Rap1 Activation in Collagen Phagocytosis Is Dependent on Nonmuscle Myosin II-A

Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or β1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.

Download Full-text

Anillin Binds Nonmuscle Myosin II and Regulates the Contractile Ring

Molecular Biology of the Cell ◽

10.1091/mbc.e04-08-0758 ◽

2005 ◽

Vol 16 (1) ◽

pp. 193-201 ◽

Cited By ~ 177

Author(s):

Aaron F. Straight ◽

Christine M. Field ◽

Timothy J. Mitchison

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Cultured Cells ◽

Contractile Activity ◽

Myosin Ii ◽

Human Cells ◽

Myosin Light Chain Phosphorylation ◽

Nonmuscle Myosin ◽

Contractile Ring ◽

Nonmuscle Myosin Ii

We demonstrate that the contractile ring protein anillin interacts directly with nonmuscle myosin II and that this interaction is regulated by myosin light chain phosphorylation. We show that despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. We propose a role for anillin in spatially regulating the contractile activity of myosin II during cytokinesis.

Download Full-text

Increased Phosphorylation of Myosin Light Chain Prevents in Vitro Decidualization

Endocrinology ◽

10.1210/en.2006-1673 ◽

2007 ◽

Vol 148 (7) ◽

pp. 3176-3184 ◽

Cited By ~ 21

Author(s):

Ivanna Ihnatovych ◽

WenYang Hu ◽

Jody L. Martin ◽

Asgerally T. Fazleabas ◽

Primal de Lanerolle ◽

...

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Smooth Muscle Actin ◽

Myosin Ii ◽

Active Form ◽

Adenoviral Infection ◽

Decidual Cells ◽

Cytoskeletal Dynamics ◽

Mlc20 Phosphorylation

Differentiation of stromal cells into decidual cells, which is critical to successful pregnancy, represents a complex transformation requiring changes in cytoskeletal architecture. We demonstrate that in vitro differentiation of human uterine fibroblasts into decidual cells includes down-regulation of α-smooth muscle actin and β-tubulin, phosphorylation of focal adhesion kinase, and redistribution of vinculin. This is accompanied by varied adhesion to fibronectin and a modified ability to migrate. Cytoskeletal organization is determined primarily by actin-myosin II interactions governed by the phosphorylation of myosin light chain (MLC20). Decidualization induced by cAMP [with estradiol-17β (E) and medroxyprogesterone acetate (P)] results in a 40% decrease in MLC20 phosphorylation and a 55% decline in the long (214 kDa) form of myosin light-chain kinase (MLCK). Destabilization of the cytoskeleton by inhibitors of MLCK (ML-7) or myosin II ATPase (blebbistatin) accelerates decidualization induced by cAMP (with E and P) but inhibits decidualization induced by IL-1β (with E and P). Adenoviral infection of human uterine fibroblast cells with a constitutively active form of MLCK followed by decidualization stimuli leads to a 30% increase in MLC20 phosphorylation and prevents decidualization. These data provide evidence that the regulation of cytoskeletal dynamics by MLC20 phosphorylation is critical for decidualization.

Download Full-text

Role of ERK1/2 in uterine contractility and preterm labor in rats

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00042.2004 ◽

2004 ◽

Vol 287 (2) ◽

pp. R328-R335 ◽

Cited By ~ 27

Author(s):

Yunping Li ◽

Hyun-Dong Je ◽

Sabah Malek ◽

Kathleen G. Morgan

Keyword(s):

