constitutively active mutants
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2021 ◽  
Vol 8 ◽  
Author(s):  
Yong Zhou ◽  
Alemayehu A. Gorfe ◽  
John F. Hancock

RAS proteins are lipid-anchored small GTPases that switch between the GTP-bound active and GDP-bound inactive states. RAS isoforms, including HRAS, NRAS and splice variants KRAS4A and KRAS4B, are some of the most frequently mutated proteins in cancer. In particular, constitutively active mutants of KRAS comprise ∼80% of all RAS oncogenic mutations and are found in 98% of pancreatic, 45% of colorectal and 31% of lung tumors. Plasma membrane (PM) is the primary location of RAS signaling in biology and pathology. Thus, a better understanding of how RAS proteins localize to and distribute on the PM is critical to better comprehend RAS biology and to develop new strategies to treat RAS pathology. In this review, we discuss recent findings on how RAS proteins sort lipids as they undergo macromolecular assembly on the PM. We also discuss how RAS/lipid nanoclusters serve as signaling platforms for the efficient recruitment of effectors and signal transduction, and how perturbing the PM biophysical properties affect the spatial distribution of RAS isoforms and their functions.



2020 ◽  
Vol 118 (3) ◽  
pp. 420a
Author(s):  
Juliana M. Larson ◽  
Robert M. Nwokonko ◽  
James H. Baraniak ◽  
Yandong Zhou ◽  
Donald L. Gill


eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Andy KM Lam ◽  
Raimund Dutzler

TMEM16A is a ligand-gated anion channel that is activated by intracellular Ca2+. This channel comprises two independent pores and closely apposed Ca2+ binding sites that are contained within each subunit of a homodimeric protein. Previously we characterized the influence of positively charged pore-lining residues on anion conduction (<xref ref-type="bibr" rid="bib20">Paulino et al., 2017a</xref>). Here, we demonstrate the electrostatic control of permeation by the bound calcium ions in mouse TMEM16A using electrophysiology and Poisson-Boltzmann calculations. The currents of constitutively active mutants lose their outward rectification as a function of Ca2+ concentration due to the alleviation of energy barriers for anion conduction. This phenomenon originates from Coulombic interactions between the bound Ca2+ and permeating anions and thus demonstrates that an electrostatic gate imposed by the vacant binding site present in the sterically open pore, is released by Ca2+ binding to enable an otherwise sub-conductive pore to conduct with full capacity.



2013 ◽  
Vol 32 (3) ◽  
pp. 389-399 ◽  
Author(s):  
Jae-Sik Shin ◽  
Seung-Woo Hong ◽  
Jai-Hee Moon ◽  
Jin-Sun Kim ◽  
Kyung-Ah Jung ◽  
...  


2011 ◽  
Vol 301 (5) ◽  
pp. L765-L771 ◽  
Author(s):  
Jianxun Lei ◽  
David H. Ingbar

We previously reported that the 3,5,3′-triiodo-l-thyronine (T3)-induced increase of Na-K-ATPase activity in rat alveolar epithelial cells (AECs) required activation of Src kinase, PI3K, and MAPK/ERK1/2. In the present study, we assessed the role of Akt in Na-K-ATPase activity and the interaction between the PI3K and MAPK in response to T3 by using MP48 cells, inhibitors, and constitutively active mutants in the MP48 (alveolar type II-like) cell line. The Akt inhibitor VIII blocked T3-induced increases in Na-K-ATPase activity and amount of plasma membrane Na-K-ATPase protein. The Akt inhibitor VIII also abolished the increase in Na-K-ATPase activity induced by constitutively active mutants of either Src kinase or PI3K. Moreover, constitutively active mutants of Akt increased Na-K-ATPase activity in the absence of T3. Thus activation of Akt was required for T3-induced Na-K-ATPase activity in AECs and is sufficient in the absence of T3. Inhibitors of Src kinase (PP1), PI3K (wortmannin), and ERK1/2 (U0126) all blocked the T3-induced Na-K-ATPase activity. PP1 blocked the activation of PI3K and also ERK1/2 by T3, whereas U0126 did not prevent T3 activation of Src kinase or PI3K activity. Wortmannin did not significantly alter T3-increased MAPK/ERK1/2 activity, suggesting that T3-activated PI3K/Akt and MAPK/ERK1/2 pathways acted downstream of the Src kinase. Furthermore, in the absence of T3, a constitutively active mutant of Src kinase increased activities of Na-K-ATPase, PI3K, and MAPK/ERK1/2. A constitutively active mutant of PI3K enhanced Na-K-ATPase activity but did not alter the MAPK/ERK1/2 activity significantly. In summary, in adult rat AECs T3-stimulated Src kinase activity can activate both PI3K/Akt and MAPK/ERK1/2, and activation of Akt is necessary for T3-induced Na-K-ATPase activity.



2011 ◽  
Vol 22 (16) ◽  
pp. 2900-2911 ◽  
Author(s):  
Yuliya Zilberman ◽  
Naila O. Alieva ◽  
Stéphanie Miserey-Lenkei ◽  
Alexandra Lichtenstein ◽  
Zvi Kam ◽  
...  

In mammalian cells, the Golgi apparatus is a ribbon-like, compact structure composed of multiple membrane stacks connected by tubular bridges. Microtubules are known to be important to Golgi integrity, but the role of the actin cytoskeleton in the maintenance of Golgi architecture remains unclear. Here we show that an increase in Rho activity, either by treatment of cells with lysophosphatidic acid or by expression of constitutively active mutants, resulted in pronounced fragmentation of the Golgi complex into ministacks. Golgi dispersion required the involvement of mDia1 formin, a downstream target of Rho and a potent activator of actin polymerization; moreover, constitutively active mDia1, in and of itself, was sufficient for Golgi dispersion. The dispersion process was accompanied by formation of dynamic F-actin patches in the Golgi area. Experiments with cytoskeletal inhibitors (e.g., latrunculin B, blebbistatin, and Taxol) revealed that actin polymerization, myosin-II–driven contractility, and microtubule-based intracellular movement were all involved in the process of Golgi dispersion induced by Rho–mDia1 activation. Live imaging of Golgi recovery revealed that fusion of the small Golgi stacks into larger compartments was repressed in cells with active mDia1. Furthermore, the formation of Rab6-positive transport vesicles derived from the Golgi complex was enhanced upon activation of the Rho–mDia1 pathway. Transient localization of mDia1 to Rab6-positive vesicles was detected in cells expressing active RhoA. Thus, the Rho–mDia1 pathway is involved in regulation of the Golgi structure, affecting remodeling of Golgi membranes.



2010 ◽  
Vol 67 (17) ◽  
pp. 2979-2989 ◽  
Author(s):  
Manuela Ambrosio ◽  
Francesca Fanelli ◽  
Silvia Brocchetti ◽  
Francesco Raimondi ◽  
Mario Mauri ◽  
...  


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