scholarly journals Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

1996 ◽  
Vol 16 (4) ◽  
pp. 1842-1850 ◽  
Author(s):  
G Baier-Bitterlich ◽  
F Uberall ◽  
B Bauer ◽  
F Fresser ◽  
H Wachter ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Pkc Alpha ◽  
Transcriptional Complex ◽  
Induced Signal ◽  
Transcription Factor Complex

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.

Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC

2002 ◽  
Vol 283 (2) ◽  
pp. E338-E345 ◽  
Author(s):  
Masatoshi Tsuru ◽  
Hideki Katagiri ◽  
Tomoichiro Asano ◽  
Tetsuya Yamada ◽  
Shigeo Ohno ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Dominant Negative ◽  
Transport Activity ◽  
Wild Type ◽  
Atypical Pkc ◽  
Endogenous Expression ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Pkc Ζ

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-α, novel PKC-δ, and atypical PKC isoforms of PKC-λ and PKC-ζ, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-α and PKC-λ/ζ, but not of PKC-δ, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-α and exogenous PKC-δ but not atypical PKC-λ/ζ. Insulin also activated the overexpressed PKC-δ but not PKC-α. Expression of the wild-type PKC-α or PKC-δ resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-α expression, which inhibited the PMA activation of PKC-α, decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-δ but not of PKC-α. These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-λ/ζ was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.

scholarly journals p110 CUX1 Cooperates with E2F Transcription Factors in the Transcriptional Activation of Cell Cycle-Regulated Genes

2008 ◽  
Vol 28 (10) ◽  
pp. 3127-3138 ◽  
Author(s):  
Mary Truscott ◽  
Ryoko Harada ◽  
Charles Vadnais ◽  
François Robert ◽  
Alain Nepveu
Keyword(s):  
Cell Cycle ◽  
Dna Polymerase ◽  
Transcriptional Activation ◽  
Affinity Purification ◽  
Gene Promoter ◽  
Dominant Negative ◽  
In Vitro Assays ◽  
Dominant Negative Mutant ◽  
Dna Polymerase Α ◽  
Reporter Assays

ABSTRACT The transcription factor p110 CUX1 was shown to stimulate cell proliferation by accelerating entry into S phase. As p110 CUX1 can function as a transcriptional repressor or activator depending on promoter context, we investigated its mechanism of transcriptional activation using the DNA polymerase α gene promoter as a model system. Linker-scanning analysis revealed that a low-affinity E2F binding site is required for transcriptional activation. Moreover, coexpression with a dominant-negative mutant of DP-1 suggested that endogenous E2F factors are indeed needed for p110-mediated activation. Tandem affinity purification, coimmunoprecipitation, chromatin immunoprecipitation, and reporter assays indicated that p110 CUX1 can engage in weak protein-protein interactions with E2F1 and E2F2, stimulate their recruitment to the DNA polymerase α gene promoter, and cooperate with these factors in transcriptional activation. On the other hand, in vitro assays suggested that the interaction between CUX1 and E2F1 either is not direct or is regulated by posttranslational modifications. Genome-wide location analysis revealed that targets common to p110 CUX1 and E2F1 included many genes involved in cell cycle, DNA replication, and DNA repair. Comparison of the degree of enrichment for various E2F factors suggested that binding of p110 CUX1 to a promoter will favor the specific recruitment of E2F1, and to a lesser extent E2F2, over E2F3 and E2F4. Reporter assays on a subset of common targets confirmed that p110 CUX1 and E2F1 cooperate in their transcriptional activation. Overall, our results show that p110 CUX1 and E2F1 cooperate in the regulation of many cell cycle genes.

PDGF and IL-1 induce and activate specific protein kinase C isoforms in mesangial cells

1996 ◽  
Vol 271 (1) ◽  
pp. F108-F113 ◽  
Author(s):  
M. B. Ganz ◽  
B. Saksa ◽  
R. Saxena ◽  
K. Hawkins ◽  
J. R. Sedor
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
De Novo ◽  
Mesangial Cells ◽  
De Novo Synthesis ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Alpha Beta ◽  
Pkc Alpha

