Myosin Regulatory Light Chain Phosphorylation Research Articles

Is Slower Myosin Cross-Bridge Kinetics in Tg-D166V Preparations Due to Decreased Myosin RLC Phosphorylation?

In this report we have investigated a particularly malignant phenotype of Familial Hypertrophic Cardiomyopathy (FHC) associated with the 166 Aspartic Acid to Valine (D166V) mutation in the ventricular myosin regulatory light chain (RLC). We show that the rates of myosin cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from transgenic (Tg)-D166V compared to Tg-WT mice. A single molecule approach was taken where the fluorescence anisotropy of rhodamine phalloidin labeled actin protomers was measured in cardiac myofibrils undergoing isometric contraction. Orientation of an actin molecule oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The rates of cross-bridge attachment as well as cross-bridge dissociation were significantly decreased in isometrically contracting Tg-D166V myofibrils (binding, 1.4 s−1; detachment, 1.2 s−1) compared to Tg-WT myofibrils (binding, 3 s−1; detachment, 1.3 s−1). The duty ratio of the cross-bridge cycle, equal to the fraction of the total cycle time that cross-bridge remains attached to actin, was 47% inTg-D166V myofibrils and 30% in Tg-WT. Immunoblotting of cardiac myofibrils used for kinetics studies demonstrated a large reduction in RLC phosphorylation in Tg-D166V vs. Tg-WT myofibrils. These data are in accord with our previous findings in skinned and intact papillary muscles showing slower fiber kinetics and prolonged force transients in Tg-D166V fibers compared to Tg-WT preparations (Kerrick et al., FASEB J. 23: 855, 2009). Similarly, the level of RLC phosphorylation in muscle extracts from Tg-D166V ventricles was decreased. Our cellular and single molecule data suggest that a mutation-dependent decrease in RLC phosphorylationcould initiate the slower kinetics of the D166V cross-bridgesand ultimately lead to abnormal cardiac muscle contraction. Supported by NIH-HL071778 (DSC), NIH-AR048622 (JB) and NIH-HL090786 (to JB and DSC).

Cdk5-Dependent Regulation of Rho Activity, Cytoskeletal Contraction, and Epithelial Cell Migration via Suppression of Src and p190RhoGAP

Cdk5 regulates adhesion and migration in a variety of cell types. We previously showed that Cdk5 is strongly activated during stress fiber formation and contraction in spreading cells. Here we determine the mechanism linking Cdk5 to stress fiber contractility and its relevance to cell migration. Immunofluorescence showed that Cdk5 colocalized with phosphorylated myosin regulatory light chain (pMRLC) on contracting stress fibers. Inhibiting Cdk5 activity by various means significantly reduced pMRLC level and cytoskeletal contraction, with loss of central stress fibers. Blocking Cdk5 activity also reduced Rho-Rho kinase (ROCK) signaling, which is the principal pathway of myosin phosphorylation under these conditions. Next, we examined the effect of Cdk5 activity on Src, a known regulator of Rho. Inhibiting Cdk5 activity increased Src activation and phosphorylation of its substrate, p190RhoGAP, an upstream inhibitor of Rho. Inhibiting both Cdk5 and Src activity completely reversed the effect of Cdk5 inhibition on Rho and prevented the loss of central stress fibers, demonstrating that Cdk5 exerts its effects on Rho-ROCK signaling by suppressing Src activity. Moreover, inhibiting either Cdk5 or ROCK activity increased cell migration to an equal extent, while inhibiting both kinases produced no additional effect, demonstrating that Cdk5-dependent regulation of ROCK activity is a physiological determinant of migration rate.

Abstract 5068: Interaction Between CD36 and Oxidized LDL Induces Loss of Cell Polarity and Inhibits Macrophage Migration: A Mechanism of Macrophage Trapping

Trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step in atherosclerosis. Previous studies from other laboratories showed that regressed atherosclerotic lesions were characterized by emigration of lipid-laden macrophages. CD36, a class B scavenger receptor expressed on macrophages is a pathogenic factor for foam cell formation and atherosclerosis. We recently showed that oxidized LDL (oxLDL) modulates macrophage cytoskeletal signaling via CD36, inhibiting migration and thus potentially contributing to intimal trapping. We now hypothesize that oxLDL-CD36 inhibition of migration is the result of specific intracellular signals that regulate cell polarity. Methods/Results: Live cell imaging of murine peritoneal macrophages showed that oxLDL, but not native LDL, induced loss of cell polarity. Macrophages from cd36 null mice did not show this effect. Similarly, macrophages from mice null for Vav, a guanine nucleotide exchange factor recently shown to be a downstream effector of CD36 signaling, did not show oxLDL-induced loss of polarity. Macrophage migration velocity and dynamic movement of macrophage membrane were decreased by oxLDL in wild type macrophages by 85% and 86% respectively, but not in cd36 null or vav null macrophages. These findings appear to be related to activity of myosin II, a recently suggested cell polarity determinant. Western blots for phosphorylated myosin regulatory light chain (MRLC) showed that oxLDL, but not native LDL, induced de-phosphorylation of MRLC by 80%. Enzyme linked immunosorbent assays for GTP-bound Rac, a regulator of MRLC phosphorylation, showed that macrophage Rac-GTPase activity was increased 2.6 fold by oxLDL. These findings were not observed in macrophages from cd36 null or vav null mice. 6-thio-GTP, a Rac-GTPase inhibitor that inhibits binding of Vav to Rac1 abrogated the effect of oxLDL on MRLC dephosphorylation. Conclusion: OxLDL interaction with CD36 inhibited macrophage migration and caused loss of cell polarity. The mechanism by which this occurs is via Vav-dependent activation of Rac-GTPase and dephosphorylation of MRLC. Activation of this pathway in the vessel wall may induce trapping of macrophages in the arterial intima and promote atherosclerosis.

Carbachol-induced rabbit bladder smooth muscle contraction: roles of protein kinase C and Rho kinase

Smooth muscle contraction is regulated by phosphorylation of the myosin light chain (MLC) catalyzed by MLC kinase and dephosphorylation catalyzed by MLC phosphatase. Agonist stimulation of smooth muscle results in the inhibition of MLC phosphatase activity and a net increase in MLC phosphorylation and therefore force. The two pathways believed to be primarily important for inhibition of MLC phosphatase activity are protein kinase C (PKC)-catalyzed CPI-17 phosphorylation and Rho kinase (ROCK)-catalyzed myosin phosphatase-targeting subunit (MYPT1) phosphorylation. The goal of this study was to determine the roles of PKC and ROCK and their downstream effectors in regulating MLC phosphorylation levels and force during the phasic and sustained phases of carbachol-stimulated contraction in intact bladder smooth muscle. These studies were performed in the presence and absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr(38)-CPI-17 and Thr(696)/Thr(850)-MYPT1 were measured at different times during carbachol stimulation using site-specific antibodies. Thr(38)-CPI-17 phosphorylation increased concurrently with carbachol-stimulated force generation. This increase was reduced by inhibition of PKC during the entire contraction but was only reduced by ROCK inhibition during the sustained phase of contraction. MYPT1 showed high basal phosphorylation levels at both sites; however, only Thr(850) phosphorylation increased with carbachol stimulation; the increase was abolished by the inhibition of either ROCK or PKC. Our results suggest that during agonist stimulation, PKC regulates MLC phosphatase activity through phosphorylation of CPI-17. In contrast, ROCK phosphorylates both Thr(850)-MYPT1 and CPI-17, possibly through cross talk with a PKC pathway, but is only significant during the sustained phase of contraction. Last, our results demonstrate that there is a constitutively activate pool of ROCK that phosphorylates MYPT1 in the basal state, which may account for the high resting levels of MLC phosphorylation measured in rabbit bladder smooth muscle.

The molecular effects of skeletal muscle myosin regulatory light chain phosphorylation

Phosphorylation of the myosin regulatory light chain (RLC) in skeletal muscle has been proposed to act as a molecular memory of recent activation by increasing the rate of force development, ATPase activity, and isometric force at submaximal activation in fibers. It has been proposed that these effects stem from phosphorylation-induced movement of myosin heads away from the thick filament backbone. In this study, we examined the molecular effects of skeletal muscle myosin RLC phosphorylation using in vitro motility assays. We showed that, independently of the thick filament backbone, the velocity of skeletal muscle myosin is decreased upon phosphorylation due to an increase in the myosin duty cycle. Furthermore, we did not observe a phosphorylation-dependent shift in calcium sensitivity in the absence of the myosin thick filament. These data suggest that phosphorylation-induced movement of myosin heads away from the thick filament backbone explains only part of the observed phosphorylation-induced changes in myosin mechanics. Last, we showed that the duty cycle of skeletal muscle myosin is strain dependent, consistent with the notion that strain slows the rate of ADP release in striated muscle.

