scholarly journals A Specific Role of Phosphatidylinositol 3–Kinase γ

2001 ◽  
Vol 152 (4) ◽  
pp. 717-728 ◽  
Author(s):  
Claire Bony ◽  
Serge Roche ◽  
Ueno Shuichi ◽  
Takehiko Sasaki ◽  
Michael A. Crackower ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cardiac Cells ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Dominant Negative Mutant ◽  
Cardiac Cell ◽  
Autonomic Activity ◽  
Cell Functions ◽  
Negative Mutant ◽  
Stimulation Of

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase–dependent pathway that stimulates PLCγ and generates IP3, a breakdown product of phosphatidylinositol 4,5–bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca2+ spiking). PIP2 is phosphorylated on 3′ by phosphoinositide 3–kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5–trisphosphate, regulates activity of PLCγ. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCγ, Ca2+ homeostasis. However, although PI3Kα and -β have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kγ. We report that neonatal rat cardiac cells in culture express PI3Kα, -β, and -γ. The purinergic agonist predominantly activates PI3Kγ. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCγ as well as IP3 generation in ATP-stimulated cells. Furthermore, an anti-PI3Kγ, but not an anti-PI3Kβ, injected in the cells prevents the effect of ATP on cell Ca2+ spiking. A dominant negative mutant of PI3Kγ transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca2+ spiking of wild-type but not of PI3Kγ2/2 embryonic stem cell–derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCγ. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca2+ spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kγ antibody and a dominant negative mutant of PI3Kγ injected or transfected into cells prevent the latter event. We conclude that PI3Kγ activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca2+ spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kγ to fully activate PLCγ in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP3 generation and thus cardiac autonomic activity.

TNF-α induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myocytes

2001 ◽  
Vol 280 (4) ◽  
pp. H1861-H1868 ◽  
Author(s):  
Eiji Hiraoka ◽  
Seinosuke Kawashima ◽  
Tomosaburo Takahashi ◽  
Yoshiyuki Rikitake ◽  
Tadahiro Kitamura ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cardiac Myocytes ◽  
Specific Inhibitor ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Dominant Negative Mutant ◽  
Tnf Α ◽  
Negative Mutant ◽  
Stimulation Of

The activation of phosphatidylinositol (PI) 3-kinase and Akt/protein kinase B (PKB) by tumor necrosis factor (TNF)-α and their roles on stimulation of protein synthesis were investigated in cultured neonatal rat cardiac myocytes. Treatment of cells with TNF-α resulted in enlargement of cell surface area and stimulation of protein synthesis without affecting myocyte viability. TNF-α induced marked activation of PI3-kinase and Akt/PKB, and the activation of PI3-kinase and Akt/PKB was rapid (maximal at 10 and 15 min, respectively) and concentration dependent. Akt/PKB activation by TNF-α was inhibited by a PI3-kinase-specific inhibitor LY-294002 and adenovirus-mediated expression of a dominant negative mutant of PI3-kinase, indicating that TNF-α activates Akt/PKB through PI3-kinase activation. Furthermore, TNF-α-induced protein synthesis was inhibited by pretreatment with LY-294002 and expression of a dominant negative mutant of PI3-kinase or Akt/PKB. These results indicate that activation of the PI3-kinase-Akt/PKB pathway plays an essential role in protein synthesis induced by TNF-α in cardiac myocytes.

scholarly journals Transcriptional Response to Calcium-Sensing Receptor Stimulation

Endocrinology ◽  
2012 ◽  
Vol 153 (10) ◽  
pp. 4716-4728 ◽  
Author(s):  
Gerald Thiel ◽  
Andrea Lesch ◽  
Anja Keim
Keyword(s):  
Gene Transcription ◽  
Ternary Complex ◽  
Dominant Negative ◽  
Signaling Cascade ◽  
Dominant Negative Mutant ◽  
Calcium Sensing Receptor ◽  
Receptor Stimulation ◽  
Calcium Sensing ◽  
Negative Mutant ◽  
Stimulation Of

Abstract Elevated extracellular Ca2+ concentrations stimulate the G-protein coupled receptor calcium-sensing receptor. Here we show that this stimulation induces the expression of biologically active early growth response protein 1 (Egr-1), a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor Ets-like protein-1 (Elk-1), a key transcriptional regulator of serum response element-driven gene transcription, prevented Egr-1 expression, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of calcium-sensing receptors with transcription of the Egr-1 gene. These data were corroborated by the fact that stimulation of calcium-sensing receptors increased the transcriptional activation potential of Elk-1. In addition, activator protein-1 (AP-1) transcriptional activity was significantly elevated after the stimulation of calcium-sensing receptors. The expression of a dominant-negative mutant of Elk-1 reduced c-Fos expression and prevented the up-regulation of AP-1 activity as a result of calcium-sensing receptor stimulation, indicating that ternary complex factors control both Egr-1- and AP-1-regulated transcription. In addition, AP-1 activity was reduced after the expression of a dominant-negative mutant of c-Jun in cells expressing an activated calcium-sensing receptor. Stimulus-transcription coupling leading to the up-regulation of Egr-1 and AP-1 controlled transcription in cells expressing calcium-sensing receptors required the protein kinases Raf and ERK, whereas the overexpression of MAPK phosphatase-1 interrupted the signaling cascade connecting calcium-sensing receptor stimulation with transcription of Egr-1 and AP-1 controlled genes. The fact that calcium-sensing receptor stimulation activates the transcription factors Egr-1, Elk-1, and AP-1 indicates that regulation of gene transcription is an integral part of calcium-sensing receptor induced signaling.

