scholarly journals Biochemical and Biological Functions of the N-Terminal, Noncatalytic Domain of Extracellular Signal-Regulated Kinase 2

2001 ◽  
Vol 21 (1) ◽  
pp. 249-259 ◽  
Author(s):  
Scott T. Eblen ◽  
Andrew D. Catling ◽  
Marcela C. Assanah ◽  
Michael J. Weber
Keyword(s):  
Kinase Activity ◽  
Mutational Analysis ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Transcriptional Responses ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

ABSTRACT Extracellular signal-regulated kinase 1 (ERK1) and ERK2 are important components in signal transduction pathways involved in many cellular processes, including cell differentiation and proliferation. These proteins consist of a central kinase domain flanked by short N- and C-terminal noncatalytic domains. While the regulation of ERK2 by sequences within the kinase domain has been extensively studied, little is known about the small regions outside of the kinase domain. We performed mutational analysis on the N-terminal, noncatalytic domain of ERK2 in an attempt to determine its role in ERK2 function and regulation. Deleting or mutating amino acids 19 to 25 (ERK2-Δ19-25) created an ERK2 molecule that could be phosphorylated in response to growth factor and serum stimulation in a MEK (mitogen-activated protein kinase kinase or ERK kinase)-dependent manner but had little kinase activity and was unable to bind to MEK in vivo. Since MEK acts as a cytoplasmic anchor for the ERKs, the lack of a MEK interaction resulted in the aberrant nuclear localization of ERK2-Δ19-25 mutants in serum-starved cells. Assaying these mutants for their ability to affect ERK signaling revealed that ERK2-Δ19-25 mutants acted in a dominant-negative manner to inhibit transcriptional signaling through endogenous ERKs to an Elk1-responsive promoter in transfected COS-1 cells. However, ERK2-Δ19-25 had no effect on the phosphorylation of RSK2, an ERK2 cytoplasmic substrate, whereas a nonactivatable ERK (T183A) that retained these sequences could inhibit RSK2 phosphorylation. These results suggest that the N-terminal domain of ERK2 profoundly affects ERK2 localization, MEK binding, kinase activity, and signaling and identify a novel dominant-negative mutant of ERK2 that can dissociate at least some transcriptional responses from cytoplasmic responses.

scholarly journals Activated R-Ras, Rac1, Pi 3-Kinase and Pkcε Can Each Restore Cell Spreading Inhibited by Isolated Integrin β1 Cytoplasmic Domains

2000 ◽  
Vol 151 (7) ◽  
pp. 1549-1560 ◽  
Author(s):  
Allison L. Berrier ◽  
Anthony M. Mastrangelo ◽  
Julian Downward ◽  
Mark Ginsberg ◽  
Susan E. LaFlamme
Keyword(s):  
Kinase Activity ◽  
Cell Spreading ◽  
Cell Types ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Signaling Proteins ◽  
Dependent Manner ◽  
Cytoplasmic Domains ◽  
Dominant Negative Inhibitor

Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact β cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKCε and Rac1; however, it is not known whether they do so through a mechanism involving integrin β cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin β cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-β1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110α-CAAX, myr-PKCε, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-β1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKCε required an intact kinase domain. Importantly, each of these signaling proteins required intact β cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKCε each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110α-CAAX and myr-PKCε to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCε require the function of integrin β cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCε in a pathway involving integrin β cytoplasmic domain function in cell spreading.

scholarly journals Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development

2007 ◽  
Vol 176 (5) ◽  
pp. 709-718 ◽  
Author(s):  
Chunxi Ge ◽  
Guozhi Xiao ◽  
Di Jiang ◽  
Renny T. Franceschi
Keyword(s):  
Transcriptional Activity ◽  
Mapk Pathway ◽  
Skeletal Development ◽  
Critical Role ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.

scholarly journals The Transcription Factor GATA4 Is Activated by Extracellular Signal-Regulated Kinase 1- and 2-Mediated Phosphorylation of Serine 105 in Cardiomyocytes

