scholarly journals Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane

2020 ◽  
Vol 117 (39) ◽  
pp. 24258-24268 ◽  
Author(s):  
Que N. Van ◽  
Cesar A. López ◽  
Marco Tonelli ◽  
Troy Taylor ◽  
Ben Niu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Signaling ◽  
Hypervariable Region ◽  
Bound State ◽  
Small Gtpase ◽  
Photochemical Oxidation ◽  
Neutron Reflectivity ◽  
Raf Kinase ◽  
Populated State ◽  
Membrane Bound

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as “membrane-distal” and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.

scholarly journals Phospholipase D1 Regulates Lymphocyte Adhesion via Upregulation of Rap1 at the Plasma Membrane

2009 ◽  
Vol 29 (12) ◽  
pp. 3297-3306 ◽  
Author(s):  
Adam Mor ◽  
Joseph P. Wynne ◽  
Ian M. Ahearn ◽  
Michael L. Dustin ◽  
Guangwei Du ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Bound State ◽  
Small Gtpase ◽  
Resting Cells ◽  
Nucleotide Exchange ◽  
Lymphocyte Adhesion ◽  
Nucleotide Exchange Factor ◽  
Phospholipase D1

ABSTRACT Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane.

scholarly journals Hindbrain GLP-1 receptor-mediated suppression of food intake requires a PI3K-dependent decrease in phosphorylation of membrane-bound Akt

2013 ◽  
Vol 305 (6) ◽  
pp. E751-E759 ◽  
Author(s):  
Laura E. Rupprecht ◽  
Elizabeth G. Mietlicki-Baase ◽  
Derek J. Zimmer ◽  
Lauren E. McGrath ◽  
Diana R. Olivos ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Food Intake ◽  
Intracellular Signaling ◽  
Nucleus Tractus Solitarius ◽  
Ex Vivo ◽  
Mapk Signaling ◽  
Current Data ◽  
Akt Phosphorylation ◽  
Membrane Bound

Glucagon-like peptide-1 (GLP-1) receptors (GLP-1R) expressed in the nucleus tractus solitarius (NTS) are physiologically required for the control of feeding. Recently, NTS GLP-1R-mediated suppression of feeding was shown to occur via a rapid PKA-induced suppression of AMPK and activation of MAPK signaling. Unknown are the additional intracellular signaling pathways that account for the long-term hypophagic effects of GLP-1R activation. Because cAMP/PKA activity can promote PI3K/PIP3-dependent translocation of Akt to the plasma membrane, we hypothesize that hindbrain GLP-1R-mediated control of feeding involves a PI3K-Akt-dependent pathway. Importantly, the novel evidence presented here challenges the dogmatic view that PI3K phosphorylation results in an obligatory activation of Akt and instead supports a growing body of literature showing that activation of cAMP/PKA can inhibit Akt phosphorylation at the plasma membrane. Behavioral data show that inhibition of hindbrain PI3K activity by a fourth icv administration of LY-294002 (3.07 μg) attenuated the food intake- and body weight-suppressive effects of a fourth icv administration of the GLP-1R agonist exendin-4 (0.3 μg) in rats. Hindbrain administration of triciribine (10 μg), an inhibitor of PIP3-dependent translocation of Akt to the cell membrane, also attenuated the intake-suppressive effects of a fourth icv injection of exendin-4. Immunoblot analyses of ex vivo NTS tissue lysates and in vitro GLP-1R-expressing neurons (GT1–7) support the behavioral findings and show that GLP-1R activation decreases phosphorylation of Akt in a time-dependent fashion. Current data reveal the requirement of PI3K activation, PIP3-dependent translocation of Akt to the plasma membrane, and suppression in phosphorylation of membrane-bound Akt to mediate the food intake-suppressive effects of hindbrain GLP-1R activation.

Exterior and Interior Events in Early Stage of Thrombin-induced Platelet Aggregation

1977 ◽  
Vol 38 (03) ◽  
pp. 0630-0639 ◽  
Author(s):  
Shuichi Hashimoto ◽  
Sachiko Shibata ◽  
Bonro Kobayashi
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Early Stage ◽  
Adenyl Cyclase ◽  
Cellular Component ◽  
Primary Event ◽  
Rabbit Platelets ◽  
Membrane Bound

SummaryTreatment of washed rabbit platelets with 1 u/ml of thrombin at 37° C resulted in a disappearance from platelets of a protein with 250,000 dalton molecular weight which was shown to be originated from plasma membrane. Parallel loss of adenyl cyclase was noted, and both reactions were complete within 30 sec. From the patterns of disc electrophoretograms, the importance of quick suppression of thrombin action in demonstrating the primary event was stressed.Thrombin induced an apparent activation of membrane bound phosphodiesterase. This reaction was also complete within 30 sec. The cellular component which contained the enzyme activity was distinct from plasma membrane. Soluble phosphodiesterase was not influenced by thrombin at all.These reactions required intact platelet cells to react with thrombin, and no reaction was detected when subcellular preparations were treated with thrombin.Possibility of collaboration of changes in externally located synthetic enzyme with those in internally located degrading enzyme in the early phase of thrombin action on platelets was suggested.

