scholarly journals Activation of IκB Kinase β by Protein Kinase C Isoforms

1999 ◽  
Vol 19 (3) ◽  
pp. 2180-2188 ◽  
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.

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

1999 ◽  
Vol 276 (2) ◽  
pp. G322-G330 ◽  
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.

Protein kinase C-ζ mediates TNF-α-induced ICAM-1 gene transcription in endothelial cells

2000 ◽  
Vol 279 (4) ◽  
pp. C906-C914 ◽  
Author(s):  
Arshad Rahman ◽  
Khandaker N. Anwar ◽  
Asrar B. Malik
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Gene Transcription ◽  
Critical Role ◽  
Dominant Negative Mutant ◽  
Kinase C ◽  
Tnf Α ◽  
Pkc Isozymes ◽  
Pkc Ζ

We addressed the role of protein kinase C (PKC) isozymes in mediating tumor necrosis factor-α (TNF-α)-induced oxidant generation in endothelial cells, a requirement for nuclear factor-κB (NF-κB) activation and intercellular adhesion molecule-1 (ICAM-1) gene transcription. Depletion of the conventional (c) and novel (n) PKC isozymes following 24 h exposure of human pulmonary artery endothelial (HPAE) cells with the phorbol ester, phorbol 12-myristate 13-acetate (500 nM), failed to prevent TNF-α-induced oxidant generation. In contrast, inhibition of PKC-ζ synthesis by the antisense oligonucleotide prevented the oxidant generation following the TNF-α stimulation. We observed that PKC-ζ also induced the TNF-α-induced NF-κB binding to the ICAM-1 promoter and the resultant ICAM-1 gene transcription. We showed that expression of the dominant negative mutant of PKC-ζ prevented the TNF-α-induced ICAM-1 promoter activity, whereas overexpression of the wild-type PKC-ζ augmented the response. These data imply a critical role for the PKC-ζ isozyme in regulating TNF-α-induced oxidant generation and in signaling the activation of NF-κB and ICAM-1 transcription in endothelial cells.

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

1993 ◽  
Vol 13 (8) ◽  
pp. 4770-4775 ◽  
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.

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

1993 ◽  
Vol 13 (8) ◽  
pp. 4770-4775
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.

scholarly journals NF-κB Activation by Double-Stranded-RNA-Activated Protein Kinase (PKR) Is Mediated through NF-κB-Inducing Kinase and IκB Kinase

2000 ◽  
Vol 20 (4) ◽  
pp. 1278-1290 ◽  
Author(s):  
Maryam Zamanian-Daryoush ◽  
Trine H. Mogensen ◽  
Joseph A. DiDonato ◽  
Bryan R. G. Williams
Keyword(s):  
Protein Kinase ◽  
Kinetic Studies ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Null Cell ◽  
Necrosis Factor Alpha ◽  
Double Stranded Rna ◽  
Iκb Kinase ◽  
Tnf Α ◽  
Factor Alpha

ABSTRACT The interferon (IFN)-inducible double-stranded-RNA (dsRNA)-activated serine-threonine protein kinase (PKR) is a major mediator of the antiviral and antiproliferative activities of IFNs. PKR has been implicated in different stress-induced signaling pathways including dsRNA signaling to nuclear factor kappa B (NF-κB). The mechanism by which PKR mediates activation of NF-κB is unknown. Here we show that in response to poly(rI) · poly(rC) (pIC), PKR activates IκB kinase (IKK), leading to the degradation of the inhibitors IκBα and IκBβ and the concomitant release of NF-κB. The results of kinetic studies revealed that pIC induced a slow and prolonged activation of IKK, which was preceded by PKR activation. In PKR null cell lines, pIC failed to stimulate IKK activity compared to cells from an isogenic background wild type for PKR in accord with the inability of PKR null cells to induce NF-κB in response to pIC. Moreover, PKR was required to establish a sustained response to tumor necrosis factor alpha (TNF-α) and to potentiate activation of NF-κB by cotreatment with TNF-α and IFN-γ. By coimmunoprecipitation, PKR was shown to be physically associated with the IKK complex. Transient expression of a dominant negative mutant of IKKβ or the NF-κB-inducing kinase (NIK) inhibited pIC-induced gene expression from an NF-κB-dependent reporter construct. Taken together, these results demonstrate that PKR-dependent dsRNA induction of NF-κB is mediated by NIK and IKK activation.

scholarly journals Syndecan-4 regulates localization, activity and stability of protein kinase C-alpha

2004 ◽  
Vol 378 (3) ◽  
pp. 1007-1014 ◽  
Author(s):  
Eunyoung KEUM ◽  
Yeonhee KIM ◽  
Jungyean KIM ◽  
Soojin KWON ◽  
Yangmi LIM ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Heparan Sulphate ◽  
Critical Role ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Prolonged Treatment ◽  
Kinase C

During cell–matrix adhesion, syndecan-4 transmembrane heparan sulphate proteoglycan plays a critical role in the formation of focal adhesions and stress fibres. We have shown previously that the syndecan-4 cytoplasmic domain directly binds to and activates PKC-α (protein kinase C-α) in vitro [Oh, Woods and Couchman (1997) J. Biol. Chem. 272, 8133–8136]. However, whether syndecan-4 has the same activity in vivo needs to be addressed. Using mammalian two-hybrid assays, we showed that syndecan-4 interacted with PKC-α in vivo and that this interaction was mediated through syndecan-4 cytoplasmic domain. Furthermore, the activation of PKC increased the extent of interaction between syndecan-4 and PKC-α. Overexpression of syndecan-4, but not a mutant lacking its cytoplasmic domain, specifically increased the level of endogenous PKC-α and enhanced the translocation of PKC-α into both detergent-insoluble and membrane fractions. In addition, rat embryo fibroblasts overexpressing syndecan-4 exhibited a slowed down-regulation of PKC-α in response either to a prolonged treatment with PMA or to maintaining cells in suspension culture. PKC-α immunocomplex kinase assays also showed that syndecan-4 overexpression increased the activity of membrane PKC-α. Taken together, these results suggest that syndecan-4 interacts with PKC-α in vivo and regulates its localization, activity and stability.

