scholarly journals The ATP-dependent Membrane Localization of Protein Kinase Cα Is Regulated by Ca2+ Influx and Phosphatidylinositol 4,5-Bisphosphate in Differentiated PC12 Cells

2005 ◽  
Vol 16 (6) ◽  
pp. 2848-2861 ◽  
Author(s):  
Consuelo Marín-Vicente ◽  
Juan C. Gómez-Fernández ◽  
Senena Corbalán-García
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Pc12 Cells ◽  
Specific Inhibitor ◽  
Enzyme Activation ◽  
C2 Domain ◽  
Differentiation Process ◽  
Rich Cluster ◽  
Differentiated Pc12 Cells ◽  
Protein Kinase Cα

Signal transduction through protein kinase Cs (PKCs) strongly depends on their subcellular localization. Here, we investigate the molecular determinants of PKCα localization by using a model system of neural growth factor (NGF)-differentiated pheochromocytoma (PC12) cells and extracellular stimulation with ATP. Strikingly, the Ca2+ influx, initiated by the ATP stimulation of P2X receptors, rather than the Ca2+ released from the intracellular stores, was the driving force behind the translocation of PKCα to the plasma membrane. Furthermore, the localization process depended on two regions of the C2 domain: the Ca2+-binding region and the lysine-rich cluster, which bind Ca2+ and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], respectively. It was demonstrated that diacylglycerol was not involved in the localization of PKCα through its C1 domain, and in lieu, the presence of PtdIns(4,5)P2 increased the permanence of PKCα in the plasma membrane. Finally, it also was shown that ATP cooperated with NGF during the differentiation process of PC12 cells by increasing the length of the neurites, an effect that was inhibited when the cells were incubated in the presence of a specific inhibitor of PKCα, suggesting a possible role for this isoenzyme in the neural differentiation process. Overall, these results show a novel mechanism of PKCα activation in differentiated PC12 cells, where Ca2+ influx, together with the endogenous PtdIns(4,5)P2, anchor PKCα to the plasma membrane through two distinct motifs of its C2 domain, leading to enzyme activation.

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 Determination of the calcium-binding sites of the C2 domain of protein kinase Cα that are critical for its translocation to the plasma membrane

1999 ◽  
Vol 337 (3) ◽  
pp. 513-521 ◽  
Author(s):  
Senena CORBALÁN-GARCÍA ◽  
José A. RODRÍGUEZ-ALFARO ◽  
Juan C. GÓMEZ-FERNÁNDEZ
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Hela Cells ◽  
Calcium Binding ◽  
Lipid Vesicles ◽  
Site Directed Mutagenesis ◽  
C2 Domain ◽  
Protein Kinase Cα

The C2 domain is a conserved protein module present in various signal-transducing proteins. To investigate the function of the C2 domain of protein kinase Cα (PKCα), we have generated a recombinant glutathione S-transferase-fused C2 domain from rat PKCα, PKC-C2. We found that PKC-C2 binds with high affinity (half-maximal binding at 0.6 µM) to lipid vesicles containing the negatively charged phospholipid phosphatidylserine. When expressed into COS and HeLa cells, most of the PKC-C2 was found at the plasma membrane, whereas when the cells were depleted of Ca2+ by incubation with EGTA and ionophore, the C2 domain was localized preferentially in the cytosol. Ca2+ titration was performed in vivo and the critical Ca2+ concentration ranged from 0.1 to 0.32 µM. We also identified, by site-directed mutagenesis, three aspartic residues critical for that Ca2+ interaction, namely Asp-187, Asp-246 and Asp-248. Mutation of these residues to asparagine, to abolish their negative charge, resulted in a domain expressed as the same extension as wild-type protein that could interact in vitro with neither Ca2+ nor phosphatidylserine. Overexpression of these mutants into COS and HeLa cells also showed that they cannot localize at the plasma membrane, as demonstrated by immunofluorescence staining and subcellular fractionation. These results suggest that the Ca2+-binding site might be involved in promoting the interaction of the C2 domain of PKCα with the plasma membrane in vivo.

scholarly journals The Simultaneous Production of Phosphatidic Acid and Diacylglycerol Is Essential for the Translocation of Protein Kinase Cϵ to the Plasma Membrane in RBL-2H3 Cells

2003 ◽  
Vol 14 (12) ◽  
pp. 4885-4895 ◽  
Author(s):  
Maria Jose Lopez-Andreo ◽  
Juan C. Gomez-Fernandez ◽  
Senena Corbalan-Garcia
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Enzyme Activation ◽  
C2 Domain ◽  
Direct Stimulation ◽  
Downstream Target ◽  
Low Concentrations ◽  
Stimulation Of

To evaluate the role of the C2 domain in protein kinase Cϵ (PKCϵ) localization and activation after stimulation of the IgE receptor in RBL-2H3 cells, we used a series of mutants located in the phospholipid binding region of the enzyme. The results obtained suggest that the interaction of the C2 domain with the phospholipids in the plasma membrane is essential for anchoring the enzyme in this cellular compartment. Furthermore, the use of specific inhibitors of the different pathways that generate both diacylglycerol and phosphatidic acid has shown that the phosphatidic acid generated via phospholipase D (PLD)-dependent pathway, in addition to the diacylglycerol generated via phosphoinosite-phospholipase C (PLC), are involved in the localization of PKCϵ in the plasma membrane. Direct stimulation of RBL-2H3 cells with very low concentrations of permeable phosphatidic acid and diacylglycerol exerted a synergistic effect on the plasma membrane localization of PKCϵ. Moreover, the in vitro kinase assays showed that both phosphatidic acid and diacylglycerol are essential for enzyme activation. Together, these results demonstrate that phosphatidic acid is an important and essential activator of PKCϵ through the C2 domain and locate this isoenzyme in a new scenario where it acts as a downstream target of PLD.

