which express only in the heart, called cardiac The large amount of involved in muscle contraction myosin light chain kinase (cMLCK). also helped the precise biochemical analysis of the proteins. In this review, I summarize the molecular mechanism of In 1954, Huxley and Hanson showed that the force of a muscle contraction and compare the specific role of MLCKs muscle was generated by slippage of the cross-bridges be- in the different muscle tissues. tween actin filaments and myosin filaments.1 Soon after this, intracellular calcium ([Ca2+]i) was discovered to be the most Intracellular Calcium Homeostasis important trigger of muscle contraction.2 Identification of the sensing protein of [Ca2+]i clarified its importance for and MLCK in Muscle Tissue muscle contraction. Ebashi and Kodama3 discovered first All 3 MLCK (smMLCK, skMLCK, cMLCK) are calmo- that purified actin and myosin only are not enough for active dulin-dependent protein kinases. The affinity for movement of the cross-bridges and tried to purify missing is strongest among the calmodulin-dependent protein kinases protein for contraction. Next, they successfully identified and is estimated to be approximately 1 nmol/L. Indeed, during troponin as a sensing protein of [Ca2+]i.3 Researchers had the purification of MLCK, calmodulin is always co-purified. already noticed that the response to [Ca2+]i differed among The activity of MLCK is enhanced approximately 1,000- the 3 types of muscles: smooth muscle, skeletal muscle and fold in response to increasing [Ca2+]i. The most important cardiac muscle. Indeed, troponin was the sensor protein of regulator of MLCK activity is [Ca2+]i and MLCK was maxi- [Ca2+]i in skeletal and cardiac muscles, but does not exist in mally activated by about 1μ mol/L of [Ca2+]i. Therefore, I smooth muscle cells, in which smooth-muscle-type myosin will first summarize the [Ca2+]i regulation of contraction in light chain kinase (smMLCK) was identified as a [Ca2+]i each muscle (Fig 1). sensor.4 In smooth muscle cells, chemical mediators such as Troponin expresses specifically in skeletal and cardiac catecholamine induce a relatively slow Ca release from muscle, whereas smMLCK expresses in almost every tissue, intracellular stores mediated by activation of the phospho- including skeletal and cardiac muscles. Moreover, skeletal inositol pathway. Smooth muscle cells gradually contract muscle expresses a specific MLCK called skeletal-muscle- according to this slow increase of [Ca2+]i. In skeletal muscle type myosin light chain kinase (skMLCK), which means cells, activation of the voltage-dependent Ca channel acti- skeletal muscle has 2 MLCK (ie, smMLCK and skMLCK). vated by terminal nerve stimuli directly opens the ryanodine receptor on the Ca store site, resulting in a rapid increase of 2+ (Received October 31, 2008; revised manuscript received November [Ca ]i. In cardiac muscle cells, electro-stimulation from the 18, 2008; accepted November 19, 2008; released online December 26, sinus node activates the voltage-dependent cation channel 2008) and increases [Ca2+]i. This increase in [Ca2+]i induces a Departments of Cardiovascular Medicine and Molecular Cardiology, large Ca release from intracellular store site named “Ca- Osaka University Graduate School of Medicine, Suita, Japan induced Ca release”. Increased [Ca2+]i in both skeletal and Mailing address: Seiji Takashima, MD, Departments of Cardiovascu- cardiac muscles binds the troponin complex and induces lar Medicine and Molecular Cardiology, Osaka University Graduate muscle contraction without delay. However, the duration of School of Medicine, 2-2 Yamada-oka, Suita 565-0871, Japan. E-mail: 2+ [email protected] increased [Ca ]i in these muscles is very short and Ca is All rights are reserved to the Japanese Circulation Society. For permis- rapidly recaptured in the store site. In cardiac muscle cells, sions, please e-mail: [email protected] after electro-stimulation [Ca2+]i increases from 0.1μ mol/L to

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Fig 1. Intracellular calcium and muscle contraction. In skeletal and cardiac muscle, electrical stimuli activate the volt- age-dependent channel and rapidly increases [Ca2+]i. After 1s, however, [Ca2+]i is rapidly absorbed into the intracellular Ca storage site. This transient increase of [Ca2+]i activates myosin ATPase via troponin and muscle briefly contracts. On the other hand, in smooth muscle cells a vasoconstrictor induces a relatively longer period of increased [Ca2+]i. This slow increase of [Ca2+]i activates MLCK by a calmodulin-dependent pathway. There is no troponin expressed in smooth muscle cells. For functional diversity, each muscle type has similar but distinct protein components, the genes for which are located on different loci. It is also evolutionarily interesting to suggest diversification of muscle cells to fit each purpose during development. Black arrow indicates increased [Ca2+]i and red arrow indicates decreased [Ca2+]i. SR, sarcoplasmic reticulum; MLC, myosin light chain; MLCK, myosin light chain kinase.

