scholarly journals Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant μ2 subunit and its effects on endocytosis

1999 ◽  
Vol 18 (9) ◽  
pp. 2489-2499 ◽  
Author(s):  
Alexandre Nesterov ◽  
Royston E. Carter ◽  
Tatiana Sorkina ◽  
Gordon N. Gill ◽  
Alexander Sorkin
Keyword(s):  
Receptor Binding ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Binding Function ◽  
Clathrin Adaptor ◽  
Negative Mutant

scholarly journals Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2–, AP-1–, and Rab22-dependent steps

2013 ◽  
Vol 24 (11) ◽  
pp. 1735-1748 ◽  
Author(s):  
Zoe G. Holloway ◽  
Antonio Velayos-Baeza ◽  
Gareth J. Howell ◽  
Clotilde Levecque ◽  
Sreenivasan Ponnambalam ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Copper Transporter ◽  
Caveolin 1 ◽  
Clathrin Adaptor ◽  
Partial Overlap ◽  
Golgi Network

The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)–mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the μ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22aQ64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22aQ64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.

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 A novel dominant-negative mutant form of Epstein–Barr virus latent membrane protein-1 (LMP1) selectively and differentially impairs LMP1 and TNF signaling pathways

Oncogene ◽  
2004 ◽  
Vol 23 (15) ◽  
pp. 2681-2693 ◽  
Author(s):  
Eric Adriaenssens ◽  
Alexandra Mougel ◽  
Gautier Goormachtigh ◽  
Estelle Loing ◽  
Véronique Fafeur ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Latent Membrane Protein ◽  
Dominant Negative ◽  
Latent Membrane Protein 1 ◽  
Dominant Negative Mutant ◽  
Mutant Form ◽  
Barr Virus ◽  
Epstein Barr ◽  
Virus Latent Membrane Protein ◽  
Negative Mutant

scholarly journals Functional Expression of Human PP2Ac in Yeast Permits the Identification of Novel C-terminal and Dominant-negative Mutant Forms

1999 ◽  
Vol 274 (34) ◽  
pp. 24038-24046 ◽  
Author(s):  
David R. H. Evans ◽  
Timothy Myles ◽  
Jan Hofsteenge ◽  
Brian A. Hemmings
Keyword(s):  
Functional Expression ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mutant Forms ◽  
Negative Mutant

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.

Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes

1996 ◽  
Vol 109 (13) ◽  
pp. 3013-3023 ◽  
Author(s):  
A.J. Zhu ◽  
F.M. Watt
Keyword(s):  
Cell Adhesion ◽  
Terminal Differentiation ◽  
Intercellular Adhesion ◽  
Dominant Negative ◽  
Epidermal Keratinocytes ◽  
Dominant Negative Mutant ◽  
Beta Catenin ◽  
Human Epidermal Keratinocytes ◽  
Negative Mutant ◽  
E Cadherin

Cell adhesion molecules are not only required for maintenance of tissue integrity, but also regulate many aspects of cell behaviour, including growth and differentiation. While the regulatory functions of integrin extracellular matrix receptors in keratinocytes are well established, such functions have not been investigated for the primary receptors that mediate keratinocyte intercellular adhesion, the cadherins. To examine cadherin function in normal human epidermal keratinocytes we used a retroviral vector to introduce a dominant negative E-cadherin mutant, consisting of the extracellular domain of H-2Kd and the transmembrane and cytoplasmic domains of E-cadherin. As a control a vector containing the same construct, but with the catenin binding site destroyed, was prepared. High levels of expression of the constructs were achieved; the dominant negative mutant, but not the control, formed complexes with alpha-, beta- and gamma-catenin. In cells expressing the dominant negative mutant there was a 5-fold decrease in the level of endogenous cadherins and a 3-fold increase in the level of beta-catenin. Cell-cell adhesion and stratification were inhibited by the dominant negative mutant and desmosome formation was reduced. Expression of the mutant resulted in reduced levels of the alpha 2 beta 1 and alpha 3 beta 1 integrins and increased cell motility, providing further evidence for cross-talk between cadherins and the beta 1 integrins. In view of the widely documented loss of E-cadherin in keratinocyte tumours it was surprising that the dominant negative mutant had an inhibitory effect on keratinocyte proliferation and stimulated terminal differentiation even under conditions in which intercellular adhesion was prevented. These results establish a role for cadherins in regulating keratinocyte growth and differentiation and raise interesting questions as to the relative importance of cell adhesion-dependent and -independent mechanisms.

scholarly journals β1-Integrin and PI 3-kinase regulate RhoA-dependent activation of skeletal α-actin promoter in myoblasts

2000 ◽  
Vol 278 (6) ◽  
pp. H1736-H1743 ◽  
Author(s):  
Lei Wei ◽  
Wei Zhou ◽  
Lu Wang ◽  
Robert J. Schwartz
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Serum Response Factor ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Muscle Gene Expression ◽  
Actin Promoter ◽  
Muscle Gene ◽  
Negative Mutant

RhoA GTPase, a regulator of actin cytoskeleton, is also involved in regulating c- fos gene expression through its effect on serum response factor (SRF) transcriptional activity. We have also shown that RhoA plays a critical role in myogenesis and regulates expression of SRF-dependent muscle genes, including skeletal α-actin. In the present study, we examined whether the RhoA signaling pathway cross talks with other myogenic signaling pathways to modulate skeletal α-actin promoter activity in myoblasts. We found that extracellular matrix proteins and the β1-integrin stimulated RhoA-dependent activation of the α-actin promoter. The muscle-specific isoform β1Dselectively activated the α-actin promoter in concert with RhoA but inhibited the c- fos promoter. In addition, focal adhesion kinase (FAK) and phosphatidylinositol (PI) 3-kinase were required for full activation of the α-actin promoter by RhoA. Expression of a dominant negative mutant of FAK, application of wortmannin to cultured myoblasts, or expression of a dominant negative mutant of PI 3-kinase inhibited α-actin promoter activity induced by RhoA. These results suggest that RhoA, β1-integrin, FAK, and PI 3-kinase serve together as an important signaling network in regulating muscle gene expression.

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