2001 ◽  
Vol 21 (21) ◽  
pp. 7460-7469 ◽  
Author(s):  
Qiangrong Liang ◽  
Russell J. Wiese ◽  
Orlando F. Bueno ◽  
Yan-Shan Dai ◽  
Bruce E. Markham ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Cardiomyocyte Hypertrophy ◽  
Hypertrophic Growth ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

ABSTRACT The zinc finger-containing transcription factor GATA4 has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Here we demonstrate that GATA4 is itself regulated by the mitogen-activated protein kinase signaling cascade through direct phosphorylation. Site-directed mutagenesis and phospho-specific GATA4 antiserum revealed serine 105 as the primary site involved in agonist-induced phosphorylation of GATA4. Infection of cultured cardiomyocytes with an activated MEK1-expressing adenovirus induced robust phosphorylation of serine 105 in GATA4, while a dominant-negative MEK1-expressing adenovirus blocked agonist-induced phosphorylation of serine 105, implicating extracellular signal-regulated kinase (ERK) as a GATA4 kinase. Indeed, bacterially purified ERK2 protein directly phosphorylated purified GATA4 at serine 105 in vitro. Phosphorylation of serine 105 enhanced the transcriptional potency of GATA4, which was sensitive to U0126 (MEK1 inhibitor) but not SB202190 (p38 inhibitor). Phosphorylation of serine 105 also modestly enhanced the DNA binding activity of bacterially purified GATA4. Finally, induction of cardiomyocyte hypertrophy with an activated MEK1-expressing adenovirus was blocked with a dominant-negative GATA4-engrailed-expressing adenovirus. These results suggest a molecular pathway whereby MEK1-ERK1/2 signaling regulates cardiomyocyte hypertrophic growth through the transcription factor GATA4 by direct phosphorylation of serine 105, which enhances DNA binding and transcriptional activation.

Ultrafine carbon black particles stimulate proliferation of human airway epithelium via EGF receptor-mediated signaling pathway

2004 ◽  
Vol 287 (6) ◽  
pp. L1127-L1133 ◽  
Author(s):  
Jun Tamaoki ◽  
Kazuo Isono ◽  
Kiyoshi Takeyama ◽  
Etsuko Tagaya ◽  
Junko Nakata ◽  
...  
Keyword(s):  
Carbon Black ◽  
Airway Epithelium ◽  
Ultrafine Particles ◽  
Egf Receptor ◽  
Kinase Inhibitor ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Dependent Manner

Exposure to ambient ultrafine particles induces airway inflammatory reactions and tissue remodeling. In this experiment, to determine whether ultrafine carbon black (ufCB) affects proliferation of airway epithelium and, if so, what the mechanism of action is, we studied human primary bronchial epithelial cell cultures. Incubation of cells in the serum-free medium with ufCB increased incorporations of [3H]thymidine and [3H]leucine into cells in a time- and dose-dependent manner. This effect was attenuated by Cu- and Zn-containing superoxide dismutase (Cu/Zn SOD) and apocynin, an inhibitor of NADPH oxidase, and completely inhibited by pretreatment with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitors AG-1478 and BIBX-1382, and the mitogen-activated protein kinase kinase inhibitor PD-98059. Transfection of a dominant-negative mutant of H-Ras likewise abolished the effect ufCB. Stimulation with ufCB also induced processing of membrane-anchored proheparin-binding (HB)-EGF, release of soluble HB-EGF into the medium, association of phosphorylated EGF-R and Shc with glutathione- S-transferase-Grb2 fusion protein, and phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment with AG-1478, [Glu52] Diphtheria toxin, a specific inhibitor of HB-EGF, neutralizing HB-EGF antibody, Cu/Zn SOD, and apocynin each inhibited ufCB-induced ERK activation. These results suggest that ufCB causes oxidative stress-mediated proliferation of airway epithelium, involving processing of HB-EGF and the concomitant activation of EGF-R and ERK cascade.

scholarly journals Potentiation of liver X receptor transcriptional activity by peroxisome-proliferator-activated receptor gamma co-activator 1alpha

