scholarly journals Cortactin and dynamin are required for the clathrin-independent endocytosis of γc cytokine receptor

2004 ◽  
Vol 168 (1) ◽  
pp. 155-163 ◽  
Author(s):  
Nathalie Sauvonnet ◽  
Annick Dujeancourt ◽  
Alice Dautry-Varsat
Keyword(s):  
Rna Interference ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
Cytokine Receptor ◽  
Actin Dynamics ◽  
Dominant Negative Mutant ◽  
Cellular Functions ◽  
Receptor Endocytosis ◽  
The Common ◽  
Negative Mutant

Endocytosis is critical for many cellular functions. We show that endocytosis of the common γc cytokine receptor is clathrin independent by using a dominant-negative mutant of Eps15 or RNA interference to knock down clathrin heavy chain. This pathway is synaptojanin independent and requires the GTPase dynamin. In addition, this process requires actin polymerization. To further characterize the function of dynamin in clathrin-independent endocytosis, in particular its connection with the actin cytoskeleton, we focused on dynamin-binding proteins that interact with F-actin. We compared the involvement of these proteins in the clathrin-dependent and -independent pathways. Thus, we observed that intersectin, syndapin, and mAbp1, which are necessary for the uptake of transferrin (Tf), a marker of the clathrin route, are not required for γc receptor endocytosis. Strikingly, cortactin is needed for both γc and Tf internalizations. These results reveal the ubiquitous action of cortactin in internalization processes and suggest its role as a linker between actin dynamics and clathrin-dependent and -independent endocytosis.

scholarly journals Amphiphysin 1 Is Important for Actin Polymerization during Phagocytosis

2007 ◽  
Vol 18 (11) ◽  
pp. 4669-4680 ◽  
Author(s):  
Hiroshi Yamada ◽  
Emiko Ohashi ◽  
Tadashi Abe ◽  
Norihiro Kusumi ◽  
Shun-AI Li ◽  
...  
Keyword(s):  
Rna Interference ◽  
Sertoli Cells ◽  
Small Interfering Rna ◽  
Actin Polymerization ◽  
Actin Dynamics ◽  
Polymerization Process ◽  
Amphiphysin 1 ◽  
Phagocytic Cups ◽  
Interfering Rna ◽  
Constitutively Active

Amphiphysin 1 is involved in clathrin-mediated endocytosis. In this study, we demonstrate that amphiphysin 1 is essential for cellular phagocytosis and that it is critical for actin polymerization. Phagocytosis in Sertoli cells was induced by stimulating phosphatidylserine receptors. This stimulation led to the formation of actin-rich structures, including ruffles, phagocytic cups, and phagosomes, all of which showed an accumulation of amphiphysin 1. Knocking out amphiphysin 1 by RNA interference in the cells resulted in the reduction of ruffle formation, actin polymerization, and phagocytosis. Phagocytosis was also drastically decreased in amph 1 (−/−) Sertoli cells. In addition, phosphatidylinositol-4,5-bisphosphate–induced actin polymerization was decreased in the knockout testis cytosol. The addition of recombinant amphiphysin 1 to the cytosol restored the polymerization process. Ruffle formation in small interfering RNA-treated cells was recovered by the expression of constitutively active Rac1, suggesting that amphiphysin 1 functions upstream of the protein. These findings support that amphiphysin 1 is important in the regulation of actin dynamics and that it is required for phagocytosis.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document