1μ mol/L and back to the basal level in 0.1 s. Taken together, this reason, in these muscles there is not a linear correlation the 3 types of muscles, especially smooth muscle, have between RLC phosphorylation and contractility. Because different molecular kinetics to increase [Ca2+]i during mus- rapid [Ca2+]i transient occurs in these cells, slow kinase cle contraction. This specific [Ca2+]i control seems to be reactions such as MLCK are not suitable as a [Ca2+]i sensor.5 necessary for the specific role of each muscle. Troponin directly binds to actin and structural change in troponin causes the release of , which inhibits the 2+ ATPase activity of myosin. Using this non-enzyme signal, [Ca ]i Increase and Muscle Contraction increased [Ca2+]i rapidly changes the contractility of these In smooth muscle cells, increased [Ca2+]i leads to the acti- muscles. However, there are kinases that specifically phos- vation of smMLCK by Ca – calmodulin pathway and myosin phorylate RLC in both cardiac and skeletal muscles. It is regulatory light chain (RLC) is phosphorylated by activated hard to believe that phosphorylation of RLC, which is the smMLCK. Phosphorylated RLC activates the ATPase of main trigger for contraction of smooth muscle cells, does not myosin and smooth muscle contracts. How the phosphory- affect contractility in these muscles. Different roles of RLC lated RLC activates the ATPase of myosin is still unknown; phosphorylation are implied in cardiac and skeletal muscles. however, there is linear correlation between the degree of How the rapid [Ca2+]i transient activates MLCK and the RLC phosphorylation and muscle contractility. In smooth degree to which RLC is phosphorylated are still controver- muscle cells, the concentration of smMLCK and its sub- sial. Before approaching these questions, the biochemical strate, RLC, is approximately 4μ mol/L and 30–40μ mol/L, character of MLCK will be summarized next. respectively. Km value and Vmax of smMLCK is 3–5μ mol/L and 10–20 /s, respectively. With these kinetics, RLC is totally phosphorylated within 1–2 s, if we hypothesize that Structure of MLCK smMLCK and RLC are located close enough to react in The myosin II molecule is a hexamer composed of 2 vivo. The affinity of Ca – calmodulin for smMLCK is very myosin heavy chains and 4 myosin light chains (MLCs). Of strong and the [Ca2+]i increase after agonist stimulation is these 6 chains, MLCKs phosphorylate RLC and regulate fast enough to negate the time of these reactions. This 1–2 s the function of myosin. There are 3 homologues of MLCK delay is well matched to the time taken from agonist stimu- that share similar catalytic domain and regulatory domain lation to RLC phosphorylation. In contrast, both skeletal (Fig 2). The smMLCK is expressed ubiquitously in every and cardiac muscles use troponin as a [Ca2+]i sensor. For tissue, whereas skMLCK or cMLCK is expressed specifical-

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Fig 2. Three subtypes of myosin light chain kinase (MLCK) have a common conserved kinase domain near the C-terminal lesion. Ig, immunoglobulin domain; Fib, fibronectin domain.

MLCKs. When cloning cMLCK, all the fractions of tissue homogenate separated by fine-column chromatography were screened and only RLC was identified as a substrate of MLCK. Structural background contributed to this speci- ficity and its analysis is expected.

