The B2 alternatively spliced isoform of nonmuscle myosin II-B lacks actin-activated MgATPase activity and in vitro motility

The study tested the hypothesis that estrogen controls epithelial paracellular resistance through modulation of myosin. The objective was to understand how estrogen modulates nonmuscle myosin-II-B (NMM-II-B), the main component of the cortical actomyosin in human epithelial cervical cells. Experiments used human cervical epithelial cells CaSki as a model, and end points were NMM-II-B phosphorylation, filamentation, and MgATPase activity. The results were as follows: 1) treatment with estrogen increased phosphorylation and MgATPase activity and decreased NMM-II-B filamentation; 2) estrogen effects could be blocked by antisense nucleotides for the estrogen receptor-α and by ICI-182,780, tamoxifen, and the casein kinase-II (CK2) inhibitor, 5,6-dichloro-1-β-(D)-ribofuranosylbenzimidazole and attenuated by AG1478 and PD98059 (inhibitors of epithelial growth factor receptor and ERK/MAPK) but not staurosporine [blocker of protein kinase C (PKC)]; 3) treatments with the PKC activator sn-1,2-dioctanoyl diglyceride induced biphasic effect on NMM-II-B MgATPase activity: an increase at 1 nm to 1 μm and a decrease in activity at more than 1 μm; 4) sn-1,2-dioctanoyl diglyceride also decreased NMM-II-B filamentation in a monophasic and saturable dose dependence (EC50 1–10 μm); 5) when coincubated directly with purified NMM-II-B filaments, both CK2 and PKC decreased filamentation and increased MgATPase activity; 6) assays done on disassembled NMM-II-B filaments showed MgATPase activity in filaments obtained from estrogen-treated cells but not estrogen-depleted cells; and 7) incubations in vitro with CK2, but not PKC, facilitated MgATPase activity, even in disassembled NMM-II-B filaments. The results suggest that estrogen, in an effect mediated by estrogen receptor-α and CK2 and involving the epithelial growth factor receptor and ERK/MAPK cascades, increases NMM-II-B MgATPase activity independent of NMM-II-B filamentation status.

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Myosin II Is Involved in the Production of Constitutive Transport Vesicles from the TGN

The Journal of Cell Biology ◽

10.1083/jcb.138.2.291 ◽

1997 ◽

Vol 138 (2) ◽

pp. 291-306 ◽

Cited By ~ 139

Author(s):

Anne Müsch ◽

David Cohen ◽

Enrique Rodriguez-Boulan

Keyword(s):

Mdck Cells ◽

Myosin Ii ◽

Nonmuscle Myosin ◽

Vesicle Release ◽

Golgi Membranes ◽

Transport Vesicles ◽

Nonmuscle Myosin Ii ◽

Vesicular Carriers ◽

Vesicular Stomatitis

The participation of nonmuscle myosins in the transport of organelles and vesicular carriers along actin filaments has been documented. In contrast, there is no evidence for the involvement of myosins in the production of vesicles involved in membrane traffic. Here we show that the putative TGN coat protein p200 (Narula, N., I. McMorrow, G. Plopper, J. Doherty, K.S. Matlin, B. Burke, and J.L. Stow. 1992. J. Cell Biol. 114: 1113–1124) is myosin II. The recruitment of myosin II to Golgi membranes is dependent on actin and is regulated by G proteins. Using an assay that studies the release of transport vesicles from the TGN in vitro, we provide functional evidence that p200/myosin is involved in the assembly of basolateral transport vesicles carrying vesicular stomatitis virus G protein (VSVG) from the TGN of polarized MDCK cells. The 50% reduced efficiency in VSVG vesicle release from the TGN in vitro after depletion of p200/myosin II could be reestablished to control levels by the addition of purified nonmuscle myosin II. Several inhibitors of the actin-stimulated ATPase activity of myosin specifically inhibited the release of VSVG-containing vesicles from the TGN.

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Role of nonmuscle myosin II in polyamine-dependent intestinal epithelial cell migration

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1996.270.2.g355 ◽

1996 ◽

Vol 270 (2) ◽

pp. G355-G362 ◽

Cited By ~ 11

Author(s):

J. Y. Wang ◽

S. A. McCormack ◽

L. R. Johnson

Keyword(s):

Cell Migration ◽

Myosin Ii ◽

Specific Inhibitor ◽

Intestinal Epithelial ◽

Nonmuscle Myosin ◽

Nonmuscle Myosin Ii ◽

Epithelial Cell Migration ◽

Vitro Model

The current study determines whether nonmuscle myosin II is involved in the process requiring polyamines for the stimulation of cell migration in an in vitro model that mimics the early stages of epithelial restitution. Treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC), for 4 days totally inhibited ODC activity and depleted intracellular polyamines in the IEC-6 cells. Nonmuscle myosin II concentrations in DFMO-treated cells were decreased by 75%, and stress fibers were sparse or absent. The most striking feature of DFMO-treated cells was the appearance of many small punctate foci of myosin II in the cell interior. Migration of DFMO-treated cells was reduced by 80%. In the presence of DFMO, exogenous putrescine not only returned nonmuscle myosin II levels and distribution toward normal but also restored cell migration to control levels. The administration of wortmannin, an inhibitor of myosin light chain kinase, significantly inhibited cell migration over the denuded area in control cells and in those treated with DFMO + polyamines. These results indicate that 1) polyamine depletion by DFMO is associated with decreased concentration and reorganization of nonmuscle myosin II in IEC-6 cells and 2) exogenous spermidine reverses the inhibitory effects of DFMO.

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A serine-kinase associated with the p127-l(2)gl tumour suppressor of Drosophila may regulate the binding of p127 to nonmuscle myosin II heavy chain and the attachment of p127 to the plasma membrane

Journal of Cell Science ◽

10.1242/jcs.109.6.1359 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1359-1368 ◽

Cited By ~ 3

Author(s):

A. Kalmes ◽

G. Merdes ◽

B. Neumann ◽

D. Strand ◽

B.M. Mechler

Keyword(s):

Plasma Membrane ◽

Heavy Chain ◽

Posttranslational Modifications ◽

Myosin Ii ◽

Gel Filtration ◽

Tumour Suppressor ◽

Nonmuscle Myosin ◽

Serine Kinase ◽

Nonmuscle Myosin Ii

The p127 tumour suppressor protein encoded by the lethal(2)giant larvae, [l(2)gl], gene of Drosophila melanogaster is a component of a cytoskeletal network distributed in both the cytoplasm and on the inner face of the plasma membrane. The p127 protein forms high molecular mass complexes consisting mainly of homo-oligomerized p127 molecules and at least ten additional proteins. One of these proteins has been recently identified as nonmuscle myosin type II heavy chain. To determine the functional interactions between p127 and other proteins present in the p127 complexes, we analyzed p127 for posttranslational modifications and found that p127 can be phosphorylated at serine residues. In this report we describe the characteristics of a serine kinase which is associated with p127, as judged by its recovery in p127 complexes purified by either gel filtration or immuno-affinity chromatography. This kinase phosphorylates p127 in vitro and its activation by supplementing ATP results in the release of p127 from the plasma membrane. Moreover, similar activation of the kinase present in immuno-purified p127 complexes dissociates nonmuscle myosin II from p127 without affecting the homo-oligomerization of p127. This dissociation can be inhibited by staurosporine and a 26mer peptide covering amino acid positions 651 to 676 of p127 and containing five serine residues which are evolutionarily conserved from Drosophila to humans. These results indicate that a serine-kinase tightly associated with p127 regulates p127 binding with components of the cytoskeleton present in both the cytoplasm and on the plasma membrane.

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