Repetitive stimulation potentiates contractile tension of fasttwitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca 2؉ ͞calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fasttwitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca 2؉ ͞calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.calcium ͉ calmodulin ͉ twitch S keletal muscle contraction depends on a voltage-driven conformational change in the L-type Ca 2ϩ channel in the transverse tubule that triggers Ca 2ϩ release from the sarcoplasmic reticulum through the intracellular ryanodine receptor (1, 2). The Ca 2ϩ binds to troponin in thin filaments, thereby allowing myosin cross bridges to bind actin and generate muscle tension (3). However, muscle contractions involve more complex mechanisms that affect performance. Since Ranke noted in 1865 (4) that, with stimuli uniform in strength the later twitch contractions were stronger than the first, there has been considerable interest in identifying the mechanisms involved in isometric twitch potentiation during trains of stimuli at low frequency (staircase) or after a tetanus (posttetanic potentiation). Considerations have been given to changes in compliance of the series elastic elements, to activation of more fibers within a muscle, to increased Ca 2ϩ release within a single fiber to activate fully the contractile proteins, and to changes in excitation-contraction coupling processes (5-8).Ca 2ϩ released during muscle contraction can also activate the dedicated protein kinase Ca 2ϩ ͞calmodulin-dependent skeletal muscle myosin light chain kinase (skMLCK) to initiate myosin regulatory light chain (RLC) phosphorylat...

Background & Aims-Smooth muscle is essential for maintaining homeostasis for many body functions and provides adaptive responses to stresses imposed by pathological disorders. Identified cell signaling networks have defined many potential mechanisms for initiating smooth muscle contraction with or without myosin regulatory light chain (RLC) phosphorylation by myosin light chain kinase (MLCK). We generate tamoxifen-inducible and smooth muscle-specific MLCK knockout (KO) mice and provide direct loss-of-function evidence that shows the primary importance of MLCK in phasic smooth muscle contractions.

Recent studies have established the involvement of the fat mass and obesity-associated gene (FTO) in metabolic disorders such as obesity and diabetes. However, the precise molecular mechanism by which FTO regulates metabolism remains unknown. Here, we used a structure-based virtual screening of U.S. Food and Drug Administration–approved drugs to identify entacapone as a potential FTO inhibitor. Using structural and biochemical studies, we showed that entacapone directly bound to FTO and inhibited FTO activity in vitro. Furthermore, entacapone administration reduced body weight and lowered fasting blood glucose concentrations in diet-induced obese mice. We identified the transcription factor forkhead box protein O1 (FOXO1) mRNA as a direct substrate of FTO, and demonstrated that entacapone elicited its effects on gluconeogenesis in the liver and thermogenesis in adipose tissues in mice by acting on an FTO-FOXO1 regulatory axis.

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca 2؉ binding site, had dramatically altered Ca 2؉ binding properties. The K Ca value for E22K was decreased by ϳ17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca 2؉ . Interestingly, Ca 2؉binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the ␣-helical content of phosphorylated R58Q greatly increased with Ca 2؉ binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K Ca than wild-type light chain, whereas phosphorylation of this mutant increased the Ca 2؉ affinity 6-fold. Whereas phosphorylation of wildtype light chain decreased its Ca 2؉ affinity, the opposite was true for A13T. The ␣-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca 2؉ binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.There is substantial evidence that myosin regulatory light chains (RLC) 1 play a primary regulatory role in scallop and smooth muscle contraction, but their functional role in mammalian striated (skeletal and cardiac) muscle contraction is unclear. RLC, together with the essential light chain, stabilizes the 8.5-nm-long ␣-helical neck of the myosin head, with the N terminus of RLC wrapped around the heavy chain (1). Smooth muscle contraction is initiated by RLC phosphorylation with a Ca 2ϩ -calmodulin-activated myosin light chain kinase (MLCK) (2, 3). However, in skeletal and cardiac muscle, RLC phosphorylation does not activate contraction but appears to play a modulatory role (4). It was shown that RLC phosphorylation increased the Ca 2ϩ sensitivity of force in skinned skeletal (5-7) and cardiac (8) muscle fibers. In the human heart, several RLC isoforms are expressed (9, 10) preferentially in the atrium and in the ventricle. Recent studies have revealed that the ventricular RLC is one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC) (11,12). FHC is an autosomal dominant disease, characterized by left ventricular hypertrophy, myofibrillar disarray, and sudden death. It is caused by missense mutations in various genes that encode for ␤-myosin heavy chain (13), myosin-binding protein C (14), ventricular RLC and essential light chain (11,12,15), troponin T (16), troponin I (17), ␣-tropomyosin (18), actin (19), and titin (20). Depending on the affected gene, and the site of the mutation, FHC has variable presentation with regard to its degree and severity and t...

