scholarly journals Regulation of the gene promoter for extracellular signal-regulated protein kinase 2 by transcription factors NF-Y and Sp3

2000 ◽  
Vol 347 (1) ◽  
pp. 155-161 ◽  
Author(s):  
Naoaki SUGIURA ◽  
Kunio TAKISHIMA
Keyword(s):  
Transcription Factors ◽  
Protein Kinase ◽  
Promoter Activity ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Extracellular Signal ◽  
Promoter Deletion Analysis ◽  
Ccaat Box

We have previously shown that the maximal promoter activity of the gene for extracellular signal-regulated protein kinase 2 (ERK2; also known as p42 mitogen-activated protein kinase) resides in the 371 bp 5ʹ-flanking sequence. In the present study we defined roles for a CCAAT box and two adjacent GC boxes in the activity of this promoter. Deletion analysis and DNase I footprinting of this 371 bp region indicated that the CCAAT box at -64 and GC boxes at -86 and -39 are crucial for promoter activity. Electrophoretic mobility-shift assays showed that transcription factor NF-Y/CBF binds to the CCAAT box. Sp1 and Sp3, members of the Sp family of transcription factors, bind to the GC boxes of the ERK2 promoter. The binding of Sp3 was predominant over that of Sp1. Disruption by mutation of any of the CCAAT box and GC boxes similarly decreased promoter activity. These three cis elements exhibited a moderate synergy in promoter function. The transactivating role of NF-Y was corroborated by the finding that a dominant-negative form of NF-YA diminished the promoter activity. These results provide clues for refining our understanding of not only the regulation of expression of the gene for ERK2 but also mechanisms by which NF-Y and Sp1/Sp3 regulate transcription.

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.

Phorbol ester-induced activation of mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase and extracellular-signal-regulated protein kinase decreases glucose-6-phosphatase gene expression

2001 ◽  
Vol 357 (3) ◽  
pp. 867-873 ◽  
Author(s):  
Dieter SCHMOLL ◽  
Rolf GREMPLER ◽  
Andreas BARTHEL ◽  
Hans-Georg JOOST ◽  
Reinhard WALTHER
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Regulation Of Gene Expression ◽  
Insulin Response ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Glucose-6-phosphatase (G6Pase) plays a central role in blood glucose homoeostasis, and insulin suppresses G6Pase gene expression by the activation of phosphoinositide 3-kinase (PI 3-kinase). Here, we show that the phorbol ester PMA decreases both basal and dexamethasone/cAMP-induced expression of a luciferase gene under the control of the G6Pase promoter in transiently transfected H4IIE hepatoma cells. This regulation was suppressed by the inhibitors of the mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK), PD98059 and U0126, but not by the inhibitor of PI 3-kinase, LY294002. The co-expression of a constitutively active mutant of MEK mimicked the regulation of G6Pase promoter activity by PMA. The effect of PMA on both basal and induced G6Pase gene transcription was impaired by the overexpression of a dominant negative MEK construct, as well as by the expression of mitogen-activated protein kinase phosphatase-1. The mutation of the forkhead-binding sites within the insulin-response unit of the G6Pase promoter, which decreases the effect of insulin on G6Pase gene expression, did not alter the regulation of gene expression by PMA. The data show that PMA decreases G6Pase gene expression by the activation of MEK and extracellular-signal regulated protein kinase. With that, PMA mimics the effect of insulin on G6Pase gene expression by a different signalling pathway.

scholarly journals Regulation of the human involucrin gene promoter by co-activator proteins

2004 ◽  
Vol 381 (1) ◽  
pp. 267-273 ◽  
Author(s):  
Nhu Q. TRAN ◽  
David L. CROWE
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Promoter Activity ◽  
Binding Protein ◽  
Regulatory Region ◽  
Terminal Differentiation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Activator Proteins ◽  
Involucrin Promoter

Stratified squamous epithelial cells undergo an orderly process of terminal differentiation that is characterized by specific molecular and morphological changes, including expression of the cornified envelope protein involucrin. Significant progress has been made in characterizing the upstream regulatory region of the involucrin gene. Binding sites for AP-1 (activator protein 1) and Sp1 transcription factors were shown to be important for involucrin promoter activity and tissue-specific expression. Defective terminal differentiation is often characterized by decreased or lack of involucrin expression. Recently, a dominant-negative construct of the transcriptional co-activator P/CAF [p300/CBP-associated factor, where CBP stands for CREB (cAMP-response-element-binding protein)-binding protein] was shown to inhibit involucrin expression in immortalized keratinocytes [Kawabata, Kawahara, Kanekura, Araya, Daitoku, Hata, Miura, Fukamizu, Kanzaki, Maruyama and Nakajima (2002) J. Biol. Chem. 277, 8099–8105]. Loss of expression or inactivation of other co-activators has also been demonstrated [Suganuma, Kawabata, Ohshima, and Ikeda (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 13073–13078]. In the present study, we re-expressed CBP and P/CAF in immortalized keratinocyte lines that had lost expression of these co-activator proteins. Re-expression of these proteins restored calcium- and RA (retinoic acid)-responsive involucrin expression in these cells. RA and calcium signalling induced exchange of CBP and P/CAF occupancy at the AP-1 sites of the involucrin promoter. CBP and P/CAF inductions of the involucrin expression were not dependent on MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase), p38, protein kinase C or CaM kinase (calcium/calmodulin-dependent kinase) signalling. Kinase-induced changes in involucrin promoter activity directly resulted from changes in AP-1 protein expression. We concluded that CBP and P/CAF are important regulators of involucrin expression in stratified squamous epithelial cells.

