Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues

1999 ◽  
Vol 112 (8) ◽  
pp. 1149-1158 ◽  
Author(s):  
K. Hayashi ◽  
S. Yonemura ◽  
T. Matsui ◽  
S. Tsukita
Keyword(s):  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Tissue Type ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Erm Proteins

Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.

scholarly journals Targeting a Lipid Desaturation Enzyme, SCD1, Selectively Eliminates Colon Cancer Stem Cells through the Suppression of Wnt and NOTCH Signaling

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 106
Author(s):  
Yeongji Yu ◽  
Hyejin Kim ◽  
SeokGyeong Choi ◽  
JinSuh Yu ◽  
Joo Yeon Lee ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Notch Signaling ◽  
Cultured Cells ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Dependent Manner ◽  
Lipid Desaturation ◽  
Novel Target

The elimination of the cancer stem cell (CSC) population may be required to achieve better outcomes of cancer therapy. We evaluated stearoyl-CoA desaturase 1 (SCD1) as a novel target for CSC-selective elimination in colon cancer. CSCs expressed more SCD1 than bulk cultured cells (BCCs), and blocking SCD1 expression or function revealed an essential role for SCD1 in the survival of CSCs, but not BCCs. The CSC potential selectively decreased after treatment with the SCD1 inhibitor in vitro and in vivo. The CSC-selective suppression was mediated through the induction of apoptosis. The mechanism leading to selective CSC death was investigated by performing a quantitative RT-PCR analysis of 14 CSC-specific signaling and marker genes after 24 and 48 h of treatment with two concentrations of an inhibitor. The decrease in the expression of Notch1 and AXIN2 preceded changes in the expression of all other genes, at 24 h of treatment in a dose-dependent manner, followed by the downregulation of most Wnt- and NOTCH-signaling genes. Collectively, we showed that not only Wnt but also NOTCH signaling is a primary target of suppression by SCD1 inhibition in CSCs, suggesting the possibility of targeting SCD1 against colon cancer in clinical settings.

CNP gene expression is activated by Wnt signaling and correlates with Wnt4 expression during renal injury

2003 ◽  
Vol 284 (4) ◽  
pp. F653-F662 ◽  
Author(s):  
Kameswaran Surendran ◽  
Theodore C. Simon
Keyword(s):  
Wnt Signaling ◽  
Renal Injury ◽  
Cultured Cells ◽  
Proximal Promoter ◽  
Dependent Manner ◽  
Binding Factor ◽  
Identical Cell ◽  
Renal Tubular

C-type natriuretic peptide (CNP) regulates salt excretion, vascular tone, and fibroblast proliferation and activation. CNP inhibits fibroblast activation in vitro and fibrosis in vivo, but endogenous CNP gene ( Nppc) expression during tissue fibrosis has not been reported. We determined that Nppc is induced in renal tubular epithelia and then in interstitial myofibroblasts after unilateral ureteral obstruction (UUO). Induction of Nppcoccurred in identical cell populations to those in which Wnt4 is induced after renal injury. In addition, Nppc was activated in Wnt4-expressing cells during nephrogenesis. Wnt signaling components β-catenin and T cell factor/lymphoid enhancer binding factor (TCF/LEF) specifically bound to cognate elements in the Nppc proximal promoter. Wnt-4, β-catenin, and LEF-1 activated an Nppc transgene in cultured cells, and transgene activation by Wnt-4 and LEF-1 was dependent on the presence of intact cognate elements. These findings suggest that Wnt-4 stimulates Nppc in a TCF/LEF-dependent manner after renal injury and thus may contribute to limiting renal fibrosis.

scholarly journals AMPK directly activates mTORC2 to promote cell survival during acute energetic stress

2019 ◽  
Vol 12 (585) ◽  
pp. eaav3249 ◽  
Author(s):  
Dubek Kazyken ◽  
Brian Magnuson ◽  
Cagri Bodur ◽  
Hugo A. Acosta-Jaquez ◽  
Deqiang Zhang ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cultured Cells ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Downstream Signaling ◽  
Energetic Stress ◽  
Diabetes Drug ◽  
Tumor Suppressive Function

AMP-activated protein kinase (AMPK) senses energetic stress and, in turn, promotes catabolic and suppresses anabolic metabolism coordinately to restore energy balance. We found that a diverse array of AMPK activators increased mTOR complex 2 (mTORC2) signaling in an AMPK-dependent manner in cultured cells. Activation of AMPK with the type 2 diabetes drug metformin (GlucoPhage) also increased mTORC2 signaling in liver in vivo and in primary hepatocytes in an AMPK-dependent manner. AMPK-mediated activation of mTORC2 did not result from AMPK-mediated suppression of mTORC1 and thus reduced negative feedback on PI3K flux. Rather, AMPK associated with and directly phosphorylated mTORC2 (mTOR in complex with rictor). As determined by two-stage in vitro kinase assay, phosphorylation of mTORC2 by recombinant AMPK was sufficient to increase mTORC2 catalytic activity toward Akt. Hence, AMPK phosphorylated mTORC2 components directly to increase mTORC2 activity and downstream signaling. Functionally, inactivation of AMPK, mTORC2, and Akt increased apoptosis during acute energetic stress. By showing that AMPK activates mTORC2 to increase cell survival, these data provide a potential mechanism for how AMPK paradoxically promotes tumorigenesis in certain contexts despite its tumor-suppressive function through inhibition of growth-promoting mTORC1. Collectively, these data unveil mTORC2 as a target of AMPK and the AMPK-mTORC2 axis as a promoter of cell survival during energetic stress.

