scholarly journals Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

2010 ◽  
Vol 21 (22) ◽  
pp. 3973-3984 ◽  
Author(s):  
Yukinao Shibukawa ◽  
Natsuko Yamazaki ◽  
Keiichi Kumasawa ◽  
Etsuko Daimon ◽  
Michiko Tajiri ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Fusion ◽  
Syncytium Formation ◽  
Terminal Region ◽  
Negative Regulator ◽  
Dominant Negative Effect ◽  
Bewo Cell ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Cytoskeleton Rearrangement

Cell–cell fusion is an intriguing differentiation process, essential for placental development and maturation. A proteomic approach identified a cytoplasmic protein, calponin 3 (CNN3), related to the fusion of BeWo choriocarcinoma cells. CNN3 was expressed in cytotrophoblasts in human placenta. CNN3 gene knockdown promoted actin cytoskeletal rearrangement and syncytium formation in BeWo cells, suggesting CNN3 to be a negative regulator of trophoblast fusion. Indeed, CNN3 depletion promoted BeWo cell fusion. CNN3 at the cytoplasmic face of cytoskeleton was dislocated from F-actin with forskolin treatment and diffused into the cytoplasm in a phosphorylation-dependent manner. Phosphorylation sites were located at Ser293/296 in the C-terminal region, and deletion of this region or site-specific disruption of Ser293/296 suppressed syncytium formation. These CNN3 mutants were colocalized with F-actin and remained there after forskolin treatment, suggesting that dissociation of CNN3 from F-actin is modulated by the phosphorylation status of the C-terminal region unique to CNN3 in the CNN family proteins. The mutant missing these phosphorylation sites displayed a dominant negative effect on cell fusion, while replacement of Ser293/296 with aspartic acid enhanced syncytium formation. These results indicated that CNN3 regulates actin cytoskeleton rearrangement which is required for the plasma membranes of trophoblasts to become fusion competent.

scholarly journals Interactions of FliJ with the Salmonella Type III Flagellar Export Apparatus

2003 ◽  
Vol 185 (18) ◽  
pp. 5546-5554 ◽  
Author(s):  
Gillian M. Fraser ◽  
Bertha González-Pedrajo ◽  
Jeremy R. H. Tame ◽  
Robert M. Macnab
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Analytical Ultracentrifugation ◽  
Terminal Region ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Size Exclusion ◽  
Dominant Negative Effect ◽  
Type Iii ◽  
Self Association

ABSTRACT FliJ, a 17-kDa protein, is a soluble component of the Salmonella type III flagellar protein export system that has antiaggregation properties and several other characteristics that suggest it may have a chaperone-like function. We have now examined this protein in detail. Ten-amino-acid scanning deletions covering the entire 147-amino-acid sequence were tested for complementation of a fliJ null strain; only the first and last deletions complemented. A few of the deletions, especially towards the C terminus, exerted a dominant negative effect on wild-type cells, indicating that they were actively interfering with function. Two truncated versions of FliJ, representing its N- and C-terminal halves, failed to complement and were not dominant. We tested for FliJ self-association by several techniques. Size-exclusion chromatography (Superdex 200) indicated an apparent molecular mass of around 50 kDa, which could reflect either multimerization or an elongated shape or both. Multiangle light scattering gave a peak value of 20 kDa, close to the molecular mass of the monomer. Analytical ultracentrifugation gave evidence for weak self-association as a trimer or tetramer. It was known from previous studies that FliJ interacts with the N-terminal region of FliH, a negative regulator of the ATPase FliI. Using both truncation and deletion versions of FliJ, we now show that it is its C-terminal region that is responsible for this interaction. We also show that FliJ interacts with the soluble cytoplasmic domain of the largest membrane component of the export apparatus, FlhA; although small deletions in FliJ did not interfere with the association, both truncated versions failed to associate, indicating that a substantial amount of the central region of the FliJ sequence participates in the association. We present a model summarizing these multiple interactions.

scholarly journals The WD-repeats of Net2p Interact with Dnm1p and Fis1p to Regulate Division of Mitochondria

2003 ◽  
Vol 14 (10) ◽  
pp. 4126-4139 ◽  
Author(s):  
Kara L. Cerveny ◽  
Robert E. Jensen
Keyword(s):  
Mitochondrial Fission ◽  
Terminal Region ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Dominant Negative Effect ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Wd Repeats