In vitro and in vivo data suggest a remarkable plasticity in the differentiated phenotype of intrinsic glomerular cells, which after injury express new structures and functions. We have shown that a protein kinase C (PKC) isoform, beta II, is expressed in diseased but not normal glomeruli. Since intrarenal cytokine synthesis has been implicated in the pathogenesis of progressive glomerular injury, we have hypothesized that these mediators induce a change in isoform profile. To test this hypothesis in vitro, we have determined whether platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) alter the expression or activation of PKC isoforms in cultured mesangial cells (MCs). By immunoblot and ribonuclease (RNase) protection assays, both PDGF and IL-1 induce as early as 2 h de novo synthesis of PKC-beta II. Since MCs constitutively express PKC-alpha, -beta I, and -zeta, we also determined whether IL-1 or PDGF alter the activity of these isoforms. PDGF maximally induced translocation of PKC-alpha (10 min), -beta I (90 min), -epsilon (120 min), and -zeta (120 min) from the cytosolic to the membrane fraction. IL-1, in contrast, did not alter the distribution of alpha, beta I, or epsilon at any time measured but did induce PKC-zeta translocation. These data suggest inflammatory mediators regulate PKC isoform activity in diseased glomeruli both by de novo synthesis of unexpressed isoforms and by activation of constitutively expressed PKC isoforms.

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.

Pivotal Role of Survivin in Leukemogenesis by E2A-HLF Chimeric Transcription Factor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2988-2988
Author(s):  
Mayuko Okuya ◽  
Hidemitsu Kurosawa ◽  
Takayuki Matsunaga ◽  
Mitsuoki Eguchi ◽  
Yusuke Furukawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Survivin Expression ◽  
Small Population ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Transcriptional Level ◽  
Survivin Promoter ◽  
M Phase ◽  
Negative Mutant

Abstract The E2A-HLF fusion transcription factor generated by the t(17;19)(q22;p13) translocation is found in a small population of pro-B cell ALL. Patients associated with this chimera share distinct clinical features such as hypercalcemia, coagulopathy and very poor prognosis due to resistance to intensive chemotherapy including aggressive conditioning for BMT, all of which are unusual for this type of ALL. We have previously demonstrated that inhibition of the trans-activation potential of the E2A-HLF chimera by the dominant negative mutant results in apoptosis in t(17;19)+ ALL cells but does not affect cell cycle. Moreover, E2A-HLF blocks apoptosis induced by cytokine deprivation in IL-3-dependent cells, suggesting that this fusion protein contributes to leukemogenesis by substituting for the anti-apoptotic function of cytokines. The present study shows that survivin is a downstream target molecule of E2A-HLF. Four t(17;19)+ ALL cell lines expressed survivin at high levels and down-regulation of E2A-HLF function by the dominant negative mutant suppressed survivin expression. In addition, forced expression of E2A-HLF in Nalm-6, a t(17;19)− ALL cell line, up-regulated survivin expression. Survivin is known to be expressed predominantly in the G2/M phase. Indeed, separation of the fractions enriched for in each phase of the cell cycle using a counterflow centrifugal elutriator revealed G2/M phase-dominant survivin expression in t(17;19) − ALL cells including Nalm-6. In t(17;19)+ ALL cells, however, survivin was expressed throughout the cell cycle. Moreover, Nalm-6 cells forced to express E2A-HLF showed cell cycle-independent survivin expression. Reporter assay revealed that E2A-HLF induced luciferase activity by transfecting with each reporter construct containing the survivin promoter at a different length from the initial ATG, suggesting that E2A-HLF induces survivin expression at the transcriptional level, but not by direct binding of E2A-HLF to the survivin promoter. To test whether survivin plays anti-apoptotic roles in t(17:19)+ cells, we used a survivin mutant lacking a phosphorylation site (T34A-survivin) and considered to inhibit survivin function in a dominant negative manner. T34A-survivin induced massive apoptosis throughout the cell cycle in t(17;19)+ cells. In contrast, T34A-survivin in t(17;19) − cells induced cell death in only a small population in G2/M phase. In addition to caspase-dependent pathways, T34A-survivin induced apoptosis in t(17;19)+ ALL cells through caspase-independent pathways, in which apoptosis-inducing factor (AIF) translocated from cytoplasm to the nucleus. These results indicate that cell cycle-independent up-regulation of survivin by the E2A-HLF chimera is indispensable for the survival of t(17;19)+ ALL cells, and that inhibition of survivin may offer an effective therapeutic strategy against this refractory ALL.

scholarly journals Smad3-Smad4 and AP-1 Complexes Synergize in Transcriptional Activation of the c-Jun Promoter by Transforming Growth Factor β

1999 ◽  
Vol 19 (3) ◽  
pp. 1821-1830 ◽  
Author(s):  
Carolyn Wong ◽  
Elissa M. Rougier-Chapman ◽  
Joshua P. Frederick ◽  
Michael B. Datto ◽  
Nicole T. Liberati ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Growth Factor ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
The Other ◽  
Promoter Activation ◽  
Complex Process ◽  
Transcription Factor Complex

