Ceramide induces translocation of protein kinase C-α to the Golgi compartment of human embryonic kidney cells by interacting with the C2 domain

ABSTRACT In Aplysia californica, the serotonin-mediated translocation of protein kinase C (PKC) Apl II to neuronal membranes is important for synaptic plasticity. The orthologue of PKC Apl II, PKCε, has been reported to require phosphatidic acid (PA) in conjunction with diacylglycerol (DAG) for translocation. We find that PKC Apl II can be synergistically translocated to membranes by the combination of DAG and PA. We identify a mutation in the C1b domain (arginine 273 to histidine; PKC Apl II-R273H) that removes the effects of exogenous PA. In Aplysia neurons, the inhibition of endogenous PA production by 1-butanol inhibited the physiological translocation of PKC Apl II by serotonin in the cell body and at the synapse but not the translocation of PKC Apl II-R273H. The translocation of PKC Apl II-R273H in the absence of PA was explained by two additional effects of this mutation: (i) the mutation removed C2 domain-mediated inhibition, and (ii) the mutation decreased the concentration of DAG required for PKC Apl II translocation. We present a model in which, under physiological conditions, PA is important to activate the novel PKC Apl II both by synergizing with DAG and removing C2 domain-mediated inhibition.

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The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser359Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity

Molecular and Cellular Biology ◽

10.1128/mcb.01436-14 ◽

2015 ◽

Vol 35 (10) ◽

pp. 1727-1740 ◽

Cited By ~ 11

Author(s):

Jianli Gong ◽

Yongneng Yao ◽

Pingbo Zhang ◽

Barath Udayasuryan ◽

Elena V. Komissarova ◽

...

Keyword(s):

Oxidative Stress ◽

Protein Kinase C ◽

Protein Kinase ◽

Kinase Domain ◽

Cellular Growth ◽

C2 Domain ◽

Kinase C ◽

High Level ◽

Docking Interaction ◽

Structural Determinant

The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr313-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδ's Tyr313-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδ's enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser359. We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser359.In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser359phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr313docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity.

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Structure of human protein kinase C eta (PKCη) C2 domain and identification of phosphorylation sites

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2006.08.160 ◽

2006 ◽

Vol 349 (4) ◽

pp. 1182-1189 ◽

Cited By ~ 15

Author(s):

Dene R. Littler ◽

John R. Walker ◽

Yi-Min She ◽

Patrick J. Finerty ◽

Elena M. Newman ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Human Protein ◽

Phosphorylation Sites ◽

C2 Domain ◽

Kinase C ◽

Human Protein Kinase

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Crystal structure of the C2 domain from protein kinase C-δ

Structure ◽

10.1016/s0969-2126(98)00090-2 ◽

1998 ◽

Vol 6 (7) ◽

pp. 885-894 ◽

Cited By ~ 72

Author(s):

H Pappa ◽

J Murray-Rust ◽

LV Dekker ◽

PJ Parker ◽

NQ McDonald

Keyword(s):

Crystal Structure ◽

Protein Kinase C ◽

Protein Kinase ◽

C2 Domain ◽

Kinase C

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00817.2010 ◽

2012 ◽

Vol 302 (3) ◽

pp. H782-H789 ◽

Cited By ~ 28

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.

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