The Net2, Fis1, and Dnm1 proteins are required for the division of mitochondria in the yeast Saccharomyces cerevisiae. Net2p has an amino-terminal region that contains predicted coiled-coil motifs and a carboxyl-terminal domain composed of WD-40 repeats. We found that the amino-terminal part of Net2p interacts with Fis1p, whereas the carboxyl-terminal region interacts with both Dnm1p and Fis1p. Overproduction of either domain of Net2p in yeast cells poisons mitochondrial fission, and the dominant-negative effect caused by the WD-repeats of Net2p is suppressed by increased levels of Dnm1p. Point mutations in the WD-region of Net2p or in the GTPase region of Dnm1p disrupt the normal Net2p-Dnm1p interaction, causing Net2p to lose its normal punctate distribution. Our results suggest that Dnm1p interacts with the WD-repeats of Net2p and in a GTP-dependent manner recruits Net2p to sites of mitochondrial division. Furthermore, our results indicate that Net2p is required for proper assembly of the mitochondrial fission components to regulate organelle division.

scholarly journals Actin Cytoskeletal Reorganizations and Coreceptor-Mediated Activation of Rac during Human Immunodeficiency Virus-Induced Cell Fusion

2004 ◽  
Vol 78 (13) ◽  
pp. 7138-7147 ◽  
Author(s):  
S. E. Pontow ◽  
N. Vander Heyden ◽  
S. Wei ◽  
L. Ratner
Keyword(s):  
Human Immunodeficiency Virus ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Actin Filament ◽  
Target Cell ◽  
Syncytium Formation ◽  
Dominant Negative ◽  
Target Cells ◽  
Dependent Manner ◽  
Immunodeficiency Virus

ABSTRACT The membrane fusion events which initiate human immunodeficiency virus type 1 (HIV-1) infection and promote cytopathic syncytium formation in infected cells commence with the binding of the HIV envelope glycoprotein (Env) to CD4 and an appropriate coreceptor. Here, we show that HIV Env-coreceptor interactions activate Rac-1 GTPase and stimulate the actin filament network reorganizations that are requisite components of the cell fusion process. Disrupting actin filament dynamics with jasplakinolide or latrunculin A arrested fusion at a late step in the formation of Env-CD4-coreceptor complexes. Time-lapse confocal microscopy of living cells revealed vigorous activity of actin-based, target cell membrane extensions at the target cell-Env-expressing cell interface. The expression of dominant-negative forms of actin-regulating Rho-family GTPases established that HIV Env-mediated syncytium formation relies on Rac-1 but not on Cdc42 or Rho activation in target cells. Similar dependencies were found when cell fusion was induced by Env expressed on viral or cellular membranes. Additionally, Rac activity was specifically upregulated in a coreceptor-dependent manner in fusion reaction cell lysates. These results define a role for HIV Env-coreceptor interactions in activating the cellular factors essential for virus-cell and cell-cell fusion and provide evidence for the participation of pertussis toxin-insensitive signaling pathways in HIV-induced membrane fusion.

scholarly journals pp60c-src associates with the SH2-containing inositol-5-phosphatase SHIP1 and is involved in its tyrosine phosphorylation downstream of αIIbβ3 integrin in human platelets

2000 ◽  
Vol 348 (1) ◽  
pp. 107-112 ◽  
Author(s):  
Sylvie GIURIATO ◽  
Stéphane BODIN ◽  
Christophe ERNEUX ◽  
Rüdiger WOSCHOLSKI ◽  
Monique PLANTAVID ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Tyrosine Phosphorylation ◽  
Blood Platelets ◽  
Human Platelets ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Cytoskeleton Reorganization ◽  
Haemopoietic Cells ◽  
Human Blood Platelets ◽  
Dependent Mechanism

SH2-containing inositol-5-phosphatase 1 (SHIP1) was originally identified as a 145 kDa protein that became tyrosine-phosphorylated in response to multiple cytokines. It is now well established that SHIP1 is specifically expressed in haemopoietic cells and is important as a negative regulator of signalling. We found recently that SHIP1 was present in human blood platelets as an Ins(1,3,4,5)P4-phosphatase and a PtdIns(3,4,5)P3-5-phosphatase that became tyrosine-phosphorylated and was relocated to the cytoskeleton in an integrin-dependent manner. Here we report biochemical and pharmacological evidence that the tyrosine kinase pp60c-src is constitutively associated with SHIP1 and is involved in its tyrosine phosphorylation downstream of integrin engagement in thrombin-activated human platelets. The use of cytochalasin D allowed us to demonstrate that the actin cytoskeleton reorganization induced on thrombin stimulation was not required for its integrin-mediated phosphorylation. Moreover, the integrin-dependent relocation of SHIP1 to the cytoskeleton did not require its tyrosine phosphorylation. These results suggest that SHIP1 is first recruited to the integrin-linked signalling complexes and then becomes tyrosine-phosphorylated through a Src-kinase-dependent mechanism but independently of the actin cytoskeleton reorganization.

scholarly journals GSK-3α Inhibition in Drug-Resistant CML Cells Promotes Susceptibility to NK Cell-Mediated Lysis in an NKG2D- and NKp30-Dependent Manner

