Phorbol Ester and Phospholipase C-Mediated Differentiated Thyroid Function In Vitro: The Effects of Protein Kinase C Inhibition and Downregulation

Thyroid ◽  
1991 ◽  
Vol 1 (2) ◽  
pp. 195-200 ◽  
Author(s):  
JODY GINSBERG ◽  
PATRICIA G. MURRAY
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Thyroid Function ◽  
Phorbol Ester ◽  
Kinase C

Phorbol ester inhibition of Na-H exchange in rabbit proximal colon

1985 ◽  
Vol 249 (5) ◽  
pp. C527-C530 ◽  
Author(s):  
J. Ahn ◽  
E. B. Chang ◽  
M. Field
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Ph ◽  
Phorbol Ester ◽  
Proximal Colon ◽  
Bathing Medium ◽  
Two Agents ◽  
Luminal Membrane ◽  
Kinase C

In rabbit proximal colon, in vitro addition of phorbol 12,13-dibutyrate (PDB, 10(-7) M) to the serosal bathing medium inhibits mucosal (m)-to-serosal (s) unidirectional Na flux (JsmNa) without altering JsmNa or unidirectional Cl fluxes. Similar results were obtained when amiloride (2 X 10(-4) M) was added to the mucosal bathing medium. No additivity of effect was seen when tissues were exposed to both agents. Measurements with carboxyfluorescein reveal that the two agents cause equal decreases of intracellular pH (pHi), an effect that is dependent on the presence of extracellular Na (Na replacement also decreases pHi). No additivity of pHi effects is seen when both agents are added together. To determine the membrane site of this PDB-inhibitable Na-H exchange, Na influx across the luminal border of proximal colon was measured and was found to be inhibited equally by PDB and amiloride. We conclude that PDB, by activation of protein kinase C, inhibits electro-neutral amiloride-sensitive Na-H exchange in the luminal membrane of proximal colon.

Effects of protein kinase C activators on the in-vitro action of thyrotrophin in pigs

1988 ◽  
Vol 118 (2) ◽  
pp. 199-203 ◽  
Author(s):  
J. Ginsberg ◽  
P. G. Murray
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Thyroid Function ◽  
Receptor Site ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Cell Extracts ◽  
Kinase C

ABSTRACT The ability of the non-phorbol protein kinase C (PKC) activator 12-hydroxy-daphnetoxin (mezerein) to modulate differentiated thyroid function was examined in vitro. A dose-dependent inhibition of TSH-stimulated iodide organification was observed in porcine thyroid cells exposed to mezerein. Under identical conditions mezerein caused the translocation of PKC from its inactive cytosolic form to an active membrane-bound form in thyroid cell extracts. The relative biological potencies of mezerein and the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), to inhibit thyroid function in vitro corresponded to their abilities to activate PKC. This effect was also observed when dibutyryl cyclic AMP was used, implying a post-receptor site of action. To provide further evidence for this concept, the effects of mezerein and TPA on receptor-related events were studied. Neither mezerein nor TPA had any effect on the binding of radiolabelled TSH to solubilized porcine thyroid membranes. However, both mezerein and TPA were capable of stimulating cyclic AMP (cAMP) production in porcine thyroid cells in the basal state but could not augment TSH or forskolin-activated cAMP release. These data provide evidence that activation of PKC plays a role in the regulation of differentiated thyroid function in vitro and suggest that the effects of PKC are complex, with independent actions on cAMP accumulation and post-receptor events. J. Endocr. (1988) 118, 199–203

scholarly journals Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of β2-chimaerin, a ‘non-protein kinase C’ phorbol ester receptor

2003 ◽  
Vol 375 (2) ◽  
pp. 313-321 ◽  
Author(s):  
Maria Jose CALOCA ◽  
HongBin WANG ◽  
Marcelo G. KAZANIETZ
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Gtpase Activating Protein ◽  
Protein Activity ◽  
Rac Gtpase ◽  
Kinase C ◽  
Phorbol Ester Receptor

The regulation and function of β2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to β2-chimaerin with high affinity and promote its subcellular distribution. β2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. β2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the β2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of β2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor β2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.

