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

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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Activation of IκB Kinase β by Protein Kinase C Isoforms

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2180 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2180-2188 ◽

Cited By ~ 281

Author(s):

Maria-José Lallena ◽

María T. Diaz-Meco ◽

Gary Bren ◽

Carlos V. Payá ◽

Jorge Moscat

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Critical Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Cell Functions ◽

Kinase C ◽

Iκb Kinase ◽

Tnf Α

ABSTRACT The atypical protein kinase C (PKC) isotypes (λ/ιPKC and ζPKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-α) and are required for the activation of NF-κB by this cytokine through a mechanism that most probably involves the phosphorylation of IκB. The inability of these PKC isotypes to directly phosphorylate IκB led to the hypothesis that ζPKC may use a putative IκB kinase to functionally inactivate IκB. Recently several groups have molecularly characterized and cloned two IκB kinases (IKKα and IKKβ) which phosphorylate the residues in the IκB molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-κB through the activation of the IKKs. We report here that αPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of ζPKC positively modulates IKKβ activity but not that of IKKα, whereas the transfection of a ζPKC dominant negative mutant severely impairs the activation of IKKβ but not IKKα in TNF-α-stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKKβ, which is entirely dependent on the activity of αPKC but not that of the atypical isoforms. In contrast, the inhibition of αPKC does not affect the activation of IKKβ by TNF-α. Interestingly, recombinant active ζPKC and αPKC are able to stimulate in vitro the activity of IKKβ but not that of IKKα. In addition, evidence is presented here that recombinant ζPKC directly phosphorylates IKKβ in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-κB pathway at the level of IKKβ activation and IκB degradation.

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Inhibition of protein kinase C zeta subspecies blocks the activation of an NF-kappa B-like activity in Xenopus laevis oocytes

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1290-1295.1993 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1290-1295

Author(s):

I Dominguez ◽

L Sanz ◽

F Arenzana-Seisdedos ◽

M T Diaz-Meco ◽

J L Virelizier ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Xenopus Laevis ◽

Critical Role ◽

Xenopus Laevis Oocytes ◽

Nuclear Factor ◽

Nf Kappa B ◽

Kinase C ◽

Ras P21 ◽

Protein Kinase C Zeta

Nuclear factor kappa B (NF-kappa B) plays a critical role in the regulation of a large variety of cellular genes. However, the mechanism whereby this nuclear factor is activated remains to be determined. In this report, we present evidence that in oocytes from Xenopus laevis, (i) ras p21- and phospholipase C (PLC)-mediated phosphatidylcholine (PC) hydrolysis activates NF-kappa B and (ii) protein kinase C zeta subspecies is involved in the activation of NF-kappa B in response to insulin/ras p21/PC-PLC. Thus, the microinjection of either ras p21 or PC-PLC, or the exposure of oocytes to insulin, promotes a significant translocation to the nucleus of an NF-kappa B-like activity. This effect is not observed when oocytes are incubated with phorbol myristate acetate or progesterone, both of which utilize a ras p21-independent pathway for oocyte activation. These data strongly suggest a critical role of the insulin/ras p21/PC-PLC/protein kinase C zeta pathway in the control of NF-kappa B activation.

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Inhibition of protein kinase C zeta subspecies blocks the activation of an NF-kappa B-like activity in Xenopus laevis oocytes.

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1290 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1290-1295 ◽

Cited By ~ 94

Author(s):

I Dominguez ◽

L Sanz ◽

F Arenzana-Seisdedos ◽

M T Diaz-Meco ◽

J L Virelizier ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Xenopus Laevis ◽

