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By admin, on March 13th, 2012
Anal Cell Pathol (Amst). 2012 Mar 9;
Ouchani F, Devy J, Rusciani A, Helesbeux JJ, Salesse S, Letinois I, Gras-Billart D, Duca L, Duval O, Martiny L, Charpentier E
Background: Leukemic cell adhesion to proteins of the bone marrow microenvironment provides signals which control morphology, motility and cell survival. We described herein the ability of ethoxyfagaronine (etxfag), a soluble synthetic derivative of fagaronine, to prevent leukemic cell adhesion to fibronectin peptide (FN/V). Methods: Phosphorylation of fak and pyk2 were evaluated by immunoblotting. Labelled proteins were localized by confocal microscopy. PI 3-kinase activity was evaluated by in vitro kinase assay. Results: Subtoxic concentration of etxfag reduced L1210 cell adhesion to FN/V dependently of β1 integrin engagement. Etxfag impaired FN-dependent formation of β1 clustering without modifying β1 expression at the cell membrane. This was accompanied by a decrease of focal adhesion number, a diminution of fak and pyk2 phosphorylation at Tyr-576, Tyr-861 and Tyr-579, respectively leading to their dissociations from β1 integrin and inhibition of PI 3-kinase activity. Etxfag also induced a cell retraction accompanied by a redistribution of phosphorylated fak and pyk2 in the perinuclear region and lipid raft relocalization. Conclusion: Through its anti-adhesive potential, etxfag, combined with conventional cytotoxic drugs could be potentially designed as a new anti-leukemic drug.
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Targeting focal adhesion assembly by ethoxyfagaronine prevents lymphoblastic cell adhesion to fibronectin.
By admin, on March 13th, 2012
Anal Cell Pathol (Amst). 2012 Mar 9;
Ouchani F, Devy J, Rusciani A, Helesbeux JJ, Salesse S, Letinois I, Gras-Billart D, Duca L, Duval O, Martiny L, Charpentier E
Background: Leukemic cell adhesion to proteins of the bone marrow microenvironment provides signals which control morphology, motility and cell survival. We described herein the ability of ethoxyfagaronine (etxfag), a soluble synthetic derivative of fagaronine, to prevent leukemic cell adhesion to fibronectin peptide (FN/V). Methods: Phosphorylation of fak and pyk2 were evaluated by immunoblotting. Labelled proteins were localized by confocal microscopy. PI 3-kinase activity was evaluated by in vitro kinase assay. Results: Subtoxic concentration of etxfag reduced L1210 cell adhesion to FN/V dependently of β1 integrin engagement. Etxfag impaired FN-dependent formation of β1 clustering without modifying β1 expression at the cell membrane. This was accompanied by a decrease of focal adhesion number, a diminution of fak and pyk2 phosphorylation at Tyr-576, Tyr-861 and Tyr-579, respectively leading to their dissociations from β1 integrin and inhibition of PI 3-kinase activity. Etxfag also induced a cell retraction accompanied by a redistribution of phosphorylated fak and pyk2 in the perinuclear region and lipid raft relocalization. Conclusion: Through its anti-adhesive potential, etxfag, combined with conventional cytotoxic drugs could be potentially designed as a new anti-leukemic drug.
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Targeting focal adhesion assembly by ethoxyfagaronine prevents lymphoblastic cell adhesion to fibronectin.
By admin, on March 10th, 2012
By admin, on March 10th, 2012
By admin, on February 25th, 2012
Mol Cell. 2012 Feb 14;
Comb WC, Hutti JE, Cogswell P, Cantley LC, Baldwin AS
The IκB kinase (IKK) pathway is an essential mediator of inflammatory, oncogenic, and cell stress pathways. Recently IKK was shown to be essential for autophagy induction in mammalian cells independent of its ability to regulate NF-κB, but the mechanism by which this occurs is unclear. Here we demonstrate that the p85 regulatory subunit of PI3K is an IKK substrate, phosphorylated at S690 in vitro and in vivo in response to cellular starvation. Cells expressing p85 S690A or inhibited for IKK activity exhibit increased Akt activity following cell starvation, demonstrating that p85 phosphorylation is required for starvation-induced PI3K feedback inhibition. S690 is in a conserved region of the p85 cSH2 domain, and IKK-mediated phosphorylation of this site results in decreased affinity for tyrosine-phosphorylated proteins and decreased PI3K membrane localization. Finally, leucine deprivation is shown to be necessary and sufficient for starvation-induced, IKK-mediated p85 phosphorylation and PI3K feedback inhibition.
