scholarly journals A Novel Mechanism for Activation of Myosin Regulatory Light Chain by Protein Kinase C-Delta in Drosophila

Genetics ◽  
2020 ◽  
Vol 216 (1) ◽  
pp. 177-190
Author(s):  
Pooneh Vaziri ◽  
Danielle Ryan ◽  
Christopher A. Johnston ◽  
Richard M. Cripps
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Flight Muscle ◽  
Wild Type ◽  
Hypomorphic Allele ◽  
Regulatory Myosin Light Chain ◽  
Kinase C ◽  
Myosin Light Chain 2 ◽  
Direct Target ◽  
And Function

Myosin is an essential motor protein, which in muscle is comprised of two molecules each of myosin heavy-chain (MHC), the essential or alkali myosin light-chain 1 (MLC1), and the regulatory myosin light-chain 2 (MLC2). It has been shown previously that MLC2 phosphorylation at two canonical serine residues is essential for proper flight muscle function in Drosophila; however, MLC2 is also phosphorylated at additional residues for which the mechanism and functional significance is not known. We found that a hypomorphic allele of Pkcδ causes a flightless phenotype; therefore, we hypothesized that PKCδ phosphorylates MLC2. We rescued flight disability by duplication of the wild-type Pkcδ gene. Moreover, MLC2 is hypophosphorylated in Pkcδ mutant flies, but it is phosphorylated in rescued animals. Myosin isolated from Pkcδ mutant flies shows a reduced actin-activated ATPase activity, and MLC2 in these myosin preparations can be phosphorylated directly by recombinant human PKCδ. The flightless phenotype is characterized by a shortened and disorganized sarcomere phenotype that becomes apparent following eclosion. We conclude that MLC2 is a direct target of phosphorylation by PKCδ, and that this modification is necessary for flight muscle maturation and function.

α1-Adrenoceptor-Gq-RhoA signaling is upregulated to increase myofibrillar Ca2+ sensitivity in failing hearts

2001 ◽  
Vol 281 (2) ◽  
pp. H637-H646 ◽  
Author(s):  
Nobuhiro Suematsu ◽  
Shinji Satoh ◽  
Shintaro Kinugawa ◽  
Hiroyuki Tsutsui ◽  
Shunji Hayashidani ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Expression ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Adrenergic Stimulation ◽  
Regulatory Myosin Light Chain ◽  
Kinase C

α1-Adrenergic stimulation, coupled to Gq, has been shown to promote heart failure. However, the role of α1-adrenergic signaling in the regulation of myocardial contractility in failing myocardium is still poorly understood. To investigate this, we observed 1) the effect of phenylephrine on myofibrillar Ca2+ sensitivity in α-toxin-skinned cardiomyocytes, and 2) protein expression of Gq, RhoA, and myosin light chain phosphorylation using tachypacing-induced canine failing hearts. Phenylephrine significantly increased myofibrillar Ca2+ sensitivity in failing but not in normal cardiomyocytes. Whereas Y-27632 (Rho kinase inhibitor) blocked the phenylephrine-induced Ca2+ sensitization in the failing myocytes, calphostin C (protein kinase C inhibitor) had no effect on Ca2+ sensitization. The protein expression of Gαq and RhoA and the phosphorylation level of regulatory myosin light chain significantly increased in the failing myocardium. Our results suggest that α1-adrenoceptor-Gq signaling is upregulated in the failing myocardium to increase the myofibrillar Ca2+sensitivity mainly through the RhoA-Rho kinase pathway rather than through the protein kinase C pathway.

scholarly journals Hypoxic modulation of fetal vascular MLCK abundance, localization, and function

2021 ◽  
Vol 320 (1) ◽  
pp. R1-R18
Author(s):  
Dane W. Sorensen ◽  
Desirelys Carreon ◽  
James M. Williams ◽  
William J. Pearce
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Carotid Arteries ◽  
Chronic Hypoxia ◽  
Contractile Apparatus ◽  
Smooth Muscle Contractility ◽  
Heterogeneous Distribution ◽  
Regulatory Myosin Light Chain ◽  
And Function

