Phosphorylation site sequence of smooth muscle myosin light chain (<i>M</i><sub>r</sub> = 20 000)

Pearson, Richard B.; Jakes, Ross; John, M.; Kendrick‐Jones, John; Kemp, Bruce E.

doi:10.1016/0014-5793(84)80216-1

Cited by 98 publications

(39 citation statements)

References 15 publications

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“…1). All MRLCs were highly homologous with each other, and showed more than 90% amino acid identity to that from chicken smooth muscle (Pearson et al, 1984). Moreover, the phosphorylation sites of MRLC by MLCK were well conserved among these three MRLC isoforms (Fig.…”

Section: Resultsmentioning

confidence: 84%

Diphosphorylated MRLC is Required for Organization of Stress Fibers in Interphase Cells and the Contractile Ring in Dividing Cells.

Iwasaki

Murata‐Hori

Ishitobi

et al. 2001

Cell Struct. Funct.

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ABSTRACT. Activity of nonmuscle myosin II is regulated by phosphorylation of its regulatory light chain (MRLC). Phosphoryration of MRLC at both Thr18 and Ser19 (diphosphorylation) results in higher MgATPase activity and in promotion of the assembly of myosin II filaments than does that of MRLC at Ser19 (monophosphorylation) in vitro. To determine the roles of the diphosphorylated MRLC in vivo, we transfected three kinds of MRLC mutants, unphosphorylated, monophosphorylated and diphosphorylated forms (MRLC2 T18AS19A , substitution of both Ser19 and Thr18 by Ala; MRLC2 T18AS19D , Ser19 by Asp and Thr18 by Ala; and MRLC2 T18DS19D , both Ser19 and Thr18 by Asp, respectively), into HeLa cells. Cells overexpressing the mutant MRLC2 T18DS19D contained a larger number of actin filament bundles than did those overexpressing the mutant MRLC2 T18AS19D . Moreover, cells overexpressing the nonphosphorylatable mutant MRLC2 T18AS19A showed a decrease in the number of actin filament bundles. Taken together, our data suggest that diphosphorylation of MRLC plays an important role in regulating actin filament assembly and reorganization in nonmuscle cells. Key words: phosphorylation/myosin II filament/myosin II regulatory light chain/cytokinesisMyosin II is an essential protein to generate force for cell locomotion (Berlot et al., 1985(Berlot et al., , 1987 and cytokinesis (Mabuchi and Okuno, 1977;De Lozanne and Spudich, 1987). In smooth muscle and vertebrate nonmuscle cells, myosin II activity is both negatively and positively regulated by phosphorylation of its regulatory light chain (MRLC). The phosphorylation sites of MRLC by Ca 2+ /calmodulindependent myosin light chain kinase (MLCK) are mainly Ser19 (monophosphorylation) and, under some conditions, both Ser19 and Thr18 (diphosphorylation) in vitro (Ikebe and Hartshorne, 1985;Ikebe et al., 1986). Phosphorylation of MRLC by MLCK enhances the actin activated Mg ATPase activity (Sellers et al., 1981;Trybus, 1989) and promotes the assembly of myosin II filaments (Suzuki et al., 1978). These effects are more effective when MRLC is diphosphorylated (Ikebe et al., 1988). On the other hand, protein kinase C (PKC) phosphorylates MRLC at Ser1/2 and Thr9. Phosphorylation of MRLC by PKC down-regulate the MgATPase activity of myosin II phosphorylated by MLCK (Nishikawa et al., 1984;Ikebe et al., 1987).It has been reported that substitution of amino acid residues on phosphorylation sites of MRLC affects the biochemical properties of myosin II in vitro. Substitution of both Ser19 and Thr18 to Asp, which mimics the diphosphorylated MRLC, exerted the actin activated MgATPase activity of myosin II and formation of myosin II filaments (Kamisoyama et al., 1994;Sweeney et al., 1994).In this study, we attempted to determine the roles of diphosphorylated MRLC in vivo. To address this, we transfected three kinds of MRLC mutants, which mimic nonphosphorylated, monophosphorylated and diphosphorylated MRLC, into HeLa cells and compared their localization in both interphase and mitotic cells. Materials ...

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Section: Resultsmentioning

confidence: 84%

Diphosphorylated MRLC is Required for Organization of Stress Fibers in Interphase Cells and the Contractile Ring in Dividing Cells.

