Browsing by Subject "Cell signaling"

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Elucidating the regulation and dynamics of [beta]-O-N-acetyl-D-glucosamine (O-GlcNAc) during signal transduction
(2010-05) Carrillo Millán, Luz Damaris; Magnus, Philip D.; Mahal, Lara K.; Iverson, Brent; Roux, Stanley; Appling, Dean; Liu, Hung-Wen
The ability of cells to respond to their microenvironment is controlled by a complex communication system. Cell signaling utilizes a series of post-translational events to regulate and coordinate cellular activities. Although phosphorylation is thought to be the key regulator of these events, recent findings implicate the O-GlcNAc modification as an additional control mechanism. Modulation of signal transduction requires compartmentalization of the kinases and phosphatases. Based on the evidence of subcellular localization of OGT isoforms, the diversity of O-GlcNAcylated proteins upon stimulation, and its role during insulin signaling, it can be hypothesized that O-GlcNAc is involved and regulates signal transduction in a compartmentalized manner. To investigate the spatio-temporal dynamics of O-GlcNAc in cell signaling, we have generated a series of genetically encoded O-GlcNAc reporters based on fluorescence resonance energy transfer (FRET). These reporters and localized variants have allowed compartment specific visualization of O-GlcNAc activity in the nucleus, cytoplasm and plasma membrane. Herein we describe these reporters and their use to examine O-GlcNAc dynamics in signaling using serum stimulation and environmentally relevant concentrations of arsenite. Acute exposure to arsenite through drinking water has become an environmental health concern worldwide. Our results imply a complex regulation of O-GlcNAc on a fast timescale that is tied to more canonical kinase pathways.
The Functions of Auxilin and Rab11 in Drosophila Suggest That the Fundamental Role of Ligand Endocytosis in Notch Signaling Cells Is Not Recycling
(Public Library of Science, 2011-03-23) Banks, Susan M. L.; Cho, Bomsoo; Eun, Suk Ho; Lee, Ji-Hoon; Windler, Sarah L.; Xie, Xuanhua; Bilder, David; Fischer, Janice A.
Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling.
The effects of dimer and oligomer separation on cell signaling in the EGFR family
(2020-02-05) Pattengale, Sarah Ruth; Leahy, Daniel J. (Ph. D. in biophysics)
The EGFR family of receptor tyrosine kinases (RTKs) includes EGFR, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB. The EGFR family members play crucial roles in development and in regulating normal cellular processes. However, these receptors are found to be overexpressed or mutated in various cancers. The complete mechanism by which the EGFR family is autoregulated is still unknown. This work focuses on the role of the extracellular domain of EGFR family members on receptor activation, dimerization/oligomerization, and downstream signaling. We generated a system using designed ankyrin repeat proteins (DARPins) to study EGFR extracellular domain dimers of varying separation distances. We biochemically, cellularly, and structurally characterize interactions between HER3 and A30, an RNA that binds to the extracellular domain of HER3 and reduces HER2 phosphorylation. This work also includes preliminary efforts to characterize HER2-HER3 oligomers using single molecule photobleaching experiments. This work lays the groundwork for future studies involving EGFR family receptor regulation