Altered growth regulation and enhanced tumorigenicity of NIH 3T3 fibroblasts transfected with protein kinase C-I cDNA

Protein kinase C (PKC), an enzyme which is believed to mediate the stimulatory effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on phospholipase D (PLD) activity, has a zinc-dependent structure required for phorbol ester binding. Accordingly, zinc or zinc chelators would be expected to promote or inhibit, respectively, the stimulatory effects of PMA on PLD-mediated phospholipid hydrolysis. Instead, treatment of [14C]choline- and [14C]ethanolamine-labelled NIH 3T3 fibroblasts with the high-affinity zinc chelator 1,10-phenanthroline (0.2-1 mM) for 20-30 min was found to enhance the stimulatory effects of PMA on PLD-mediated hydrolysis of phosphatidylcholine and phosphatidylethanolamine. In [14C]palmitic acid-labelled fibroblasts, in the presence of ethanol, phenanthroline also enhanced the stimulatory effect of PMA on the synthesis of phosphatidylethanol, a marker of PLD activity. Addition of zinc (250 microM) to phenanthroline-treated fibroblasts reversed the stimulatory effects of the chelator. The potentiating effects of phenanthroline were also partially reversed by cadmium, whereas iron, lead, copper, magnesium and calcium were without effects. Of the other activators of PLD tested, phenanthroline also enhanced the stimulatory effects of platelet-derived growth factor and staurosporine, but not that of sphingosine and H2O2, on the hydrolysis of both phospholipids. These results suggest that regulation of PLD by PKC activators and staurosporine involves a common intermediate step, which is inhibited by a chelatable cellular pool of zinc.

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Regulation of phospholipase D by sphingosine involves both protein kinase C-dependent and -independent mechanisms in NIH 3T3 fibroblasts

Biochemical Journal ◽

10.1042/bj2880853 ◽

1992 ◽

Vol 288 (3) ◽

pp. 853-858 ◽

Cited By ~ 27

Author(s):

Z Kiss ◽

E Deli

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase D ◽

Activity Data ◽

Pkc Inhibitor ◽

Phospholipid Hydrolysis ◽

3T3 Fibroblasts ◽

Kinase C ◽

Nih 3T3 ◽

Hydrolysis Of

Previously, the protein kinase C (PKC) inhibitor sphingosine was found to stimulate phospholipase D (PLD)-mediated hydrolysis of both phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) in NIH 3T3 fibroblasts [Kiss & Anderson (1990) J. Biol. Chem. 265, 7345-7350]. Here we examined the possible relationship between the opposite effects of sphingosine on PKC-mediated protein phosphorylation and PLD activation. After treatments for 3-5 min, sphingosine (25 microM) and the PKC activators phorbol 12-myristate 13-acetate (PMA) (100 nM), bryostatin (100 nM) or platelet-derived growth factor (50 ng/ml) synergistically stimulated the hydrolysis of both PtdEtn and PtdCho in NIH 3T3 fibroblasts prelabelled with [14C]ethanolamine or [14C]choline. Inhibition of PMA-induced phospholipid hydrolysis could also be elicited by sphingosine, but this process required prolonged (60 min) treatments of fibroblasts with 40-60 microM-sphingosine. Similarly to sphingosine, the protein phosphatase inhibitor okadaic acid also had either potentiating or inhibitory effects on PMA-stimulated PLD activity, depending on the length of incubation time and the concentration of PMA. Consistent with the presence of an inhibitory component in the overall action of PKC, the PKC inhibitor staurosporine and down-regulation of PKC activity by prolonged (24 h) treatment with PMA similarly enhanced PLD activity. Data suggest that (a) sphingosine may enhance PMA-mediated phospholipid hydrolysis by neutralizing the action of an inhibitory PKC isoform, and that (b) the stimulatory PKC isoform is less sensitive to the inhibitory action of sphingosine.

