The C2 domains of classical/conventional PKCs are specific PtdIns(4,5)P2-sensing domains

2007 ◽  
Vol 35 (5) ◽  
pp. 1046-1048 ◽  
Author(s):  
S. Corbalán-García ◽  
M. Guerrero-Valero ◽  
C. Marín-Vicente ◽  
J.C. Gómez-Fernández
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Living Cells ◽  
Model Membranes ◽  
Membrane Localization ◽  
Dependent Manner ◽  
C2 Domain ◽  
C2 Domains ◽  
Local Densities

The C2 domains of cPKCs [classical/conventional PKCs (protein kinase Cs)] bind to membranes in a Ca2+-dependent manner and thereby act as cellular Ca2+ effectors. Recent findings have demonstrated that the C2 domain of cPKCs interacts specifically with PtdIns(4,5)P2 through its polybasic cluster located in the β3–β4-strands, this interaction being critical for the membrane localization of these enzymes in living cells. In addition, these C2 domains exhibit higher affinity to bind PtdIns(4,5)P2 than any other polyphosphate phosphatidylinositols. It has also been shown that the presence of PtdIns(4,5)P2 in model membranes decreases the Ca2+ concentration required for classical C2 domains to bind them. Overall, the studies reviewed here suggest a new mechanism of membrane docking by the C2 domains of cPKCs in which the local densities of phosphatidylserine and PtdIns(4,5)P2 on the inner leaflet of the plasma membrane are sufficient to drive Ca2+-activated membrane docking during a physiological Ca2+ signal.

scholarly journals Real-time measurement of E2:ERα transcriptional activity in living cells

2019 ◽  
Author(s):  
Manuela Cipolletti ◽  
Stefano Leone ◽  
Stefania Bartoloni ◽  
Claudia Busonero ◽  
Filippo Acconcia
Keyword(s):  
Plasma Membrane ◽  
Real Time ◽  
Cell Biology ◽  
Transcriptional Activity ◽  
Receptor Activation ◽  
Living Cells ◽  
Membrane Localization ◽  
Dependent Manner ◽  
Plasma Membrane Localization ◽  
The Impact

AbstractKinetic analyses of diverse physiological processes have the potential to unveil new aspects of the molecular regulation of cell biology at temporal levels. 17β-estradiol (E2) regulates diverse physiological effects by binding to the estrogen receptor α (ERα), which primarily works as a transcription factor. Although many molecular details of the modulation of ERα transcriptional activity have been discovered including the impact of receptor plasma membrane localization and its relative E2-evoked signalling, the knowledge of real-time ERα transcriptional dynamics in living cells is lacking. Here, we report the generation of MCF-7 and HeLa cells stably expressing a modified luciferase under the control of an E2-sensitive promoter, which activity can be continuously monitored in living cells and show that E2 induces a linear increase in ERα transcriptional activity. Ligand-independent (e.g., epidermal growth factor) receptor activation was also detected in a time-dependent manner. Kinetic profiles of ERα transcriptional activity measured in the presence of both receptor antagonists and inhibitors of ERα plasma membrane localization reveals a biphasic dynamic of receptor behaviour underlying novel aspects of receptor-regulated transcriptional effects. Finally, analysis of the rate of the dose-dependent E2 induction of ERα transcriptional activity demonstrates that low doses of E2 induce an effect identical to that determined by high concentrations of E2 as a function of the duration of hormone administration. Overall, we present the characterization of sensitive stable cell lines where to study the kinetic of E2 transcriptional signaling and to identify new aspects of ERα function in different physiological or pathophysiological conditions.

scholarly journals Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca2+mobilization and degranulation in mast cells

2011 ◽  
Vol 22 (24) ◽  
pp. 4908-4917 ◽  
Author(s):  
Deepti Gadi ◽  
Alice Wagenknecht-Wiesner ◽  
David Holowka ◽  
Barbara Baird
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Time Course ◽  
Fluorescent Protein ◽  
Immunoglobulin E ◽  
Dependent Manner ◽  
Effector Domain ◽  
Granule Exocytosis ◽  
Kinase C

Protein kinase C β (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, FcεRI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca2+concentrations and oscillatory association of PKCβ–enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKCβ. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca2+mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca2+entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.

scholarly journals Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+ and PIP2 Recognition by Its C2 Domain

2006 ◽  
Vol 17 (1) ◽  
pp. 56-66 ◽  
Author(s):  
John H. Evans ◽  
Diana Murray ◽  
Christina C. Leslie ◽  
Joseph J. Falke
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Membrane Binding ◽  
Membrane Lipid ◽  
C2 Domain ◽  
Plasma Membrane Lipid ◽  
Protein Kinase Cα ◽  
Golgi Network

The C2 domain of protein kinase Cα (PKCα) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCα and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCα Ca2+-binding loops (CBLs) and PIP2 binding site (β-strands 3–4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCα C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on β-strands 3–4, which bind to plasma membrane PIP2.

scholarly journals Role of the Ca2+/Phosphatidylserine Binding Region of the C2 Domain in the Translocation of Protein Kinase Cα to the Plasma Membrane

2003 ◽  
Vol 278 (12) ◽  
pp. 10282-10290 ◽  
Author(s):  
Stephen R. Bolsover ◽  
Juan C. Gomez-Fernandez ◽  
Senena Corbalan-Garcia
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
C2 Domain ◽  
Binding Region ◽  
Protein Kinase Cα

scholarly journals Electrostatic and Hydrophobic Interactions Differentially Tune Membrane Binding Kinetics of the C2 Domain of Protein Kinase Cα