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p85α SH2 Domain Phosphorylation by IKK Promotes Feedback Inhibition of PI3K and Akt in Response to Cellular Starvation.
By admin, on February 25th, 2012
Mol Cell. 2012 Feb 14;
Comb WC, Hutti JE, Cogswell P, Cantley LC, Baldwin AS
The IκB kinase (IKK) pathway is an essential mediator of inflammatory, oncogenic, and cell stress pathways. Recently IKK was shown to be essential for autophagy induction in mammalian cells independent of its ability to regulate NF-κB, but the mechanism by which this occurs is unclear. Here we demonstrate that the p85 regulatory subunit of PI3K is an IKK substrate, phosphorylated at S690 in vitro and in vivo in response to cellular starvation. Cells expressing p85 S690A or inhibited for IKK activity exhibit increased Akt activity following cell starvation, demonstrating that p85 phosphorylation is required for starvation-induced PI3K feedback inhibition. S690 is in a conserved region of the p85 cSH2 domain, and IKK-mediated phosphorylation of this site results in decreased affinity for tyrosine-phosphorylated proteins and decreased PI3K membrane localization. Finally, leucine deprivation is shown to be necessary and sufficient for starvation-induced, IKK-mediated p85 phosphorylation and PI3K feedback inhibition.
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p85α SH2 Domain Phosphorylation by IKK Promotes Feedback Inhibition of PI3K and Akt in Response to Cellular Starvation.
By admin, on February 16th, 2012
J Biol Chem. 2012 Feb 9;
Choi HS, Su WM, Han GS, Plote D, Xu Z, Carman GM
The yeast Pah1p phosphatidate phosphatase, which catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes, is a cytosolic enzyme that associates with the nuclear/endoplasmic reticulum membrane to catalyze the dephosphorylation of phosphatidate to yield diacylglycerol. Pah1p is phosphorylated on seven (Ser(110), Ser(114), Ser(168), Ser(602), Thr(723), Ser(744), and Ser(748)) sites that are targets for proline-directed proteins kinases. In this work, we showed that the seven sites are phosphorylated by Pho85p-Pho80p, a protein kinase-cyclin complex known to regulate a variety of cellular processes. The phosphorylation of recombinant Pah1p was time- and dose-dependent, and dependent on the concentrations of ATP (3.7 μM) and Pah1p (0.25 μM). Phosphorylation reduced (6-fold) the catalytic efficiency (Vmax/Km) of Pah1p and reduced (3-fold) its interaction (Kd) with liposomes. Alanine mutations of the seven sites ablated the inhibitory effect that Pho85p-Pho80p had on Pah1p activity and on the interaction with liposomes. Analysis of pho85Δ mutant cells, phosphate-starved wild type cells, and cells expressing phosphorylation-deficient forms of Pah1p indicated that loss of Pho85p-Pho80p phosphorylation reduced Pah1p abundance. In contrast, lack of Nem1p-Spo7p, the phosphatase complex that dephosphorylates Pah1p at the nuclear/endoplasmic reticulum membrane, stabilized Pah1p abundance. While loss of phosphorylation caused a decrease in abundance, a greater amount of Pah1p was associated with membranes when compared with phosphorylated enzyme, and the loss of phosphorylation allowed bypass of the Nem1p-Spo7p requirement for Pah1p function in the synthesis of triacylglycerol.
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Pho85p-Pho80p phosphorylation of yeast Pah1p phosphatidate phosphatase regulates its activity, location, abundance, and function in lipid metabolism.
By admin, on February 15th, 2012
Spermatogenesis. 2011 10; 1(4): 318-324
Lucas TF, Pimenta MT, Pisolato R, Lazari MF, Porto CS
In this review, we will present an overview of estrogen actions in the testis from immature and adult animals, with special emphasis on signaling mechanisms involved in the 17β-estradiol regulation of Sertoli cell function in immature rats. 17β-estradiol activates Sertoli cell proliferation in immature rats by a mechanism that involves the translocation of the estrogen receptors ESR1 and ESR2 to the plasma membrane, phosphorylation of epidermal growth factor receptor and activation of mitogen-activated protein kinase 3/1. Activation of the G protein-coupled estrogen receptor (GPER) also induces phosphorylation of mitogen-activated protein kinase 3/1 via epidermal growth factor receptor transactivation, which in turn increases expression of the antiapoptotic protein BCL2 and decreases the expression of proapoptotic protein BAX, indicating an antiapoptotic role of E2-GPER in immature rat Sertoli cells. In conclusion, ESRs and GPER can mediate rapid 17β-estradiol signaling in Sertoli cells, and modulate transcriptional events important for Sertoli cell function and maintenance of normal testis development and homeostasis. Our findings are important to clarify the role of estrogen in a critical period of testicular development and to direct further studies, which may contribute to better understand the causes of male infertility.