Regulatory mechanism of smooth muscle contraction studied with gelsolin-treated strips of taenia caeci in guinea pig

The potential roles of the regulatory proteins actin, tropomyosin (Tm), and caldesmon (CaD), i.e., the components of the thin filament, in smooth muscle have been extensively studied in several types of smooth muscles. However, controversy remains on the putative physiological significance of these proteins. In this study, we intended to determine the functional roles of Tm and CaD in the regulation of smooth muscle contraction by using a reconstitution system of the thin filaments. At appropriate conditions, the thin (actin) filaments within skinned smooth muscle strips of taenia caeci in guinea pigs could be selectively removed by an actin-severing protein, gelsolin, without irreversible damage to the contractile apparatus, and then the thin filaments were reconstituted with purified components of thin filaments, i.e., actin, Tm, and CaD. We found that the structural remodeling of actin filaments or thin filaments was functionally linked to the Ca(2+)-induced force development and reduction in muscle cross-sectional area (CSA). That is, after the reconstitution of the gelsolin-treated skinned smooth muscle strips with pure actin, the Ca(2+)-dependent force development was partially restored, but the Ca(2+)-induced reduction in CSA occurred once. In contrast, the reconstitution with actin, followed by Tm and CaD, restored not only the force generation but also both its Ca(2+) sensitivity and the reversible Ca(2+)-dependent reduction in CSA. We confirmed that both removal of the thin filaments by gelsolin treatment and reconstitution of the actin (thin) filaments with Tm and CaD caused no significant changes in the level of myosin regulatory light chain phosphorylation. We thus conclude that Tm and CaD are necessary for the full regulation of smooth muscle contraction in addition to the other regulatory systems, including the myosin-linked one.

Thromboxane A2-induced Bi-directional Regulation of Cerebral Arterial Tone

Myosin light chain phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin phosphatase targeting subunit (MYPT1). As cerebral blood flow is highly spatio-temporally modulated under normal physiologic conditions, severe perturbations in normal cerebral blood flow, such as in cerebral vasospasm, can induce neurological deficits. In nonpermeabilized cerebral vessels stimulated with U-46619, a stable mimetic of endogenous thromboxane A2 implicated in the etiology of cerebral vasospasm, we observed significant increases in contractile force, RhoA activation, regulatory light chain phosphorylation, as well as phosphorylation of MYPT1 at Thr-696, Thr-853, and surprisingly Ser-695. Inhibition of nitric oxide signaling completely abrogated basal MYPT1 Ser-695 phosphorylation and significantly increased and potentiated U-46619-induced MYPT1 Thr-853 phosphorylation and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of calcium sensitization. Surprisingly, a fall in Ser-695 phosphorylation did not result in an increase in phosphorylation of the Thr-696 site. Although activation of cGKI with exogenous cyclic nucleotides inhibited thromboxane A2-induced MYPT1 membrane association, RhoA activation, contractile force, and regulatory light chain phosphorylation, the anticipated decreases in MYPT1 phosphorylation at Thr-696/Thr-853 were not observed, indicating that the vasorelaxant effects of cGKI are not through dephosphorylation of MYPT1. Thus, thromboxane A2 signaling within the intact cerebral vasculature induces "buffered" vasoconstrictions, in which both the RhoA/Rho-kinase calcium-sensitizing and the NO/cGMP/cGKI calcium-desensitizing pathways are activated.