Rho-dependent and -independent activation mechanisms of ezrin/radixin/moesin proteins: an essential role for polyphosphoinositides in vivo

2002 ◽  
Vol 115 (12) ◽  
pp. 2569-2580 ◽  
Author(s):  
Shigenobu Yonemura ◽  
Takeshi Matsui ◽  
Shoichiro Tsukita ◽  
Sachiko Tsukita
Keyword(s):  
Plasma Membranes ◽  
Kinase Inhibitor ◽  
Mdck Cells ◽  
Aminoglycoside Antibiotic ◽  
Activation Mechanism ◽  
Dependent Manner ◽  
A431 Cells ◽  
Erm Proteins ◽  
Protein Activation

Ezrin/radixin/moesin (ERM) proteins crosslink actin filaments to plasma membranes and are involved in the organization of the cortical cytoskeleton,especially in the formation of microvilli. ERM proteins are reported to be activated as crosslinkers in a Rho-dependent manner and are stabilized when phosphorylated at their C-terminal threonine residue to create C-terminal threonine-phosphorylated ERM proteins (CPERMs). Using a CPERM-specific mAb, we have shown, in vivo, that treatment with C3 transferase (a Rho inactivator) or staurosporine (a protein kinase inhibitor) leads to the dephosphorylation of CPERMs, the translocation of ERM proteins from plasma membranes to the cytoplasm and microvillar breakdown. We further elucidated that ERM protein activation does not require C-terminal phosphorylation in A431 cells stimulated with epidermal growth factor. In certain types of kidney-derived cells such as MDCK cells, however, ERM proteins appear to be activated in the absence of Rho activation and remain active without C-terminal phosphorylation. Interestingly, microinjection of an aminoglycoside antibiotic, neomycin, which binds to polyphosphoinositides, such as phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2],affected the activation of ERM proteins regardless of cell type. These findings not only indicate the existence of a Rho-independent activation mechanism of ERM proteins but also suggest that both Rho-dependent and-independent activation of ERM proteins require a local elevation of PtdIns(4,5)P2 concentration in vivo.

scholarly journals Ponticulin is an atypical membrane protein.

1994 ◽  
Vol 126 (6) ◽  
pp. 1421-1431 ◽  
Author(s):  
A L Hitt ◽  
T H Lu ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Plasma Membranes ◽  
Signal Sequence ◽  
Metabolic Labeling ◽  
Cortical Actin ◽  
Intact Cells ◽  
Membrane Spanning

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

scholarly journals Brefeldin A-dependent Membrane Tubule Formation Reconstituted In Vitro Is Driven by a Cell Cycle–regulated Microtubule Motor

2000 ◽  
Vol 11 (3) ◽  
pp. 941-955 ◽  
Author(s):  
Alasdair M. Robertson ◽  
Victoria J. Allan
Keyword(s):  
Cell Cycle ◽  
Cultured Cells ◽  
Brefeldin A ◽  
Binding Activity ◽  
Membrane Dynamics ◽  
Dependent Manner ◽  
Tubule Formation ◽  
Early Endosomes

Treatment of cultured cells with brefeldin A (BFA) induces the formation of extensive membrane tubules from the Golgi apparatus,trans-Golgi network, and early endosomes in a microtubule-dependent manner. We have reconstituted this transport process in vitro using Xenopus egg cytosol and a rat liver Golgi-enriched membrane fraction. The presence of BFA results in the formation of an intricate, interconnected tubular membrane network, a process that, as in vivo, is inhibited by nocodazole, the H1 anti-kinesin monoclonal antibody, and by membrane pretreatment with guanosine 5′-O-(3-thiotriphosphate). Surprisingly, membrane tubule formation is not due to the action of conventional kinesin or any of the other motors implicated in Golgi membrane dynamics. Two candidate motors of ∼100 and ∼130 kDa have been identified using the H1 antibody, both of which exhibit motor properties in a biochemical assay. Finally, BFA-induced membrane tubule formation does not occur in metaphase cytosol, and because membrane binding of both candidate motors is not altered after incubation in metaphase compared with interphase cytosol, these results suggest that either the ATPase or microtubule-binding activity of the relevant motor is cell cycle regulated.