Iwasaki

Murata‐Hori

Ishitobi

et al. 2001

Cell Struct. Funct.

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“…MLCK can also phosphorylate Thr18 of rMLC, but this occurs at a 1,000-fold lower rate. MLCK does not phosphorylate other sites of rMLC, the heavy chain, or the essential light chain (2,19). Consequently, the rate of 32 P release from this phosphorylated myosin only comes from the rMLC, and therefore represents the specific MLCP activity.…”

Section: Protein Phosphatase and Kinase Activity Assaymentioning

confidence: 99%

Human Airway Contraction and Formoterol-Induced Relaxation Is Determined by Ca²⁺ Oscillations and Ca²⁺ Sensitivity

Ressmeyer¹,

Bai²,

Delmotte³

et al. 2010

Am J Respir Cell Mol Biol

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The etiology of airway hyperresponsiveness associated with asthma requires an understanding of the regulatory mechanisms mediating human airway smooth muscle cell (SMC) contraction. The objective of this study was to determine how human airway SMC contraction (induced by histamine) and relaxation (induced by formoterol) are regulated by Ca 21 oscillations and Ca 21 sensitivity. The responses of human small airways and their associated SMCs were studied in human lung slices cut from agarose-inflated lungs. Airway contraction was measured with phase-contrast video microscopy. Ca 21 signaling and Ca 21 sensitivity of airway SMCs were measured with two-photon fluorescence microscopy and Ca 21 -permeabilized lung slices. The agonist histamine induced contraction of human small airways by stimulating both an increase in intracellular Ca 21 concentration in the SMCs in the form of oscillatory Ca 21 waves and an increase in Ca 21 sensitivity. The frequency of the Ca 21 oscillations increased with histamine concentration, and correlated with increased contraction. Formoterol induced airway relaxation at low concentrations by initially decreasing SMC Ca 21 sensitivity. At higher concentrations, formoterol additionally slowed or inhibited the Ca 21 oscillations of the SMCs to relax the airways. The action of formoterol was only slowly reversed. Human lung slices provide a powerful experimental assay for the investigation of small airway physiology and pharmacology. Histamine induces contraction by simultaneously increasing SMC Ca 21 signaling and Ca 21 sensitivity. Formoterol induces long-lasting relaxation by initially reducing the Ca 21 sensitivity and, subsequently, the frequency of the Ca 21 oscillations of the airway SMCs.Keywords: lung slice; smooth muscle cell; two-photon microscopy; hyperresponsiveness; histamine Because increased airway smooth muscle cell (SMC) contraction is a major characteristic of airway hyperresponsiveness (AHR) associated with asthma (1), a prerequisite to address AHR is an understanding of the mechanisms that regulate airway SMC contraction. It is well established that airway SMC contraction is regulated by the phosphorylation of the regulatory myosin light chain (rMLC) (2, 3) to induce its interaction with adjacent actin filaments via the classical mechanism of cross-bridge cycling to generate force (4). (5)(6)(7)(8). Although the frequency of these Ca 21 oscillations has been found to increase with agonist concentration, and that this correlates with increased contraction, the frequency of the Ca 21 oscillations generated by similar agonist concentrations differs considerably between species (9).To induce SMC relaxation, the increase in [Ca 21 ] i is generally reversed to inactivate MLCK, but the cessation of the cross-bridge cycle requires that the rMLC is dephosphorylated; this reaction is mediated by MLC phosphatase (MLCP). With the exception of mice (10), the activity of MLCP appears to be Ca 21 independent, but is regulated by the same agonists that stimulate increases in [Ca 21 ] ...

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“…We obtained further evidence that Mbs acts through Sqh by looking at the effects of sqh transgenes with phosphorylation site mutations on the Mbs mutant phenotype. Previous biochemical and genetic studies have shown that serine 21 and threonine 20 are the conserved primary and secondary phosphorylation sites, respectively (Pearson et al, 1984; a The number of nonbalancer flies reaching pupation or adulthood is shown over the total number of flies at that stage. Survival is shown as a percentage of the expected number of nonbalancer flies in parentheses.…”

Section: Mbs Mediates Its Effects On the Eye Through Sqh Dephosphorylmentioning

confidence: 99%

Excessive Myosin Activity inMbsMutants Causes Photoreceptor Movement Out of theDrosophilaEye Disc Epithelium

