A ternary complex comprising FAK, PTPα and IP3 receptor 1 functionally engages focal adhesions and the endoplasmic reticulum to mediate IL-1-induced Ca2+ signalling in fibroblasts

2016 ◽  
Vol 473 (4) ◽  
pp. 397-410 ◽  
Author(s):  
Qin Wang ◽  
Yongqiang Wang ◽  
Gregory P. Downey ◽  
Sergey Plotnikov ◽  
Christopher A. McCulloch
Keyword(s):  
Endoplasmic Reticulum ◽  
Ternary Complex ◽  
Focal Adhesions ◽  
Eukaryotic Cells ◽  
Temporal Control ◽  
Ip3 Receptor ◽  
Cell Adhesions ◽  
Spatio Temporal ◽  
Mechanistic Basis

Our studies provide a mechanistic basis by which Ca2+ release is linked to the formation of cell adhesions, thereby providing a tightly sequestered system in eukaryotic cells that enables fine spatio-temporal control of signalling.

scholarly journals Computational design and experimental characterization of a photo-controlled mRNA-cap guanine-N7 methyltransferase

2021 ◽  
Author(s):  
Dennis Reichert ◽  
Helena Schepers ◽  
Julian Simke ◽  
Horst Lechner ◽  
Wolfgang Dörner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Computational Design ◽  
Eukaryotic Cells ◽  
Transcriptional Level ◽  
Experimental Characterization ◽  
Temporal Control ◽  
Control Of Gene Expression ◽  
Multicellular Organisms

The spatial and temporal control of gene expression at the post-transcriptional level is essential in eukaryotic cells and developing multicellular organisms. In recent years optochemical and optogenetic tools have enabled...

Intramembrane proteolysis and post-targeting functions of signal peptides

2003 ◽  
Vol 31 (6) ◽  
pp. 1243-1247 ◽  
Author(s):  
B. Martoglio
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Bilayer ◽  
Signal Peptide ◽  
Eukaryotic Cells ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Peptides ◽  
Peptide Fragments ◽  
Intramembrane Proteolysis ◽  
Signal Sequences ◽  
Membrane Spanning

Signal sequences are the addresses of proteins destined for secretion. In eukaryotic cells, they mediate targeting to the endoplasmic reticulum membrane and insertion into the translocon. Thereafter, signal sequences are cleaved from the pre-protein and liberated into the endoplasmic reticulum membrane. We have recently reported that some liberated signal peptides are further processed by the intramembrane-cleaving aspartic protease signal peptide peptidase. Cleavage in the membrane-spanning portion of the signal peptide promotes the release of signal peptide fragments from the lipid bilayer. Typical processes that include intramembrane proteolysis is the regulatory or signalling function of cleavage products. Likewise, signal peptide fragments liberated upon intramembrane cleavage may promote such post-targeting functions in the cell.

scholarly journals Overexpression of CALNUC (Nucleobindin) Increases Agonist and Thapsigargin Releasable Ca2+ Storage in the Golgi

1999 ◽  
Vol 145 (2) ◽  
pp. 279-289 ◽  
Author(s):  
Ping Lin ◽  
Yong Yao ◽  
Robert Hofmeister ◽  
Roger Y. Tsien ◽  
Marilyn Gist Farquhar
Keyword(s):  
Endoplasmic Reticulum ◽  
Cho Cells ◽  
Binding Capacity ◽  
Binding Protein ◽  
Ip3 Receptor ◽  
Transfected Cells ◽  
Permeabilized Cells ◽  
Endoplasmic Reticulum Calcium ◽  
Golgi Protein

We previously demonstrated that CALNUC, a Ca2+-binding protein with two EF-hands, is the major Ca2+-binding protein in the Golgi by 45Ca2+ overlay (Lin, P., H. Le-Niculescu, R. Hofmeister, J.M. McCaffery, M. Jin, H. Henneman, T. McQuistan, L. De Vries, and M. Farquhar. 1998. J. Cell Biol. 141:1515–1527). In this study we investigated CALNUC's properties and the Golgi Ca2+ storage pool in vivo. CALNUC was found to be a highly abundant Golgi protein (3.8 μg CALNUC/mg Golgi protein, 2.5 × 105 CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca2+-binding site (Kd = 6.6 μM, binding capacity = 1.1 μmol Ca2+/μmol CALNUC). 45Ca2+ storage was increased by 2.5- and 3-fold, respectively, in HeLa cells transiently overexpressing CALNUC-GFP and in EcR-CHO cells stably overexpressing CALNUC. Deletion of the first EF-hand α helix from CALNUC completely abolished its Ca2+-binding capability. CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, α-mannosidase II (Man II). Approximately 70% of the 45Ca2+ taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca2+ pump) blocker. Stimulation of transfected cells with the agonist ATP or IP3 alone (permeabilized cells) also resulted in a significant increase in Ca2+ release from Golgi stores. By immunofluorescence, the IP3 receptor type 1 (IP3R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi. These results provide direct evidence that CALNUC binds Ca2+ in vivo and together with SERCA and IP3R is involved in establishment of the agonist-mobilizable Golgi Ca2+ store.

scholarly journals Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Mun-Teng Wong ◽  
Steve S. Chen
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Ternary Complex ◽  
Choline Kinase ◽  
Replication Complex ◽  
Viral Replication Complex ◽  
Phosphatidylinositol 4