Changes in vascular contractility are among the most important physiological effects of acute and chronic fetal hypoxia. Given the essential role of myosin light-chain kinase (MLCK) in smooth muscle contractility and its heterogeneous distribution, this study explores the hypothesis that subcellular changes in MLCK distribution contribute to hypoxic modulation of fetal carotid artery contractility. Relative to common carotid arteries from normoxic term fetal lambs (FN), carotids from fetal lambs gestated at high altitude (3,802 m) (FH) exhibited depressed contractility without changes in MLCK mRNA or protein abundance. Patterns of confocal colocalization of MLCK with α-actin and 20-kDa regulatory myosin light chain (MLC20) enabled calculation of subcellular MLCK fractions: 1) colocalized with the contractile apparatus, 2) colocalized with α-actin distant from the contractile apparatus, and 3) not colocalized with α-actin. Chronic hypoxia did not affect MLCK abundance in the contractile fraction, despite a concurrent decrease in contractility. Organ culture for 72 h under 1% O2 decreased total MLCK abundance in FN and FH carotid arteries, but decreased the contractile MLCK abundance only in FH carotid arteries. Correspondingly, culture under 1% O2 depressed contractility more in FH than FN carotid arteries. In addition, hypoxia appeared to attenuate ubiquitin-independent proteasomal degradation of MLCK, as reported for other proteins. In aggregate, these results demonstrate that the combination of chronic hypoxia followed by hypoxic culture can induce MLCK translocation among at least three subcellular fractions with possible influences on contractility, indicating that changes in MLCK distribution are a significant component of fetal vascular responses to hypoxia.

Effect of phorbol esters on Ca2+ sensitivity and myosin light-chain phosphorylation in airway smooth muscle

1998 ◽  
Vol 274 (5) ◽  
pp. C1253-C1260 ◽  
Author(s):  
Dorothee H. Bremerich ◽  
Tetsuya Kai ◽  
David O. Warner ◽  
Keith A. Jones
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Isometric Force ◽  
Calphostin C ◽  
Regulatory Myosin Light Chain ◽  
Kinase C ◽  
The Relationship

We studied in β-escin-permeabilized canine tracheal smooth muscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol 12,13-dibutyrate (PDBu) on isometric force at a constant submaximal Ca2+ concentration (i.e., the effect on Ca2+ sensitivity) and regulatory myosin light-chain (rMLC) phosphorylation. PDBu increased Ca2+sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered the relationship between rMLC phosphorylation and isometric force such that the increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC, PKC peptide-(19—31) (PSSI), and staurosporine] on PDBu-induced Ca2+ sensitization as well as the effect of calphostin C and PSSI on rMLC phosphorylation were determined. Whereas none of these compounds prevented or reversed the PDBu-induced increase in Ca2+sensitivity, the PDBu-induced increase in rMLC phosphorylation was inhibited. We conclude that PDBu increases rMLC phosphorylation by activation of PKC but that the associated PDBu-induced increases in Ca2+ sensitivity are mediated by mechanisms other than activation of PKC in permeabilized airway smooth muscle.

scholarly journals Role of protein kinase C in the phosphorylation of cardiac myosin light chain 2

1993 ◽  
Vol 294 (2) ◽  
pp. 401-406 ◽  
Author(s):  
R C Venema ◽  
R L Raynor ◽  
T A Noland ◽  
J F Kuo
Keyword(s):  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Heart Cells ◽  
Adult Rat ◽  
Kinase C ◽  
Myosin Light Chain 2

