The protein kinase Pelle mediates feedback regulation in the Drosophila Toll signaling pathway

Development ◽  
2001 ◽  
Vol 128 (23) ◽  
pp. 4729-4736 ◽  
Author(s):  
Par Towb ◽  
Andreas Bergmann ◽  
Steven A. Wasserman
Keyword(s):  
Protein Kinase ◽  
Immunofluorescence Microscopy ◽  
Nuclear Translocation ◽  
Adaptor Protein ◽  
Feedback Regulation ◽  
Kinase Domain ◽  
Drosophila Embryo ◽  
Confocal Immunofluorescence Microscopy ◽  
Toll Signaling ◽  
Confocal Immunofluorescence

Dorsoventral polarity in the Drosophila embryo is established through a signal transduction cascade triggered in ventral and ventrolateral regions. Activation of a transmembrane receptor, Toll, leads to localized recruitment of the adaptor protein Tube and protein kinase Pelle. Signaling through these components directs degradation of the IκB-like inhibitor Cactus and nuclear translocation of the Rel protein Dorsal. Here we show through confocal immunofluorescence microscopy that Pelle functions to downregulate the signal-dependent relocalization of Tube. Inactivation of the Pelle kinase domain, or elimination of the Tube-Pelle interaction, dramatically increases Tube recruitment to the ventral plasma membrane in regions of active signaling. We also characterize a large collection of pelle alleles, identifying the molecular lesions in these alleles and their effects on Pelle autophosphorylation, Tube phosphorylation and Tube relocalization. Our results point to a mechanism operating to modulate the domain or duration of signaling downstream from Tube and Pelle.

Recruitment of Tube and Pelle to signaling sites at the surface of the Drosophila embryo

Development ◽  
1998 ◽  
Vol 125 (13) ◽  
pp. 2443-2450 ◽  
Author(s):  
P. Towb ◽  
R.L. Galindo ◽  
S.A. Wasserman
Keyword(s):  
Plasma Membrane ◽  
Intracellular Signaling ◽  
Immunofluorescence Microscopy ◽  
Nuclear Translocation ◽  
Signal Transduction Pathway ◽  
Drosophila Embryo ◽  
Transduction Pathway ◽  
Entire Surface ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence

A signaling pathway initiated by activation of the transmembrane receptor Toll defines dorsoventral polarity in the Drosophila embryo. Toll, which is present over the entire surface of the embryo, is activated ventrally by interaction with a spatially restricted, extracellular ligand. Tube and Pelle then transduce the signal from activated Toll to a complex of Dorsal and Cactus. Here we demonstrate by an mRNA microinjection assay that targeting of either Tube or Pelle to the plasma membrane by myristylation is sufficient to activate the signal transduction pathway that leads to Dorsal nuclear translocation. Using confocal immunofluorescence microscopy we also show that activated Toll induces a localized recruitment of Tube and Pelle to the plasma membrane. Together, these results strongly support the hypothesis that intracellular signaling requires the Toll-mediated formation of a membrane-associated complex containing both Tube and Pelle.

scholarly journals Multiple mechanisms collectively regulate clathrin-mediated endocytosis of the epidermal growth factor receptor

2010 ◽  
Vol 189 (5) ◽  
pp. 871-883 ◽  
Author(s):  
Lai Kuan Goh ◽  
Fangtian Huang ◽  
Woong Kim ◽  
Steven Gygi ◽  
Alexander Sorkin
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Epidermal Growth ◽  
Egfr Mutant

Endocytosis of the epidermal growth factor receptor (EGFR) is important for the regulation of EGFR signaling. However, EGFR endocytosis mechanisms are poorly understood, which precludes development of approaches to specifically inhibit EGFR endocytosis and analyze its impact on signaling. Using a combination of receptor mutagenesis and RNA interference, we demonstrate that clathrin-dependent internalization of activated EGFR is regulated by four mechanisms, which function in a redundant and cooperative fashion. These mechanisms involve ubiquitination of the receptor kinase domain, the clathrin adaptor complex AP-2, the Grb2 adaptor protein, and three C-terminal lysine residues (K1155, K1158, and K1164), which are acetylated, a novel posttranslational modification for the EGFR. Based on these findings, the first internalization-defective EGFR mutant with functional kinase and normal tyrosine phosphorylation was generated. Analysis of the signaling kinetics of this mutant revealed that EGFR internalization is required for the sustained activation of protein kinase B/AKT but not for the activation of mitogen-activated protein kinase.

scholarly journals Human Rhinovirus Type 2 Is Internalized by Clathrin-Mediated Endocytosis

2003 ◽  
Vol 77 (9) ◽  
pp. 5360-5369 ◽  
Author(s):  
Luc Snyers ◽  
Hannes Zwickl ◽  
Dieter Blaas
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Immunofluorescence Microscopy ◽  
Dominant Negative ◽  
Human Rhinovirus ◽  
Coated Pits ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Transient Overexpression

ABSTRACT Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K+ depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-β-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.

Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus

2008 ◽  
Vol 86 (6) ◽  
pp. 509-519 ◽  
Author(s):  
Magdalena Sobczak ◽  
Anna Wasik ◽  
Wanda Kłopocka ◽  
Maria Jolanta Rędowicz
Keyword(s):  
Electron Microscopy ◽  
Immunofluorescence Microscopy ◽  
Amoeba Proteus ◽  
Myosin Vi ◽  
Filamentous Actin ◽  
Confocal Immunofluorescence Microscopy ◽  
Endogenous Protein ◽  
Confocal Immunofluorescence ◽  
Pinocytotic Vesicles ◽  
Phagocytic Cups

Recently, we found a 130-kDa myosin VI immunoanalog in amoeba, which bound to actin in an ATP-sensitive manner and in migrating amoebae colocalized to filamentous actin and dynamin II-containing vesicular structures. To further characterize this protein, we assessed its involvement in amoeba pinocytosis and phagocytosis. Confocal immunofluorescence microscopy and electron microscopy of immunogold-stained cells revealed that, in pinocytotic and phagocytotic amoebae, the myosin VI immunoanalog was visible throughout the cells, including pinocytotic channels and pinocytotic vesicles as well as phagosomes and emerging phagocytic cups. Blocking endogenous protein with anti-porcine myosin VI antibody (introduced into cells by means of microinjection) caused severe defects in pinocytosis and phagocytosis. In comparison with control cells, the treated amoebae formed ~75% less pinocytotic channels and phagocytosed ~65% less Tetrahymena cells. These data indicate that the myosin VI immunoanalog has an important role in pinocytosis and phagocytosis in Amoeba proteus (Pal.).

scholarly journals Rab11 regulates recycling through the pericentriolar recycling endosome.

1996 ◽  
Vol 135 (4) ◽  
pp. 913-924 ◽  
Author(s):  
O Ullrich ◽  
S Reinsch ◽  
S Urbé ◽  
M Zerial ◽  
R G Parton
Keyword(s):  
Immunofluorescence Microscopy ◽  
Bound State ◽  
Small Gtpases ◽  
Recycling Endosome ◽  
Functional Studies ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Recycling Pathway ◽  
And Function ◽  
Perinuclear Area

Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rab11. Rab11 was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rab11 mutants that are preferentially in the GTP- or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rab11-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rab11-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rab11 proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rab11-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rab11 in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive recycling endosome. These results suggest a novel role for rab11 in controlling traffic through the recycling endosome.

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