scholarly journals Unveiling the Dynamics of KRAS4b on Lipid Model Membranes

2021 ◽  
Author(s):  
Cesar A. López ◽  
Animesh Agarwal ◽  
Que N. Van ◽  
Andrew G. Stephen ◽  
S. Gnanakaran
Keyword(s):  
Molecular Mechanisms ◽  
Hypervariable Region ◽  
Small Gtpase ◽  
Model Systems ◽  
Coarse Grained ◽  
Regulatory Function ◽  
Limited Information ◽  
Long Time ◽  
Cell Growth And Differentiation ◽  
State Models

AbstractSmall GTPase proteins are ubiquitous and responsible for regulating several processes related to cell growth and differentiation. Mutations that stabilize their active state can lead to uncontrolled cell proliferation and cancer. Although these proteins are well characterized at the cellular scale, the molecular mechanisms governing their functions are still poorly understood. In addition, there is limited information about the regulatory function of the cell membrane which supports their activity. Thus, we have studied the dynamics and conformations of the farnesylated KRAS4b in various membrane model systems, ranging from binary fluid mixtures to heterogeneous raft mimics. Our approach combines long time-scale coarse-grained (CG) simulations and Markov state models to dissect the membrane-supported dynamics of KRAS4b. Our simulations reveal that protein dynamics is mainly modulated by the presence of anionic lipids and to some extent by the nucleotide state (activation) of the protein. In addition, our results suggest that both the farnesyl and the polybasic hypervariable region (HVR) are responsible for its preferential partitioning within the liquid-disordered (Ld) domains in membranes, potentially enhancing the formation of membrane-driven signaling platforms. Graphic Abstract

scholarly journals PROBLEMS ASSOCIATED WITH THE DEMONSTRATION BY LEAD METHODS OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN RESIDENT PERITONEAL MACROPHAGES AND EXUDATE MONOCYTES OF THE GUINEA PIG

1973 ◽  
Vol 21 (5) ◽  
pp. 488-498 ◽  
Author(s):  
R. E. POELMANN ◽  
W. T. DAEMS ◽  
E. J. VAN LOHUIZEN
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Microscopic Study ◽  
Heterogeneous Nucleation ◽  
Adenosine Triphosphatase ◽  
Peritoneal Macrophages ◽  
Electron Microscopic ◽  
Adenosine Triphosphatase Activity ◽  
Eosinophilic Granulocytes ◽  
Membrane Bound

This cytochemical and electron microscopic study on peritoneal macrophages of the guinea pig has raised doubts concerning the validity of lead methods for the demonstration of plasma membrane-bound adenosine triphosphatase activity. The problems encountered are inherent in the use of lead ions as a capture reagent. The nonenzymatically formed precipitates reflect sites of heterogeneous nucleation specific for certain kinds of cells, e.g., resident peritoneal macrophages, eosinophilic granulocytes and, to a lesser degree, exudate monocytes. This type of precipitation is also catalyzed on the surface of nonbiologic matrices such as latex particles. Enzymatic processes may well occur, but they cannot be distinguished from nonenzymatic processes.

scholarly journals A constitutive, plasma-membrane bound β-glucosidase inTrichoderma reesei

1986 ◽  
Vol 34 (3) ◽  
pp. 291-295 ◽  
Author(s):  
Claudio Umile ◽  
Christian P. Kubicek
Keyword(s):  
Plasma Membrane ◽  
Membrane Bound

scholarly journals Advances in understanding the role of cardiac glycosides in control of sodium transport in renal tubules

2014 ◽  
Vol 222 (1) ◽  
pp. R11-R24 ◽  
Author(s):  
Syed Jalal Khundmiri
Keyword(s):  
Heart Failure ◽  
Intracellular Signaling ◽  
Cardiac Contractility ◽  
Myocardial Cell ◽  
Renal Tubules ◽  
Intracellular Signaling Pathway ◽  
Body Sodium ◽  
Membrane Bound ◽  
Total Body

Cardiotonic steroids have been used for the past 200 years in the treatment of congestive heart failure. As specific inhibitors of membrane-bound Na+/K+ATPase, they enhance cardiac contractility through increasing myocardial cell calcium concentration in response to the resulting increase in intracellular Na concentration. The half-minimal concentrations of cardiotonic steroids required to inhibit Na+/K+ATPase range from nanomolar to micromolar concentrations. In contrast, the circulating levels of cardiotonic steroids under physiological conditions are in the low picomolar concentration range in healthy subjects, increasing to high picomolar levels under pathophysiological conditions including chronic kidney disease and heart failure. Little is known about the physiological function of low picomolar concentrations of cardiotonic steroids. Recent studies have indicated that physiological concentrations of cardiotonic steroids acutely stimulate the activity of Na+/K+ATPase and activate an intracellular signaling pathway that regulates a variety of intracellular functions including cell growth and hypertrophy. The effects of circulating cardiotonic steroids on renal salt handling and total body sodium homeostasis are unknown. This review will focus on the role of low picomolar concentrations of cardiotonic steroids in renal Na+/K+ATPase activity, cell signaling, and blood pressure regulation.

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