The Molecular Effects of Skeletal Muscle Fatigue on Myosin Mechanics and Kinetics

Skeletal muscle, during periods of exertion, experiences several different fatigue based changes in contraction including reductions in force, velocity, power output, and energy usage. The physiological bases of fatigue induced changes in contractility stem from many different factors including changes in neuronal activity, excitation-contraction coupling, and actomyosin contraction. The direct changes in the actomyosin contractile complex have previously been shown to be the result of alterations in the levels of metabolites, oxidative damage, and an increase in myosin regulatory light chain phosphorylation. Here, we measured the direct molecular effects of fatigue-like conditions on actomyosin velocity and force generation using the in vitro motility assay. We examined how changes in ATP, ADP, Pi, and pH affect the ability of the myosin to translocate actin. We show that fatigue-induced increases in metabolites decrease unloaded shortening velocity, with ADP and pH contributing the most to the observed changes in velocity. We also examined whether myosin regulatory light chain phosphorylation alters the sensitivity of the myosin to fatigue-like conditions. We show that under fatigue-like conditions, phosphorylation of the myosin regulatory light chian enhances force production and reduces actin sliding velocity, similar to the effects of phosphorylation under fatigue-like conditions observed in both muscle fiber and in vitro motility studies. Furthermore, we found that force production by dephosphorylated myosin is very sensitive to fatigue-like conditions whereas force production by phosphorylated myosin is rather fatigue insensitive. These results suggest that phosphorylation of the myosin regulatory light chain in skeletal muscle may serve as a protective mechanism against fatigue. Supported by NIH-HL077280, AHA-0435434T (J.R.M.), AHA-0815704D (to M.J.G.), and NIH-HL071778 (D.S-C.).

Ablation of Ventricular Myosin Regulatory Light Chain Phosphorylation in Mice Causes Cardiac Dysfunction in Situ and Affects Neighboring Myofilament Protein Phosphorylation

There is little direct evidence on the role of myosin regulatory light chain phosphorylation in ejecting hearts. In studies reported here we determined the effects of regulatory light chain (RLC) phosphorylation on in situ cardiac systolic mechanics and in vitro myofibrillar mechanics. We compared data obtained from control nontransgenic mice (NTG) with a transgenic mouse model expressing a cardiac specific nonphosphorylatable RLC (TG-RLC(P-). We also determined whether the depression in RLC phosphorylation affected phosphorylation of other sarcomeric proteins. TG-RLC(P-) demonstrated decreases in base-line load-independent measures of contractility and power and an increase in ejection duration together with a depression in phosphorylation of myosin-binding protein-C (MyBP-C) and troponin I (TnI). Although TG-RLC(P-) displayed a significantly reduced response to beta(1)-adrenergic stimulation, MyBP-C and TnI were phosphorylated to a similar level in TG-RLC(P-) and NTG, suggesting cAMP-dependent protein kinase signaling to these proteins was not disrupted. A major finding was that NTG controls were significantly phosphorylated at RLC serine 15 following beta(1)-adrenergic stimulation, a mechanism prevented in TG-RLC(P-), thus providing a biochemical difference in beta(1)-adrenergic responsiveness at the level of the sarcomere. Our measurements of Ca(2+) tension and Ca(2+)-ATPase rate relations in detergent-extracted fiber bundles from LV trabeculae demonstrated a relative decrease in maximum Ca(2+)-activated tension and tension cost in TG-RLC(P-) fibers, with no change in Ca(2+) sensitivity. Our data indicate that RLC phosphorylation is critical for normal ejection and response to beta(1)-adrenergic stimulation. Our data also indicate that the lack of RLC phosphorylation promotes compensatory changes in MyBP-C and TnI phosphorylation, which when normalized do not restore function.

Centrosome/Spindle Pole–associated Protein Regulates Cytokinesis via Promoting the Recruitment of MyoGEF to the Central Spindle

Cooperative communications between the central spindle and the contractile ring are critical for the spatial and temporal regulation of cytokinesis. Here we report that MyoGEF, a guanine nucleotide exchange factor that localizes to the central spindle and cleavage furrow, interacts with centrosome/spindle pole-associated protein (CSPP), which is concentrated at the spindle pole and central spindle during mitosis and cytokinesis. Both in vitro and in vivo pulldown assays show that MyoGEF interacts with CSPP. The C-terminus of MyoGEF and N-terminus of CSPP are required for their interaction. Immunofluorescence analysis indicates that MyoGEF and CSPP colocalize at the central spindle. Depletion of CSPP or MyoGEF by RNA-interference (RNAi) not only causes defects in mitosis and cytokinesis, such as metaphase arrest and furrow regression, but also mislocalization of nonmuscle myosin II with a phosphorylated myosin regulatory light chain (p-MRLC). Importantly, CSPP depletion by RNAi interferes with MyoGEF localization at the central spindle. Finally, MyoGEF interacts with ECT2, and RNAi-mediated depletion of MyoGEF leads to mislocalization of ECT2 and RhoA during cytokinesis. Therefore, we propose that CSPP interacts with and recruits MyoGEF to the central spindle, where MyoGEF contributes to the spatiotemporal regulation of cytokinesis.