2003 ◽  
Vol 371 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Hannes OBERKOFLER ◽  
Elisabeth SCHRAML ◽  
Franz KREMPLER ◽  
Wolfgang PATSCH
Keyword(s):  
Mutational Analysis ◽  
Liver X Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Brown Adipocyte ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Transcriptional Responses ◽  
Response Element ◽  
Peroxisome Proliferator Activated Receptor ◽  
Co Activation

Peroxisome-proliferator-activated receptor (PPAR) γ co-activator 1α (PGC-1α/PPARGC1) plays an important role in energy metabolism by co-ordinating transcriptional programmes of mitochondrial biogenesis, adaptive thermogenesis and fatty acid β-oxidation. PGC-1α has also been identified to play a role in the intermediary metabolism by co-activating key transcription factors of hepatic gluconeogenesis and glucose uptake in muscles. In the present study, we show that PGC-1α serves as a co-activator for the liver X receptor (LXR) α, known to contribute to the regulation of cellular cholesterol homoeostasis. In transient transfection studies, PGC-1α amplified the LXR-mediated autoregulation of the LXRα promoter in a human brown adipocyte line and in 3T3-L1 cells via an LXR response element described previously. LXR-mediated transactivation via a natural LXR response element from the cholesteryl ester transfer-protein gene promoter was also enhanced by PGC-1α in a ligand-dependent manner. Mutational analysis showed that the LXXLL signature motif (L2) of PGC-1α was essential for co-activation of LXR-mediated transcriptional responses. This motif is located in the vicinity of the binding region for a putative repressor described previously. The repressor sequesters PGC-1α from PPARα and the glucocorticoid receptor, and this repressor did not interfere with PGC-1α-mediated co-activation of LXR-dependent gene transcription. Moreover, inhibition of p38 mitogen-activated protein kinase signalling, shown to abolish the co-activation of PPARα by PGC-1α, had only a moderate inhibitory effect on the co-activation of LXR. These results identify PGC-1α as a bona fide LXR co-activator and implicate distinct interfaces of PGC-1α and/or additional cofactors in the modulation of LXR and PPARα transcriptional activities.

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.

MAPK-induced Ser727 phosphorylation promotes SUMOylation of STAT1

2007 ◽  
Vol 409 (1) ◽  
pp. 179-185 ◽  
Author(s):  
Sari Vanhatupa ◽  
Daniela Ungureanu ◽  
Maija Paakkunainen ◽  
Olli Silvennoinen
Keyword(s):  
Interferon Γ ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Microbial Infections ◽  
Mitogen Activated Protein ◽  
P38mapk Pathway ◽  
Ifn Γ

STAT1 (signal transducer and activator of transcription 1) is a critical mediator of IFN-γ (interferon-γ)-induced gene responses, and its function is regulated through phosphorylation of Tyr701 and Ser727. MAPK (mitogen-activated protein kinase) pathways mediate phosphorylation of Ser727 in response to microbial infections, stress stimuli and growth factors. Recently, STAT1 was found to become modified by PIAS (protein inhibitor of activated STAT)-mediated SUMO-1 (small ubiquitin-related modifier-1) conjugation at Lys703, but the regulation of this modification is largely unknown. Here, we have investigated the role of MAPK-induced Ser727 phosphorylation in regulation of STAT1 SUMOylation. Activation of the p38MAPK pathway by upstream activating kinase, MKK6 (MAPK kinase-6) or osmotic stress enhanced the SUMOylation of STAT1, which was counteracted by the p38MAPK inhibitor SB202190 or by dominant-negative p38MAPK. Activation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway by Raf-1 also enhanced Ser727 phosphorylation and SUMOylation of STAT1, and this induction was counteracted by PD98059 inhibitor. Mutation of Ser727 to alanine abolished the p38MAPK-induced SUMOylation. Furthermore, S727D and S727E mutations, which mimic the phosphorylation of Ser727, enhanced the basal SUMOylation of STAT1 and interaction between PIAS1 and STAT1. Taken together, these results identify Ser727 phosphorylation as a regulator of STAT1 SUMOylation and highlight the central role of Ser727 in co-ordination of STAT1 functions in cellular responses.