Light Chain ◽

Rat Model ◽

Preterm Labor ◽

Myosin Light Chain ◽

Myosin Light Chain Phosphorylation ◽

Uterine Contractility ◽

Erk Activation ◽

Ru 486 ◽

Erk Phosphorylation

The present study tested the hypothesis that ERK activation is an essential step in the onset of labor in a rat model of preterm labor. The administration of RU-486, an antiprogesterone agent, to rats induced preterm delivery 22.2 ± 0.24 h after treatment. Changes in basal signaling events were studied in myometrial tissue from CO2-euthanized rats. Rats treated with RU-486 displayed a dramatically increased in vitro uterine contractility compared with gestational stage-matched, sham-treated rats. In vitro contractility was not significantly different from that during spontaneous labor. During RU-486-induced preterm labor, as previously described for spontaneous labor, ERK phosphorylation levels increased, as did phosphorylation of caldesmon at Ser789, an ERK phosphorylation site. Also, a small but significant increase in 20-kDa myosin light chain phosphorylation was seen at a constant intracellular pCa of 7. When rats were chronically treated with an agent that prevents ERK activation, U-0126, the onset of RU-486-induced preterm labor was delayed in a statistically significant manner. Chronic in vivo treatment with U-0126 also significantly inhibited the RU-486-induced increase in in vitro contractility and ERK and caldesmon phosphorylation but did not alter the RU-486-induced increase in 20-kDa myosin light chain phosphorylation. These data indicate that ERK activation is a component of the multiple events leading to the development of labor in this rat model. We suggest that the ERK pathway could possibly be used to identify targets for the development of a novel class of tocolytic agents.

Download Full-text

Blebbistatin, a myosin II inhibitor, suppresses contraction and disrupts contractile filaments organization of skinned taenia cecum from guinea pig

AJP Cell Physiology ◽

10.1152/ajpcell.00269.2009 ◽

2010 ◽

Vol 298 (5) ◽

pp. C1118-C1126 ◽

Cited By ~ 15

Author(s):

Masaru Watanabe ◽

Masatoshi Yumoto ◽

Hideyuki Tanaka ◽

Hon Hui Wang ◽

Takeshi Katayama ◽

...

Keyword(s):

Smooth Muscle ◽

Guinea Pig ◽

Muscle Contraction ◽

Light Chain ◽

Myosin Light Chain ◽

Myosin Ii ◽

Smooth Muscle Contraction ◽

Myosin Light Chain Phosphorylation ◽

Thin Filaments ◽

Tension Development

To explore the precise mechanisms of the inhibitory effects of blebbistatin, a potent inhibitor of myosin II, on smooth muscle contraction, we studied the blebbistatin effects on the mechanical properties and the structure of contractile filaments of skinned (cell membrane permeabilized) preparations from guinea pig taenia cecum. Blebbistatin at 10 μM or higher suppressed Ca2+-induced tension development at any given Ca2+ concentration but had little effects on the Ca2+-induced myosin light chain phosphorylation. Blebbistatin also suppressed the 10 and 2.75 mM Mg2+-induced, “myosin light chain phosphorylation-independent” tension development at more than 10 μM. Furthermore, blebbistatin induced conformational change of smooth muscle myosin (SMM) and disrupted arrangement of SMM and thin filaments, resulting in inhibition of actin-SMM interaction irrespective of activation with Ca2+. In addition, blebbistatin partially inhibited Mg2+-ATPase activity of native actomyosin from guinea pig taenia cecum at around 10 μM. These results suggested that blebbistatin suppressed skinned smooth muscle contraction through disruption of structure of SMM by the agent.

Download Full-text

Role of myosin II activity and the regulation of myosin light chain phosphorylation in astrocytomas

Cell Motility and the Cytoskeleton ◽

10.1002/cm.20240 ◽

2007 ◽

Vol 65 (1) ◽

pp. 12-24 ◽

Cited By ~ 22

Author(s):

Bodour Salhia ◽

Jeong Hyun Hwang ◽

Christian A. Smith ◽

Mitsutoshi Nakada ◽

Fiona Rutka ◽

...

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Myosin Ii ◽

Myosin Light Chain Phosphorylation

Download Full-text

An Alternatively Spliced Isoform of Non-muscle Myosin II-C Is Not Regulated by Myosin Light Chain Phosphorylation

Journal of Biological Chemistry ◽

10.1074/jbc.m806574200 ◽

2009 ◽

Vol 284 (17) ◽

pp. 11563-11571 ◽

Cited By ~ 26

Author(s):

Siddhartha S. Jana ◽

Kye-Young Kim ◽

Jian Mao ◽

Sachiyo Kawamoto ◽

James R. Sellers ◽

...