scholarly journals Contrasting effects of ERK on tight junction integrity in differentiated and under-differentiated Caco-2 cell monolayers

2010 ◽  
Vol 433 (1) ◽  
pp. 51-63 ◽  
Author(s):  
Sudhir Aggarwal ◽  
Takuya Suzuki ◽  
William L. Taylor ◽  
Aditi Bhargava ◽  
Radhakrishna K. Rao
Keyword(s):  
Protein Kinase ◽  
Tight Junction ◽  
Tight Junctions ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Cell Monolayers ◽  
Differentiated Cells ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ERK (extracellular-signal-regulated kinase) activation leads to disruption of tight junctions in some epithelial monolayers, whereas it prevents disruption of tight junctions in other epithelia. The factors responsible for such contrasting influences of ERK on tight junction integrity are unknown. The present study investigated the effect of the state of cell differentiation on ERK-mediated regulation of tight junctions in Caco-2 cell monolayers. EGF (epidermal growth factor) potentiated H2O2-induced tight junction disruption in under-differentiated cell monolayers, which was attenuated by the MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitor U0126. In contrast, EGF prevented H2O2-induced disruption of tight junctions in differentiated cell monolayers, which was also attenuated by U0126. Knockdown of ERK1/2 enhanced tight junction integrity and accelerated assembly of tight junctions in under-differentiated cell monolayers, whereas it had the opposite effect in differentiated cell monolayers. Regulated expression of wild-type and constitutively active MEK1 disrupted tight junctions, and the expression of dominant-negative MEK1 enhanced tight junction integrity in under-differentiated cells, whereas contrasting responses were recorded in differentiated cells. EGF prevented both H2O2-induced association of PP2A (protein phosphatase 2A), and loss of association of PKCζ (protein kinase Cζ), with occludin by an ERK-dependent mechanism in differentiated cell monolayers, but not in under-differentiated cell monolayers. Active ERK was distributed in the intracellular compartment in under-differentiated cell monolayers, whereas it was localized mainly in the perijunctional region in differentiated cell monolayers. Thus ERK may exhibit its contrasting influences on tight junction integrity in under-differentiated and differentiated epithelial cells by virtue of differences in its subcellular distribution and ability to regulate the association of PKCζ and PP2A with tight junction proteins.

scholarly journals Rac-PAK Signaling Stimulates Extracellular Signal-Regulated Kinase (ERK) Activation by Regulating Formation of MEK1-ERK Complexes

2002 ◽  
Vol 22 (17) ◽  
pp. 6023-6033 ◽  
Author(s):  
Scott T. Eblen ◽  
Jill K. Slack ◽  
Michael J. Weber ◽  
Andrew D. Catling
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Signaling Complex ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-Δ19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-Δ19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.

scholarly journals Ras triggers acidosis-induced activation of the extracellular-signal-regulated kinase pathway in cardiac myocytes

2006 ◽  
Vol 399 (3) ◽  
pp. 493-501 ◽  
Author(s):  
Robert S. Haworth ◽  
Semjidmaa Dashnyam ◽  
Metin Avkiran
Keyword(s):  
Protein Kinase ◽  
Cardiac Myocytes ◽  
Intracellular Ph ◽  
Ph Dependence ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Nucleotide Exchange ◽  
Intracellular Acidosis ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