Lee

Treisman

2004

MBoC

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Neuronal cells must extend a motile growth cone while maintaining the cell body in its original position. In migrating cells, myosin contraction provides the driving force that pulls the rear of the cell toward the leading edge. We have characterized the function of myosin light chain phosphatase, which down-regulates myosin activity, in Drosophila photoreceptor neurons. Mutations in the gene encoding the myosin binding subunit of this enzyme cause photoreceptors to drop out of the eye disc epithelium and move toward and through the optic stalk. We show that this phenotype is due to excessive phosphorylation of the myosin regulatory light chain Spaghetti squash rather than another potential substrate, Moesin, and that it requires the nonmuscle myosin II heavy chain Zipper. Myosin binding subunit mutant cells continue to express apical epithelial markers and do not undergo ectopic apical constriction. In addition, mutant cells in the wing disc remain within the epithelium and differentiate abnormal wing hairs. We suggest that excessive myosin activity in photoreceptor neurons may pull the cell bodies toward the growth cones in a process resembling normal cell migration. INTRODUCTIONThe cytoskeleton plays a variety of roles during development, allowing cells to change their shape, adhesive properties, and motility (Jamora and Fuchs, 2002). Two critical components of the cytoskeleton are actin and nonmuscle myosin II. The contractile activity of actomyosin complexes has been implicated in cell migration, epithelial sheet movements, cytokinesis, axon outgrowth, and cell adhesion (Maciver, 1996;Jacinto et al., 2002;Dent and Gertler, 2003). During migration of several cell types, myosin activity is required to retract the rear of the cell (Ridley et al., 2003).Nonmuscle myosin II consists of a hexamer of two myosin heavy chains (MHC), two myosin light chains (MLC), and two myosin regulatory light chains (MRLC) (Korn and Hammer, 1988). Phosphorylation of key serine and threonine residues on MRLC stimulates the ATPase activity of MHC and promotes its assembly into filaments, leading to stress fiber contraction (Adelstein and Conti, 1975;Craig et al., 1983;Umemoto et al., 1989;Katoh et al., 2001). Mutations in the Drosophila orthologs of these myosin subunits have provided insight into the developmental functions of myosin II. Mutations in zipper (zip), which encodes MHC, cause defects in cytokinesis, closure of the dorsal embryonic epidermis over the amnioserosa, axon patterning, and myofibril formation (Zhao et al., 1988;Young et al., 1993;Bloor and Kiehart, 2001). spaghetti squash (sqh), encoding MRLC, is required for cytokinesis, oogenesis, and imaginal disc eversion (Karess et al., 1991;Wheatley et al., 1995;Edwards and Kiehart, 1996;Jordan and Karess, 1997).Actin-binding proteins of the ezrin, radixin, and moesin (ERM) family are thought to link transmembrane proteins to the actin cytoskeleton (Bretscher, 1999). ERM proteins are activated by phosphorylation of a conserved threonine residue, which inhibits associati...

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Phosphorylation site sequence of smooth muscle myosin light chain (M_r = 20 000)

Cited by 98 publications

References 15 publications

Diphosphorylated MRLC is Required for Organization of Stress Fibers in Interphase Cells and the Contractile Ring in Dividing Cells.

Diphosphorylated MRLC is Required for Organization of Stress Fibers in Interphase Cells and the Contractile Ring in Dividing Cells.

Human Airway Contraction and Formoterol-Induced Relaxation Is Determined by Ca²⁺ Oscillations and Ca²⁺ Sensitivity

Excessive Myosin Activity inMbsMutants Causes Photoreceptor Movement Out of theDrosophilaEye Disc Epithelium

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Phosphorylation site sequence of smooth muscle myosin light chain (Mr = 20 000)

Cited by 98 publications

References 15 publications

Diphosphorylated MRLC is Required for Organization of Stress Fibers in Interphase Cells and the Contractile Ring in Dividing Cells.

Diphosphorylated MRLC is Required for Organization of Stress Fibers in Interphase Cells and the Contractile Ring in Dividing Cells.

Human Airway Contraction and Formoterol-Induced Relaxation Is Determined by Ca2+ Oscillations and Ca2+ Sensitivity

Excessive Myosin Activity inMbsMutants Causes Photoreceptor Movement Out of theDrosophilaEye Disc Epithelium

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Phosphorylation site sequence of smooth muscle myosin light chain (M_r = 20 000)

Human Airway Contraction and Formoterol-Induced Relaxation Is Determined by Ca²⁺ Oscillations and Ca²⁺ Sensitivity