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 Phorbol Esters and Related Analogs Regulate the Subcellular Localization of β2-Chimaerin, a Non-protein Kinase C Phorbol Ester Receptor

2001 ◽  
Vol 276 (21) ◽  
pp. 18303-18312 ◽  
Author(s):  
Maria J. Caloca ◽  
HongBin Wang ◽  
Andrew Delemos ◽  
Shaomeng Wang ◽  
Marcelo G. Kazanietz
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Subcellular Localization ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Kinase C ◽  
Phorbol Ester Receptor

Tamoxifen inhibits phorbol ester stimulated osteoclastic bone resorption: An effect mediated by calmodulin

2000 ◽  
Vol 78 (6) ◽  
pp. 715-723 ◽  
Author(s):  
John P Williams ◽  
Margaret A McKenna ◽  
Allyn M Thames III ◽  
Jay M McDonald
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Bone Resorption ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Fold Increase ◽  
Dependent Manner ◽  
Osteoclast Activity ◽  
Kinase C ◽  
Protein Kinase Cα

Tamoxifen inhibits bone resorption by disrupting calmodulin-dependent processes. Since tamoxifen inhibits protein kinase C in other cells, we compared the effects of tamoxifen and the phorbol ester, phorbol myristate acetate, on osteoclast activity. Phorbol esters stimulate bone resorption and calmodulin levels four-fold (k0.5 = 0.1–0.3 µM). In contrast, tamoxifen inhibited osteoclast activity ~60% with an IC50 of 1.5 µM, had no apparent effect on protein kinase C activity in whole-cell lysates, and reduced protein kinase Cα recovered by immunoprecipitation 75%. Phorbol esters stimulated resorption in a time-dependent manner that was closely correlated with a similar-fold increase in calmodulin. Protein kinase Cα, β, δ, ε, and ζ were all down-regulated in response to phorbol ester treatment. Tamoxifen and trifluoperazine inhibited PMA-dependent increases in bone resorption and calmodulin by 85 ± 10%. Down-regulation of protein kinase C isoforms by phorbol esters suggests that the observed increases in bone resorption and calmodulin levels are most likely due to a mechanism independent of protein kinase C and dependent on calmodulin. In conclusion, the data suggest that protein kinase C negatively regulates calmodulin expression and support the hypothesis that the effects of both phorbol esters and tamoxifen on osteoclast activity is mediated by calmodulin.Key words: osteoclast, calmodulin, tamoxifen, osteoporosis, protein kinase C.

Regulation of protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC-12 cells

1990 ◽  
Vol 10 (6) ◽  
pp. 2983-2990
Author(s):  
J C Lacal ◽  
A Cuadrado ◽  
J E Jones ◽  
R Trotta ◽  
D E Burstein ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Neuronal Differentiation ◽  
Phorbol Esters ◽  
Ras Oncogene ◽  
Intact Cells ◽  
Pc 12 Cells ◽  
Kinase C ◽  
Ras P21

Expression of the N-ras oncogene under the control of the glucocorticoid-responsive promoter in the pheochromocytoma cell line UR61, a subline of PC-12 cells, has been used to investigate the differentiation process to neuronal cells triggered by ras oncogenes (I. Guerrero, A. Pellicer, and D. E. Burstein, Biochem. Biophys. Res. Commun. 150:1185-1192, 1988). Using ras-inducible cell lines, we observed that expression of the oncogenic N-ras p21 protein interferes with the ability of phorbol esters to induce downregulation of protein kinase C. This effect was associated with the appearance of immunologically detectable protein kinase C as well as the activity of the enzyme as analyzed either by binding of [3H]phorbol-12,13-dibutyrate in intact cells or by in vitro kinase activity. These results indicate a relationship between ras p21 and protein kinase C in neuronal differentiation in this model system. Comparison to the murine fibroblast system suggests that this relationship may be functional.

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.