In cardiac myocytes, sustained (3 min) intracellular acidosis activates the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway and, through this pathway, increases sarcolemmal NHE (Na+/H+ exchanger) activity [Haworth, McCann, Snabaitis, Roberts and Avkiran (2003) J. Biol. Chem. 278, 31676–31684]. In the present study, we aimed to determine the time-dependence, pH-dependence and upstream signalling mechanisms of acidosis-induced ERK1/2 activation in ARVM (adult rat ventricular myocytes). Cultured ARVM were subjected to intracellular acidosis for up to 20 min by exposure to NH4Cl, followed by washout with a bicarbonate-free Tyrode solution containing the NHE1 inhibitor cariporide. After the desired duration of intracellular acidosis, the phosphorylation status of ERK1/2 and its downstream effector p90RSK (90 kDa ribosomal S6 kinase) were determined by Western blotting. This revealed a time-dependent transient phosphorylation of both ERK1/2 and p90RSK by intracellular acidosis (intracellular pH ∼6.6), with maximum activation occurring at 3 min and a return to basal levels by 20 min. When the degree of intracellular acidosis was varied from ∼6.8 to ∼6.5, maximum ERK1/2 phosphorylation was observed at an intracellular pH of 6.64. Inhibition of MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK kinase 1/2) by pre-treatment of ARVM with U0126 or adenoviral expression of dominant-negative D208A-MEK1 protein prevented the phosphorylation of ERK1/2 by sustained intracellular acidosis, as did inhibition of Raf-1 with GW 5074 or ZM 336372. Interference with Ras signalling by the adenoviral expression of dominant-negative N17-Ras protein or with FPT III (farnesyl protein transferase inhibitor III) also prevented acidosis-induced ERK1/2 phosphorylation, whereas inhibiting G-protein signalling [by adenoviral expression of RGS4 or Lsc, the RGS domain of p115 RhoGEF (guanine nucleotide-exchange factor)] or protein kinase C (with bisindolylmaleimide I) had no effect. Our data show that, in ARVM, sustained intracellular acidosis activates ERK1/2 through proximal activation of the classical Ras/Raf/MEK pathway.

scholarly journals Regulation of Na+/H+exchanger gene expression: mitogenic stimulation increases NHE1 promoter activity

1998 ◽  
Vol 274 (3) ◽  
pp. C831-C839 ◽  
Author(s):  
Pierre Besson ◽  
Francoise Fernandez-Rachubinski ◽  
Weidong Yang ◽  
Larry Fliegel
Keyword(s):  
Protein Kinase ◽  
Promoter Activity ◽  
Phorbol Esters ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitors ◽  
Proximal Fragment ◽  
Mobility Shift ◽  
Regulation Of Expression ◽  
Mitogenic Stimulation ◽  
Promoter Deletion Analysis

We examined factors important in regulation of expression of the Na+/H+exchanger gene in NIH/3T3 cells. A stable fibroblast cell line was generated that contained a 1.1-kb proximal fragment of the mouse NHE1 promoter. The addition of serum to serum-starved cells resulted in an increase in activity of the NHE1 promoter. The mitogenic agonists insulin, thrombin, and epidermal growth factor also increased transcription from the NHE1 promoter. Phorbol esters also increased NHE1 promoter-directed transcription, whereas the serine/threonine protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine inhibited this stimulation. The protein kinase inhibitors GF-109203X, PD-98059, and genistein all stimulated promoter activity. Promoter deletion analysis and gel mobility shift assays showed that a region between 0.9 and 1.1 kb from the start site was involved in mediating the effect of mitogenic stimulation. The results show that a variety of mitogenic factors can activate the NHE1 promoter during cell growth and proliferation.

Regulation and composition of activator protein 1 (AP-1) transcription factors controlling collagenase and c-Jun promoter activities

2001 ◽  
Vol 360 (3) ◽  
pp. 599-607 ◽  
Author(s):  
Lars STEINMÜLLER ◽  
Giuseppe CIBELLI ◽  
Jonathan R. MOLL ◽  
Charles VINSON ◽  
Gerald THIEL
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Leucine Zipper ◽  
Dominant Negative ◽  
Activator Protein 1 ◽  
Activator Protein ◽  
Extracellular Signal ◽  
Enhancer Binding Protein ◽  
Activating Transcription Factor

The activator protein 1 (AP-1) transcription factor is composed of heterodimers of the Fos/activating transcription factor (ATF) and Jun subfamilies of basic-region leucine-zipper (B-ZIP) proteins. In order to determine the identities of individual B-ZIP proteins in various AP-1 complexes we tested the effect of dominant-negative mutants to the B-ZIP proteins c-Fos, ATF2, ATF4 and CCAAT-enhancer-binding protein (C/EBP) on the activities of the collagenase and c-Jun promoters. These dominant-negative mutants inhibit DNA binding of wild-type B-ZIP proteins in a leucine-zipper-dependent fashion. Transcription of a collagenase promoter/reporter gene was induced in HepG2 hepatoma cells by expression of c-Fos and c-Jun, administration of PMA (‘TPA’) or by expression of a truncated form of MEK (mitogen-activated/extracellular-signal-regulated kinase kinase) kinase-1, MEKK1Δ. In all cases, the dominant-negative mutants A-Fos and A-ATF2 decreased collagenase promoter activity. However, A-ATF4 and A-C/EBP had no effect. A-Fos and A-ATF2 also reduced MEKK1Δ-induced stimulation of the c-Jun promoter. In contrast, constitutive c-Jun promoter activity was blocked solely by A-ATF2, strongly suggesting that ATF2 and/or an ATF2-dimerizing protein are of major importance for c-Jun transcription in unstimulated cells. These results demonstrate that AP-1 transcription factors of different compositions control c-jun gene transcription in resting or stimulated cells.