scholarly journals Diacylglycerol generated by exogenous phospholipase C activates the mitogen-activated protein kinase pathway independent of Ras- and phorbol ester-sensitive protein kinase C: dependence on protein kinase C-ζ

1997 ◽  
Vol 323 (3) ◽  
pp. 693-699 ◽  
Author(s):  
Marc C. M. van DIJK ◽  
Francisco J. G. MURIANA ◽  
Paul C. J. van der HOEVEN ◽  
John de WIDT ◽  
Dick SCHAAP ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phorbol Ester ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activation ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Pkc Ζ

The role of diacylglycerol (DG) formation from phosphatidylcholine in mitogenic signal transduction is poorly understood. We have generated this lipid at the plasma membrane by treating Rat-1 fibroblasts with bacterial phosphatidylcholine-specific phospholipase C (PC-PLC). This treatment leads to activation of mitogen-activated protein kinase (MAPK). However, unlike platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), PC-PLC fails to activate Ras and to induce DNA synthesis, and activates MAPK only transiently (< 45 min). Down-regulation of protein kinase C (PKC) -α, -Δ and -ε isotypes has little or no effect on MAPK activation by either PC-PLC or growth factors. However, Ro 31-8220, a highly selective inhibitor of all PKC isotypes, including atypical PKC-ζ but not Raf-1, blocks MAPK activation by PDGF and PC-PLC, but not that by EGF, suggesting that atypical PKC mediates the PDGF and PC-PLC signal. In line with this, PKC-ζ is activated by PC-PLC and PDGF, but not by EGF, as shown by a kinase assay in vitro, using biotinylated ε-peptide as a substrate. Furthermore, dominant-negative PKC-ζ inhibits, while (wild-type) PKC-ζ overexpression enhances MAPK activation by PDGF and PC-PLC. The results suggest that DG generated by PC-PLC can activate the MAPK pathway independent of Ras and phorbol-ester-sensitive PKC but, instead, via PKC-ζ.

scholarly journals G-protein βγ subunits antagonize protein kinase C-dependent phosphorylation and inhibition of phospholipase C-β1

1997 ◽  
Vol 326 (3) ◽  
pp. 701-707 ◽  
Author(s):  
Irene LITOSCH
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
G Protein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Negative Feedback Regulation ◽  
Kinase C ◽  
Stimulation Of

Protein kinase C (PKC) isoforms phosphorylated phospholipase C-β1 (PLC-β1) in vitro as follows: PKCα ≫ PKCϵ; not PKCζ. PLC-β3 was not phosphorylated by PKCα. G-protein βγ subunits inhibited the PKCα phosphorylation of PLC-β1 in a concentration-dependent manner. Half-maximal inhibition occurred with 500 nM βγ. G-protein βγ subunits also antagonized the PKCα-mediated inhibition of PLC-β1 enzymic activity. PKCα, in turn, inhibited the stimulation of PLC-β1 activity by βγ. There was little effect of PKCα on the stimulation of PLC-β1 by αq/11–guanosine 5′[γ-thio]triphosphate (GTP[S]). These findings demonstrate that G protein βγ subunits antagonize PKCα regulation of PLC-β1. Thus βγ subunits might have a role in modulating the negative feedback regulation of this signalling system by PKC.

FLUORESCENT PHORBOL ESTER BINDING TO THE RECEPTOR PROTEIN KINASE C IN-VITRO AND IN-VIVO

1986 ◽  
Vol 38 (S12) ◽  
pp. 30P-30P ◽  
Author(s):  
N. A. Morrice ◽  
C. Ellis ◽  
A. T. Evans ◽  
F. J. Evans ◽  
A. Drummond ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Receptor Protein ◽  
Kinase C

Bryostatin 1, an activator of protein kinase C, mimics as well as inhibits biological effects of the phorbol ester TPA in vivo and in vitro

1988 ◽  
Vol 9 (4) ◽  
pp. 555-562 ◽  
Author(s):  
M. Gschwendt ◽  
G. Fürstenberger ◽  
S. Rose-John ◽  
M. Rogers ◽  
W. Kittstein ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Biological Effects ◽  
Bryostatin 1 ◽  
Kinase C
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