Regulation of p-aminohippurate transport by protein kinase C in OK kidney epithelial cells

1996 ◽  
Vol 271 (2) ◽  
pp. F469-F475 ◽  
Author(s):  
M. Takano ◽  
J. Nagai ◽  
M. Yasuhara ◽  
K. Inui
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Basolateral Membrane ◽  
Significant Inhibition ◽  
Regulatory Control ◽  
Ok Cells ◽  
Kinase C ◽  
Apical Side

We studied the effect of phorbol 12-myristate 13-acetate (PMA), a phorbol ester which activates protein kinase C, on p-aminohippurate (PAH) transport in OK cells. PMA (10(-7) M) almost completely inhibited the transcellular transport of PAH across OK cell monolayers from the basal to the apical side, as well as the accumulation of PAH in the cells. The uptake of PAH across the basolateral membrane of OK cells was inhibited by PMA in a time-and dose-dependent fashion. Exposing the cells with other protein kinase C activators such as active phorbol esters and diacylglycerols also resulted in a significant inhibition of basolateral PAH uptake, but the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, had no effect. The inhibition of basolateral PAH uptake by PMA was blocked by staurosporine, an inhibitor of protein kinase C. Cycloheximide, actinomycin D, colchicine, and cytochalasin D did not affect the inhibitory effect of PMA on basolateral PAH uptake. These results suggested that the PAH transport system in OK cells is under the regulatory control of protein kinase C.

scholarly journals Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.

1989 ◽  
Vol 108 (2) ◽  
pp. 553-567 ◽  
Author(s):  
V Papadopoulos ◽  
P F Hall
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Intact Cells ◽  
Adrenal Cells ◽  
Isolation And Characterization ◽  
Kinase C

The cytoskeletons of Y-1 mouse adrenal tumor cells contain a calcium and phospholipid-dependent protein kinase (protein kinase C) that is bound sufficiently tight to resist extraction by 0.5% Triton but not by 1.0% Triton. The enzyme has been purified to near homogeneity from cytoskeleton and cytosol. It shows features typical of this type of kinase, namely a requirement for Ca2+ and phospholipid, stimulation by tumor promoters but not by nontumor-promoting phorbol esters, and inhibition by trifluoperazine. The enzyme shows specificity for four substrates found in the cytoskeleton, namely 80, 33, 20, and 18 kD. The first three substrates are phosphorylated by the enzyme; the fourth is dephosphorylated and is therefore affected by the kinase indirectly. The 80-kD protein is the kinase enzyme itself which is autophosphorylated in vitro and in the cytoskeleton. The 20-kD protein is myosin light chain. The 33- and 18-kD proteins are unidentified. The same substrates were phosphorylated when Y-1 cells were permeabilized with digitonin and incubated with [gamma-32P]ATP and phorbol-12-myristate-13-acetate. Partly purified protein kinase C changes the extent of phosphorylation of the same substrates when added to cytoskeletons previously extracted to remove endogenous protein kinase C. Addition of Ca2+, phosphatidylserine, and phorbol-12-myristate-13-acetate to cytoskeletons, and addition of these three agents plus protein kinase C to extracted cytoskeletons, causes these structures to undergo a rapid and extensive rounding. A similar change is induced in intact cells by addition of phorbol ester. It is concluded that protein kinase C is capable of changing the shape of adrenal cells by an action that involves autophosphorylation and phosphorylation of myosin light chain. This response may in turn be related to the steroidogenic responses to ACTH and cyclic AMP.

Conformationally constrained analogs of diacylglycerol. Interaction of .gamma.-lactones with the phorbol ester receptor of protein kinase C

1992 ◽  
Vol 114 (3) ◽  
pp. 1059-1070 ◽  
Author(s):  
Kelly Teng ◽  
Victor E. Marquez ◽  
George W. A. Milne ◽  
Joseph J. Barchi ◽  
Marcelo G. Kazanietz ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Conformationally Constrained ◽  
Kinase C ◽  
Phorbol Ester Receptor
Sign in / Sign up

Export Citation Format

Share Document