Restoration of Phosphorylation-dependent Regulation to the Skeletal Muscle Myosin Regulatory Light Chain

Yang, Zhaohui; Sweeney, H. Lee

doi:10.1074/jbc.270.42.24646

Cited by 37 publications

(26 citation statements)

References 21 publications

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“…These mechanisms participate also in a variety of acto-myosin based cellular functions of non-muscle cells, including cell migration and invasion, stress fiber and focal adhesion formation, neurite retraction, and membrane ruffling (Narumiya et al, 1997). More recently, it has been shown that phosphorylation of MLC, although not necessary for activating the contractile apparatus in striated skeletal/cardiac fibers, may play an important modulatory role in the muscle force development (Yang and Sweeney, 1995;Davis et al, 2001;Suematsu et al, 2001;Szczesna et al, 2002). In fact, MLC phosphorylation has been shown to correlate with potentiation of force development in skeletal muscle and with positive inotropic response of the heart (Andersen et al, 2002;Davis et al, 2002).…”

mentioning

confidence: 98%

Sphingosine 1‐phosphate induces cell contraction via calcium‐independent/Rho‐dependent pathways in undifferentiated skeletal muscle cells

Formigli

Meacci²,

Vassalli

et al. 2003

Journal Cellular Physiology

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We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca(2+) mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca(2+)-independent mechanisms of cell contraction have been the focus of numerous studies on Ca(2+) sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca(2+)-independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca(2+), by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca(2+) transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca(2+) and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC alpha and PKC delta, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca(2+)-independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction.

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mentioning

confidence: 98%

Sphingosine 1‐phosphate induces cell contraction via calcium‐independent/Rho‐dependent pathways in undifferentiated skeletal muscle cells

Formigli

Meacci²,

Vassalli

et al. 2003

Journal Cellular Physiology

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“…Phosphorylation of HMM WT and mutant constructs was performed as described in ref. 41. Both unphosphorylated and phosphorylated forms of WT and mutant human smooth myosin 2 HMM constructs were assayed at 0.2 nM final concentration.…”

Section: Construction Expression and Purification Of Human Smooth Mmentioning

confidence: 99%

Unregulated smooth-muscle myosin in human intestinal neoplasia

Alhopuro¹,

Phichith

Tuupanen³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt. Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia.colorectal cancer ͉ microsatellite instability ͉ Peutz-Jeghers syndrome ͉ smmhc/myh11 G ermline mutations in human myosin heavy chain genes contribute to multiple inherited human diseases (1). In addition, smooth-muscle myosin (MYH11) participates in formation of an oncogenic fusion gene CBFB/MYH11 in acute myeloid leukaemia (2).Microsatellite instability (MSI) occurs in Ϸ15% of colorectal cancers (CRC), and is a consequence of defects in the DNA mismatch repair (MMR) system (3). Mutations occurring during DNA replication are not repaired, resulting in genetic instability that particularly affects short repetitive DNA sequences. Mutations that result in malignant progression are selected and thus detected in surgically removed MSI cancers. Passenger mutations also occur frequently in this mutator tumor type (4-6).By examining gene expression data (7), we had identified MYH11 as a gene with reduced expression in MSI CRCs compared with normal colon samples. The reduced expression of MYH11 in CRCs could simply reflect a larger smooth-muscle component in our normal colon specimens. Nevertheless, the differential expression prompted us to examine whether MYH11 has a mononucleotide tract in the coding sequence, because mononucleotide tracts are prone to mutations under MSI. Two splice variants, SM1 and SM2, which are distinct in the Cterminal tailpiece, are produced from the MYH11 gene. Indeed, the SM2 isoform of MYH11 was observed to harbor a mononucleotide repeat of 8 cytosines (C8). Thus, MYH11 was identified as a candidate MSI colon cancer gene. Dominant-negative germ-line mutations in MYH11 occur in a syndrome predisposing to thoracic aortic aneurysm/aortic dissection (TAAD) and patent ductus arteriosus (8). The TAAD phenotype closely resembles the on...