The role of protein kinase C (PKC) in the phosphorylation of myosin light chain 2 (MLC2) in adult rat heart cells has been investigated. PKC-mediated phosphorylation of MLC2 in adult rat cardiac myofibrils in vitro occurs with a stoichiometry (0.7 mol of phosphate/mol of protein) similar to that mediated by myosin light chain kinase (MLCK). Two-dimensional tryptic phosphopeptide mapping of MLC2 following phosphorylation by PKC or MLCK in vitro yields the same major phosphopeptides for each protein kinase. These sites are also 32P-labelled in situ when isolated cardiomyocytes are incubated with [32P]P(i). 32P labelling of MLC2 in cardiomyocytes is increased by 5-fold in 10 min upon incubation with the phosphatase inhibitor calyculin A, demonstrating the existence of a rapidly turning over component of MLC2 phosphorylation in these cells. 32P label is completely removed from MLC2 when myocytes are exposed to 2,3-butanedione monoxime, an inhibitor of cardiac contraction known to desensitize the myofilaments to activation by Ca2+. 32P labelling of MLC2 is also decreased by 50-100% following exposure to the PKC-selective inhibitors calphostin C and chelerythrine, suggesting that PKC, and not MLCK, is primarily responsible for incorporation of rapidly turning over phosphate into MLC2 in situ. Taken together, these data implicate PKC in the phosphorylation of MLC2 in heart cells and support the hypothesis that phosphorylation of cardiac MLC2 has a role in determining myofibrillar Ca2+ sensitivity.

scholarly journals Myosin light chain-2 mutation affects flight, wing beat frequency, and indirect flight muscle contraction kinetics in Drosophila.

1992 ◽  
Vol 119 (6) ◽  
pp. 1523-1539 ◽  
Author(s):  
J Warmke ◽  
M Yamakawa ◽  
J Molloy ◽  
S Falkenthal ◽  
D Maughan
Keyword(s):  
Myosin Light Chain ◽  
Flight Muscle ◽  
Beat Frequency ◽  
Indirect Flight Muscle ◽  
Wing Beat ◽  
Wild Type ◽  
Wing Beat Frequency ◽  
Myosin Light Chain 2 ◽  
Kinetics Of ◽  
Contraction Kinetics

We have used a combination of classical genetic, molecular genetic, histological, biochemical, and biophysical techniques to identify and characterize a null mutation of the myosin light chain-2 (MLC-2) locus of Drosophila melanogaster. Mlc2E38 is a null mutation of the MLC-2 gene resulting from a nonsense mutation at the tenth codon position. Mlc2E38 confers dominant flightless behavior that is associated with reduced wing beat frequency. Mlc2E38 heterozygotes exhibit a 50% reduction of MLC-2 mRNA concentration in adult thoracic musculature, which results in a commensurate reduction of MLC-2 protein in the indirect flight muscles. Indirect flight muscle myofibrils from Mlc2E38 heterozygotes are aberrant, exhibiting myofilaments in disarray at the periphery. Calcium-activated Triton X-100-treated single fiber segments exhibit slower contraction kinetics than wild type. Introduction of a transformed copy of the wild type MLC-2 gene rescues the dominant flightless behavior of Mlc2E38 heterozygotes. Wing beat frequency and single fiber contraction kinetics of a representative rescued line are not significantly different from those of wild type. Together, these results indicate that wild type MLC-2 stoichiometry is required for normal indirect flight muscle assembly and function. Furthermore, these results suggest that the reduced wing beat frequency and possibly the flightless behavior conferred by Mlc2E38 is due in part to slower contraction kinetics of sarcomeric regions devoid or partly deficient in MLC-2.

The expression of the regulatory myosin light chain 2 gene during mouse embryogenesis

Development ◽  
1993 ◽  
Vol 118 (3) ◽  
pp. 919-929 ◽  
Author(s):  
A. Faerman ◽  
M. Shani
Keyword(s):  
Skeletal Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Fast Skeletal Muscle ◽  
Mouse Embryogenesis ◽  
Skeletal Muscle Myosin ◽  
Adult Mice ◽  
Regulatory Myosin Light Chain ◽  
Myosin Light Chain 2 ◽  
Muscle Groups