smMLCK Compared with the other 2 MLCKs, smMLCK is ex- pressed ubiquitously in almost all tissues and is involved not only in contraction of smooth muscle cells but also many cellular activities. In non-muscle cells the rate of phosphory- lation of RLC is small and other kinases can phosphorylate RLC. The role of smMLCK in non-muscle cells is reviewed in another article.6 In smooth muscle cells, the importance of smMLCK is obvious because there is a direct link between the activity of smMLCK and the contractility of the cell. Be- cause inhibitors of smMLCK relax the smooth muscle, and are possible candidate antihypertensive drugs, physiological and biochemical analysis of smMLCK has been intensive. After phosphorylation of smooth muscle RLC by smMLCK, Fig 3. Tissue distribution of 3 MLCK subtypes shown by northern blotting. Cardiac (cMLCK) and skeletal (skMCK) forms are exclu- RLC is dephosphorylated by myosin phosphatase the activi- sively expressed in heart and skeletal muscle, respectively. MLCK, ty of which is regulated by Rho kinase. Rho kinase phos- myosin light chain kinase. phorylates myosin phosphatase and inhibits its activity. Therefore, activation of Rho kinase leads to muscle contrac- tion.7 Other kinases, such as PKA, p21-activated kinase 1 ly in skeletal muscle or cardiac muscle, respectively (Fig 3). (PAK1), p21-activated kinase 2 (PAK2), protein kinase C, The regulatory mechanism of MLCK activity is also con- mitogen-activated protein kinase, and calmodulin kinase II, served in all MLCKs, which are all Ca–calmodulin-depen- are also reported to phosphorylate RLC and regulate muscle dent kinases. Although the complete structure of MLCK contraction; however, whether this activity exceeds the has not been elucidated, the regulatory mechanism has been strong activation of smMLCK by Ca–calmodulin pathway speculated from its similarity to the structure of protein is still questionable. kinase A (PKA). When [Ca2+]i is low, the regulatory domain can bind to the catalytic domain to inhibit kinase activity. Binding the Ca–calmodulin complex to this regulatory skMLCK domain releases the regulatory domain from catalytic Compared with smMLCK, the investigation of skMLCK domain and MLCK is activated. If the complete structure has been limited because in skeletal muscle contraction does were available, the precise regulatory mechanism could not directly link to the phosphorylation of MLC. However, be clarified. The intriguing thing is that MLKC has only 1 Persechini and Stull in Texas University have been inten- substrate: RLC. This specificity is common among the 3 sively analyzing the specific role of skMLCK. The Km

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Fig 4. Phosphorylation of MLC by MLCK is important for establishment of the sarcomere structure in rat cardiomyocytes. Rat cardiomyocytes treated with siRNA of MLCK show less phosphorylation of cMCL and cannot form the sarcomere structure after phenylephrine treatment. MLC, myosin light chain; MLCK, myosin light chain kinase. value of skMLCK to skeletal muscle type MLC (skMLC) is ranging from 5% to 40%. This wide range implies the diffi- approximately 5μ mol/L, which is far below the Km value of culty of measuring the phosphorylation rate of MLC in smMLCK to skMLC, which is approximately 100μ mol/L. vivo. Considering the dominancy of phosphatase in tissue, Therefore, in skeletal muscle, skMLCK is the main kinase it is speculated that 30–40% of skMLC is phosphorylated. It for the phosphorylation of skMLC. is essential for a future study to determine the accurate ratio In an experiment using skinned fibers, the phosphorylation of phospho-MLC and clarify how the phosphorylation of of skMLC by skMLCK was reported to enhance contractility skMLC affects the contractility of skeletal muscle. under the condition of submaximal [Ca2+]i.8 However, in skinned fibers it is difficult to evaluate the function of skMLCK, because the MLC phosphorylation rate of the cMLCK initial preparation is rather different from that of the various cMLCK was first identified in 2007;11 however, before following preparations. Also, assaying the level of phosphor- then several papers reported the importance of phosphoryla- ylation by adding external MLCK can not be done in real tion of cardiac MLC (cMLC) in cardiac structure and con- time. These experimental difficulties are the reason for the