scholarly journals A Spatiotemporally Coordinated Cascade of Protein Kinase C Activation Controls Isoform-Selective Translocation

2006 ◽  
Vol 26 (6) ◽  
pp. 2247-2261 ◽  
Author(s):  
Alejandra Collazos ◽  
Barthélémy Diouf ◽  
Nathalie C. Guérineau ◽  
Corinne Quittau-Prévostel ◽  
Marion Peter ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Fluorescent Protein ◽  
Functional Dependence ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Cell Contacts ◽  
Kinase C ◽  
Trh Receptor ◽  
Cell Cell

ABSTRACT In pituitary GH3B6 cells, signaling involving the protein kinase C (PKC) multigene family can self-organize into a spatiotemporally coordinated cascade of isoform activation. Indeed, thyrotropin-releasing hormone (TRH) receptor activation sequentially activated green fluorescent protein (GFP)-tagged or endogenous PKCβ1, PKCα, PKCε, and PKCδ, resulting in their accumulation at the entire plasma membrane (PKCβ and -δ) or selectively at the cell-cell contacts (PKCα and -ε). The duration of activation ranged from 20 s for PKCα to 20 min for PKCε. PKCα and -ε selective localization was lost in the presence of Gö6976, suggesting that accumulation at cell-cell contacts is dependent on the activity of a conventional PKC. Constitutively active, dominant-negative PKCs and small interfering RNAs showed that PKCα localization is controlled by PKCβ1 activity and is calcium independent, while PKCε localization is dependent on PKCα activity. PKCδ was independent of the cascade linking PKCβ1, -α, and -ε. Furthermore, PKCα, but not PKCε, is involved in the TRH-induced β-catenin relocation at cell-cell contacts, suggesting that PKCε is not the unique functional effector of the cascade. Thus, TRH receptor activation results in PKCβ1 activation, which in turn initiates a calcium-independent but PKCβ1 activity-dependent sequential translocation of PKCα and -ε. These results challenge the current understanding of PKC signaling and raise the question of a functional dependence between isoforms.

Role of protein kinase C-η in cigarette smoke extract-induced apoptosis in MRC-5-cells

2014 ◽  
Vol 34 (9) ◽  
pp. 869-877 ◽  
Author(s):  
ES Son ◽  
SY Kyung ◽  
SP Lee ◽  
SH Jeong ◽  
JY Shin ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cigarette Smoke ◽  
Caspase 3 ◽  
Dominant Negative ◽  
Lung Fibroblasts ◽  
Apoptotic Pathway ◽  
Kinase C ◽  
Extrinsic Apoptotic Pathway ◽  
Infected Cells

Cigarette smoke (CS) is a major risk factor for emphysema, which causes cell death in structural cells of the lung by mechanisms that are still not completely understood. We demonstrated previously that CS extract (CSE) induces caspase activation in MRC-5 human lung fibroblasts, activated protein kinase C-η (PKC-η), and translocated PKC-η from the cytosol to the membrane. The objective of this study was to investigate the involvement of PKC-η activation in a CSE-induced extrinsic apoptotic pathway. We determined that CSE increases expression of caspase 3 and 8 cleavage in MRC-5 cells and overexpression of PKC-η significantly increased expression of caspase 3 and 8 cleavage compared with control LacZ-infected cells. In contrast, dominant negative (dn) PKC-η inhibited apoptosis in MRC-5 cells exposed to CSE and decreased expression of caspase 3 and 8 compared with control cells. Exposure to 10% CSE for >8 h significantly increased lactate dehydrogenase release in PKC-η-infected cells compared with LacZ-infected cells. Additionally, PKC-η-infected cells had an increased number of Hoechst 33342 stained nuclei compared with LacZ-infected cells, while dn PKC-η-infected cells exhibited fewer morphological changes than LacZ-infected cells under phase-contrast microscopy. In conclusion, PKC-η activation plays a pro-apoptotic role in CSE-induced extrinsic apoptotic pathway in MRC-5 cells. These results suggest that modulation of PKC-η may be a useful tool for regulating the extrinsic apoptosis of MRC-5 cells by CSE and may have therapeutic potential in the treatment of CS-induced lung injury.

Protein kinase C induces endocytosis of the sodium taurocholate cotransporting polypeptide

2010 ◽  
Vol 299 (2) ◽  
pp. G320-G328 ◽  
Author(s):  
Claudia Stross ◽  
Angelika Helmer ◽  
Katrin Weissenberger ◽  
Boris Görg ◽  
Verena Keitel ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Bile Salt ◽  
Fluorescent Protein ◽  
Membrane Surface ◽  
Sodium Taurocholate ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Salt Uptake ◽  
Kinase C

Bile salts influence signaling and metabolic pathways. In hepatocytes, the sodium taurocholate cotransporting polypeptide (Ntcp) is a major determinant of intracellular bile salt levels. Short-term downregulation of Ntcp is not well characterized to date. FLAG and enhanced green fluorescent protein (EGFP) tags were cloned to the extra- and intracellular termini of Ntcp. Endocytosis of Ntcp in transfected HepG2 cells was visualized by fluorescence of EGFP, and membrane surface expression of Ntcp was quantified by flow cytometry with fluorochrome-labeled FLAG antibodies. Activation of protein kinase C (PKC) by phorbolester or thymeleatoxin an activator of Ca2+-dependent conventional PKCs (cPKCs), induced endocytosis of Ntcp, whereas the Na+-K+-ATPase remained in the plasma membrane. The PKC inhibitor BIM I and the cPKC-selective inhibitor Gö6976 abolished PMA-induced endocytosis. Because of this internalization, cell surface expression of Ntcp was reduced by 36 ± 7%, bile salt uptake was decreased by 25%, and taurolithocholate sulfate-induced cell toxicity was prevented. In conclusion, Ca2+-dependent PKCs induce vesicular retrieval of Ntcp, thereby reducing bile salt uptake. This mechanism may protect hepatocytes from toxic intracellular bile salt concentrations.