Myosin regulatory light chain phosphorylation inhibits shortening velocities of skeletal muscle fibers in the presence of the myosin inhibitor blebbistatin

Phosphorylation of skeletal myosin regulatory light chain (RLC) occurs in fatigue and may play a role in the inhibition of shortening velocities observed in vivo. Forces and shortening velocities were measured in permeabilized rabbit psoas fibers with either phosphorylated or dephosphorylated RLCs and in the presence or absence of the myosin inhibitor blebbistatin. Addition of 20 microM blebbistatin decreased tensions by approximately 80% in fibers, independent of phosphorylation. In blebbistatin maximal shortening velocities (V(max)) at 30 degrees C, were decreased by 45% (3.2 +/- 0.34 vs. 5.8 +/- 0.18 lengths/s) in phosphorylated fibers but were not inhibited in dephosphorylated fibers (6.0 +/- 0.30 vs. 5.4 +/- 0.30). In the presence of 20 microM blebbistatin, K(m) for V(max) as a function of [ATP] was lower for phosphorylated fibers than for dephosphorylated fibers (50 +/- 20 vs. 330 +/- 84 microM) indicating that the apparent binding of ATP is stronger in these fibers. Phosphorylation of RLC in situ during fiber preparation or by addition of myosin light chain kinase yielded similar data. RLC phosphorylation inhibited velocity in blebbistatin at both 30 and 10 degrees C, unlike previous reports where RLC phosphorylation only affected shortening velocities at higher temperatures.

Myosin light chains are not a physiological substrate of AMPK in the control of cell structure changes

The kinetics of myosin regulatory light chain (MLC) phosphorylation by recombinant AMP-activated protein kinase (AMPK) were compared with commercial AMPK from rat liver and smooth muscle myosin light chain kinase (smMLCK). With identical amounts of activity units, initial rates of phosphorylation of MLC were at least 100-fold less with recombinant AMPK compared to smMLCK, whereas with rat liver AMPK significant phosphorylation was seen. In Madin-Darby Canine Kidney cells, AMPK activation led to an increase in MLC phosphorylation, which was decreased by a Rho kinase inhibitor without affecting AMPK activation. Therefore, MLC phosphorylation during energy deprivation does not result from direct phosphorylation by AMPK. Structured summaryMINT-6800264: smMLCK (uniprotkb:P11799) phosphorylates (MI:0217) MLC (uniprotkb:P08590) by protein kinase assay (MI:0424)MINT-6800252: AMPK (uniprotkb:Q13131) phosphorylates (MI:0217) ACC2 (uniprotkb:000763) by protein kinase assay (MI:0424)

Actin Depolymerization Factor/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.

Helicobacter pylori Dysregulation of Gastric Epithelial Tight Junctions by Urease-Mediated Myosin II Activation

Helicobacter pylori-induced gastritis predisposes to the development of gastric cancer. Increased epithelial tight junction permeability and alterations in apical-junctional complexes are also associated with an increased risk of carcinogenesis. Phosphorylation of myosin regulatory light chain (MLC) by MLC kinase (MLCK) regulates tight junction function. We determined whether MLCK was activated by H pylori and defined the mechanisms through which such activation dysregulates gastric epithelial barrier function. MKN28 gastric epithelial cells were cocultured with the H pylori cag(+) strain 60190 or cagA(-), cagE(-), ureB(-), or vacA(-) mutants. MLC phosphorylation and barrier integrity were determined by immunoblot analysis and transepithelial electrical resistance measurements, respectively. Localization of the tight junction protein occludin was determined by immunocytochemistry in MKN28 cells and INS-GAS mice. H pylori induced a progressive loss of barrier function that was attenuated by inactivation of ureB, but not cagA, cagE, or vacA. Reductions in transepithelial electrical resistance were also dependent on functional urease activity. H pylori increased MLC phosphorylation in epithelial monolayers; this was significantly decreased by inhibition of MLCK or Rho kinase or by loss of UreB. H pylori infection of either cultured monolayers or hypergastrinemic INS-GAS mice induced occludin endocytosis, reflecting cytoskeletally mediated disruption of tight junctions. H pylori increases MLC phosphorylation, occludin internalization and barrier dysfunction in gastric epithelial cells. This process requires functional urease activity and is independent of the cag pathogenicity island or VacA. These data provide new insights into the mechanisms by which H pylori disrupts gastric barrier function.