tractility. cMLC is also 1 of the genes causing hypertrophic different reports on the physiological function of skMLCK. cardiomyopathy.12 Almost all the causative genes of hyper- However, enhancement of contractility by phosphorylation trophic cardiomyopathy13 reported so far are coding proteins of MLC is obvious under extreme experimental conditions, related to heart muscle contraction, such as the myosin such as the comparison of completely unphosphorylated heavy chain, troponin.14 These genetic findings imply the MLC by phosphatase and sufficiently phosphorylated MLC important role of cMLC in contraction of the heart. Also, by skMLCK.9 These data suggest that the basic function of the difference in the phosphorylation rate of cMLC in the MLCK as enhancing contraction is still preserved in outer and inner layers of the heart muscle contributes to the skMLCK. efficient contraction of the whole heart.15 As in the case of In 2005, a knock-out mouse model of skMLCK was skeletal muscle, phosphorylation of cMLC in cardiac muscle made.10 In skMLCK-deficient mice there is difference in also increases muscle contraction at submaximal levels of contractility after 15 Hz tetanus stimulation; however, almost [Ca2+]i. To evaluate the role the cMLC phosphorylation, normal development and function of skeletal muscle are knowing the precise rate of phosphorylation of cMLC is also observed. These data suggest that the response to unphysio- essential. In cardiac muscle, 20–40% of cMLC is reported to logical stimulation is not important for the physiological be phosphorylated.16 Repeated contraction might cause the function of skeletal muscle. Also in these mice, some high phosphorylation rate of cMLC induced by transient skMLC phosphorylation is observed, which might be caused increase of [Ca2+]i with each heart beat. On the other hand, by redundancy between skMLCK and smMLCK, even the phosphorylation of cMLC is important for the establish- though a compensatory increase in smMLCK expression is ment of the sarcomere structure of the heart. An inhibitor of not observed in skMLCK-deficient mice. Because skMLC MLCK, ML-7, impairs the establishment of sarcomere is not a good substrate for smMLCK, this discrepancy may structure in the early embryo of Xenopus17 or the chicken.18 be explained by the specific localization of smMLCK on In mammalian cardiomyocytes, ML-7 also impairs the estab- the Z-band of the actomyosin fiber. However, the in vivo lishment of sarcomere structure. Moreover, mice lacking function of skMLCK is still unknown and only a complete atrial or ventricular cMLC die in the early embryo stage be- in vitro assay system could clarify the function of skMLCK cause of poor development of the heart. Impaired cardiac in skeletal muscle. Another important question is how much development because of the lack of MLC is also verified in MLC is phosphorylated in skeletal muscle cells. Several zebra fish, which can survive for 7 days without blood reports calculate the phosphorylated ratio of skMLC as flow. One of the ENU mutant zebra fish, called tel mutant,

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Fig 6. Inhibition of cardiac myosin light chain kinase (cMLCK) impairs cardiac development. Injection of the antisense morpholino of Fig 5. In situ hybridization of cardiac myosin light chain kinase in cMLCK caused heart dilatation and reduced contractility at 48 h after zebra fish. fertilization. processes this phenotype. Cells transplanted from the tel with actin to form mature muscle. This pathway is easily mutant to the wild-type zebra fish also cannot establish the blocked by inhibitors of MLCK, suggesting that embedding sarcomere structure, suggesting the important autonomous of myosin in the actin structure may require phosphoryla- cell function of cMLC in heart muscle development.19 tion of MLC. cMLCK has been identified as a novel kinase, the expres- In this review, I have focused on 3 MCLK homologs sion of which is enhanced in severe human heart failure.11 Its (smMLCK, skMLCK, cMLCK) and described the bio- substrate was purified, which turned out to be cMLC. There- chemical role of each in smooth muscle, skeletal muscle fore, this kinase was named as cMLCK and it is expressed and heart muscle. Each muscle has similar components of exclusively in the heart (Fig 3). The relationship between the contraction machinery; however, every