2013 ◽  
Vol 288 (23) ◽  
pp. 16905-16915 ◽  
Author(s):  
Angela M. Scott ◽  
Corina E. Antal ◽  
Alexandra C. Newton
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Rate Constants ◽  
Electrostatic Interactions ◽  
Hydrophobic Interactions ◽  
Dissociation Rate ◽  
C2 Domain ◽  
Pkc Isozymes ◽  
Dissociation Rate Constants

The cellular activation of conventional protein kinase C (PKC) isozymes is initiated by the binding of their C2 domains to membranes in response to elevations in intracellular Ca2+. Following this C2 domain-mediated membrane recruitment, the C1 domain binds its membrane-embedded ligand diacylglycerol, resulting in activation of PKC. Here we explore the molecular mechanisms by which the C2 domain controls the initial step in the activation of PKC. Using stopped-flow fluorescence spectroscopy to measure association and dissociation rate constants, we show that hydrophobic interactions are the major driving force in the binding of the C2 domain to anionic membranes, whereas electrostatic interactions dominate in membrane retention. Specifically, mutation of select hydrophobic or select basic residues in the Ca2+-binding loops reduces membrane affinity by distinct mechanisms; mutation of hydrophobic residues primarily alters association rate constants, whereas mutation of charged residues affects dissociation rate constants. Live cell imaging reveals that introduction of these mutations into full-length PKCα not only reduces the Ca2+-dependent translocation to plasma membrane but, by impairing the plasma membrane-sensing role of the C2 domain, causes phorbol ester-triggered redistribution of PKCα to other membranes, such as the Golgi. These data underscore the key role of the C2 domain in driving conventional PKC isozymes to the plasma membrane and reveal that not only the amplitude but also the subcellular location of conventional PKC signaling can be tuned by altering the affinity of this module for membranes.

scholarly journals Apical Membrane Targeting of Nedd4 Is Mediated by an Association of Its C2 Domain with Annexin Xiiib

2000 ◽  
Vol 149 (7) ◽  
pp. 1473-1484 ◽  
Author(s):  
Pamela J. Plant ◽  
Frank Lafont ◽  
Sandra Lecat ◽  
Paul Verkade ◽  
Kai Simons ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Immunogold Electron Microscopy ◽  
Apical Plasma Membrane ◽  
Apical Region ◽  
Apical Surface ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
C2 Domain

Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca2+-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca2+, a ∼35–40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca2+, and the interaction is direct and optimal at 1 μM Ca2+. Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca2+-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.

scholarly journals Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels

2012 ◽  
Vol 302 (3) ◽  
pp. H782-H789 ◽  
Author(s):  
Haifa Hallaq ◽  
Dao W. Wang ◽  
Jennifer D. Kunic ◽  
Alfred L. George ◽  
K. Sam Wells ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Oxidase Inhibitor ◽  
Intracellular Distribution ◽  
Human Embryonic Kidney Cells ◽  
Kinase C ◽  
Pkc Activation

Na+ current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na+ channels at the plasma membrane in this effect, but the results are controversial. In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na+ currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na+ channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca2+-sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser1503 or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleaching demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na+ channels in living cells.

scholarly journals Palmitoylation-dependent Estrogen Receptor α Membrane Localization: Regulation by 17β-Estradiol

2005 ◽  
Vol 16 (1) ◽  
pp. 231-237 ◽  
Author(s):  
Filippo Acconcia ◽  
Paolo Ascenzi ◽  
Alessio Bocedi ◽  
Enzo Spisni ◽  
Vittorio Tomasi ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Estrogen Receptor ◽  
Plasma Membrane ◽  
Estrogen Receptor Α ◽  
Membrane Localization ◽  
Target Cells ◽  
Dependent Manner ◽  
Caveolin 1 ◽  
17Β Estradiol

A fraction of the nuclear estrogen receptor α (ERα) is localized to the plasma membrane region of 17β-estradiol (E2) target cells. We previously reported that ERα is a palmitoylated protein. To gain insight into the molecular mechanism of ERα residence at the plasma membrane, we tested both the role of palmitoylation and the impact of E2 stimulation on ERα membrane localization. The cancer cell lines expressing transfected or endogenous human ERα (HeLa and HepG2, respectively) or the ERα nonpalmitoylable Cys447Ala mutant transfected in HeLa cells were used as experimental models. We found that palmitoylation of ERα enacts ERα association with the plasma membrane, interaction with the membrane protein caveolin-1, and nongenomic activities, including activation of signaling pathways and cell proliferation (i.e., ERK and AKT activation, cyclin D1 promoter activity, DNA synthesis). Moreover, E2 reduces both ERα palmitoylation and its interaction with caveolin-1, in a time- and dose-dependent manner. These data point to the physiological role of ERα palmitoylation in the receptor localization to the cell membrane and in the regulation of the E2-induced cell proliferation.

scholarly journals Optogenetic inhibition and activation of Rac and Rap1 using a modified iLID system

2020 ◽  
Author(s):  
Nick R. Elston ◽  
Michael Pablo ◽  
Fred Pimenta ◽  
Klaus M. Hahn ◽  
Takashi Watanabe
Keyword(s):  
Plasma Membrane ◽  
Living Cells ◽  
Small Gtpases ◽  
Spatiotemporal Dynamics ◽  
Membrane Localization ◽  
Cellular Functions ◽  
Activation Kinetics ◽  
System 1 ◽  
Control Plasma

The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatiotemporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light induced dimerization (iLID) system [1] was used to control plasma membrane localization of protein domains that specifically activate or inactivate Rap1 and Rac (Tiam1 and Chimerin for Rac, RasGRP2 and Rap1GAP for Rap1 [2, 3, 4, 5]). Irradiation yielded a 50% to 230% increase in the concentration of these domains at the membrane, leading to effects on cell morphodynamics consistent with the known roles of Rac1 and Rap1.

Sign in / Sign up

Export Citation Format

Share Document