Myosin phosphorylation triggers actin polymerization in vascular smooth muscle

A variety of contractile stimuli increases actin polymerization, which is essential for smooth muscle contraction. However, the mechanism(s) of actin polymerization associated with smooth muscle contraction is not fully understood. We tested the hypothesis that phosphorylated myosin triggers actin polymerization. The present study was conducted in isolated intact or beta-escin-permeabilized rat small mesenteric arteries. Reductions in the 20-kDa myosin regulatory light chain (MLC20) phosphorylation were achieved by inhibiting MLC kinase with ML-7. Increases in MLC20 phosphorylation were achieved by inhibiting myosin light chain phosphatase with microcystin. Isometric force, the degree of actin polymerization as indicated by the F-actin-to-G-actin ratio, and MLC20 phosphorylation were determined. Reductions in MLC20 phosphorylation were associated with a decreased force development and actin polymerization. Increased MLC20 phosphorylation was associated with an increased force generation and actin polymerization. We also found that a heptapeptide that mimics the actin-binding motif of myosin II enhanced microcystin-induced force generation and actin polymerization without affecting MLC20 phosphorylation in beta-escin-permeabilized vessels. Collectively, our data demonstrate that MLC20 phosphorylation is capable of triggering actin polymerization. We further suggest that the binding of myosin to actin triggers actin polymerization and enhances the force development in arterial smooth muscle.

Myosin Regulatory Light Chain Phosphorylation Attenuates Cardiac Hypertrophy

Hyperphosphorylation of myosin regulatory light chain (RLC) in cardiac muscle is proposed to cause compensatory hypertrophy. We therefore investigated potential mechanisms in genetically modified mice. Transgenic (TG) mice were generated to overexpress Ca2+/calmodulin-dependent myosin light chain kinase specifically in cardiomyocytes. Phosphorylation of sarcomeric cardiac RLC and cytoplasmic nonmuscle RLC increased markedly in hearts from TG mice compared with hearts from wild-type (WT) mice. Quantitative measures of RLC phosphorylation revealed no spatial gradients. No significant hypertrophy or structural abnormalities were observed up to 6 months of age in hearts of TG mice compared with WT animals. Hearts and cardiomyocytes from WT animals subjected to voluntary running exercise and isoproterenol treatment showed hypertrophic cardiac responses, but the responses for TG mice were attenuated. Additional biochemical measurements indicated that overexpression of the Ca2+/calmodulin-binding kinase did not perturb other Ca2+/calmodulin-dependent processes involving Ca2+/calmodulin-dependent protein kinase II or the protein phosphatase calcineurin. Thus, increased myosin RLC phosphorylation per se does not cause cardiac hypertrophy and probably inhibits physiological and pathophysiological hypertrophy by contributing to enhanced contractile performance and efficiency.

H89 ( N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) induces reduction of myosin regulatory light chain phosphorylation and inhibits cell proliferation

H89 ( N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) is a compound characterized in vitro as a potent and selective inhibitor of protein kinase A (PKA). In this study, we found that H89 reduced the phosphorylation of the myosin regulatory light chain (MRLC) at Thr-18/Ser-19 and induced disassembly of stress fibers in HeLa cells. In addition, we found that H89 induced not only reduction of the MRLC phosphorylation but also cell growth inhibition in several human cancer cell lines. Recently H89 has been found to inhibit Rho-kinase with potency similar to or greater than that for inhibition of PKA. Indeed, the effects of H89 on both the MRLC phosphorylation and actin cytoskeleton organization were nearly identical to those of Rho-kinase inhibitor Y-27632. However, unlike H89, Y-27632 did not affect cell growth of HeLa cells. Further, when the myosin phosphatase targeting subunit 1 (MYPT1) expression was silenced by RNA interference in HeLa cells, the suppressive effect of H89 on the MRLC phosphorylation was not affected, while H89-induced cell growth inhibition was blocked. These results suggest that H89-induced reduction of the MRLC phosphorylation results from inhibition of Rho-kinase and that H89-induced cell growth inhibition is independent of reduction of the MRLC phosphorylation.