scholarly journals Mechanism of E47-Pip Interaction on DNA Resulting in Transcriptional Synergy and Activation of Immunoglobulin Germ Line Sterile Transcripts

2002 ◽  
Vol 22 (20) ◽  
pp. 7337-7350 ◽  
Author(s):  
Sujatha Nagulapalli ◽  
Aisha Goheer ◽  
Leslie Pitt ◽  
Lawrence P. McIntosh ◽  
Michael L. Atchison
Keyword(s):  
B Cell ◽  
Germ Line ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Cell Functions ◽  
Helix Loop Helix ◽  
Spatial Alignment ◽  
Negative Mutant

ABSTRACT E47 and Pip are proteins crucial for proper B-cell development. E47 and Pip cooperatively bind to adjacent sites in the immunoglobulin kappa chain 3′ enhancer and generate a potent transcriptional synergy. We generated protein-DNA computer models to visualize E47 and Pip bound to DNA. These models predict precise interactions between the two proteins. We tested predictions deduced from these models by mutagenesis studies and found evidence for novel direct interactions between the E47 helix-loop-helix domain (Arg 357 or Asp 358) and the Pip N terminus (Leu 24). We also found that precise spatial alignment of the binding sites was necessary for transcriptional synergy and cooperative DNA binding. A Pip dominant negative mutant that cannot synergize with E47 inhibited enhancer activity in plasmacytoma cells and could not activate transcription in pre-B cells. Using electrophoretic mobility shift assays, we found that Pip can bind to the heavy-chain intron enhancer region. In addition, we found that in fibroblasts Pip greatly increased E47 induction of germ line Iμ transcripts associated with somatic rearrangement and isotype class switching. However, a Pip dominant negative mutant inhibited germ line Iμ transcripts. The importance of these results for late B-cell functions is discussed.

scholarly journals A Dominant-negative Mutant of Androgen Receptor Coregulator ARA54 Inhibits Androgen Receptor-mediated Prostate Cancer Growth

2001 ◽  
Vol 277 (7) ◽  
pp. 4609-4617 ◽  
Author(s):  
Hiroshi Miyamoto ◽  
Mujib Rahman ◽  
Hiroshi Takatera ◽  
Hong-Yo Kang ◽  
Shuyuan Yeh ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Dominant Negative ◽  
Cancer Growth ◽  
Dominant Negative Mutant ◽  
Negative Mutant

scholarly journals UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53

2002 ◽  
Vol 365 (1) ◽  
pp. 133-145 ◽  
Author(s):  
Nadine CHOUINARD ◽  
Kristoffer VALERIE ◽  
Mahmoud ROUABHIA ◽  
Jacques HUOT
Keyword(s):  
Adaptive Response ◽  
Human Keratinocytes ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Normal Human Keratinocytes ◽  
Mitogen Activated Protein ◽  
Normal Human ◽  
Induced Apoptosis ◽  
Negative Mutant

Human keratinocytes respond to UV rays by developing a fast adaptive response that contributes to maintaining their functions and survival. We investigated the role of the mitogen-activated protein kinase pathways in transducing the UV signals in normal human keratinocytes. We found that UVA, UVB or UVC induced a marked and persistent activation of p38, whereas c-Jun N-terminal kinase or extracellular signal-regulated kinase were less or not activated respectively. Inhibition of p38 activity by expression of a dominant-negative mutant of p38 or with SB203580 impaired cell viability and led to an increase in UVB-induced apoptosis. This sensitization to apoptosis was independent of caspase activities. Inhibition of p38 did not sensitize transformed HaCaT keratinocytes to UVB-induced apoptosis. In normal keratinocytes, expression of a dominant-negative mutant of p53 increased UVB-induced cell death, pointing to a role for p53. In these cells, UVB triggered a p38-dependent phosphorylation of p53 on Ser-15. This phosphorylation was associated with an SB203580-sensitive accumulation of p53, even in the presence of a serine phosphatase inhibitor. Accumulated p53 was localized mainly in the cytoplasm, independently of CRM1 nuclear export. In HaCaT cells, p53 was localized exclusively in the nucleus and its distribution and level were not affected by UVB or p38 inhibition. However, UVB induced an SB203580-insensitive phosphorylation on Ser-15 of mutated p53. Overall, our results suggest that, in normal human keratinocytes, protection against UVB depends on p38-mediated phosphorylation and stabilization of p53 and is tightly associated with the cytoplasmic sequestration of wild-type p53. We conclude that the p38/p53 pathway plays a key role in the adaptive response of normal human keratinocytes against UV stress.

scholarly journals Role of Nectin in Formation of E-Cadherin–based Adherens Junctions in Keratinocytes: Analysis with the N-Cadherin Dominant Negative Mutant

2003 ◽  
Vol 14 (4) ◽  
pp. 1597-1609 ◽  
Author(s):  
Yoshinari Tanaka ◽  
Hiroyuki Nakanishi ◽  
Shigeki Kakunaga ◽  
Noriko Okabe ◽  
Tomomi Kawakatsu ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Adherens Junctions ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Adhesion Activity ◽  
Negative Mutant ◽  
E Cadherin ◽  
Cell Cell

E-Cadherin is a Ca2+-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin–based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin–based AJs in keratinocytes.

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