A primary mechanism for initiating smooth muscle contraction involves an increase in [Ca 2ϩ ] i , leading to myosin regulatory light chain (RLC) phosphorylation, crossbridge cycling, and force development (1, 2). Phosphorylation of Ser-19 on RLC of myosin II changes the orientation of myosin crossbridges, allowing actin activation of myosin ATPase activity. A similar mechanism occurs with nonmuscle myosin II with effects on many cellular actomyosin-dependent functions. The Ca 2ϩ -dependent phosphorylation of RLC is mediated by Ca 2ϩ ͞cal-modulin (CaM)-dependent myosin light chain kinase (MLCK), whereas myosin light chain phosphatase dephosphorylates RLC. In smooth muscles, agonists stimulate greater RLC phosphorylation and force than do depolarizing stimuli at comparable [Ca 2ϩ ] i because of Ca We developed a different CaM-sensor MLCK capable of monitoring MLCK activation to obtain temporal and quantitative information on Ca 2ϩ ͞CaM binding to MLCK where Ca 2ϩ -dependent CaM binding increased kinase activity coincident with a decrease in FRET (11). The CaM-sensor MLCK was expressed in smooth muscle tissues of transgenic mice to obtain quantitative insights on CaM activation of MLCK relative to [Ca 2ϩ ] i and RLC phosphorylation and force development. These results show that genetically encoded biosensors may be used to investigate physiological processes in tissues of transgenic mice. MethodsConstruction of SM8 35 KCS Plasmid. The pSM8 35 KCS construct was prepared by subcloning the 1.6-kb cDNA of Ca 2ϩ ͞CaM-sensor containing the MLCK CaM-binding sequence flanked by enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) (12) into the pSM8-CAT vector, which contains the smooth muscle ␣-actin promoter (13, 14). The 3.1-kb cDNA fragment of short rabbit smooth muscle MLCK was further subcloned into the site between the Ca 2ϩ ͞ CaM-sensor gene and the smooth muscle ␣-actin promoter in pSM8-CAT vector to produce pSM8 35 KCS construct. Correct This paper was submitted directly (Track II) to the PNAS office.

Phosphorylation of the myosin regulatory light chains (RLCs) activates contraction in smooth muscle and modulates force production in striated muscle. RLC phosphorylation changes the net charge in a critical region of the N terminus and thereby may alter interactions between the RLC and myosin heavy chain. A series of N-terminal charge mutations in the human smooth muscle RLC has been engineered, and the mutants have been evaluated for their ability to mimic the phosphorylated form of the RLC when reconstituted into scallop striated muscle bundles or into isolated smooth muscle myosin. Changing the net charge in the region from Arg-13 to Ser-19 potentiates force in scallop striated muscle and maintains smooth muscle myosin in an unfolded framentous state without affecting ATPase activity or motility of smooth muscle myosin.Thus, the effect of RLC phosphorylation in striated muscle and its ability to regulate the folded-'to-extended conformational transition in smooth muscle may be due to a simple reduction of net charge at the N terminus of the light chain. The ability of phosphorylation to regulate smooth muscle myosin's ATPase activity and motility involves a more complex mechanism.

The interaction of activated Ras with Raf initiates signaling cascades that contribute to a significant percentage of human tumors, suggesting that agents that specifically disrupt this interaction might have desirable chemotherapeutic properties. We used a subtractive forward two-hybrid approach to identify small molecule compounds that block the interaction of Ras with Raf. These compounds (

The role of phosphorylation of the myosin regulatory light chains (RLC) is well established in smooth muscle contraction, but in striated (skeletal and cardiac) muscle its role is still controversial. We have studied the effects of RLC phosphorylation in reconstituted myosin and in skinned skeletal muscle fibers where Ca2+ sensitivity and the kinetics of steady-state force development were measured. Skeletal muscle myosin reconstituted with phosphorylated RLC produced a much higher Ca2+ sensitivity of thin filament-regulated ATPase activity than nonphosphorylated RLC (change in -log of the Ca2+ concentration producing half-maximal activation = approximately 0.25). The same was true for the Ca2+ sensitivity of force in skinned skeletal muscle fibers, which increased on reconstitution of the fibers with the phosphorylated RLC. In addition, we have shown that the level of endogenous RLC phosphorylation is a crucial determinant of the Ca2+ sensitivity of force development. Studies of the effects of RLC phosphorylation on the kinetics of force activation with the caged Ca2+, DM-nitrophen, showed a slight increase in the rates of force development with low statistical significance. However, an increase from 69 to 84% of the initial steady-state force was observed when nonphosphorylated RLC-reconstituted fibers were subsequently phosphorylated with exogenous myosin light chain kinase. In conclusion, our results suggest that, although Ca2+ binding to the troponin-tropomyosin complex is the primary regulator of skeletal muscle contraction, RLC play an important modulatory role in this process.