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.

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.

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.

scholarly journals Differential Regulation of Angiotensin II-induced Expression of Connective Tissue Growth Factor by Protein Kinase C Isoforms in the Myocardium

2005 ◽  
Vol 280 (16) ◽  
pp. 15719-15726 ◽  
Author(s):  
Zhiheng He ◽  
Kerrie J. Way ◽  
Emi Arikawa ◽  
Eva Chou ◽  
Darren M. Opland ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Growth Factor ◽  
Protein Kinase ◽  
Connective Tissue ◽  
Mrna Expression ◽  
Tissue Growth ◽  
Dominant Negative ◽  
Wild Type ◽  
Kinase C

Protein kinase C (PKC) and angiotensin II (AngII) can regulate cardiac function in pathological conditions such as in diabetes or ischemic heart disease. We have reported that expression of connective tissue growth factor (CTGF) is increased in the myocardium of diabetic mice. Now we showed that the increase in CTGF expression in cardiac tissues of streptozotocin-induced diabetic rats was reversed by captopril and islet cell transplantation. Infusion of AngII in rats increased CTGF mRNA expression by 15-fold, which was completely inhibited by co-infusion with AT1 receptor antagonist, candesartan. Similarly, incubation of cultured cardiomyocytes with AngII increased CTGF mRNA expression by 2-fold, which was blocked by candesartan and a general PKC inhibitor, GF109203X. The role of PKC isoform-dependent action was further studied using adenoviral vector-mediated gene transfer of dominant negative (dn) PKC or wild type PKC isoforms. Expression of dnPKCα, -ϵ, and -ζ isoforms suppressed AngII-induced CTGF expression in cardiomyocytes. In contrast, expression of dominant negative PKCδ significantly increased AngII-induced CTGF expression, whereas expression of wild type PKCδ inhibited this induction. This inhibitory effect was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of PKCδ (δTg mice). Thus, AngII can regulate CTGF expression in cardiomyocytes through a PKC activation-mediated pathway in an isoform-selective manner both in physiological and diabetic states and may contribute to the development of cardiac fibrosis in diabetic cardiomyopathy.

scholarly journals The Small Mr Ras-like GTPase Rap1 and the Phospholipase C Pathway Act to Regulate Phagocytosis inDictyostelium discoideum

1999 ◽  
Vol 10 (2) ◽  
pp. 393-406 ◽  
Author(s):  
David J. Seastone ◽  
Linyi Zhang ◽  
Greg Buczynski ◽  
Patrick Rebstein ◽  
Gerald Weeks ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Protein Tyrosine Kinase ◽  
Dominant Negative ◽  
Wild Type ◽  
Cortical Actin ◽  
Calphostin C ◽  
Kinase C ◽  
Protein Kinase C Inhibitor

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes inDictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C–specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.

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 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 Identification of protein kinase C phosphorylation sites in the angiotensin II (AT1A) receptor

1999 ◽  
Vol 343 (3) ◽  
pp. 637-644 ◽  
Author(s):  
Hongwei QIAN ◽  
Luisa PIPOLO ◽  
Walter G. THOMAS
Keyword(s):  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Single Mutation ◽  
Wild Type ◽  
Receptor Phosphorylation ◽  
Kinase C ◽  
C Terminus ◽  
At1a Receptor ◽  
Mutant Receptors

Protein kinase C (PKC) phosphorylates the C-terminus of the type 1 angiotensin II receptor (AT1), although the exact site(s) of phosphorylation are unidentified. In the present study, we examined the phosphorylation of epitope-tagged wild-type AT1A receptors, transiently expressed in Chinese hamster ovary K1 cells, in response to angiotensin II (AngII) and following selective activation and inhibition of PKC. This phosphorylation was compared with mutant receptors where C-terminal serine residues (Ser331, Ser338 and Ser348) within three putative PKC consensus sites were replaced with alanine, either individually or in combination. Stimulation by AngII or the phorbol ester PMA to activate PKC induced an increase in phosphorylation of the wild-type AT1A receptor, which was prevented by truncation of the receptor C-terminus to remove the last 34 amino acids, including Ser331, Ser338 and Ser348. Whereas single alanine mutation (Ser331Ala, Ser338Ala and Ser348Ala) resulted in decreased receptor phosphorylation, no single mutant completely inhibited either AngII- or PMA-induced phosphorylation. Combined mutation of the three PKC consensus sites caused an ≈ 70% reduction in PMA-mediated phosphorylation. The ≈ 60% reduction in AngII (1 μM)-induced phosphorylation of this triple mutant and the partial inhibition of wild-type receptor phosphorylation by bisindolylmaleimide, a specific PKC inhibitor, suggest a significant contribution of PKC to agonist-stimulated regulation. The ratio of PKC to total receptor phosphorylation was greatest at low doses of AngII (1 nM), consistent with the idea that PKC phosphorylates and regulates receptor function at low levels of stimulation, whereas phosphorylation by other kinases is more prevalent at high levels of agonist stimulation. To determine if a single PKC site is favoured when the contribution of PKC varies, the phosphorylation of wild-type and mutant receptors was examined over a range of AngII concentrations (0, 1, 10 and 100 nM). At all AngII concentrations, single mutation of Ser331, Ser338 or Ser348 was incapable of completely preventing receptor phosphorylation, suggesting no clear preference for PKC consensus-site utilization. Together, these results indicate a redundancy in PKC phosphorylation of the AT1A receptor, whereby all three consensus sites are utilized to some degree following homologous (AngII) and heterologous (PMA) stimulation. The contribution of PKC phosphorylation to receptor regulation is unclear, but multiple PKC phosphorylation of the AT1A receptor may allow independent and/or complementary events to occur at the three separate sites of the C-terminus.

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.

Sign in / Sign up

Export Citation Format

Share Document