The fast skeletal muscle myosin light chain 2 (MLC2) gene is expressed specifically in skeletal muscles of newborn and adult mice, and has no detectable sequence homology with any of the other MLC genes including the slow cardiac MLC2 gene. The expression of the fast skeletal muscle MLC2 gene during early mouse embryogenesis was studied by in situ hybridization. Serial sections of embryos from 8.5 to 12.5 days post coitum (d.p.c.) were hybridized to MLC2 cRNA and to probes for the myogenic regulatory genes MyoD1 and myogenin. The results revealed different temporal and spatial patterns of hybridization for different muscle groups. MLC2 transcripts were first detected 9.5 d.p.c. in the myotomal regions of rostral somites, already expressing myogenin. Surprisingly, at the same stage, a weak MLC2 signal was also detected in the cardiomyocytes. The cardiac expression was transient and could not be detected at later stages while the myotomal signal persisted and spread to the more caudal somites, very similar to the expression of myogenin. Beginning from 10.5 d.p.c., several extramyotomal premuscle cells masses have been demarcated by MyoD1 expression. MLC2 transcripts were detected in only one of these cell masses. Although, transcripts of myogenin were detected in all these cell masses, the number of expressing cells was significantly lower than that observed for MyoD1. By 11.5 d.p.c., all three hybridization signals colocalized in most extramyotomal muscle-forming regions, with the exception of the diaphragm and the hindlimb buds, where only few cells expressed MLC2 and more cells expressed MyoD1 than myogenin. At 12.5 d.p.c., all three studied genes displayed a similar spatial pattern of expression in most muscle-forming regions. However, in some muscles, the MyoD1 signal spread over more cells compared to myogenin or MLC2. Our results are consistent with the suggestion that multiple myogenic programs exist for myoblasts differentiating in the myotome and extramyotomal regions.

Propofol Increases Phosphorylation of Troponin I and Myosin Light Chain 2 via  Protein Kinase C Activation in Cardiomyocytes

2003 ◽  
Vol 98 (6) ◽  
pp. 1363-1371 ◽  
Author(s):  
Noriaki Kanaya ◽  
Brad Gable ◽  
Paul A. Murray ◽  
Derek S. Damron
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Adenosine Triphosphatase ◽  
Myosin Light Chain ◽  
Troponin I ◽  
Adenosine Triphosphatase Activity ◽  
Kinase C ◽  
Myosin Light Chain 2 ◽  
Pkc Alpha

Background Troponin I (TnI) and myosin light chain 2 (MLC2) are important myofibrillar proteins involved in the regulation of myofilament calcium (Ca2+) sensitivity and cardiac inotropy. The objectives of this study were to determine the role of protein kinase C (PKC) in mediating propofol-induced changes in actomyosin adenosine triphosphatase activity in cardiac myofibrils and to examine the extent to which propofol alters the phosphorylation of TnI and MLC2 in cardiomyocytes. Methods Freshly isolated adult rat ventricular myocytes were used for the study. Cardiac myofibrils were extracted for assessment of actomyosin adenosine triphosphatase activity and phosphorylation of TnI and MLC2. Western blot analysis for PKC-alpha was performed on cardiomyocyte subcellular fractions. Simultaneous measurement of intracellular free Ca2+ concentration ([Ca2+](i)) and myocyte shortening was assessed using fura-2 and video edge detection, respectively. Results Propofol (30 microM) reduced the Ca2+ concentration required for activation of actomyosin adenosine triphosphatase activity, and this effect was abolished by bisindolylmaleimide I. In addition, propofol stimulated dose-dependent phosphorylation of TnI and MLC2. PKC activation with phorbol myristic acetate also stimulated an increase in phosphorylation of TnI and MLC2. The actions of propofol and phorbol myristic acetate together on phosphorylation of TnI and MLC2 were not additive. PKC inhibition with bisindolylmaleimide I attenuated phorbol myristic acetate- and propofol-induced phosphorylation of TnI and MLC2. Propofol stimulated translocation of PKC-alpha from cytosolic to membrane fraction. Propofol caused a shift in the extracellular Ca2+-shortening relationship, and this effect was abolished by bisindolylmaleimide I. Conclusions These results suggest that propofol increases myofilament Ca2+ sensitivity via a PKC-dependent pathway involving the phosphorylation of MLC2.

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