component of the establishment of sarcomere structure and the phosphory- each muscle type has a specific subtype and those genes are lation of cMLC was first analyzed in rat cultured cardiomyo- located in different genome positions. Also, each muscle cytes. The sarcomere structure is disrupted by removing components seems to develop distinct and indispensable serum from the culture media and the phosphorylation rate functions. These diversified processes must be necessary of cMLC reduces. Treatment with reagents that increase for each muscle to have its specific function. This precise [Ca2+]i, such as catecholamines, re-establishes the sarcomere adaptation of each component is mysterious and splendid. structure with an increased phosphorylation ratio of cMLC. I hope the force–energy relationship hypothesized by Frank This re-establishment of the sarcomere structure is blocked and Starling connects with the model of 1 molecule force by RNAi pretreatment of cMLCK (Fig 4). This implies that development proposed by Yanagida et al20 for analyzing cMLCK activation by increased [Ca2+]i is indispensable MLCKs. signaling for the establishment of the sarcomere structure under these conditions. In zebra fish, 3 MLCK orthologs Acknowledgments (cardiac-, skeletal-, smooth-muscle-type MLCK), exist and My thanks to all the following collaborators: Masafumi Kitakaze, Naoki have a similar gene location as the respective mammalian Mochizuki, Hitonobu Tomoike, Soichiro Kitamura, Masanori Asakura, MLCKs. Zebra fish cMLCK also exclusively expresses in Satoru Yamazaki at the National Cardiovascular Center; Osamu Seguchi, the heart from the early embryo (Fig 5). Reduced cMLCK Yoshihiro Asano, Tetsuo Minamino, Yasunori Shintani, Masakatsu Wakeno at Osaka University; Hidehiko Furukawa, Kenji Nakamura, Asuka Naito, expression by the antisense morpholino causes severely im- Tomoko Takahashi, Toshiaki Ohtsuka at Daiichi Sankyo; Koichi Kawakami paired heart development. After 72 h fertilization, reduced at the National Institute of Genetics; Tadashi Isomura at the Hayama Heart expression of cMLCK caused cardiac arrest following Center. reduced contractility (Fig 6). Histologically, the structure of the sarcomere was poorly developed compared with control References zebra fish. Taken together with the similar phenotype of the 1. Huxley H, Hanson J. Changes in the cross-striations of muscle during tel mutant (cMLC mutant), cMLC phosphorylation by contraction and stretch and their structural interpretation. Nature cMLCK is an essential signaling pathway of the establish- 1954; 173: 973 – 976. 2. Ebashi F, Ebashi S. Removal of calcium and relaxation in actomyosin ment of sarcomere structure. Compared with the mice systems. Nature 1962; 194: 378 – 379. lacking skMLCK, these phenotypes of the cMLCK knock- 3. Ebashi S, Kodama A. Interaction of troponin with F-actin in the pres- out gene are very severe, even though both are expressed ence of . J Biochem 1966; 59: 425 – 426. specifically in each organ and smMLCK expresses almost 4. Barany K, Barany M, Gillis JM, Kushmerick MJ. Phosphorylation- equally in both organs. It is still unclear whether this differ- dephosphorylation of the 18,000-dalton light chain of myosin during the contraction-relaxation cycle of frog muscle. J Biol Chem 1979; ence is a species difference of cMLCK or cMLC. Also, early 254: 3617 – 3623. lethality of cMLCK knock-out zebra fish made it difficult 5. Koizumi K, Hoshiai M, Ishida H, Ohyama K, Sugiyama H, Naito A, to assess the specific role of cMLCK in cardiac contractili- et al. Stanniocalcin 1 prevents cytosolic Ca2+ overload and cell hyper- ty. My group are now making Cre-lox mediated knock-out contracture in cardiomyocytes. Circ J 2007; 71: 796 – 801. 6. Kamm KE, Stull JT. Dedicated myosin light chain kinases with of cMLCK mice, which should help in the analysis of the diverse cellular functions. J Biol Chem 2001; 276: 4527 – 4530. role of cMLCK in the contractility of the developed heart. 7. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, et al. At which developmental stage cMLCK contributes to the Regulation of myosin phosphatase by Rho and Rho-associated kinase sarcomere structure is also unknown. In avian heart devel- (Rho-kinase). Science 1996; 273: 245 – 248. 8. Persechini A, Stull JT. 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Circulation Journal Vol.73, February 2009