Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 9

1.
Figure 3.

Figure 3. From: Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells.

Myosin-IIA is phosphorylated on both the heavy and light chains. (A) Left, Coomassie-stained gel of myosin-IIA immunoprecipitates from unstimulated cells and cells 3 min after EGF stimulation. Right, corresponding autoradiogram. (B) Immunoblot of 3-min immunoprecipitate with a P-T18/S19 RLC antibody. Left, Coomassie-stained gel; middle, corresponding autoradiogram; right, immunoblot with P-T18/S19 antibody. The migration positions of the myosin-IIA heavy chain and RLC, and IgG heavy and light chains are indicated.

Natalya G. Dulyaninova, et al. Mol Biol Cell. 2007 August;18(8):3144-3155.
2.
Figure 5.

Figure 5. From: Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells.

CK2 associates with myosin-IIA in response to EGF stimulation. The endogenous CK2 displayed increased association with His-tagged myosin-IIA rods in EGF-stimulated cells (3 min) compared with unstimulated cells. The Ni2+-resin alone did not pull down CK2 from the whole cell lysates (w.c.l.). Protein complexes were detected by immunoblot with antibody against CK2 alpha subunits. Five percent of the whole cell lysate was run on the gel.

Natalya G. Dulyaninova, et al. Mol Biol Cell. 2007 August;18(8):3144-3155.
3.
Figure 7.

Figure 7. From: Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells.

Expression of myosin-IIA S1943 mutants affects cell migration in a wound-healing assay. (A) Confluent monolayers of untransfected or stable MDA-MB-231 transfectants expressing either wild-type GFP-NMHC-IIA or S1943 mutants were wounded at time 0. Bar, 250 μm. (B) The average rate of wound closure during the first 4 h of wound healing was calculated from three independent experiments. Values represent the mean ± SD. Asterisk (*) denotes a statistically significant difference compared with wild-type GFP-NMHC-IIA cells.

Natalya G. Dulyaninova, et al. Mol Biol Cell. 2007 August;18(8):3144-3155.
4.
Figure 2.

Figure 2. From: Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells.

Localization of the endogenous myosin II isoforms in MDA-MB-231 cells. (A) Cells in normal growth medium. Myosin IIA (a and a′) and myosin IIB (b and b′) were detected by direct immunofluorescence using isoform-specific polyclonal antibodies. (c and c′) An overlay of the two images. Arrows indicate cell protrusions. Bar, 10 μm. (B) Localization of myosin-IIA and myosin-IIB in EGF-stimulated MDA-MB-231 cells. Arrows indicate cortical structures Bar, 50 μm. (C) Localization of myosin-IIA and myosin-IIB 2 min after EGF stimulation in MDA-MB-231 cells. Bar, 10 μm. Arrowheads and arrows indicate cellular regions undergoing active protrusions and regions not actively extending, respectively.

Natalya G. Dulyaninova, et al. Mol Biol Cell. 2007 August;18(8):3144-3155.
5.
Figure 4.

Figure 4. From: Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells.

The myosin-IIA heavy chain is phosphorylated in vivo on serine residues at the CK2 site. (A) In vitro sites of NMHC-IIA phosphorylation. Myosin-IIA is phosphorylated on Ser-1916 near the C-terminal end of the α-helical coiled coil by PKC and on Ser-1943 in the tailpiece by CK2. (B) Phosphoamino acid analysis of the myosin-IIA heavy chain immunoprecipitated from EGF-stimulated cells showed that the heavy chain is phosphorylated on serine residues. Circles indicate the positions of phosphoamino acid standards. (C) Two-dimensional tryptic phosphopeptide maps of the myosin-IIA rod phosphorylated in vitro by (a) PKC alpha or (b) CK2, (c) endogenous myosin-IIA heavy chain from EGF-stimulated cells, and (d) mixture of in vitro– and in vivo–derived phosphopeptides. Based on the comigration of phosphopeptides from the CK2-phosphorylated myosin-IIA rod, the myosin-IIA heavy chain is phosphorylated in vivo on the CK2 site after EGF stimulation.

Natalya G. Dulyaninova, et al. Mol Biol Cell. 2007 August;18(8):3144-3155.
6.
Figure 1.

Figure 1. From: Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells.

EGF stimulation of MDA-MB-231 cells resulted in transient increases in both the insolubility and phosphorylation of the myosin-II heavy chain isoforms. (A) Representative immunoblot of myosin-IIA in Triton-insoluble fractions at different times after stimulation with EGF. EGF-mediated increases in (B) myosin-IIA and (C) myosin-IIB insolubility and heavy-chain phosphorylation. Myosin-II assembly was assessed by isolating Tx-100–resistant cytoskeletons from EGF-stimulated cells. To examine the phosphorylation status on the myosin-II heavy chain, cells were serum-starved, metabolically labeled with 32P-orthophosphate, and stimulated with EGF. At different times after stimulation, the myosin-II heavy chain isoforms were immunoprecipitated and analyzed by SDS-PAGE and autoradiography. (D) EGF-mediated increases in total T18/S19 (solid line) or S19 (dotted line) RLC phosphorylation. Cells were serum-starved and stimulated with EGF. The phosphorylation status of the RLC in whole cell lysates was examined by immunoblot with P-T18/S19 and P-S19 antibodies. For all curves, values represent the mean and SE of the mean for three to four independent experiments.