Critical Role ◽

Xenopus Laevis Oocytes ◽

Nuclear Factor ◽

Nf Kappa B ◽

Kinase C ◽

Ras P21 ◽

Protein Kinase C Zeta

Nuclear factor kappa B (NF-kappa B) plays a critical role in the regulation of a large variety of cellular genes. However, the mechanism whereby this nuclear factor is activated remains to be determined. In this report, we present evidence that in oocytes from Xenopus laevis, (i) ras p21- and phospholipase C (PLC)-mediated phosphatidylcholine (PC) hydrolysis activates NF-kappa B and (ii) protein kinase C zeta subspecies is involved in the activation of NF-kappa B in response to insulin/ras p21/PC-PLC. Thus, the microinjection of either ras p21 or PC-PLC, or the exposure of oocytes to insulin, promotes a significant translocation to the nucleus of an NF-kappa B-like activity. This effect is not observed when oocytes are incubated with phorbol myristate acetate or progesterone, both of which utilize a ras p21-independent pathway for oocyte activation. These data strongly suggest a critical role of the insulin/ras p21/PC-PLC/protein kinase C zeta pathway in the control of NF-kappa B activation.

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Epithelial injury induces Egr-1 and Fos expression by a pathway involving protein kinase C and ERK

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.276.2.g322 ◽

1999 ◽

Vol 276 (2) ◽

pp. G322-G330 ◽

Cited By ~ 7

Author(s):

Brian K. Dieckgraefe ◽

Danielle M. Weems

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Dominant Negative Mutant ◽

Mrna Levels ◽

Mobility Shift ◽

Erk Activation ◽

Kinase C

The signaling pathways activated in response to gastrointestinal injury remain poorly understood. Previous work has implicated the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase as a mediator of wound-signal transduction and a possible regulator of epithelial restitution. Monolayer injury resulted in rapid activation of p42 and p44 ERK. Injury-induced ERK activation was blocked by protein kinase C inhibition or by disruption of the cell cytoskeleton. Significant increases in Fos and early growth response (Egr)-1 mRNA levels were stimulated by injury, peaking by 20 min. ERK activation and the induction of Egr-1 mRNA were inhibited in a dose-dependent fashion with PD-98059. Fos mRNA expression was partially blocked by PD-98059. Western blot analysis demonstrated strong expression and nuclear localization of Fos and Egr after wounding. Electrophoretic mobility shift assays demonstrated that nuclear extracts contained a protein that specifically bound double-stranded oligonucleotides containing the Egr consensus binding element. Gel supershift assays demonstrated that the protein-DNA complexes were recognized by anti-Egr antibody. Inhibition of injury-induced ERK activation by PD-98059 or direct interference with Egr by expression of a dominant negative mutant led to significantly reduced in vitro monolayer restitution.

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

Cell ◽

10.1016/s0092-8674(88)80037-0 ◽

1988 ◽

Vol 52 (3) ◽

pp. 447-458 ◽

Cited By ~ 207

Author(s):

Derek A. Persons ◽

William O. Wilkison ◽

Robert M. Bell ◽

Olivera J. Finn

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Growth Regulation ◽

3T3 Fibroblasts ◽

Kinase C ◽

Nih 3T3

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The zinc chelator 1,10-phenanthroline enhances the stimulatory effects of protein kinase C activators and staurosporine, but not sphingosine and H2O2, on phospholipase D activity in NIH 3T3 fibroblasts

Biochemical Journal ◽

10.1042/bj2980093 ◽

1994 ◽

Vol 298 (1) ◽

pp. 93-98 ◽

Cited By ~ 14

Author(s):

Z Kiss

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase D ◽

Intermediate Step ◽

Phospholipid Hydrolysis ◽

3T3 Fibroblasts ◽

Kinase C ◽

Zinc Chelator ◽

Nih 3T3 ◽

Hydrolysis Of

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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Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3776 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3776-3783 ◽

Cited By ~ 118

Author(s):

I Dominguez ◽

M T Diaz-Meco ◽

M M Municio ◽

E Berra ◽

A García de Herreros ◽

...

Keyword(s):

Protein Kinase C ◽

Growth Factors ◽

Protein Kinase ◽

Xenopus Laevis ◽

Critical Role ◽

Study Data ◽

Xenopus Laevis Oocytes ◽

Kinase C ◽

Protein Kinase C Zeta

A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways.

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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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