PKC-ε regulation of extracellular signal-regulated kinase: a potential role in phenylephrine-induced cardiocyte growth

2004 ◽  
Vol 286 (6) ◽  
pp. H2195-H2203 ◽  
Author(s):  
Vijay U. Rao ◽  
Hirokazu Shiraishi ◽  
Paul J. McDermott
Keyword(s):  
Growth Regulation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Neonatal Rat ◽  
Inhibitory Effects ◽  
Hypertrophic Growth ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Acute Increase

Hypertrophic growth of cardiac muscle is dependent on activation of the PKC-ε isoform. To define the effectors of PKC-ε involved in growth regulation, recombinant adenoviruses were used to overexpress either wild-type PKC-ε (PKC-ε/WT) or dominant negative PKC-ε (PKC-ε/DN) in neonatal rat cardiocytes. PKC-ε/DN inhibited acute activation of PKC-ε produced in response to phorbol ester and reduced ERK1/2 activity as measured by the phosphorylation of p42 and p44 isoforms. The inhibitory effects were specific to PKC-ε because PKC-ε/DN did not prevent translocation of either PKC-α or PKC-δ. Overexpression of PKC-ε/DN blunted the acute increase in ERK1/2 phorphorylation induced by the α1-adrenergic agonist phenylephrine (PE ). Inhibition of PKC-δ with rottlerin potentiated the effects of PE on ERK1/2 phosphorylation. PKC-ε/DN adenovirus also blocked cardiocyte growth as measured after 48 h of PE treatment, although the multiplicity of infection was lower than that required to block acute ERK1/2 activation. PE activated p38 mitogen-activated protein kinase as measured by its phosphorylation, but the response was not blocked by PKC inhibitors or by overexpression of PKC-ε/DN. Taken together, these studies show that the hypertrophic agonist PE regulates ERK1/2 activity in cardiocytes by a pathway dependent on PKC-ε and that PE-induced growth is mediated by PKC-ε.

scholarly journals Impact of Feedback Phosphorylation and Raf Heterodimerization on Normal and Mutant B-Raf Signaling

2009 ◽  
Vol 30 (3) ◽  
pp. 806-819 ◽  
Author(s):  
Daniel A. Ritt ◽  
Daniel M. Monson ◽  
Suzanne I. Specht ◽  
Deborah K. Morrison
Keyword(s):  
Binding Site ◽  
Developmental Disorders ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Prolyl Isomerase ◽  
Ras Pathway ◽  
Extracellular Signal Regulated Kinase ◽  
Raf Kinase ◽  
Extracellular Signal ◽  
Signaling Activity

ABSTRACT The B-Raf kinase is a Ras pathway effector activated by mutation in numerous human cancers and certain developmental disorders. Here we report that normal and oncogenic B-Raf proteins are subject to a regulatory cycle of extracellular signal-regulated kinase (ERK)-dependent feedback phosphorylation, followed by PP2A- and Pin1-dependent dephosphorylation/recycling. We identify four S/TP sites of B-Raf phosphorylated by activated ERK and find that feedback phosphorylation of B-Raf inhibits binding to activated Ras and disrupts heterodimerization with C-Raf, which is dependent on the B-Raf pS729/14-3-3 binding site. Moreover, we find that events influencing Raf heterodimerization can alter the transforming potential of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity but have no significant effect on proteins with high kinase activity, such as V600E B-Raf. Mutation of the feedback sites or overexpression of the Pin1 prolyl-isomerase, which facilitates B-Raf dephosphorylation/recycling, resulted in increased transformation, whereas mutation of the S729/14-3-3 binding site or expression of dominant negative Pin1 reduced transformation. Mutation of each feedback site caused increased transformation and correlated with enhanced heterodimerization and activation of C-Raf. Finally, we find that B-Raf and C-Raf proteins containing mutations identified in certain developmental disorders constitutively heterodimerize and that their signaling activity can also be modulated by feedback phosphorylation.

Sign in / Sign up

Export Citation Format

Share Document