scholarly journals Interferon-γ inhibits enterocyte migration by reversibly displacing connexin43 from lipid rafts

2008 ◽  
Vol 295 (3) ◽  
pp. G559-G569 ◽  
Author(s):  
Cynthia L. Leaphart ◽  
Shipan Dai ◽  
Steven C. Gribar ◽  
Ward Richardson ◽  
John Ozolek ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Gap Junctions ◽  
Lipid Rafts ◽  
Interferon Γ ◽  
Dependent Manner ◽  
Wild Type ◽  
Kinase C ◽  
Intercellular Channels ◽  
Ifn Treatment

Necrotizing enterocolitis (NEC) is associated with the release of interferon-γ (IFN) by enterocytes and delayed intestinal restitution. Our laboratory has recently demonstrated that IFN inhibits enterocyte migration by impairing enterocyte gap junctions, intercellular channels that are composed of connexin43 (Cx43) monomers and that are required for enterocyte migration to occur. The mechanisms by which IFN inhibits gap junctions are incompletely understood. Lipid rafts are cholesterol-sphingolipid-rich microdomains of the plasma membrane that play a central role in the trafficking and signaling of various proteins. We now hypothesize that Cx43 is present on enterocyte lipid rafts and that IFN inhibits enterocyte migration by displacing Cx43 from lipid rafts in enterocytes. We now confirm our previous observations that intestinal restitution is impaired in NEC and demonstrate that Cx43 is present on lipid rafts in IEC-6 enterocytes. We show that lipid rafts are required for enterocyte migration, that IFN displaces Cx43 from lipid rafts, and that the phorbol ester phorbol 12-myristate 13-acetate (PMA) restores Cx43 to lipid rafts after treatment with IFN in a protein kinase C-dependent manner. IFN also reversibly decreased the phosphorylation of Cx43 on lipid rafts, which was restored by PMA. Strikingly, restoration of Cx43 to lipid rafts by PMA or by transfection of enterocytes with adenoviruses expressing wild-type Cx43 but not mutant Cx43 is associated with the restoration of enterocyte migration after IFN treatment. Taken together, these findings suggest an important role for lipid raft-Cx43 interactions in the regulation of enterocyte migration during exposure to IFN, such as NEC.

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.

Phospholipase C-γ1 is required for cell survival in oxidative stress by protein kinase C

2002 ◽  
Vol 363 (2) ◽  
pp. 395-401 ◽  
Author(s):  
Xiao-Chun BAI ◽  
Fan DENG ◽  
An-Ling LIU ◽  
Zhi-Peng ZOU ◽  
Yu WANG ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Survival ◽  
Phospholipase C ◽  
Cellular Response ◽  
Caspase 3 ◽  
Dependent Manner ◽  
Wild Type ◽  
Kinase C

Phospholipase C-γ1 (PLC-γ1) activation has been reported to enhance cell survival during the cellular response to oxidative stress. We studied the role of protein kinase C (PKC) pathways in mediating PLC-γ1 survival signalling in oxidative stress by using mouse embryonic fibroblasts genetically deficient in PLC-γ1 (Plcg1−/−) and its wild type (Plcg1+/+). PLC-γ1 was activated by H2O2 treatment in a dose- and time-dependent manner. Activation of PKC was also markedly increased in both cell lines treated with H2O2 (1–5mM), but with low doses (50–200μM), PKC activation was considerably decreased in Plcg1−/− cells. After treatment with H2O2, PKC-dependent phosphorylation of Bcl-2 and cell viability of Plcg1−/− cells decreased dramatically and caspase-3-like activity increased significantly compared with that of the wild-type cells. Furthermore, pretreatment of Plcg1+/+ cells with PKC-specific inhibitor decreased levels of PKC-dependent Bcl-2 phosphorylation, enhanced caspase-3 activity and their sensitivity to H2O2. On the contrary, treatment of Plcg1−/− cells with PKC-specific activator increased the Bcl-2 phosphorylation, decreased caspase-3 activity and improved their survival. These results suggest that PLC-γ1 mediates survival signalling in oxidative-stress response by PKC-dependent phosphorylation of Bcl-2 and inhibition of caspase-3.

Phospholipase A(2) and its products are involved in the purinergic receptor-mediated translocation of protein kinase C in CHO-K1 cells

2000 ◽  
Vol 113 (8) ◽  
pp. 1335-1343
Author(s):  
Y. Shirai ◽  
K. Kashiwagi ◽  
N. Sakai ◽  
N. Saito
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Purinergic Receptor ◽  
Phospholipase A ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Phospholipase A 2

The signal transduction involved in the purinergic stimuli-induced activation of protein kinase C (PKC) in CHO-K1 cells was investigated. Purinergic stimuli such as adenosine triphosphate and uridine triphosphate induced a transient translocation of PKC epsilon, gamma, and delta from the cytoplasm to the plasma membrane. These translocations were blocked by an inhibitor of phosphatidylinositol-specific phospholipase C (PLC), but not by an inhibitor of phosphatidylcholine-specific PLC. A diacylglycerol (DAG) analogue also induced reversible translocations of PKC gamma, epsilon, and delta from the cytoplasm to the plasma membrane, while the calcium ionophore A23187 caused a similar translocation of only the gamma subtype. These results confirm that the hydrolysis of phosphatidylinositol-2-phosphate by PLC and the subsequent generation of DAG and increase in Ca(2+)are involved in the purinergic stimuli-induced translocation of PKC. A DAG antagonist, 1-o-hexadecyl-2-o-acetyl-glycerol, blocked the DAG analogue-induced translocations of all PKC subtypes tested but failed to inhibit the purinergic stimuli-induced translocations of PKC epsilon and gamma. The DAG antagonist could not block the ATP- and UTP-induced translocation of PKC epsilon even in the absence of extracellular Ca(2+). Co-application of the DAG antagonist and a phospholipase A(2) (PLA(2)) inhibitor such as aristolochic acid, arachidonyltrifluoromethyl ketone, or bromoenol lactone inhibited the purinergic receptor-mediated translocation of PKC epsilon although each PLA(2) inhibitor alone did not block the translocation. In contrast to the epsilon subtype, ATP-induced translocation of PKC gamma was observed in the presence of both the PLA(2) inhibitor and the DAG antagonist. However, it is noteworthy that re-translocation of PKC gamma was hastened by the PLA(2) inhibitor. Furthermore products of PLA(2), such as lysophospholipids and fatty acids, induced the translocation of PKC gamma and epsilon in a dose dependent manner, but not delta. These results indicate that, in addition to PLC and DAG, PLA(2) and its products are involved in the purinergic stimuli-induced translocation of PKC epsilon and gamma in CHO-K1 cells. Each subtype of PKC in CHO-K1 cell is individually activated in response to a purinergic stimulation.

Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

1993 ◽  
Vol 70 (05) ◽  
pp. 800-806 ◽  
Author(s):  
C Ternisien ◽  
M Ramani ◽  
V Ollivier ◽  
F Khechai ◽  
T Vu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tissue Factor ◽  
Factor Ix ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Kinase C ◽  
Pkc Activation

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

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