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Myosin Ii ◽

Myosin Light Chain Phosphorylation ◽

Alternatively Spliced ◽

Muscle Myosin

Download Full-text

Phosphorylation of myosin-II regulatory light chain by cyclin-p34cdc2: a mechanism for the timing of cytokinesis.

The Journal of Cell Biology ◽

10.1083/jcb.118.3.595 ◽

1992 ◽

Vol 118 (3) ◽

pp. 595-605 ◽

Cited By ~ 123

Author(s):

L L Satterwhite ◽

M J Lohka ◽

K L Wilson ◽

T Y Scherson ◽

L J Cisek ◽

...

Keyword(s):

Smooth Muscle ◽

Chromosome Segregation ◽

Light Chain ◽

Kinase Activity ◽

Myosin Ii ◽

Regulatory Light Chain ◽

Nonmuscle Myosin ◽

Xenopus Egg ◽

Muscle Myosin

To understand how cytokinesis is regulated during mitosis, we tested cyclin-p34cdc2 for myosin-II kinase activity, and investigated the mitotic-specific phosphorylation of myosin-II in lysates of Xenopus eggs. Purified cyclin-p34cdc2 phosphorylated the regulatory light chain of cytoplasmic and smooth muscle myosin-II in vitro on serine-1 or serine-2 and threonine-9, sites known to inhibit the actin-activated myosin ATPase activity of smooth muscle and nonmuscle myosin (Nishikawa, M., J. R. Sellers, R. S. Adelstein, and H. Hidaka. 1984. J. Biol. Chem. 259:8808-8814; Bengur, A. R., A. E. Robinson, E. Appella, and J. R. Sellers. 1987. J. Biol. Chem. 262:7613-7617; Ikebe, M., and S. Reardon. 1990. Biochemistry. 29:2713-2720). Serine-1 or -2 of the regulatory light chain of Xenopus cytoplasmic myosin-II was also phosphorylated in Xenopus egg lysates stabilized in metaphase, but not in interphase. Inhibition of myosin-II by cyclin-p34cdc2 during prophase and metaphase could delay cytokinesis until chromosome segregation is initiated and thus determine the timing of cytokinesis relative to earlier events in mitosis.

Download Full-text

Parameters That Specify the Timing of Cytokinesis

The Journal of Cell Biology ◽

10.1083/jcb.146.5.981 ◽

1999 ◽

Vol 146 (5) ◽

pp. 981-992 ◽

Cited By ~ 39

Author(s):

Charles B. Shuster ◽

David R. Burgess

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Kinase Activity ◽

Myosin Ii ◽

Cell Activity ◽

Spindle Poles ◽

Close Proximity ◽

Cortical Cytoskeleton

One model for the timing of cytokinesis is based on findings that p34cdc2 can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595–605), and this inhibition is proposed to delay cytokinesis until p34cdc2 activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373–1386). Among these kinases and substrates is p34cdc2 and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34cdc2 influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [32P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.

Download Full-text

Decreased Phosphatase Activity, Increased Ca2+ Sensitivity, and Myosin Light Chain Phosphorylation in Urinary Bladder Smooth Muscle of Newborn Mice

The Journal of General Physiology ◽

10.1085/jgp.200409212 ◽

2005 ◽

Vol 125 (2) ◽

pp. 187-196 ◽

Cited By ~ 26

Author(s):

Mari Ekman ◽

Katarina Fagher ◽

Mia Wede ◽

Karolina Stakeberg ◽

Anders Arner

Keyword(s):

Smooth Muscle ◽

Urinary Bladder ◽

Phosphatase Activity ◽

Light Chain ◽

Myosin Light Chain ◽

Calcium Sensitivity ◽

Myosin Light Chain Phosphorylation ◽

Nonmuscle Myosin ◽

Bladder Smooth Muscle ◽

Urinary Bladder Smooth Muscle

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 ± 0.01, 1.14 ± 0.12, and 1.31 ± 0.08 mM. Force of the newborn tissue was inhibited by ∼45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 ± 0.07, 5.77 ± 0.08, and 5.55 ± 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase–induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.

Download Full-text