scholarly journals Distinct Effects of Fatty Acids on Translocation of γ- and ε-Subspecies of Protein Kinase C

1998 ◽  
Vol 143 (2) ◽  
pp. 511-521 ◽  
Author(s):  
Yasuhito Shirai ◽  
Kaori Kashiwagi ◽  
Keiko Yagi ◽  
Norio Sakai ◽  
Naoaki Saito
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Plasma Membrane ◽  
Protein Kinase C ◽  
Linoleic Acid ◽  
Protein Kinase ◽  
Fluorescent Protein ◽  
Target Site ◽  
Simultaneous Application ◽  
Kinase C

Effects of fatty acids on translocation of the γ- and ε-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). γ-PKC–GFP and ε-PKC–GFP predominated in the cytoplasm, but only a small amount of γ-PKC–GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of γ-PKC–GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on γ-PKC–GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of ε-PKC–GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of ε-PKC–GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of ε-PKC–GFP than that of γ-PKC–GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of γ-PKC–GFP. Simultaneous application of arachidonic acid (25 μM) and DiC8 (10 μM) elicited a slow, irreversible translocation of γ-PKC– GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation. These findings confirm the involvement of fatty acids in the translocation of γ- and ε-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.

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 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 Phospholipase C and Protein Kinase C-β 2 Mediate Insulin-Like Growth Factor II-Dependent Sphingosine Kinase 1 Activation

2011 ◽  
Vol 25 (12) ◽  
pp. 2144-2156 ◽  
Author(s):  
Hesham M. El-Shewy ◽  
Souzan A. Abdel-Samie ◽  
Abdelmohsen M. Al Qalam ◽  
Mi-Hye Lee ◽  
Kazuyuki Kitatani ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Fluorescent Protein ◽  
Sphingosine Kinase ◽  
Sphingosine Kinase 1 ◽  
Membrane Translocation ◽  
Kinase C ◽  
Sphingosine 1 Phosphate

Abstract We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.

Arachidonic acid and free fatty acids as second messengers and the role of protein kinase C

1995 ◽  
Vol 7 (3) ◽  
pp. 171-184 ◽  
Author(s):  
Wasiuddin A. Khan ◽  
Gerard C. Blobe ◽  
Yusuf A. Hannun
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Free Fatty Acids ◽  
Second Messengers ◽  
Kinase C

scholarly journals Lipoxygenase metabolites of arachidonic and linoleic acids modulate the adhesion of tumor cells to endothelium via regulation of protein kinase C.

1991 ◽  
Vol 2 (12) ◽  
pp. 1045-1055 ◽  
Author(s):  
B Liu ◽  
J Timar ◽  
J Howlett ◽  
C A Diglio ◽  
K V Honn
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Cell Adhesion ◽  
Linoleic Acid ◽  
Protein Kinase ◽  
Tumor Cell ◽  
Cell Monolayer ◽  
Protein Kinase Inhibitors ◽  
Tumor Cell Adhesion ◽  
Kinase C

12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) and 13(S)-hydroxyoctadecadienoic acid (13[S]-HODE), lipoxygenase metabolites of arachidonic acid and linoleic acid, respectively, previously have been suggested to regulate tumor cell adhesion to endothelium during metastasis. Adhesion of rat Walker carcinosarcoma (W256) cells to a rat endothelial cell monolayer was enhanced after treatment with 12(S)-HETE and this 12(S)-HETE enhanced adhesion was blocked by 13(S)-HODE. Protein kinase inhibitors, staurosporine, calphostin C, and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, inhibited the 12(S)-HETE enhanced W256 cell adhesion. Depleting W256 cells of protein kinase C (PKC) with phorbol 12-myristate-13-acetate abolished their ability to respond to 12(S)-HETE. Treatment of W256 cells with 12(S)-HETE induced a 100% increase in membrane-associated PKC activity whereas 13(S)-HODE inhibited the effect of 12(S)-HETE on PKC translocation. High-performance liquid chromatographic analysis revealed that in W256 cells 12-HETE and 13-HODE were two of the major lipoxygenase metabilites of arachidonic acid and linoleic acid, respectively. Therefore, these two metabolites may provide an alternative signaling pathway for the regulation of PKC. Further, these findings suggest that the regulation of tumor cell adhesion to endothelium by 12(S)-HETE and 13(S)-HODE may be a PKC-dependent process.

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