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

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m113.467456 ◽

2013 ◽

Vol 288 (23) ◽

pp. 16905-16915 ◽

Cited By ~ 19

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.

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Membrane docking of the C2 domain from protein kinase Cα as seen by polarized ATR-IR. The role of PIP2

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2010.11.035 ◽

2011 ◽

Vol 1808 (3) ◽

pp. 684-695 ◽

Cited By ~ 15

Author(s):

Alessio Ausili ◽

Senena Corbalán-García ◽

Juan C. Gómez-Fernández ◽

Derek Marsh

Keyword(s):

Protein Kinase ◽

C2 Domain ◽

Protein Kinase Cα

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

Molecular Biology of the Cell ◽

10.1091/mbc.e05-01-0067 ◽

2005 ◽

Vol 16 (6) ◽

pp. 2848-2861 ◽

Cited By ~ 39

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.

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

Biochemical Journal ◽

10.1042/bj3370513 ◽

1999 ◽

Vol 337 (3) ◽

pp. 513-521 ◽

Cited By ~ 28

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.

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Role of Protein Kinase Cα in Signaling from the Histamine H1 Receptor to the Nucleus

Molecular Pharmacology ◽

10.1124/mol.59.5.1012 ◽

2001 ◽

Vol 59 (5) ◽

pp. 1012-1021 ◽

Cited By ~ 31

Author(s):

A. C. Megson ◽

E. M. Walker ◽

S. J. Hill

Keyword(s):

Protein Kinase ◽

H1 Receptor ◽

Histamine H1 Receptor ◽

Protein Kinase Cα

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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

Journal of Cell Science ◽

10.1242/jcs.115.15.3083 ◽

2002 ◽

Vol 115 (15) ◽

pp. 3083-3092 ◽

Cited By ~ 1

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.

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Action of a phorbol ester on B-cells: potentiation of stimulant-induced electrical activity

AJP Cell Physiology ◽

10.1152/ajpcell.1985.248.5.c527 ◽

1985 ◽

Vol 248 (5) ◽

pp. C527-C534 ◽

Cited By ~ 33

Author(s):

C. S. Pace ◽

K. T. Goldsmith

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Electrical Activity ◽

Electrical Response ◽

Tumor Promoter ◽

Kinase C ◽

Cell Plasma ◽

Lag Period

The possible role of protein kinase c in regulating the electrical events in the B-cell plasma membrane was examined by using the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a known activator of this enzyme. TPA has been found to enhance glucose- and sulfonylurea-induced insulin secretion with little or no effect on the fluxes of 86Rb+ or 45Ca2+ across the plasma membrane. TPA, 0.2 microM, did not influence the membrane potential from 0 to 5.6 mM glucose but increased by two- to threefold the fraction of the plateau phase of the oscillatory electrical activity induced by 7.0-11.1 mM glucose. This effect of TPA was completely blocked by 0.5 mM spermidine, an inhibitor of protein kinase c. However, spermidine had no influence on the electrical activity elicited by glucose alone. Glyburide, 10 nM, initiated slow depolarization and constant spike activity after about 18 and 25 min, respectively. TPA or 2.8 mM glucose reduced the lag period for glyburide to elicit an electrical response by about 75%. The duration of the spikes was increased two- to threefold by the presence of glucose or TPA with glyburide. There were also characteristic differences in the shape of the spikes under each experimental condition. Spermidine inhibited the influence of glucose, but not TPA, on the glyburide-induced electrical response. These results indicate that TPA may influence stimulant-induced electrical events via protein kinase c or by directly altering the ionic permeability of the plasma membrane.

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Counteractive Control of Polarized Morphogenesis during Mating by Mitogen-activated Protein Kinase Fus3 and G1 Cyclin-dependent Kinase

Molecular Biology of the Cell ◽

10.1091/mbc.e07-08-0757 ◽

2008 ◽

Vol 19 (4) ◽

pp. 1739-1752 ◽

Cited By ~ 20

Author(s):

Lu Yu ◽

Maosong Qi ◽

Mark A. Sheff ◽

Elaine A. Elion

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Cell Polarization ◽

Mitogen Activated Protein Kinase ◽

G1 Arrest ◽

Cyclin Dependent Kinase ◽

Cycle Arrest ◽

Mitogen Activated Protein ◽

Cdc28 Kinase

Cell polarization in response to external cues is critical to many eukaryotic cells. During pheromone-induced mating in Saccharomyces cerevisiae, the mitogen-activated protein kinase (MAPK) Fus3 induces polarization of the actin cytoskeleton toward a landmark generated by the pheromone receptor. Here, we analyze the role of Fus3 activation and cell cycle arrest in mating morphogenesis. The MAPK scaffold Ste5 is initially recruited to the plasma membrane in random patches that polarize before shmoo emergence. Polarized localization of Ste5 is important for shmooing. In fus3 mutants, Ste5 is recruited to significantly more of the plasma membrane, whereas recruitment of Bni1 formin, Cdc24 guanine exchange factor, and Ste20 p21-activated protein kinase are inhibited. In contrast, polarized recruitment still occurs in a far1 mutant that is also defective in G1 arrest. Remarkably, loss of Cln2 or Cdc28 cyclin-dependent kinase restores polarized localization of Bni1, Ste5, and Ste20 to a fus3 mutant. These and other findings suggest Fus3 induces polarized growth in G1 phase cells by down-regulating Ste5 recruitment and by inhibiting Cln/Cdc28 kinase, which prevents basal recruitment of Ste5, Cdc42-mediated asymmetry, and mating morphogenesis.

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