Involvement of protein kinase C in crystalline silica-induced activation of the MAP kinase and AP-1 pathway

2006 ◽  
Vol 290 (2) ◽  
pp. L291-L297 ◽  
Author(s):  
Min Ding ◽  
Chuanshu Huang ◽  
Yongju Lu ◽  
Linda Bowman ◽  
Vince Castranova ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Map Kinase ◽  
Crystalline Silica ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
P38 Kinase ◽  
Kinase C ◽  
Pkc Inhibitors

Crystalline silica has long been well established as a fibrogenic agent, and recent evidence has implicated it as a potential human carcinogen. However, the mechanisms of silica-induced disease development and progression are not well understood. Our previous studies demonstrated that crystalline silica is able to activate activator protein-1 (AP-1) through mitogen-activated protein kinase (MAPK) pathways. The present study investigates the possible involvement of protein kinase C (PKC) in silica-induced activation of the MAPK/AP-1 signal transduction pathway. Treatment of mouse epidermal cells (JB6 cell line) with freshly fractured silica stimulated translocation of PKCα and PKCε from the cytosol to the membrane and activated AP-1 transcription activity. Pretreatment of cells with PKC inhibitors, including RO-32-0432, calphostin C, and bisindolylmaleimide I, inhibited silica-induced AP-1 activation and phosphorylation of ERKs and p38 kinase. These inhibitory effects by PKC inhibitors were dose dependent. Furthermore, overexpression of dominant negative mutant (DNM) of PKCα or PKCε markedly blocked AP-1 activation as well as phosphorylation of ERKs and p38 kinase induced by freshly fractured silica. These results demonstrate that PKCα and PKCε are essential in silica-induced AP-1 activation through the MAP kinase (ERKs and p38 kinases) pathway.

scholarly journals Emerging and diverse roles of protein kinase C in immune cell signalling

2003 ◽  
Vol 376 (3) ◽  
pp. 545-552 ◽  
Author(s):  
Seng-Lai TAN ◽  
Peter J. PARKER
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Immune Responses ◽  
Immune Cell ◽  
Cell Signalling ◽  
Dominant Negative ◽  
In Vivo Studies ◽  
Kinase C

Members of the protein kinase C (PKC) family are expressed in many different cell types, where they are known to regulate a wide variety of cellular processes that impact on cell growth and differentiation, cytoskeletal remodelling and gene expression in the response to diverse stimuli. The broad tissue distribution and redundancy of in vitro function have often hampered the identification of definitive roles for each PKC family member. However, recent in vivo studies of PKC isoenzyme-selective knockout and transgenic mice have highlighted distinct functions of individual PKCs in the immune system. These genetic analyses, along with biochemical studies utilizing PKC isoenzyme-specific cDNA (wild-type, constitutively active and dominant-negative), antisense oligonucleotides (ASO), RNA interference (RNAi), and pharmacological inhibitors, indicate that PKC-regulated signalling pathways play a significant role in many aspects of immune responses, from development, differentiation, activation and survival of lymphocytes to macrophage activation. The importance of PKCs in cellular immune responses suggests that improved understanding of the molecular events that govern their actions could point to new avenues for development of treatments for immune disorders.

scholarly journals Modulation of mouse estrogen receptor transcription activity by protein kinase C delta

1998 ◽  
Vol 20 (2) ◽  
pp. 245-259 ◽  
Author(s):  
H Lahooti ◽  
T Thorsen ◽  
A Aakvaag
Keyword(s):  
Estrogen Receptor ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Transcriptional Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Receptor Phosphorylation ◽  
Pkc Delta ◽  
Kinase C

The effect of protein kinase C (PKC) delta on the transcriptional activity of the mouse estrogen receptor was investigated. The receptor was expressed transiently in Cos-1 and NIH3T3 cells in the presence of wild-type, dominant negative or constitutively active forms of PKC delta. Transfection experiments demonstrated that PKC delta stimulated both unliganded and liganded estrogen receptor transcriptional activity. This stimulatory effect was not observed using PKC alpha or PKC epsilon. 4-Hydroxytamoxifen and the pure anti-estrogen ICI 164,384 reduced receptor transcriptional activity in the presence of PKC delta. The stimulatory effect of PKC delta on estrogen receptor transcriptional activity was mediated by the N-terminal activation function 1 (AF-1) domain. The reduced stimulatory effect of PKC delta on transcriptional activity of the phosphorylation defective mutant of estrogen receptor suggests that phosphorylation of serine 122 in the AF-1 region may mediate the modulatory effect of PKC delta. Wild-type PKC delta caused a twofold increase in estrogen receptor phosphorylation, while a dominant negative mutant of PKC delta reduced the receptor phosphorylation to five percent of that caused by wild-type PKC delta. Our results suggest that PKC delta participates in the signaling pathways that lead to estrogen receptor phosphorylation and its effect on estrogen receptor transcriptional activation is both cell type and promoter specific.

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