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1802
Author(s):  
Nayoung Kim ◽  
Mi Yeon Kim ◽  
Woo Seon Choi ◽  
Eunbi Yi ◽  
Hyo Jung Lee ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Negative Regulator ◽  
Target Cells ◽  
Dependent Manner ◽  
Cell Functions ◽  
Cell Based Therapy ◽  
Activating Receptors ◽  
Gsk 3Β

Natural killer (NK) cells are innate cytotoxic lymphocytes that provide early protection against cancer. NK cell cytotoxicity against cancer cells is triggered by multiple activating receptors that recognize specific ligands expressed on target cells. We previously demonstrated that glycogen synthase kinase (GSK)-3β, but not GSK-3α, is a negative regulator of NK cell functions via diverse activating receptors, including NKG2D and NKp30. However, the role of GSK-3 isoforms in the regulation of specific ligands on target cells is poorly understood, which remains a challenge limiting GSK-3 targeting for NK cell-based therapy. Here, we demonstrate that GSK-3α rather than GSK-3β is the primary isoform restraining the expression of NKG2D ligands, particularly ULBP2/5/6, on tumor cells, thereby regulating their susceptibility to NK cells. GSK-3α also regulated the expression of the NKp30 ligand B7-H6, but not the DNAM-1 ligands PVR or nectin-2. This regulation occurred independently of BCR-ABL1 mutation that confers tyrosine kinase inhibitor (TKI) resistance. Mechanistically, an increase in PI3K/Akt signaling in concert with c-Myc was required for ligand upregulation in response to GSK-3α inhibition. Importantly, GSK-3α inhibition improved cancer surveillance by human NK cells in vivo. Collectively, our results highlight the distinct role of GSK-3 isoforms in the regulation of NK cell reactivity against target cells and suggest that GSK-3α modulation could be used to enhance tumor cell susceptibility to NK cells in an NKG2D- and NKp30-dependent manner.

scholarly journals Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

1997 ◽  
Vol 8 (3) ◽  
pp. 533-545 ◽  
Author(s):  
T Harder ◽  
R Kellner ◽  
R G Parton ◽  
J Gruenberg
Keyword(s):  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Annexin Ii ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Independent Manner ◽  
Low Concentrations ◽  
Early Endosomes ◽  
Associated Proteins ◽  
Cytoskeletal Elements

Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton.

Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2648-2656 ◽  
Author(s):  
Juan A. Rosado ◽  
Else M. Y. Meijer ◽  
Karly Hamulyak ◽  
Irena Novakova ◽  
Johan W. M. Heemskerk ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding

Abstract Effects of the occupation of integrin αIIbβ3 by fibrinogen on Ca++signaling in fura-2–loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca++] concentrations ([Ca++]i) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca++ but not in the absence of external Ca++ or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca++entry. Fibrinogen also inhibited store-mediated Ca++ entry (SMCE) activated after Ca++ store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to αIIbβ3 was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca++ chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60src to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin αIIbβ3 inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60src. This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected to low-level stimuli in vivo.

scholarly journals Hyperphosphorylation of the retinoblastoma gene product is determined by domains outside the simian virus 40 large-T-antigen-binding regions.

1990 ◽  
Vol 10 (12) ◽  
pp. 6586-6595 ◽  
Author(s):  
P A Hamel ◽  
B L Cohen ◽  
L M Sorce ◽  
B L Gallie ◽  
R A Phillips
Keyword(s):  
Cell Cycle ◽  
Complex Formation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Large T Antigen ◽  
T Complex ◽  
Cycle Dependent

With the murine retinoblastoma (RB) cDNA, a series of RB mutants were expressed in COS-1 cells and the pRB products were assessed for their ability (i) to bind to large T antigen (large T), (ii) to become modified by phosphorylation, and (iii) to localize in the nucleus. All point mutations and deletions introduced into regions previously defined as contributing to binding to large T abolished pRB-large T complex formation and prevented hyperphosphorylation of the RB protein. In contrast, a series of deletions 5' to these sites did not interfere with binding to large T. While some of the 5' deletion mutants were clearly phosphorylated in a cell cycle-dependent manner, one, delta Pvu, failed to be phosphorylated depsite binding to large T. pRB with mutations created at three putative p34cdc2 phosphorylation sites in the N-terminal region behaved similarly to wild-type pRB, whereas the construct delta P5-6-7-8, mutated at four serine residues C terminal to the large T-binding site, failed to become hyperphosphorylated despite retaining the ability to bind large T. All of the mutants described were also found to localize in the nucleus. These results demonstrate that the domains in pRB responsible for binding to large T are distinct from those recognized by the relevant pRB-specific kinase(s) and/or those which contain cell cycle-dependent phosphorylation sites. Furthermore, these data are consistent with a model in which cell cycle-dependent phosphorylation of pRB requires complex formation with other cellular proteins.

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