ABSTRACT In this study, we elucidated the mechanism by which human choline kinase-α (hCKα) interacts with nonstructural protein 5A (NS5A) and phosphatidylinositol-4-kinase IIIα (PI4KIIIα), the lipid kinase crucial for maintaining the integrity of virus-induced membranous webs, and modulates hepatitis C virus (HCV) replication. hCKα activity positively modulated phosphatidylinositol-4-phosphate (PI4P) levels in HCV-expressing cells, and hCKα-mediated PI4P accumulation was abolished by AL-9, a PI4KIIIα-specific inhibitor. hCKα colocalized with NS5A and PI4KIIIα or PI4P; NS5A expression increased hCKα and PI4KIIIα colocalization; and hCKα formed a ternary complex with PI4KIIIα and NS5A, supporting the functional interplay of hCKα with PI4KIIIα and NS5A. PI4KIIIα inactivation by AL-9 or hCKα inactivation by CK37, a specific hCKα inhibitor, impaired the endoplasmic reticulum (ER) localization and colocalization of these three molecules. Interestingly, hCKα knockdown or inactivation inhibited PI4KIIIα-NS5A binding. In an in vitro PI4KIIIα activity assay, hCKα activity slightly increased PI4KIIIα basal activity but greatly augmented NS5A-induced PI4KIIIα activity, supporting the essential role of ternary complex formation in robust PI4KIIIα activation. Concurring with the upregulation of PI4P production and viral replication, overexpression of active hCKα-R (but not the D288A mutant) restored PI4KIIIα and NS5A translocation to the ER in hCKα stable knockdown cells. Furthermore, active PI4KIIIα overexpression restored PI4P production, PI4KIIIα and NS5A translocation to the ER, and viral replication in CK37-treated cells. Based on our results, hCKα functions as an indispensable regulator that bridges PI4KIIIα and NS5A and potentiates NS5A-stimulated PI4KIIIα activity, which then facilitates the targeting of the ternary complex to the ER for viral replication. IMPORTANCE The mechanisms by which hCKα activity modulates the transport of the hCKα-NS5A complex to the ER are not understood. In the present study, we investigated how hCKα interacts with PI4KIIIα (a key element that maintains the integrity of the “membranous web” structure) and NS5A to regulate viral replication. We demonstrated that HCV hijacks hCKα to bridge PI4KIIIα and NS5A, forming a ternary complex, which then stimulates PI4KIIIα activity to produce PI4P. Pronounced PI4P synthesis then redirects the translocation of the ternary complex to the ER-derived, PI4P-enriched membrane for assembly of the viral replication complex and viral replication. Our study provides novel insights into the indispensable modulatory role of hCKα in the recruitment of PI4KIIIα to NS5A and in NS5A-stimulated PI4P production and reveals a new perspective for understanding the impact of profound PI4KIIIα activation on the targeting of PI4KIIIα and NS5A to the PI4P-enriched membrane for viral replication complex formation.

scholarly journals The Spatio-temporal Control of the Expression of Glutamine Synthetase in the Liver Is Mediated by Its 5′-Enhancer

1995 ◽  
Vol 270 (47) ◽  
pp. 28251-28256 ◽  
Author(s):  
Heleen Lie-Venema ◽  
T. Labruyère Wil ◽  
A. van Roon Marian ◽  
A. J. de Boer Piet ◽  
Antoon F.M. Moorman ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Temporal Control ◽  
Spatio Temporal

scholarly journals The P5A ATPase Spf1p is stimulated by phosphatidylinositol 4-phosphate and influences cellular sterol homeostasis

2019 ◽  
Vol 30 (9) ◽  
pp. 1069-1084 ◽  
Author(s):  
Danny Mollerup Sørensen ◽  
Henrik Waldal Holen ◽  
Jesper Torbøl Pedersen ◽  
Helle Juel Martens ◽  
Daniele Silvestro ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Marked Change ◽  
Hydrolytic Activity ◽  
Genetic Interactions ◽  
Eukaryotic Cells ◽  
Lipid Bodies ◽  
Increased Sensitivity ◽  
Phosphatidylinositol 4 ◽  
P Type

P5A ATPases are expressed in the endoplasmic reticulum (ER) of all eukaryotic cells, and their disruption results in severe ER stress. However, the function of these ubiquitous membrane proteins, which belong to the P-type ATPase superfamily, is unknown. We purified a functional tagged version of the Saccharomyces cerevisiae P5A ATPase Spf1p and observed that the ATP hydrolytic activity of the protein is stimulated by phosphatidylinositol 4-phosphate (PI4P). Furthermore, SPF1 exhibited negative genetic interactions with SAC1, encoding a PI4P phosphatase, and with OSH1 to OSH6, encoding Osh proteins, which, when energized by a PI4P gradient, drive export of sterols and lipids from the ER. Deletion of SPF1 resulted in increased sensitivity to inhibitors of sterol production, a marked change in the ergosterol/lanosterol ratio, accumulation of sterols in the plasma membrane, and cytosolic accumulation of lipid bodies. We propose that Spf1p maintains cellular sterol homeostasis by influencing the PI4P-induced and Osh-mediated export of sterols from the ER.

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