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“…However, a hybrid smooth muscle HMM containing the smooth RLC and skeletal essential light chain was well regulated in the actin filament motility assay, as was an essential light chain-less molecule (7), refuting the role of the latter subunit. Other studies focusing on the RLC showed that complete activation of smooth muscle myosin requires specific arrangements of charge both at the N terminus (8,9) and C terminus of the molecule (10). However, the manner in which these charged groups contribute to activation remains unknown.…”

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confidence: 99%

A Long, Weakly Charged Actin-binding Loop Is Required for Phosphorylation-dependent Regulation of Smooth Muscle Myosin

Rovner

1998

Journal of Biological Chemistry

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Chimeric substitution of the weak actin-binding loop (ABL) from chicken skeletal muscle myosin for that of gizzard smooth muscle heavy meromyosin (HMM) causes activation of the dephosphorylated mutant (SABL HMM; Rovner, A. S., Freyzon, Y., and Trybus, K. M. (1995) J. Biol. Chem. 270, 30260 -30263). The present study determined whether this loss of regulation is due to the greater positive charge density (5 versus 3 clustered lysine residues) or lesser length (14 versus 26 residues) of the mutant ABL. Charge augmentation had little effect on regulation of expressed mutants, but elimination of the 12 N-terminal amino acids from the wild-type ABL significantly increased actin-activated ATPase activity of the dephosphorylated relative to the phosphorylated molecule while conferring the ability to move actin filaments in vitro on the former. Addition of the same 12 residues to the SABL mutant increased the ratio of phosphorylated to dephosphorylated ATPase activity while imparting wild type-like regulation to motility. However, full actin activation of dephosphorylated ATPase activity required both the shorter length and greater positive charge density found in the SABL loop. These results demonstrate that, compared with skeletal, both the greater length and lesser positive charge density of the smooth muscle myosin ABL are required for proper phosphorylation-mediated regulation of the molecule.Unlike the myosins from striated muscle, vertebrate smooth muscle myosin requires phosphorylation of the regulatory light chain (RLC) 1 to activate its enzymatic and mechanical activities (reviewed in Ref. 1). Early theories suggested that activation of the molecule is caused by a change in shape that occurs in response to RLC phosphorylation (2, 3), but this idea was refuted by separating its effects on molecular folding and activity (4). It is now widely accepted that phosphorylation activates the molecule by accelerating the rate-limiting step of the acto-myosin ATPase cycle independent of any gross changes in shape (5).Since the discovery of this "thick filament-linked" form of regulation, there have been intensive efforts to determine the mechanism whereby RLC phosphorylation in the neck region of myosin causes activation of the 80-angstrom distant nucleotide-binding cleft. Experiments with chimeric RLCs suggested that the regulatory signal is propagated to the active site by physical overlap between the RLC and the essential light chain (6). However, a hybrid smooth muscle HMM containing the smooth RLC and skeletal essential light chain was well regulated in the actin filament motility assay, as was an essential light chain-less molecule (7), refuting the role of the latter subunit. Other studies focusing on the RLC showed that complete activation of smooth muscle myosin requires specific arrangements of charge both at the N terminus (8, 9) and C terminus of the molecule (10). However, the manner in which these charged groups contribute to activation remains unknown.Additional important information shows that proper re...

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Restoration of Phosphorylation-dependent Regulation to the Skeletal Muscle Myosin Regulatory Light Chain

Cited by 37 publications

References 21 publications

Sphingosine 1‐phosphate induces cell contraction via calcium‐independent/Rho‐dependent pathways in undifferentiated skeletal muscle cells

Sphingosine 1‐phosphate induces cell contraction via calcium‐independent/Rho‐dependent pathways in undifferentiated skeletal muscle cells

Unregulated smooth-muscle myosin in human intestinal neoplasia

A Long, Weakly Charged Actin-binding Loop Is Required for Phosphorylation-dependent Regulation of Smooth Muscle Myosin

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