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

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.

In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C

1993 ◽  
pp. 293-307 ◽  
Author(s):  
Angela J. Godwin ◽  
Lora M. Green ◽  
Michael P. Walsh ◽  
John R. McDonald ◽  
Donal A. Walsh ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Communication ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Kinase C ◽  
Dependent Protein ◽  
Cell Cell

scholarly journals Essential role of protein kinase C ζ in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP

2007 ◽  
Vol 176 (7) ◽  
pp. 1049-1060 ◽  
Author(s):  
Kageaki Kuribayashi ◽  
Kiminori Nakamura ◽  
Maki Tanaka ◽  
Tsutomu Sato ◽  
Junji Kato ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Morphological Changes ◽  
Specific Inhibitor ◽  
Squamous Carcinoma ◽  
Inflammatory Cells ◽  
Dominant Negative ◽  
Chemotactic Peptide ◽  
Kinase C ◽  
Pkc Ζ

Under various pathological conditions, including infection, malignancy, and autoimmune diseases, tissues are incessantly exposed to reactive oxygen species produced by infiltrating inflammatory cells. We show augmentation of motility associated with morphological changes of human squamous carcinoma SASH1 cells, human peripheral monocytes (hPMs), and murine macrophage-like cell line J774.1 by superoxide stimulation. We also disclose that motility of hPMs and J774.1 induced by a chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) was inhibited by superoxide dismutase or N-acetylcystein, indicating stimulation of motility by superoxide generated by fMLP stimulation. In these cells, protein kinase C (PKC) ζ was activated to phosphorylate RhoGDI-1, which liberated RhoGTPases, leading to their activation. These events were inhibited by dominant-negative PKCζ in SASH1 cells, myristoylated PKCζ peptides in hPMs and J774.1, or a specific inhibitor of RhoGTPase in SASH1, hPMs, and J774.1. These results suggest a new approach for manipulation of inflammation as well as tumor cell invasion by targeting this novel signaling pathway.

scholarly journals RACK1 Regulates Integrin-mediated Adhesion, Protrusion, and Chemotactic Cell Migration via Its Src-binding Site

2003 ◽  
Vol 14 (2) ◽  
pp. 658-669 ◽  
Author(s):  
Elisabeth A. Cox ◽  
David Bennin ◽  
Ashley T. Doan ◽  
Timothy O'Toole ◽  
Anna Huttenlocher
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Site ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Expression Cloning ◽  
Chemotactic Migration ◽  
Cell Protrusion ◽  
Kinase C ◽  
Green Fluorescent

Mammalian cDNA expression cloning was used to identify novel regulators of integrin-mediated cell-substratum adhesions. Using a focal adhesion morphology screen, we identified a cDNA with homology to a receptor for activated protein kinase C (RACK1) that induced a loss of central focal adhesions and stress fibers in CHO-K1 cells. The identified cDNA was a C-terminal truncated form of RACK1 that had one of the putative protein kinase C binding sites but lacked the region proposed to bind the β integrin cytoplasmic domain and the tyrosine kinase Src. To investigate the role of RACK1 during cell spreading and migration, we tagged RACK1, a C-terminal truncated RACK1 and a point mutant that does not bind Src (RACK Y246F) with green fluorescent protein and expressed them in CHO-K1 cells. We found that RACK1 regulates the organization of focal adhesions and that it localizes to a subset of nascent focal complexes in areas of protrusion that contain paxillin but not vinculin. We also found that RACK1 regulates cell protrusion and chemotactic migration through its Src binding site. Together, these findings suggest that RACK1 regulates adhesion, protrusion, and chemotactic migration through its interaction with Src.

Arachidonic acid enhances contraction and intracellular Ca2+ transients in individual rat ventricular myocytes

1997 ◽  
Vol 272 (1) ◽  
pp. H350-H359 ◽  
Author(s):  
D. S. Damron ◽  
B. A. Summers
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cardiac Muscle ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Receptor Activation ◽  
K Channels ◽  
Rat Ventricular Myocytes ◽  
Kinase C

Modulation of intracellular free Ca2+ concentration ([Ca2+]i) by inotropic stimuli alters contractility in cardiac muscle. Arachidonic acid (AA), a precursor for eicosanoid formation, is released in response to receptor activation and myocardial ischemia and has been demonstrated to alter K+ and Ca2+ channel activity. We investigated the effects of AA on contractility by simultaneously measuring [Ca2+]i and shortening in single field-stimulated rat ventricular myocytes. [Ca2+]i transients were measured using fura 2, and myocyte shortening was assessed using video edge detection. AA stimulated a doubling in the amplitude of the [Ca2+]i transient and a twofold increase in myocyte shortening. In addition, AA stimulated a 30% increase in the time to 50% diastolic [Ca2+]i and a 35% increase in the time to 50% relengthening. These effects of AA were mediated by AA itself (56 +/- 5%) and by cyclooxygenase metabolites. Pretreatment with the protein kinase C inhibitors staurosporine and chelerythrine nearly abolished (> 90% inhibition) these AA-induced effects. Inhibition of voltagegated K+ channels with 4-aminopyridine mimicked the effects of AA. Addition of AA to the 4-aminopyridine-treated myocyte had no additional effect on parameters of contractile function. These data indicate that AA alters the amplitude and duration of Ca2- transients and myocyte shortening via protein kinase C-dependent inhibition of voltage-gated K+ channels. Release of AA by phospholipases in response to receptor activation by endogenous mediators or pathological stimuli may be involved in mediating inotropic responses in cardiac muscle.

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