Myosin Light Chain Kinase Is Central to Smooth Muscle Contraction and Required for Gastrointestinal Motility in Mice

Smooth muscle is essential for maintaining homeostasis for many body functions and provides adaptive responses to stresses imposed by pathologic 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 generated 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. We used the Cre-loxP system to establish Mlck floxed mice in which exons 23, 24, and 25 were flanked by 2 loxP sites. Smooth muscle-specific MLCK KO mice were generated by crossing Mlck floxed mice with SM-CreER(T2) (ki) mice followed by tamoxifen treatment. The phenotype was assessed by histologic, biochemical, molecular, cell biological, and physiologic analyses. Targeted deletion of MLCK in adult mouse smooth muscle resulted in severe gut dysmotility characterized by weak peristalsis, dilation of the digestive tract, and reduction of feces excretion and food intake. There was also abnormal urinary bladder function and lower blood pressure. Isolated muscles showed a loss of RLC phosphorylation and force development induced by K(+)-depolarization. The kinase knockout also markedly reduced RLC phosphorylation and force development with acetylcholine which activates Ca(2+)-sensitizing signaling pathways. MLCK and its phosphorylation of RLC are required physiologically for smooth muscle contraction and are essential for normal gastrointestinal motility.

Involvement of 67-kDa laminin receptor-mediated myosin phosphatase activation in antiproliferative effect of epigallocatechin-3- O-gallate at a physiological concentration on Caco-2 colon cancer cells

Previously we reported that 67-kDa laminin receptor (67LR) mediates epigallocatechin-3- O-gallate (EGCG)-induced cell growth inhibition and reduction of myosin regulatory light chain (MRLC) phosphorylation at Thr-18/Ser-19, which is important for cytokinesis. Here, we found that human colon adenocarcinoma Caco-2 cells exhibited higher expression level of 67LR and EGCG at a physiologically achievable concentration (1 μM) significantly accumulated the cells in G 2/M phase without affecting expression of Wnt-signaling components. We also found that myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation at Thr-696, which inhibits myosin phosphatase and promotes MRLC phosphorylation, was reduced in response to 1 μM EGCG. 67LR knockdown by RNA interference abolished the inhibitory effects of 1 μM EGCG on cell cycle progression and the phosphorylation of MRLC and MYPT1. These results suggest that through 67LR, EGCG at a physiological concentration can activate myosin phosphatase by reducing MYPT1 phosphorylation and that may be involved in EGCG-induced cell growth inhibition.

A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles

Phosphorylation of the 20-kDa myosin regulatory light chains (LC(20)) plays a key role in the regulation of smooth muscle contraction. The level of LC(20) phosphorylation is governed by the relative activities of myosin light chain kinase and phosphatase pathways. The regulation of these two pathways differs in different smooth muscle types and in the actions of different vasoactive stimuli. Little is known concerning the regulation of LC(20) phosphorylation in the renal microcirculation. The available pharmacological probes are often nonspecific, and current techniques to directly measure LC(20) phosphorylation are not sensitive enough for quantification in small arterioles. We describe here a novel approach to address this important issue. Using SDS-PAGE with polyacrylamide-bound Mn(2+)-phosphate-binding tag and enhanced Western blot analysis, we were able to detect LC(20) phosphorylation using as little as 5 pg (250 amol) of isolated LC(20). Phosphorylated and unphosphorylated LC(20) were detected in single isolated afferent arterioles, and LC(20) phosphorylation levels could be accurately quantified in pooled samples of three arterioles (<300 cells). The phosphorylation level of LC(20) in the afferent arteriole was 6.8 +/- 1.7% under basal conditions and increased to 34.7 +/- 5.1% and 44.6 +/- 6.6% in response to 30 mM KCl and 10(-8) M angiotensin II, respectively. The application of this technique will enable investigations of the different determinants of LC(20) phosphorylation in afferent and efferent arterioles and provide insights into the signaling pathways that regulate LC(20) phosphorylation in the renal microvasculature under physiological and pathophysiological conditions.