Suppression of Synaptotagmin II restrains phorbolester-induced downregulation of protein kinase Cα by diverting the kinase from a degradative pathway to the recycling endocytic compartment

2002 ◽  
Vol 115 (15) ◽  
pp. 3083-3092 ◽  
Author(s):  
Ze Peng ◽  
Elena Grimberg ◽  
Ronit Sagi-Eisenberg
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Secretory Granules ◽  
Critical Factor ◽  
Cellular Level ◽  
Endocytic Compartment ◽  
Early Endosomes ◽  
Protein Kinase Cα ◽  
Rbl Cells

Downregulation of protein kinase Cα (PKCα) following long-term exposure to phorbol esters such as TPA is traffic dependent and involves delivery of the active, membrane-associated PKCα to endosomes. In this study, we show that synaptotagmin II (Syt II), a member of the Syt family of proteins, is required for TPA-induced degradation of PKCα. Thus, whereas the kinase half-life in TPA-treated cultured mast cells (the mast cell line rat basophilic leukemia RBL-2H3) is 2 hours, it is doubled in RBL-Syt II- cells, in which the cellular level of Syt II is reduced by>95% by transfection with Syt II antisense cDNA. We demonstrate that in TPA-treated RBL cells, PKCα travels from the cytosol to the plasma membrane, where it is delivered to early endosomes on its route to degradation. By contrast, in TPA-treated RBL-Syt II- cells,PKCα is diverted to recycling endosomes and remains distributed between the plasma membrane and the perinuclear recycling endocytic compartment. Notably, in both RBL and RBL-Syt II- cells, a fraction of PKCα is delivered and maintained in the secretory granules (SG). These results implicate Syt II as a critical factor for the delivery of internalized cargo for degradation. As shown here, one consequence of Syt II suppression is a delay in PKCα downregulation, resulting in its prolonged signaling.

scholarly journals Hydrogen Gas Attenuates Hypoxic-Ischemic Brain Injury via Regulation of the MAPK/HO-1/PGC-1a Pathway in Neonatal Rats

2020 ◽  
Vol 2020 ◽  
pp. 1-16 ◽  
Author(s):  
Peipei Wang ◽  
Mingyi Zhao ◽  
Zhiheng Chen ◽  
Guojiao Wu ◽  
Masayuki Fujino ◽  
...  
Keyword(s):  
Brain Injury ◽  
Protein Kinase ◽  
Pc12 Cells ◽  
Heme Oxygenase 1 ◽  
Ischemic Brain Injury ◽  
Protective Effects ◽  
Neonatal Rats ◽  
Ischemic Brain ◽  
Differentiated Pc12 Cells ◽  
Hypoxic Ischemic Brain Injury

Neonatal hypoxic-ischemic encephalopathy (HIE) is a leading cause of death in neonates with no effective treatments. Recent advancements in hydrogen (H2) gas offer a promising therapeutic approach for ischemia reperfusion injury; however, the impact of this approach for HIE remains a subject of debate. We assessed the therapeutic effects of H2 gas on HIE and the underlying molecular mechanisms in a rat model of neonatal hypoxic-ischemic brain injury (HIBI). H2 inhalation significantly attenuated neuronal injury and effectively improved early neurological outcomes in neonatal HIBI rats as well as learning and memory in adults. This protective effect was associated with initiation time and duration of sustained H2 inhalation. Furthermore, H2 inhalation reduced the expression of Bcl-2-associated X protein (BAX) and caspase-3 while promoting the expression of Bcl-2, nuclear factor erythroid-2-related factor 2, and heme oxygenase-1 (HO-1). H2 activated extracellular signal-regulated kinase and c-Jun N-terminal protein kinase and dephosphorylated p38 mitogen-activated protein kinase (MAPK) in oxygen-glucose deprivation/reperfusion (OGD/R) nerve growth factor-differentiated PC12 cells. Inhibitors of MAPKs blocked H2-induced HO-1 expression. HO-1 small interfering RNA decreased the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and sirtuin 1 (SIRT1) and reversed the protectivity of H2 against OGD/R-induced cell death. These findings suggest that H2 augments cellular antioxidant defense capacity through activation of MAPK signaling pathways, leading to HO-1 expression and subsequent upregulation of PGC-1α and SIRT-1 expression. Thus, upregulation protects NGF-differentiated PC12 cells from OGD/R-induced oxidative cytotoxicity. In conclusion, H2 inhalation exerted protective effects on neonatal rats with HIBI. Early initiation and prolonged H2 inhalation had better protective effects on HIBI. These effects of H2 may be related to antioxidant, antiapoptotic, and anti-inflammatory responses. HO-1 plays an important role in H2-mediated protection through the MAPK/HO-1/PGC-1α pathway. Our results support further assessment of H2 as a potential therapeutic for neurological conditions in which oxidative stress and apoptosis are implicated.

scholarly journals A New Phosphatidylinositol 4,5-Bisphosphate-binding Site Located in the C2 Domain of Protein Kinase Cα

2002 ◽  
Vol 278 (7) ◽  
pp. 4972-4980 ◽  
Author(s):  
Senena Corbalán-Garcı́a ◽  
Josefa Garcı́a-Garcı́a ◽  
José A. Rodrı́guez-Alfaro ◽  
Juan C. Gómez-Fernández
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
C2 Domain ◽  
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.

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.

Sign in / Sign up

Export Citation Format

Share Document