ABSTRACT Transcriptional regulation by transforming growth factor β (TGF-β) is a complex process which is likely to involve cross talk between different DNA responsive elements and transcription factors to achieve maximal promoter activation and specificity. Here, we describe a concurrent requirement for two discrete responsive elements in the regulation of the c-Jun promoter, one a binding site for a Smad3-Smad4 complex and the other an AP-1 binding site. The two elements are located 120 bp apart in the proximal c-Jun promoter, and each was able to independently bind its corresponding transcription factor complex. The effects of independently mutating each of these elements were nonadditive; disruption of either sequence resulted in complete or severe reductions in TGF-β responsiveness. This simultaneous requirement for two distinct and independent DNA binding elements suggests that Smad and AP-1 complexes function synergistically to mediate TGF-β-induced transcriptional activation of the c-Jun promoter.

scholarly journals Involvement of JunD in transcriptional activation of the orphan receptor gene nur77 by nerve growth factor and membrane depolarization in PC12 cells

1994 ◽  
Vol 14 (12) ◽  
pp. 7731-7743
Author(s):  
J K Yoon ◽  
L F Lau
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Transcriptional Activation ◽  
Membrane Depolarization ◽  
Dominant Negative ◽  
Orphan Receptor ◽  
Early Gene ◽  
Dominant Negative Mutant ◽  
Transcription Activator ◽  
Nerve Growth

nur77, an immediate-early gene that encodes an orphan nuclear receptor, is rapidly and transiently induced by nerve growth factor (NGF) stimulation or membrane depolarization in the rat pheochromocytoma-derived cell line PC12. The Nur77 protein can act as a potent transcription activator and may function to regulate the expression of downstream genes in response to extracellular stimuli. We show here that activation of nur77 by NGF treatment and membrane depolarization is signalled through distinct pathways. These distinct signals appear to converge on the same transcription factors acting on the same promoter elements. We show that nur77 activation by both processes requires two cis-acting AP1-like elements, NAP1 and NAP2, which contain the core sequence TGCGTCA centered at 67 and 38 nucleotides upstream of the transcription start site. The NAP elements can confer inducibility by NGF and membrane depolarization on an otherwise unresponsive heterologous promoter. We identified JunD as a key mediator of nur77 activation by reason of the following observations. (i) JunD, but not CREB or other members of the Fos/Jun family, is a component of NAP binding activity in PC12 cell nuclear extracts. (ii) JunD, but not other Fos/Jun family members, specifically transactivates the nur77 promoter through the NAP elements (iii) A dominant-negative mutant of JunD effectively abolishes the activation of nur77 by either NGF treatment or membrane depolarization. These data draw a contrast between the regulation of nur77 with that of c-fos, in which the sequence requirements for activation by NGF treatment and membrane depolarization appear separable, and CREB appears to play a role in activation by both NGF and membrane depolarization.

scholarly journals Zyxin regulates migration of renal epithelial cells through activation of hepatocyte nuclear factor-1β

2013 ◽  
Vol 305 (1) ◽  
pp. F100-F110 ◽  
Author(s):  
Yun-Hee Choi ◽  
Brian T. McNally ◽  
Peter Igarashi
Keyword(s):  
Transcription Factor ◽  
Epithelial Cells ◽  
Nuclear Translocation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Epithelial Tissue ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Lim Domain ◽  
Renal Epithelial Cells

Hepatocyte nuclear factor-1β (HNF-1β) is an epithelial tissue-specific transcription factor that regulates gene expression in the kidney, liver, pancreas, intestine, and other organs. Mutations of HNF-1β in humans produce renal cysts and congenital kidney anomalies. Here, we identify the LIM-domain protein zyxin as a novel binding partner of HNF-1β in renal epithelial cells. Zyxin shuttles to the nucleus where it colocalizes with HNF-1β. Immunoprecipitation of zyxin in leptomycin B-treated cells results in coprecipitation of HNF-1β. The protein interaction requires the second LIM domain of zyxin and two distinct domains of HNF-1β. Overexpression of zyxin stimulates the transcriptional activity of HNF-1β, whereas small interfering RNA silencing of zyxin inhibits HNF-1β-dependent transcription. Epidermal growth factor (EGF) induces translocation of zyxin into the nucleus and stimulates HNF-1β-dependent promoter activity. The EGF-mediated nuclear translocation of zyxin requires activation of Akt. Expression of dominant-negative mutant HNF-1β, knockdown of zyxin, or inhibition of Akt inhibits EGF-stimulated cell migration. These findings reveal a novel pathway by which extracellular signals are transmitted to the nucleus to regulate the activity of a transcription factor that is essential for renal epithelial differentiation.

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