scholarly journals The 14-3-3 Protein Translates the NA+,K+-ATPase α1-Subunit Phosphorylation Signal into Binding and Activation of Phosphoinositide 3-Kinase during Endocytosis

2005 ◽  
Vol 280 (16) ◽  
pp. 16272-16277 ◽  
Author(s):  
Riad Efendiev ◽  
Zongpei Chen ◽  
Rafael T. Krmar ◽  
Sabine Uhles ◽  
Adrian I. Katz ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Α Subunit ◽  
Opossum Kidney ◽  
Rich Domain ◽  
Opossum Kidney Cells ◽  
Basolateral Plasma Membrane ◽  
Functional Relevance ◽  
Phosphoinositide 3 Kinase ◽  
Negative Mutant

Clathrin-dependent endocytosis of Na+,K+-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the α-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na+,K+-ATPase α-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na+,K+-ATPase α-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na+,K+-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the α-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na+,K+-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the α-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na+,K+-ATPase endocytosis.

scholarly journals Role of EGR-1 in thapsigargin-inducible apoptosis in the melanoma cell line A375-C6.

1995 ◽  
Vol 15 (11) ◽  
pp. 6262-6272 ◽  
Author(s):  
S Muthukkumar ◽  
P Nair ◽  
S F Sells ◽  
N G Maddiwar ◽  
R J Jacob ◽  
...  
Keyword(s):  
Actinomycin D ◽  
Interleukin 1 ◽  
Growth Control ◽  
Melanoma Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Functional Relevance ◽  
Antisense Oligomer ◽  
Negative Mutant

Induction of apoptosis by diverse exogenous signals is dependent on elevation of intracellular Ca2+. This process of cell death can be blocked by actinomycin D, indicating that it requires gene transcription events. To identify genes that are required for apoptosis, we used thapsigargin (TG), which inhibits endoplasmic reticulum-dependent Ca(2+)-ATPase and thereby increases cytosolic Ca2+. Exposure to TG led to induction of the zinc finger transcription factor, EGR-1, and apoptosis in human melanoma cells, A375-C6. To determine the functional relevance of EGR-1 expression in TG-inducible apoptosis, we employed a dominant negative mutant which functionally competes with EGR-1 in these cells. Interestingly, the dominant negative mutant inhibited TG-inducible apoptosis. Consistent with this observation, an antisense oligomer directed against Egr-1 also led to a diminution of the number of cells that undergo TG-inducible apoptosis. These results suggest a novel regulatory role for EGR-1 in mediating apoptosis that is induced by intracellular Ca2+ elevation. We have previously shown that in these melanoma cells, EGR-1 acts to inhibit the growth arresting action of interleukin-1. Together, these results imply that EGR-1 plays inducer-specific roles in growth control.

scholarly journals Phosphoinositide 3 Kinase Mediates Toll-Like Receptor 4-Induced Activation of NF-κB in Endothelial Cells

2003 ◽  
Vol 71 (8) ◽  
pp. 4414-4420 ◽  
Author(s):  
Xianwu Li ◽  
Joan C. Tupper ◽  
Douglas D. Bannerman ◽  
Robert K. Winn ◽  
Christopher J. Rhodes ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Kinase Activity ◽  
Luciferase Activity ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Toll Like Receptor 4 ◽  
Kinase Activation ◽  
Phosphoinositide 3 Kinase ◽  
Negative Mutant

ABSTRACT Many of the proinflammatory effects of gram-negative bacteria are elicited by the interaction of bacterial lipopolysaccharide (LPS) with Toll-like receptor 4 (TLR4) expressed on host cells. TLR4 signaling leads to activation of NF-κB and transcription of many genes involved in the inflammatory response. In this study, we examined the signaling pathways involved in NF-κB activation by TLR4 signaling in human microvascular endothelial cells. Akt is a major downstream target of phosphoinositide 3 kinase (PI3-kinase), and PI3-kinase activation is necessary and sufficient for Akt phosphorylation. Consequently, Akt kinase activation was used as a measure of PI3-kinase activity. In a stable transfection system, dominant-negative mutants of myeloid differentiation factor 88 (MyD88) and interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK-1) (MyD88-TIR and IRAK-DD, respectively) blocked Akt kinase activity in response to LPS and IL-1β. A dominant-negative mutant (Mal-P/H) of MyD88 adapter-like protein (Mal), a protein with homology to MyD88, failed to inhibit LPS- or IL-1β-induced Akt activity. Moreover, a dominant-negative mutant of p85 (p85-DN) inhibited the NF-κB luciferase activity, IL-6 production, and IκBα degradation elicited by LPS and IL-1β but not that stimulated by tumor necrosis factor alpha. The dominant-negative mutant of Akt partially inhibited the NF-κB luciferase activity evoked by LPS and IL-1β. However, expression of a constitutively activated Akt failed to induce NF-κB luciferase activity. These findings indicate that TLR4- and IL-1R-induced PI3-kinase activity is mediated by the adapter proteins MyD88 and IRAK-1 but not Mal. Further, these studies suggest that PI3-kinase is an important mediator of LPS and IL-1β signaling leading to NF-κB activation in endothelial cells and that Akt is necessary but not sufficient for NF-κB activation by TLR4.

scholarly journals Dynamin 2 Is Required for Phagocytosis in Macrophages

1999 ◽  
Vol 190 (12) ◽  
pp. 1849-1856 ◽  
Author(s):  
Elizabeth S. Gold ◽  
David M. Underhill ◽  
Naomi S. Morrissette ◽  
Jian Guo ◽  
Mark A. McNiven ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Membrane Extension ◽  
Large Particles ◽  
Particle Binding ◽  
Phosphoinositide 3 Kinase ◽  
Endocytic Vesicles ◽  
Particle Internalization ◽  
Dynamin 2 ◽  
Negative Mutant

Cells internalize soluble ligands through endocytosis and large particles through actin-based phagocytosis. The dynamin family of GTPases mediates the scission of endocytic vesicles from the plasma membrane. We report here that dynamin 2, a ubiquitously expressed dynamin isoform, has a role in phagocytosis in macrophages. Dynamin 2 is enriched on early phagosomes, and expression of a dominant-negative mutant of dynamin 2 significantly inhibits particle internalization at the stage of membrane extension around the particle. This arrest in phagocytosis resembles that seen with inhibitors of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K prevents the recruitment of dynamin to the site of particle binding. Although expression of mutant dynamin in macrophages inhibited particle internalization, it had no effect on the production of inflammatory mediators elicited by particle binding.

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.

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