Natalya G. Dulyaninova, et al. Mol Biol Cell. 2007 August;18(8):3144-3155.
7.
Figure 9.

Figure 9. From: Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells.

Expression of GFP-NMHC-IIA S1943 mutants affects EGF-induced lamellipod extension. MDA-MB-231 cells, parental or expressing the GFP-NMHC-IIA constructs, were stimulated with EGF and changes in cell shape were monitored by phase-contrast and fluorescence microscopy. (A) Representative phase-contrast (a–d) and fluorescent (a′–d′) micrographs of an untransfected cell and cell expressing wild-type GFP-NMHC-IIA responding to chemoattractant. The cells are shown before EGF stimulation (time 0) and at several times after EGF treatment. Cell trace in the fluorescence panel (a′–d′) demonstrates the extending edge of an untransfected cell. Bar, 50 μm. (B) Quantitation of the changes in cell shape at 15 min after EGF stimulation. Lamellipod extension of stable or transient transfectants was evaluated as the overall increase in the total area of the cells after EGF stimulation. The area before stimulation was set as 100%, and the relative area change was calculated as a percentage of the initial cell area. Error bar, SE. Asterisk (*) denotes a statistically significant difference as compared with untransfected cells.

Natalya G. Dulyaninova, et al. Mol Biol Cell. 2007 August;18(8):3144-3155.
8.
Figure 6.

Figure 6. From: Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells.

Biochemical characterization and cellular localization of the myosin-IIA CK2 site (S1943) mutants. (A) The assembly properties of wild-type, S1943A, S1943D, and S1943E myosin-IIA rods were monitored in a standard sedimentation assay. The assembly of the S1943D or E myosin-IIA rods was comparable to those of CK2-phosphorylated rods, whereas the S1943A rod assembled similarly to wild-type, unphosphorylated rods. ○, unphosphorylated myosin-IIA rods; •, S1943A myosin-IIA rods; ▴, S1943D myosin-IIA rods; and ◇, S1943E myosin-IIA rods. The solid lines represent the best fit to the Hill equation. (B) Detection of full-length GFP-NMHC-IIA in MDA-MB-231 cells. Whole cell lysates from stable transfectants were subjected to immunoblot analysis with an antibody against the C-terminus of the myosin-IIA heavy chain. The lower bands represent the endogenous myosin-IIA. (C) Distribution of GFP-NMHC-IIA constructs was visualized by green fluorescence in transient (top) or stable (bottom) transfectants in normal growth medium. The wild-type NMHC-IIA displayed both cytoplasmic and stress fiber localization. The S1943A NMHC-IIA demonstrated increased assembly as evidenced by the prominent stress fibers and strong cortical localization (arrows). The S1943E and S1943D NMHC-IIA were primarily cytoplasmic with minor assembly into stress fibers and the cell cortex. Bar, 50 μm. (D) Wild-type mCherry-NMHC-IIA colocalizes with wild-type, S1943A, or S1943D GFP-NMHC-IIA in EGF-stimulated cells. Transient transfectants were fixed 4–6 min after EGF stimulation. Both S1943A and S1943D GFP-NMHC-IIA form puncta, which localize with wild-type mCherry-NMHC-IIA puncta in extending lamellae. Bar, 10 μm.

Natalya G. Dulyaninova, et al. Mol Biol Cell. 2007 August;18(8):3144-3155.
9.
Figure 8.

Figure 8. From: Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells.

Stable focal adhesions are observed in GFP-NMHC-IIA S1943A–expressing cells. (A) Dynamic alteration of focal adhesions is observed in response to EGF stimulation in untransfected MDA-MB-231 cells by immunostaining with a paxillin pY118 phosphotyrosine antibody. (B) In transient transfectants expressing the GFP-NMHC-IIA S1943A, EGF-stimulated destabilization of pre-existing focal adhesions due to removal of phosphotyrosine is inhibited. Top, paxillin pY118 phosphotyrosine immunostaining; bottom, GFP fluorescence. Arrows indicate small, new focal contacts that are visible in the extending lamellae of untransfected cells. Bar, 50 μm. (C) Cell detachment assay on MDA-MB-231 cells stably expressing GFP-NMHC-IIA S1943 mutants. Adhesion was determined by calculating the percentage of total cells that detached in response to trypsin treatment. Expression of the GFP-NMHC-IIA S1943A resulted in ∼30% increase in resistance to detachment as compared with wild-type, S1943D- and S1943E-expressing cells. Asterisk (*) denotes a statistically significant difference compared with wild-type GFP-NMHC-IIA cells. (D) Immunoblots of GFP-NMHC-IIA stable transfectants 2 min after EGF addition with antibodies to vinculin, pY118 paxillin, total paxillin, CK2α, or actin. Relative to cells expressing wild-type GFP-MHC-IIA, S1943A cells display increased pY118 paxillin incorporation, and S1943D/E cells show decreased phosphotyrosine content. The levels of vinculin and CK2 were not altered in the different transfectants. Actin was used as a loading control.

Natalya G. Dulyaninova, et al. Mol Biol Cell. 2007 August;18(8):3144-3155.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk