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

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

Journal of Biological Chemistry ◽

10.1074/jbc.m209385200 ◽

2002 ◽

Vol 278 (7) ◽

pp. 4972-4980 ◽

Cited By ~ 73

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

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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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Inorganic lead stimulates DNA synthesis in human astrocytoma cells: role of protein kinase Cα

Journal of Neurochemistry ◽

10.1046/j.1471-4159.2001.00434.x ◽

2001 ◽

Vol 78 (3) ◽

pp. 590-599 ◽

Cited By ~ 29

Author(s):

Hailing Lu ◽

Marina Guizzetti ◽

Lucio G. Costa

Keyword(s):

Protein Kinase ◽

Dna Synthesis ◽

Astrocytoma Cells ◽

Inorganic Lead ◽

Human Astrocytoma ◽

Protein Kinase Cα ◽

Human Astrocytoma Cells

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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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