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17β-estradiol signaling and regulation of Sertoli cell function.
By admin, on February 14th, 2012
J Cell Sci. 2012 Feb 10;
Liu M, Huang C, Polu SR, Schneiter R, Chang A
Sphingolipids are critical components of membranes and sphingolipid metabolites also serve as signaling molecules. Yeast Orm1 and Orm2 belong to a conserved family of ER membrane proteins that regulate serine palmitoyltransferase, catalyzing the first and rate-limiting step in sphingolipid synthesis. We now show that sphingolipid synthesis via Orm1 is a target of TOR signaling which regulates cell growth in response to nutritional signals. Orm1 phosphorylation is dependent on the Tap42-phosphatase complex which acts downstream of TOR protein kinase complex 1; in temperature-sensitive tap42-11 cells, impaired Orm1 phosphorylation occurs concomitantly with reduced sphingolipid synthesis. A second mechanism regulating sphingolipid synthesis is via controlling Orm2 protein level. Orm2 protein level responds to ER stress conditions, increasing when cells are treated with tunicamycin or DTT, agents that induce the unfolded protein response (UPR). The sphingolipid intermediates, long chain base and ceramide, are decreased when ORM2 is overexpressed, suggesting sphingolipid synthesis is repressed under ER stress conditions. Finally, in the absence of the Orms, the UPR is constitutively activated. Lipid dysregulation in the absence of the Orms may signal to the ER from the plasma membrane as UPR activation is dependent on a cell surface sensor and the MAPK cell wall integrity pathway. Thus, sphingolipid synthesis and the UPR are coordinately regulated.
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Regulation of sphingolipid synthesis via Orm1 and Orm2 in yeast.
By admin, on February 14th, 2012
J Cell Sci. 2012 Feb 10;
Liu M, Huang C, Polu SR, Schneiter R, Chang A
Sphingolipids are critical components of membranes and sphingolipid metabolites also serve as signaling molecules. Yeast Orm1 and Orm2 belong to a conserved family of ER membrane proteins that regulate serine palmitoyltransferase, catalyzing the first and rate-limiting step in sphingolipid synthesis. We now show that sphingolipid synthesis via Orm1 is a target of TOR signaling which regulates cell growth in response to nutritional signals. Orm1 phosphorylation is dependent on the Tap42-phosphatase complex which acts downstream of TOR protein kinase complex 1; in temperature-sensitive tap42-11 cells, impaired Orm1 phosphorylation occurs concomitantly with reduced sphingolipid synthesis. A second mechanism regulating sphingolipid synthesis is via controlling Orm2 protein level. Orm2 protein level responds to ER stress conditions, increasing when cells are treated with tunicamycin or DTT, agents that induce the unfolded protein response (UPR). The sphingolipid intermediates, long chain base and ceramide, are decreased when ORM2 is overexpressed, suggesting sphingolipid synthesis is repressed under ER stress conditions. Finally, in the absence of the Orms, the UPR is constitutively activated. Lipid dysregulation in the absence of the Orms may signal to the ER from the plasma membrane as UPR activation is dependent on a cell surface sensor and the MAPK cell wall integrity pathway. Thus, sphingolipid synthesis and the UPR are coordinately regulated.
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Regulation of sphingolipid synthesis via Orm1 and Orm2 in yeast.
By admin, on February 11th, 2012
J Immunol. 2012 Feb 8;
Lin Q, Liu Y, Moore DJ, Elizer SK, Veach RA, Hawiger J, Ruley HE
The expression of proinflammatory cytokines and chemokines in response to TCR agonists is regulated by the caspase-recruitment domain membrane-associated guanylate kinase 1 (CARMA1) signalosome through the coordinated assembly of complexes containing the BCL10 adaptor protein. We describe a novel mechanism to negatively regulate the CARMA1 signalosome by the “death” adaptor protein caspase and receptor interacting protein adaptor with death domain (CRADD)/receptor interacting protein-associated ICH-1/CED-3 homologous protein with a death domain. We show that CRADD interacts with BCL10 through its caspase recruitment domain and suppresses interactions between BCL10 and CARMA1. TCR agonist-induced interaction between CRADD and BCL10 coincides with reduction of its complex formation with CARMA1 in wild-type, as compared with Cradd-deficient, primary cells. Finally, Cradd-deficient spleen cells, CD4(+) T cells, and mice respond to T cell agonists with strikingly higher production of proinflammatory mediators, including IFN-γ, IL-2, TNF-α, and IL-17. These results define a novel role for CRADD as a negative regulator of the CARMA1 signalosome and suppressor of Th1- and Th17-mediated inflammatory responses.