The Ser186 phospho-acceptor site within ERK4 is essential for its ability to interact with and activate PRAK/MK5

2008 ◽  
Vol 411 (3) ◽  
pp. 613-622 ◽  
Author(s):  
Maria Perander ◽  
Espen Åberg ◽  
Bjarne Johansen ◽  
Bo Dreyer ◽  
Ingrid J. Guldvik ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Acceptor Site ◽  
Wild Type ◽  
Mobility Shift ◽  
Extracellular Signal ◽  
Sds Page ◽  
Mitogen Activated Protein ◽  
Reduced Mobility

ERK (extracellular-signal-regulated kinase) 4 [MAPK (mitogen-activated protein kinase) 4] and ERK3 (MAPK6) are atypical MAPKs. One major difference between these proteins and the classical MAPKs is substitution of the conserved T-X-Y motif within the activation loop by a single phospho-acceptor site within an S-E-G motif. In the present study we report that Ser186 of the S-E-G motif in ERK4 is phosphorylated in vivo. Kinase-dead ERK4 is also phosphorylated on Ser186, indicating that an ERK4 kinase, rather than autophosphorylation, is responsible. Co-expression of MK5 [MAPK-activated protein kinase 5; also known as PRAK (p38-regulated/activated kinase)], a physiological target of ERK4, increases phosphorylation of Ser186. This is not dependent on MK5 activity, but does require interaction between ERK4 and MK5 suggesting that MK5 binding either prevents ERK4 dephosphorylation or facilitates ERK4 kinase activity. ERK4 mutants in which Ser186 is replaced with either an alanine residue or a phospho-mimetic residue (glutamate) are unable to activate MK5 and Ser186 is also required for cytoplasmic anchoring of MK5. Both defects seem to reflect an impaired ability of the ERK4 mutants to interact with MK5. We find that there are at least two endogenous pools of wild-type ERK4. One form exhibits reduced mobility when analysed using SDS/PAGE. This is due to MK5-dependent phosphorylation and only this retarded ERK4 species is both phosphorylated on Ser186 and co-immunoprecipitates with wild-type MK5. We conclude that binding between ERK4 and MK5 facilitates phosphorylation of Ser186 and stabilization of the ERK4–MK5 complex. This results in phosphorylation and activation of MK5, which in turn phosphorylates ERK4 on sites other than Ser186 resulting in the observed mobility shift.

scholarly journals Role of phosphoinositide 3-kinase and the Cbl adaptor protein in coupling the α4β1 integrin to mitogen-activated protein kinase signalling

2000 ◽  
Vol 345 (2) ◽  
pp. 385-392 ◽  
Author(s):  
Lisa D. FINKELSTEIN ◽  
Yoji SHIMIZU
Keyword(s):  
Protein Kinase ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Hl60 Cells ◽  
Intracellular Signals ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Phosphoinositide 3 Kinase ◽  
Stimulation Of

Cell adhesion mediated by β1 integrin receptors leads to the initiation of intracellular signals that affect cell differentiation and survival. Here we have analysed the mechanism by which the α4β1 integrin activates the mitogen-activated protein kinase pathway in HL60 cells, a myelomonocytic cell line that lacks the expression of focal adhesion kinase. A role for phosphoinositide 3-kinase (PI-3K) in α4 integrin-mediated activation of extracellular signal-regulated protein kinase 2 (ERK2) is suggested by the ability of PI-3K inhibitors and a dominant-negative form of the p85 subunit of PI-3K to block the activation of ERK2 by integrin. Stimulation of α4β1 integrins on HL60 cells also leads to increased tyrosine phosphorylation of the 120 kDa adaptor protein Cbl. PI-3K activity associated with Cbl also increases on the stimulation of α4β1 integrins, although immunodepletion experiments suggest that Cbl-associated PI-3K does not account for all of the PI-3K activity induced on the stimulation of integrins in these cells. The expression of wild-type Cbl or the 70Z/3 Cbl mutant enhances basal ERK2 activity in transfectants with a minimal effect on α4 integrin-mediated ERK2 activity. In contrast, overexpression of the Hut Cbl truncation mutant, which does not associate with p85, has no effect on the ERK2 pathway. These results suggest that PI-3K has a major role in coupling α4β1 integrins to ERK2 activation in myeloid cells and that the Cbl adaptor protein has a role in basal, but not α4β1 integrin-mediated, activation of ERK2.

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