Antiangiogenic kringles derived from human plasminogen and apolipoprotein(a) inhibit fibrinolysis through a mechanism that requires a functional lysine-binding site

2011 ◽  
Vol 392 (4) ◽  
Author(s):  
Jin-Hyung Ahn ◽  
Ho-Jeong Lee ◽  
Eun-Kyoung Lee ◽  
Hyun-Kyung Yu ◽  
Tae-Ho Lee ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Safety Concern ◽  
Tissue Type ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Kringle Domains ◽  
Apolipoprotein A

AbstractMany proteins in the fibrinolysis pathway contain antiangiogenic kringle domains. Owing to the high degree of homology between kringle domains, there has been a safety concern that antiangiogenic kringles could interact with common kringle proteins during fibrinolysis leading to adverse effectsin vivo. To address this issue, we investigated the effects of several antiangiogenic kringle proteins including angiostatin, apolipoprotein(a) kringles IV9-IV10-V (LK68), apolipoprotein(a) kringle V (rhLK8) and a derivative of rhLK8 mutated to produce a functional lysine-binding site (Lys-rhLK8) on the entire fibrinolytic processin vitroand analyzed the role of lysine binding. Angiostatin, LK68 and Lys-rhLK8 increased clot lysis time in a dose-dependent manner, inhibited tissue-type plasminogen activator-mediated plasminogen activation on a thrombin-modified fibrinogen (TMF) surface, showed binding to TMF and significantly decreased the amount of plasminogen bound to TMF. The inhibition of fibrinolysis by these proteins appears to be dependent on their functional lysine-binding sites. However, rhLK8 had no effect on these processes owing to an inability to bind lysine. Collectively, these results indicate that antiangiogenic kringles without lysine binding sites might be safer with respect to physiological fibrinolysis than lysine-binding antiangiogenic kringles. However, the clinical signi-ficance of these findings will require further validationin vivo.

scholarly journals Androgen Receptor Acetylation Governs trans Activation and MEKK1-Induced Apoptosis without Affecting In Vitro Sumoylation and trans-Repression Function

2002 ◽  
Vol 22 (10) ◽  
pp. 3373-3388 ◽  
Author(s):  
Maofu Fu ◽  
Chenguang Wang ◽  
Jian Wang ◽  
Xueping Zhang ◽  
Toshiyuki Sakamaki ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Cultured Cells ◽  
Nuclear Hormone Receptor ◽  
Dependent Manner ◽  
Wild Type ◽  
Acetylation Site ◽  
Induced Apoptosis

ABSTRACT The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both trans repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. The AR acetylation site point mutants showed wild-type trans repression of NF-κB, AP-1, and Sp1 activity; wild-type sumoylation in vitro; wild-type ligand binding; and ligand-induced conformational changes. However, acetylation-deficient AR mutants were selectively defective in DHT-induced trans activation of androgen-responsive reporter genes and coactivation by SRC1, Ubc9, TIP60, and p300. The AR acetylation site mutant showed 10-fold increased binding of the N-CoR corepressor compared with the AR wild type in the presence of ligand. Furthermore, histone deacetylase 1 (HDAC1) bound the AR both in vivo and in cultured cells and HDAC1 binding to the AR was disengaged in a DHT-dependent manner. MEKK1 induced AR-dependent apoptosis in prostate cancer cells. The AR acetylation mutant was defective in MEKK1-induced apoptosis, suggesting that the conserved AR acetylation site contributes to a pathway governing prostate cancer cellular survival. As AR lysine residue mutations that abrogate acetylation correlate with enhanced binding of the N-CoR repressor in cultured cells, the conserved AR motif may directly or indirectly regulate ligand-dependent corepressor disengagement and, thereby, ligand-dependent trans activation.

scholarly journals The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle

1999 ◽  
Vol 147 (2) ◽  
pp. 351-366 ◽  
Author(s):  
Vicki Mountain ◽  
Calvin Simerly ◽  
Louisa Howard ◽  
Asako Ando ◽  
Gerald Schatten ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Cultured Cells ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Cross Linking ◽  
Simultaneous Inhibition ◽  
Balance Of Forces ◽  
And Function

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.

scholarly journals Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry.

1990 ◽  
Vol 110 (1) ◽  
pp. 13-25 ◽  
Author(s):  
T Nakata ◽  
K Sobue ◽  
N Hirokawa
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Plasma Membranes ◽  
Cross Linking ◽  
Calcium Dependent ◽  
Chromaffin Vesicles ◽  
Ultrathin Sections ◽  
In Vitro Data

Calpactin I complex, a calcium-dependent phospholipid-binding protein, promotes aggregation of chromaffin vesicles at physiological micromolar calcium ion levels. Calpactin I complex was found to be a globular molecule with a diameter of 10.7 +/- 1.7 (SD) nm on mica. When liposomes were aggregated by calpactin, quick-freeze, deep-etching revealed fine thin strands (6.5 +/- 1.9 [SD] nm long) cross-linking opposing membranes in addition to the globules on the surface of liposomes. Similar fine strands were also observed between aggregated chromaffin vesicles when they were mixed with calpactin in the presence of Ca2+ ion. In cultured chromaffin cells, similar cross-linking short strands (6-10 nm) were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structures approximately 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy on frozen ultrathin sections showed that calpactin I was closely associated with the inner face of the plasma membranes and was especially conspicuous between plasma membranes and adjacent vesicles in chromaffin cells. These in vivo and in vitro data strongly suggest that calpactin I complex changes its conformation to cross-link vesicles and the plasma membrane after stimulation of cultured chromaffin cells.

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