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Immunocytochemical Localization of Eight Protein Kinase C Isozymes Overexpressed in NIH 3T3 Fibroblasts

Journal of Biological Chemistry ◽

10.1074/jbc.270.17.9991 ◽

1995 ◽

Vol 270 (17) ◽

pp. 9991-10001 ◽

Cited By ~ 222

Author(s):

JoAnne Goodnight ◽

Harald Mischak ◽

Walter Kolch ◽

J. Frederic Mushinski

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Immunocytochemical Localization ◽

3T3 Fibroblasts ◽

Kinase C ◽

Nih 3T3

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Stimulation of phosphatidylcholine synthesis by activators of protein kinase C is dissociable from increased phospholipid hydrolysis

Biochemical Journal ◽

10.1042/bj2730189 ◽

1991 ◽

Vol 273 (1) ◽

pp. 189-194 ◽

Cited By ~ 16

Author(s):

Z Kiss ◽

J Chattopadhyay ◽

G R Pettit

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Lines ◽

3T3 Cells ◽

Phospholipid Hydrolysis ◽

3T3 Fibroblasts ◽

Kinase C ◽

Protein Kinase C Activators ◽

Nih 3T3 ◽

Hydrolysis Of

The aim of this study was to clarify the relationship between the stimulatory effects of protein kinase C activators, including phorbol 12-myristate 13-acetate (PMA) and bryostatin, on the hydrolysis of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) and on PtdCho synthesis. The cell lines used were selected because of their differential responses to protein kinase C activators and included rat-1 fibroblasts, untransformed and A-raf-transformed NIH 3T3 fibroblasts and human HL60 leukaemia cells. Exposure of rat-1 and NIH 3T3 fibroblasts to 100 nM-PMA stimulated phospholipase D-mediated hydrolysis of phospholipids about 2- and 6-fold respectively. In contrast, 100 nM-PMA had similar (2.5-3.0-fold) stimulatory effects on PtdCho synthesis in these cell lines. In the untransformed NIH 3T3 cells, both PMA and bryostatin stimulated both phospholipid hydrolysis and PtdCho synthesis, with 100 nM-bryostatin being somewhat less potent than 100 nM-TPA. In contrast, in A-raf-transformed NIH 3T3 cells or in HL60 cells, only TPA, but not bryostatin, stimulated PtdCho synthesis. In these transformed cells, bryostatin had 3-fold, or higher, stimulatory effects on phospholipid hydrolysis. Addition of ionomycin, a Ca2(+)-elevating agent, partially restored the stimulatory effect of bryostatin on PtdCho synthesis, but it failed to modify the effect of bryostatin on phospholipid hydrolysis. These data indicate that increased phospholipid hydrolysis is not necessarily associated with increased PtdCho synthesis.

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A dominant negative protein kinase C zeta subspecies blocks NF-kappa B activation.

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4770 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4770-4775 ◽

Cited By ~ 172

Author(s):

M T Diaz-Meco ◽

E Berra ◽

M M Municio ◽

L Sanz ◽

J Lozano ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Critical Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Xenopus Laevis Oocytes ◽

Nf Kappa B ◽

3T3 Fibroblasts ◽

Kinase C ◽

Nih 3T3

Nuclear factor kappa B (NF-kappa B) plays a critical role in the regulation of a number of genes. NF-kappa B is a heterodimer of 50- and 65-kDa subunits sequestered in the cytoplasm complexed to inhibitory protein I kappa B. Following stimulation of cells, I kappa B dissociates from NF-kappa B, allowing its translocation to the nucleus, where it carries out the transactivation function. The precise mechanism controlling NF-kappa B activation and the involvement of members of the protein kinase C (PKC) family of isotypes have previously been investigated. It was found that phorbol myristate acetate, (PMA) which is a potent stimulant of phorbol ester-sensitive PKC isotypes, activates NF-kappa B. However, the role of PMA-sensitive PKCs in vivo is not as apparent. It has recently been demonstrated in the model system of Xenopus laevis oocytes that the PMA-insensitive PKC isotype, zeta PKC, is a required step in the activation of NF-kappa B in response to ras p21. We demonstrate here that overexpression of zeta PKC is by itself sufficient to stimulate a permanent translocation of functionally active NF-kappa B into the nucleus of NIH 3T3 fibroblasts and that transfection of a kinase-defective dominant negative mutant of zeta PKC dramatically inhibits the kappa B-dependent transactivation of a chloramphenicol acetyltransferase reporter plasmid in NIH 3T3 fibroblasts. All these results support the notion that zeta PKC plays a decisive role in NF-kappa B regulation in mammalian cells.

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