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Cutting Edge: The "Death" Adaptor CRADD/RAIDD Targets BCL10 and Suppresses Agonist-Induced Cytokine Expression in T Lymphocytes.
By admin, on January 24th, 2012
By admin, on January 24th, 2012
By admin, on January 24th, 2012
By admin, on January 18th, 2012
J Biol Chem. 2012 Jan 13;
Chaudhri RA, Olivares-Navarrete R, Cuenca N, Hadadi A, Boyan BD, Schwartz Z
Protein kinase C (PKC) signaling can be activated rapidly by 17β-estradiol (E(2)) via non-traditional signaling in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells and is associated with tumorigenicity. Additionally, E(2) has been shown to elicit anti-apoptotic effects in cancer cells counteracting pro-apoptotic effects of chemotherapeuties. Supporting evidence suggests the existence of a membrane-associated ER that differs from the traditional receptors, ERα and ERβ. Our aim was to identify the ER responsible for rapid PKC activation and to evaluate downstream effects, such as proliferation, apoptosis and metastasis. RT-PCR, western blot, and immunofluorescence were used to determine presence of ER splice variants in multiple cell lines. E(2) effects on PKC activity were measured with and without ER blocking antibodies. Cell proliferation was determined by [(3)H]-thymidine incorporation, cell viability was measured by MTT, while apoptosis was determined by DNA fragmentation and TUNEL. Quantitative RT-PCR (qPCR) and sandwich ELISA were used to determine effects on metastatic factors. The role of membrane-dependent signaling in cancer cell invasiveness was examined using an in vitro assay. The results indicate the presence of an ERα splice variant, ERα36, in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells, which localized to plasma membranes and rapidly activated PKC in response to E(2), leading to deleterious effects such as enhancement of proliferation, protection against apoptosis, and enhancement of metastatic factors. These findings propose ERα36 as a novel target for the development of therapies that can prevent progression of breast cancer in the primary tumor as well as during metastasis.
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Membrane estrogen signaling enhances tumorigenesis and metastatic potential of breast cancer cells via estrogen receptor-α36 (ERα36).
By admin, on January 18th, 2012
J Biol Chem. 2012 Jan 13;
Chaudhri RA, Olivares-Navarrete R, Cuenca N, Hadadi A, Boyan BD, Schwartz Z
Protein kinase C (PKC) signaling can be activated rapidly by 17β-estradiol (E(2)) via non-traditional signaling in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells and is associated with tumorigenicity. Additionally, E(2) has been shown to elicit anti-apoptotic effects in cancer cells counteracting pro-apoptotic effects of chemotherapeuties. Supporting evidence suggests the existence of a membrane-associated ER that differs from the traditional receptors, ERα and ERβ. Our aim was to identify the ER responsible for rapid PKC activation and to evaluate downstream effects, such as proliferation, apoptosis and metastasis. RT-PCR, western blot, and immunofluorescence were used to determine presence of ER splice variants in multiple cell lines. E(2) effects on PKC activity were measured with and without ER blocking antibodies. Cell proliferation was determined by [(3)H]-thymidine incorporation, cell viability was measured by MTT, while apoptosis was determined by DNA fragmentation and TUNEL. Quantitative RT-PCR (qPCR) and sandwich ELISA were used to determine effects on metastatic factors. The role of membrane-dependent signaling in cancer cell invasiveness was examined using an in vitro assay. The results indicate the presence of an ERα splice variant, ERα36, in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells, which localized to plasma membranes and rapidly activated PKC in response to E(2), leading to deleterious effects such as enhancement of proliferation, protection against apoptosis, and enhancement of metastatic factors. These findings propose ERα36 as a novel target for the development of therapies that can prevent progression of breast cancer in the primary tumor as well as during metastasis.
The rest is here:
Membrane estrogen signaling enhances tumorigenesis and metastatic potential of breast cancer cells via estrogen receptor-α36 (ERα36).
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